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### FIFRA SCIENTIFIC ADVISORY PANEL (SAP)

OPEN MEETING

SCIENTIFIC REVIEW OF THE ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP); TIER I SCREENING ASSAY AND BATTERY PERFORMANCE

DOCKET NUMBER: EPA-HQ-OPP-2013-0075

FIFRA SAP WEB SITE http://www.epa.gov/scipoly/sap/ OPP Docket Telephone: (703) 305-5805

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY CONFERENCE CENTER LOBBY LEVEL ONE POTOMAC YARD (SOUTH BUILDING) 2777 SOUTH CRYSTAL DRIVE ARLINGTON, VIRGINIA 22202 MAY 21 - 23, 2013

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1 **DR. FRED JENKINS:**If I could ask everyone to 

### 2 please get seated, we're going to go ahead and get started. 

### 3 Just give us one second to get the panel 

### 4 members all seated. 

### 5 Good morning again, everyone. I want to 

### 6 welcome everyone to this FIFRA Scientific Advisory Panel 

### 7 meeting. The topic of this meeting is the Endocrine Disruptor 

### 8 Screening Program: Tier 1 Screening Assays Battery Performance. 

### 9 My name is Fred Jenkins, and I am your 

### 10 Designated Federal Official for this meeting. Before we get 

### 11 started, I want to take just a couple of minutes to go over a 

### 12 few administrative items. As the DFO, I serve as the liaison 

### 13 between the panel and the Agency, and I'm responsible for 

### 14 ensuring that the provisions of the Federal Advisory Committee 

### 15 Act are met. I want to extend my thanks to the panel for 

### 16 agreeing to serve on this committee and as well to the public 

### 17 for attending this meeting. 

### 18 The FIFRA Scientific Advisory Panel Committee 

### 19 provides independent scientific peer review and advice to the 

### 20 Agency on pesticide issues. It's important to note that the 

### 21 panel only provides advice and recommendations to the EPA and 

### 22 that all regulatory decision making and implementation authority 

### 23 remains with the Agency. We have worked with appropriate Agency 

### 24 officials to ensure that all appropriate ethics regulations are 

### 25 satisfied for this meeting. Panel members have been provided 

### 26 the provisions of the Federal Conflict of Interest laws, and

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1 each participant has filed a financial disclosure report. I, 

### 2 along with our Deputy Ethics Officer and in consultation with 

### 3 the Office of General Counsel, have reviewed those reports to 

### 4 ensure all ethics requirements are met. 

### 5 This meeting provides for an opportunity for 

### 6 public comments. We will have several people who will be 

### 7 speaking during the public comment period, if you have not made 

### 8 prior arrangements and wish to make public comments at this 

### 9 meeting during the public comment period, please let me know or 

### 10 someone else on our SAP staff, who will be seated here to my 

### 11 right, know that you would like to provide public comments if 

### 12 you haven't made arrangements with me. Now it's important to 

### 13 note that without having made prior arrangements to provide your 

### 14 oral comments, we ask that you please limit your comments to no 

### 15 more than five minutes. 

### 16 There is a docket for this meeting. The 

### 17 docket number is listed on the agenda. All background materials 

### 18 and other related dockets are available in the docket. Slides 

### 19 of EPA presentations that you will see should be available in 

### 20 the docket very shortly. 

### 21 At this point, I want to introduce and extend 

### 22 my thanks to Dr. Daniel Schlenk, to my left, who is the FIFRA 

### 23 Scientific Advisory Panel Chair who is chairing this meeting for 

### 24 this week. Thank you. 

### 25 **DR. DANIEL SCHLENK:**Thanks, Fred. Good 

### 26 morning, everyone. Welcome to our panel this bright Tuesday,

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1 titled Scientific Review of the Endocrine Disruptor Screening 

### 2 Program: Tier 1 Screening Assay and Battery Performance. My 

### 3 name is Dan Schlenk. I'm a professor of environmental sciences 

### 4 at the University of California Riverside, and I'll be chairing 

### 5 the panel this week. 

### 6 What I'd like to do at this particular point 

### 7 in time is to go around the panel and have you state your name, 

### 8 your affiliation and a brief description of your expertise so 

### 9 that everyone can be familiar with you. So let me begin with my 

### 10 left with Dr. Klaine. 

### 11 **DR. STEPHEN KLAINE:**I'm Steve Klaine. I'm a 

### 12 professor at Clemson University, and I direct the Institute of 

### 13 Environmental Toxicology and the Aquatic Ecoducts Colleges. And 

### 14 I'm a member of the permanent panel. 

### 15 **DR. MARION EHRICH:**I'm Marion Ehrich from 

### 16 Virginia Tech. I teach pharmacology and toxicology to 

### 17 veterinary and medical students, general toxicology. I'm a 

### 18 member of the permanent panel. 

### 19 **DR. JAMES McMANAMAN:**Hi. Good morning. I'm 

### 20 Jim McManaman. I'm a professor of reproductive biology at the 

### 21 University of Colorado, School of Medicine and a member of the 

### 22 permanent panel. 

### 23 **DR. BARRY DELCLOS:**I'm Barry Delclos. I'm 

### 24 from the National Center for Toxicological Research, the FDA. I 

### 25 work in reproductive toxicology and carcinogenesis, and I am 

### 26 also on the permanent panel.

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1 **DR. GEOFFREY SCOTT:**I'm Geoff Scott with the 

### 2 National Oceanic and Atmospheric Administration, Director of the 

### 3 Center for Coastal Environmental Health & Biomolecular Research. 

### 4 My expertise is in aquatic toxicology. 

### 5 **DR. DANIEL SELVAGE:**I'm Dan Selvage. I'm an 

### 6 associate professor at the University of New England, and I 

### 7 specialize in endocrinology and neuroendocrinology. 

### 8 **DR. TERRY SCHULTZ:**Good morning. I'm Terry 

### 9 Schultz, professor emeritus, Department of Comparative Medicine 

### 10 in the College of Veterinary Medicine at the University of 

### 11 Tennessee. My expertise is in adverse outcome pathways and 

### 12 screening protocols for endocrine disruption, among other 

### 13 things. Thank you. 

### 14 **DR. DONALD TILLITT:**Morning. My name is Don 

### 15 Tillitt. I'm with the U.S. Geological Survey, Department of the 

### 16 Interior in Columbia, Missouri. My expertise is contaminant 

### 17 effects on fish development and early life stages. 

### 18 DR. KATHERINE ROBY: Hi. I'm Kathy Roby from 

### 19 the University of Kansas Medical Center and Department of 

### 20 Anatomy and Cell Biology, and I'm a reproductive 

### 21 endocrinologist. 

### 22 **DR. ALDERT PIERSMA:**Good morning. My name is 

### 23 Aldert Piersma. I work in the Netherlands in the National 

### 24 Institute of Public Health as a reproductive and developmental 

### 25 toxicologist, and I'm a professor in Utrecht University. 

### 26 **DR. REYNALDO PATINO:**Reynaldo Patino. I'm

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1 with USGS Texas Cooperative Fish and Wildlife Research Unit at 

### 2 Texas Tech University. My expertise is in aquatic toxicology, 

### 3 endocrine disruption in fish and amphibians primarily. 

### 4 **DR. DEBORAH MacLATCHY:**I'm Deborah MacLatchy. 

### 5 I'm a professor of biology at Wilfrid Laurier University in 

### 6 Waterloo, Ontario, Canada and a comparative endocrinologist and 

### 7 ecotoxicologist, particularly in fish. 

### 8 **DR. JACK LEONARD:**I'm Jack Leonard from the 

### 9 University of Massachusetts Medical School. I'm a professor of 

### 10 microbiology and physiological systems, and I'm an expert in 

### 11 thyroid and steroid hormone action. 

### 12 **DR. NELSON HORSEMAN:**Good morning. Nelson 

### 13 Horseman. I'm at University of Cincinnati in the College of 

### 14 Medicine, a professor there, endocrinologist. Over the years, 

### 15 I've worked on everything from fish and frogs to pigeons and 

### 16 people and currently work primarily in the area of lactation and 

### 17 breast milk. 

### 18 **DR. SHERRIL GREEN:**Good morning. I'm Sherril 

### 19 Green. I am a professor in comparative medicine at the Stanford 

### 20 University School of Medicine, and my expertise is in the health 

### 21 and disease of laboratory Xenopus laevis. 

### 22 **DR. DANIEL SCHLENK:**Thank you, everyone. 

### 23 Let's begin this morning with some opening remarks. And our 

### 24 first presenters are Dr. David Dix. He's the Acting Director of 

### 25 the Office of Science Coordination and Policy. 

### 26 **DR. DAVID DIX:**Thank you, Dr. Schlenk.

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1 Welcome to the panel. I appreciate you all being here very 

### 2 much. I'm the newly appointed Acting Director of OSCP. I'm 

### 3 coming over from the Office of Research and Development within 

### 4 EPA. I've been involved in toxicology, reproductive toxicology, 

### 5 toxicogenomics, and most recently I was the Acting Director of 

### 6 the National Center for Computational Toxicology prior to this 

### 7 latest appointment. 

### 8 But within OSCP, the Office of Science 

### 9 Coordination and Policy, our focus is on providing coordination, 

### 10 leadership, peer review and ensuring that sound scientific 

### 11 decisions are being made across the Office of Chemical Safety 

### 12 and Pollution Prevention. Our goal is to coordinate across new 

### 13 and emerging scientific assessments such as within the EDSP 

### 14 program, the Endocrine Disruptive Screening Program, and to 

### 15 continue to bring the best science from within and without the 

### 16 Agency to bear on meeting EPA's mission. 

### 17 Today's Science Advisory Panel is the second 

### 18 EDSP-related meeting this calendar year and is focused on 

### 19 evaluating performance of the EDSP Tier 1 assays, as we well 

### 20 know, and looking across the battery at the performance of these 

### 21 assays for the initial List 1 of EDSP chemicals. 

### 22 This review, I think, is informed very much by 

### 23 the 2008 SAP review on the Tier 1 battery for selective 

### 24 reference compounds. So today we have information from the 

### 25 initial list of chemicals to evaluate the performance of the 

### 26 Tier 1 battery in the context of these actual test chemicals

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1 with unknown endocrine modes of action. 

### 2 So on behalf of OSCP and OCSPP, I acknowledge 

### 3 and thank your scientific expertise here on the panel as well as 

### 4 the scientific expertise and hard work that has been put forth 

### 5 by members of the Office of Pesticide Programs, the Office of 

### 6 Pollution Prevention and Toxics and the Office of Research and 

### 7 Development here within EPA. I thank you for the time you've 

### 8 committed this week and to look across this work and provide us 

### 9 your insights. I hope the meeting is successful and enjoyable 

### 10 for all of you, and we look forward to your feedback and your 

### 11 input in this important process. Thank you very much. 

### 12 **DR. DANIEL SCHLENK:**Thank you. Additional 

### 13 opening remarks from Dr. Steve Bradbury who is the Director of 

### 14 the Office of Pesticide Programs. 

### 15 **DR. STEVEN BRADBURY:**Good morning, everyone. 

### 16 I'm pleased to see some of you again from our meeting last 

### 17 January as well as some other faces I remember from other 

### 18 peer-review meetings that we've had over the years. And I want 

### 19 to stress the importance of the SAP, the FIFRA SAP panel, to the 

### 20 work that we do not only in OPP but as this peer review 

### 21 reflects, work across the Agency and how we move forward in our 

### 22 scientific processes that underlie our decision making. 

### 23 And the role of the Science Advisory Panel 

### 24 most in terms of external peer review but having a public 

### 25 transparent process throughout the peer-review process is really 

### 26 fundamental to the work we do and the business we do. Our

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1 regulations are science based, and having said that, peer review 

### 2 is an important part of that process. 

### 3 So I just want to thank you again for the hard 

### 4 work leading up to getting here today, the hard work you'll have 

### 5 over the next three days here and the hard work it takes to put 

### 6 the report together. So I want to thank you in advance for all 

### 7 your efforts. 

### 8 Also want to thank the public for 

### 9 participating in the process. As indicated before, all these 

### 10 peer reviews reflecting on a White Paper that scientists, my 

### 11 colleagues across the Agency have put together, we also greatly 

### 12 value the input from the public in terms of scientific 

### 13 approaches and views that the Agency has, and we look forward to 

### 14 you taking a hard look at and a good look at those comments and 

### 15 weave that into your thoughts as we move forward with the 

### 16 program. 

### 17 I also want to thank Fred and all his 

### 18 colleagues in the SAP office and Designated Federal Officials 

### 19 for all the work it takes to put on these meetings, everything 

### 20 from logistics to all the ethics stuff you all had to go through 

### 21 and just sort of getting everything organized and the work it 

### 22 takes to put the report together. Without Fred and his 

### 23 colleagues in the SAP office, it would be almost impossible to 

### 24 pull a meeting like this off and the many meetings that we have. 

### 25 I'd like to spend just a couple of minutes 

### 26 following up on some of David's points for context before we get

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1 into the real meat of the next three days. 

### 2 And as you know from the background materials 

### 3 and the SAP we had in January and at least from historical 

### 4 perspective previous SAPs looking at the endocrine program, this 

### 5 effort flows out of two Acts, the Federal Food, Drug and 

### 6 Cosmetic Act when it was amended in 1996 by the Food Quality 

### 7 Protection Act; which basically said to the Agency take a look 

### 8 at all pesticide chemicals, and make a determination as to 

### 9 whether or not these chemicals have the potential to interact. 

### 10 It said estrogen in the Statute, but subsequently, through peer 

### 11 review and advice, we expanded that to a broader interpretation 

### 12 of endocrine systems. 

### 13 But take a look at pesticides, and determine 

### 14 whether or not they have the potential to interact with 

### 15 endocrine systems, and if so, gather the appropriate information 

### 16 so that you can make an informed risk assessment and an 

### 17 appropriate regulatory decision. The lawyers will be all upset 

### 18 as to how I synthesized that, but that's essentially the 

### 19 message. And the Safe Drinking Water Act had sort of comparable 

### 20 updates to it in 1996 in terms of taking a look at chemicals 

### 21 that could be in drinking water, and Agency, make sure you're 

### 22 regulating these chemicals properly using a sound scientific 

### 23 approach to do so. 

### 24 And through the years from 1996 on, peer 

### 25 review, public participation input has been critical. The 

### 26 EDSTAC, Endocrine Disruptor Screening and Testing Committee of

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1 FACA that provided advice to the Agency -- and I think it was 

### 2 around 1998 -- it provided some advice on a tiering process to 

### 3 go through this evaluation of all these compounds. 

### 4 And then in 1999, there was a combined 

### 5 scientific advisory board, Science Advisory Panel peer review on 

### 6 basically the framework of going forward. And the framework 

### 7 essentially laid out a two-tiered process. Well, actually it 

### 8 laid out more than that. It had a prioritization process which 

### 9 I'll run through in a second. 

### 10 But related to this specific SAP, it laid out 

### 11 two tiers. And it said the first tier use a set of validated 

### 12 assays to determine whether or not a chemical has the potential 

### 13 to interact with the estrogen, androgen or thyroid system. And 

### 14 it concurred with the EDSTAC to say take a look across a variety 

### 15 of taxa and take a look across various assays, some which may be 

### 16 very diagnostic, some which may be more in terms of frank 

### 17 effects, apical outcomes. In those days, adverse outcome 

### 18 pathways wasn't jargon we used but basically the concept of an 

### 19 adverse outcome pathway were reflected in the logic of the 

### 20 framework. 

### 21 And so then in 2008, the Agency brought to the 

### 22 SAP 11 assays that had gone through validation processes through 

### 23 OECD, Organization for Economic Cooperation and Development, and 

### 24 other national and international groups, to get feedback on 

### 25 those 11 assays and get advice as to whether or not these assays 

### 26 were ready to go forward.

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1 And the SAP concurred but gave us a really 

### 2 great framework to use as data would start to come in to 

### 3 evaluate how well those validated assays worked as laboratories 

### 4 started to undertake the efforts. And that was consistent with 

### 5 the SAB/SAP review on 1999 which said Agency, after you've 

### 6 issued test orders for maybe 50 to 100 chemicals and you start 

### 7 to gets some of that data coming in, it would be good to have a 

### 8 check in and see how well those assays are working as we move 

### 9 forward. 

### 10 And that's essentially what this first SAP in 

### 11 spring/summer is about, is getting feedback on how well those 11 

### 12 validated assays were executed looking at a cross-section of the 

### 13 chemicals that we're issuing test orders three years ago; and 

### 14 also to get some advice on how well the battery is performing as 

### 15 a whole, consistent with the 2008 SAP recommendations and how to 

### 16 sort of get your head around that and think those things 

### 17 through. 

### 18 So the development of those assays, as David 

### 19 indicated, was the result of a lot of hard work not only in EPA 

### 20 but EPA with colleagues in NIH, with colleagues in Europe who 

### 21 are doing the research on some of these same methods, colleagues 

### 22 in Japan who are working on a number of these assays. And so in 

### 23 many ways, these assays reflect an international effort in terms 

### 24 of their development and ultimate validation. The Organization 

### 25 of Economic Cooperation and Development, is a sort of 

### 26 international body that lots of member countries work together

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1 on harmonized methods, was a body that sort of helps us go 

### 2 through that validation process back in the 2000s. 

### 3 So as we go through this SAP, we're hoping to 

### 4 get feedback on how well those assays perform and how to take a 

### 5 look at the battery as a whole. Later this summer, we'll have 

### 6 another SAP that will give us advice on our proposed method of 

### 7 how to take that Tier 1 information, combine it with other 

### 8 information that may be available, and then integrate that 

### 9 information to determine whether or not a chemical has the 

### 10 potential to interact with the estrogen, androgen or thyroid 

### 11 pathway. 

### 12 So we sort of broke up getting feedback from 

### 13 you all in two steps. First step is give us some feedback on 

### 14 how we're thinking about how well the assays are working, how 

### 15 well they seem to be working together, and kind of absorb that 

### 16 feedback from you all. And then see again -- maybe not all of 

### 17 you -- but see the panel again at the end of July and give us 

### 18 advice on how we're taking that information and integrating it 

### 19 with other information to determine whether or not a chemical 

### 20 has the potential to interact. We kind of broke up in two steps 

### 21 the recommendations from the 2008 SAP. 

### 22 And just to wrap up quickly how this 

### 23 information then feeds into our overall program, it gets back to 

### 24 what I discussed in terms of the statute. So we have this 

### 25 charge to take a look at pesticide chemicals, certain chemicals 

### 26 that can be in drinking water, and make a determination as to

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1 whether or not they can interact with an endocrine system, and 

### 2 if so, do the proper risk assessment and make the appropriate 

### 3 regulatory decision. 

### 4 So the way this tiered process is working, is 

### 5 first, does the chemical have the potential to interact? If no, 

### 6 done; realizing as science evolves there may be reasons to come 

### 7 back and revisit that question, but these are based on today's 

### 8 science. 

### 9 If yes, then how to take that information, 

### 10 combine it with other information to determine if it does and if 

### 11 so what kinds of additional tests would be needed to get the 

### 12 dose response curve to do the risk assessment to make the 

### 13 appropriate regulatory decision. 

### 14 And in the case of FFDCA is if it's a 

### 15 food-used pesticide is there reasonable certainty of no harm; 

### 16 same kind of risk assessments we do all the time in the 

### 17 pesticide program, but making sure we're taking a look at this 

### 18 specific toxicological pathway appropriately. And in the case 

### 19 of ecological issues, whether or not there could be unreasonable 

### 20 adverse effects associated with this chemical, but making that 

### 21 risk assessment we need that dose response curve to go forward. 

### 22 So Tier 1, does it have the potential? If no, 

### 23 done. Keep track of the science so you may come back and 

### 24 revisit that. If yes, how? How do you make that determination, 

### 25 and if so, then what kind of information do you ask afterwards. 

### 26

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1 But this week's SAP is focusing on how well 

### 2 does that Tier 1 battery work as it was executed on these 

### 3 chemicals in List 1? And then how the battery as a whole seem 

### 4 to be hanging together. In July we'll talk about how to 

### 5 integrate that information to make a determination if a chemical 

### 6 can interact. 

### 7 So with that, I thank you all again. I also 

### 8 want to thank all the members of the pesticide program team and 

### 9 the OSCP because it's a lot of hard work, as you can imagine, 

### 10 pulling this together. And I want to thank the team. Mary 

### 11 Manibusan, as the Director of the Endocrine Program, will give 

### 12 you a little more background and be introducing the team members 

### 13 as well. 

### 14 With that, just want to thank you all again 

### 15 for your time and effort in this review. 

### 16 **DR. DANIEL SCHLENK:**Thank you, Dr. Bradbury. 

### 17 So to give an overview of the EDSP program, let me introduce 

### 18 Mary Manibusan who is the Director of Exposure Assessment 

### 19 Coordination and Policy Division, Office of Science Coordination 

### 20 and Policy. 

### 21 **DR. MARY MANIBUSAN:**So good morning 

### 22 everybody, and a warm welcome to everyone on the permanent 

### 23 panel, welcome back, and to those who are new to the panel, we 

### 24 welcome you and thank you for all your efforts that you've 

### 25 already done so far in preparation for today's meeting. 

### 26 Let me start by thanking the opening remarks

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1 provided by Dr. Dix and Dr. Bradbury, welcoming Dr. Dix, and we 

### 2 are very much excited that his expertise and his background will 

### 3 be a warm welcome to the endocrine program in moving us forward 

### 4 in the area of computational toxicology as well as reproductive 

### 5 toxicology. 

### 6 So with that, my job this morning is to 

### 7 provide a really brief overview of the Endocrine Disruptor 

### 8 Screening Program. How I'd like to do that is by focusing on 

### 9 the mission and vision of the program and then how that mission 

### 10 and vision is implemented in the program. So I'll provide a 

### 11 roadmap in terms of how a chemical moves from prioritization to 

### 12 screening and testing. And as Dr. Bradbury indicated, we do the 

### 13 work of risk assessment in our programs, so I'll focus on a 

### 14 little bit of that. And overlaid on top of that roadmap, I'll 

### 15 give you a sense of where we're headed in terms of future 

### 16 Scientific Advisory Panel reviews. 

### 17 The EDSP program has a unique mission, and 

### 18 that is to protect public health and wildlife by screening and 

### 19 testing chemicals. But we don't stop there in terms of hazard 

### 20 identification. If indeed there is hazard, we do take 

### 21 appropriate actions for those chemicals in terms of our risk 

### 22 assessment and put forward risk mitigation options if they're 

### 23 found to have endocrine effects and be protective again of 

### 24 public health and wildlife. 

### 25 How we operate is under two legislative 

### 26 mandates. The 1996 Federal Food, Drug and Cosmetic Act requires

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1 the Agency to have developed a screening program, and then 

### 2 underscoring the word "validation" here, using validated test 

### 3 systems and other scientifically relevant methods to determine 

### 4 certain substances may have an effect on humans and effects that 

### 5 produce and actually create estrogen and other endocrine effects 

### 6 as the administrator may designate. Just want to focus on 

### 7 validated test systems focusing on humans as well as estrogenic 

### 8 pathway. 

### 9 Under the FFDCA, the Agency is required to 

### 10 screen all pesticide chemicals inclusive of active ingredients 

### 11 as well as inert compounds. Different from the FFDCA is the 

### 12 1996 Safe Drinking Water Act Amendment. This is in Section 

### 13 1457. Here it explicitly states that the endocrine program is 

### 14 allowed to screen chemicals only if they meet the exposure 

### 15 finding, and that is that they're found in sources of drinking 

### 16 water and if there are substantial human populations of 

### 17 exposure, very different, again, from FFDCA, but certainly we've 

### 18 looked to that to establish our Universe of Chemicals. 

### 19 Shortly after the 1996 amendments, the Agency 

### 20 had looked to scientific expertise through development of the 

### 21 Federal Advisory Committee. This was called the Endocrine 

### 22 Disruptor Screening and Testing Advisory Committee. This 

### 23 committee provided some really critical recommendations that 

### 24 really formed the look and feel of our program to date. Certain 

### 25 key recommendations are highlighted here, and that is the first 

### 26 being the expansion of protection to be not only inclusive of

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1 human health but also of wildlife, that being more sensitive 

### 2 sometimes to human adverse effects. And you'll see that 

### 3 demonstrated in the range of Tier 1 assays as well as the 

### 4 proposed Tier 2 assays as we move forward. 

### 5 We also look to expand beyond the estrogen 

### 6 pathway to be inclusive of the androgen and the thyroid pathways 

### 7 because those pathways were clearly most common and most known 

### 8 at that time. Again, we look to the advancement of science to 

### 9 be inclusive of other modes of action. 

### 10 But more critical of those recommendations is 

### 11 this development of a two-tiered screening and testing program. 

### 12 The founding members of the EDSTAC group actually had a 

### 13 conceptual framework that produced chemicals that led from 

### 14 prioritization into a Tier 1 screen, and that is to answer the 

### 15 question of whether that chemical even has the potential to 

### 16 interact with the endocrine system, specifically estrogen, 

### 17 androgen and thyroid hormone, allowing for some tolerated 

### 18 uncertainty. 

### 19 As chemicals are evaluated, and a weight of 

### 20 evidence indicates that it warrants additional testing through 

### 21 Tier 2, the Tier 2 tests are really to determine whether that 

### 22 interaction actually occurs in the endocrine system, and if so, 

### 23 the ability to quantify that dose-response relationship is 

### 24 really important as we move it forward into our risk assessment 

### 25 paradigm. 

### 26 Here is what the endocrine program Tier 1

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1 screening battery looks like. It's comprised of five in vitro 

### 2 assays and six in vivo assays. And the thought here is that it 

### 3 forms a battery of assays, and so they're intended to be 

### 4 complementary. And I put forward this visual of a puzzle piece 

### 5 because they're meant to interconnect. They're not duplicative, 

### 6 but they're meant to inform each other in terms of the certainty 

### 7 in which they interact with the endocrine system. 

### 8 Of course, the in vivo assays, because they're 

### 9 multi-parameter, can cover many more modes of action, even those 

### 10 that are not yet known in a wholistic, whole-animal system where 

### 11 you're capturing not only the toxicodynamics but also the 

### 12 toxicokinetic features of metabolism and compensatory 

### 13 mechanisms; while the in vitro assays give you a lot more 

### 14 specificity in terms of mode of action, in terms of binding 

### 15 potential and activation assays as well as steroidogenesis and 

### 16 aromatase assays. 

### 17 Here's just another look at the Tier 1 assays 

### 18 together, and here, while in 1998 the terms of mode f action or 

### 19 adverse outcome pathway were not common at the time, you can see 

### 20 just looking at the estrogen agonist pathway that that's how 

### 21 it's laid out in terms of toxicity and our understanding of 

### 22 molecular initiating events occurring early on in terms of ER 

### 23 binding and transactivation occurring subsequent to that. 

### 24 We're also looking at organ responses in terms 

### 25 of uterotrophic assays; and again, in the whole animal, do we 

### 26 see apical endpoints that match up to what we would expect to

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1 see when you do have the estrogenic pathway triggered? And that 

### 2 is exemplified in the pubertal female and the fish reproductive 

### 3 for the estrogen agonist. 

### 4 We look to the three pathways, estrogen, 

### 5 androgen and the thyroid HPT, but you'll note here when you look 

### 6 across the battery that that redundancy, that understanding of 

### 7 adverse outcome pathway, is not clearly laid out as for estrogen 

### 8 as compared to the thyroid. So thyroid, we really just have the 

### 9 rat pubertals and the amphibian metamorphosis assay. And that 

### 10 was clearly noted in the 1998 report from EDSTAC as well as the 

### 11 2008 SAP report. 

### 12 Combining that Tier 1 data alongside with 

### 13 other relevant scientific information, if we conclude that 

### 14 chemical warrants the movement forward to Tier 2 testing, these 

### 15 are the Tier 2 tests that are being proposed. Starting from the 

### 16 top, we have the mammalian two-generation reproduction study 

### 17 that's already a validated study as well as the extended 

### 18 one-generation reproduction study. 

### 19 What we're currently working on in terms of 

### 20 validation effort, and what we plan to bring before the SAP next 

### 21 month in June, are four remaining ecological Tier 2 test 

### 22 methods. They run across a broad range of taxa, as you can see 

### 23 here, starting with the avian, moving forward to the larval 

### 24 amphibian growth and development, the fish multi-generation 

### 25 study and the invertebrate multi-generation reproduction study. 

### 26

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1 Similar to the mammalian studies, we'll look 

### 2 for opportunities, and we'll ask for SAP's recommendations on 

### 3 moving from a multi-generation reproduction study protocol to a 

### 4 single-generation reproduction study but not losing the 

### 5 sensitivity in terms of identifying sensitive endocrine 

### 6 endpoints. 

### 7 Here's just a quick snapshot. I think it's 

### 8 really important as the EDSTAC committee had focused on, there 

### 9 is a clear distinction between Tier 1 screening and Tier 2 

### 10 testing. So Tier 1 screens are intended to be shorter term 

### 11 assays and done at a very much lower cost. It is intended 

### 12 because it service as a screen to minimize false negatives but 

### 13 also balancing false positive, so the ability to really operate 

### 14 and function as an adequate screen. 

### 15 The real question for the screen is again does 

### 16 that chemical have the potential to interact with the endocrine 

### 17 system, and is this customarily a binary response, yes, no. And 

### 18 because of that, you'll see that a lot of the Tier 1 assays have 

### 19 limited doses. Some of the assays have only two doses, and 

### 20 that's intended, again, to push the system so that we're seeing 

### 21 an effect of the screen but not chase lower-dose effects as you 

### 22 would in a Tier 2 test. 

### 23 The combination of the in vitro/in vivo assays 

### 24 are intended, again, to complement and create that power in 

### 25 terms of the screen and intended to work together. And that's 

### 26 why we ask for all 11 assays in a Tier 1 screening battery so

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1 that we can get information in order to decide whether that 

### 2 chemical can and should be moved forward to Tier 2, or we can 

### 3 decide that it does not exemplify the potential to interact with 

### 4 the endocrine system, and we put it in a hold box. 

### 5 If we move forward to Tier 2 testing, these 

### 6 are again similar to definitive studies that we use in typical 

### 7 risk assessments of longer term. They have many more doses, 

### 8 many more animals per dose so that you have the statistical 

### 9 power to identify any subtle changes or effects for apical 

### 10 endpoints upon which to establish your risk assessment. 

### 11 Here the question to be answered is again 

### 12 whether that chemical has, indeed, the ability to interact with 

### 13 the endocrine system, and then more importantly, what is the 

### 14 dose at which you want to set your point of departure for risk 

### 15 assessment. The collection of Tier 2 tests are primarily in 

### 16 vivo for that reason, but they cover multiple taxa, so they 

### 17 cover the rat for the human risk assessment and then the four 

### 18 ecological assessments intended to capture the most sensitive 

### 19 ecological species. 

### 20 And these differences from the Tier 1 screen 

### 21 are not intended to be a battery but intended to be a targeted 

### 22 testing approach. So again, asking for only that Tier 2 study 

### 23 that we need in order to conduct our risk assessment to be 

### 24 protective of human health and wildlife. 

### 25 So how do we put the Tier 1 and Tier 2 assays 

### 26 into an operational program? This slide is intended to do that,

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1 to capture how a chemical moves from prioritization to screening 

### 2 and testing, and then overlaid on top of that is our projected 

### 3 SAP schedule for all these fundamental steps. 

### 4 So starting from the left, step one is the 

### 5 prioritization of chemicals. For the initial list of chemicals, 

### 6 we prioritize that pesticide chemical list by exposure pathways. 

### 7 So based on exposure, they were put onto our initial list. 

### 8 Going forward to the second step, which is 

### 9 where we are today, when the data is submitted to the Agency, 

### 10 the Agency conducts a very thorough review inclusive of 

### 11 performing a primary, secondary, as well as a tertiary view for 

### 12 consistency across the assays and across chemicals. And that is 

### 13 what we're bringing forward to you today. 

### 14 Step three involves bringing that Tier 1 assay 

### 15 data and its conclusions collectively with integration of other 

### 16 scientifically relevant data. This is inclusive of the 

### 17 pesticide submission information in terms of the developmental 

### 18 studies, if there's existent multi-reproduction assay that's 

### 19 been conducted as well as the chronic assays and any repeat 

### 20 systemic dosing study, is very informative to the weight of 

### 21 evidence in terms of balancing what is biologically plausible to 

### 22 what we're seeing in the Tier 1 assays. 

### 23 If the determination from that weight of 

### 24 evidence allows the agency to decide that we need to move that 

### 25 chemical forward for additional testing, for example, Tier 2, 

### 26 then we would move that forward based on the effects that we're

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1 seeing and the potential effects that we're acknowledging in the 

### 2 Tier 1 assays. That would move into the multi-generation 

### 3 reproduction studies and then embed it into the risk assessment. 

### 4 In terms of the SAP timeframe, in January we 

### 5 brought before the panel a prioritization method that was 

### 6 inclusive of physicochemical properties as well as (Q)SAR and 

### 7 high-throughput computational toxicological methods in order to 

### 8 strategically prioritize chemicals. 

### 9 We are at the second step, and the second step 

### 10 is really again meant to anchor down the Tier 1 assays. Are the 

### 11 Tier 1 assays performing as intended? Is the battery as a whole 

### 12 operating as it was intended? As Dr. Dix had indicated, this 

### 13 time we're bringing back to the panel some real-world chemicals 

### 14 with unknown endocrine modes of action. So really testing the 

### 15 systems and asking the question can we do better; can we 

### 16 optimize the assays and still answer the question without 

### 17 sacrificing any certainty. 

### 18 And then, of course, the third step which is 

### 19 weight of evidence we're going to bring back to the panel in 

### 20 July, and that will be bringing forward some specific case 

### 21 studies exemplifying, again, the methodology of weight of 

### 22 evidence. How do we bring forward the Tier 1 alongside with the 

### 23 158 data alongside with published information, and put together 

### 24 an integrated assessment to help us decide whether that chemical 

### 25 moves forward? 

### 26 Next month, in June, we'll be bringing forward

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1 the work that our Office of Science Coordination and Policy in 

### 2 conjunction with Office of Research Development and Office of 

### 3 Pesticide Program have been working on for probably over the 

### 4 last decade now in completing the validation effort for the 

### 5 remaining ecological Tier 2 assays. And these are, again, 

### 6 primarily multi-generation reproduction studies. 

### 7 So that's to just quickly give you a sense of 

### 8 where we're headed, where we are today and where we'd like to 

### 9 be. 

### 10 Just as a quick reminder, back in January, 

### 11 that chemical prioritization methodology was brought before the 

### 12 SAP. At that time, the focus was to prioritize the Universe of 

### 13 Chemicals using the estrogen receptor adverse outcome pathway as 

### 14 a specific example or model for other pathways, and in 

### 15 particular, focusing on the use of computational toxicology 

### 16 tools to make our program much more efficient and effective as 

### 17 we move forward. 

### 18 We sought to obtain the SAP's input or 

### 19 recommendations on the scientific concepts and the principles 

### 20 and processes used to prioritize chemicals for EDSP screening. 

### 21 At the beginning of this month, we received the final report. 

### 22 We thank the panel very much for their recommendations. They 

### 23 will be very critical as we move forward to prioritizing our 

### 24 10,341 chemical comprised Universe of Chemicals. 

### 25 Here's just a quick snapshot, just again as a 

### 26 reminder that what we brought before the panel in January was a

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1 methodology that looked to prioritize strategically our Universe 

### 2 of Chemicals using a combination of various filters. On your 

### 3 right, it's demonstrated the different filters, the use of 

### 4 physicochemical properties such as acidity, whether a chemical 

### 5 is too basic, whether it's a long-chain polymer and not able to 

### 6 be bio-available systemically to introduce any endocrine 

### 7 activity. That's a filter that could preclude chemicals from 

### 8 endocrine testing. 

### 9 The next being a very important filter, the 

### 10 exposure potential filter, and their chemicals would then filter 

### 11 into the expert system and high-throughput box which is our 

### 12 computational toxicology tools in parallel. And as chemicals 

### 13 move forward, based on its potential through this particular 

### 14 prioritization, it moves into a list for EDSP Tier 1 screen. 

### 15 This is where the January SAP ended, and this 

### 16 is where our SAP today starts. And that is the focus on the 

### 17 data that is received having gone through the screen. Here the 

### 18 focus for the SAP panel is not only on the performance of the 

### 19 Tier 1 individual assays but how laboratories are able to 

### 20 execute these study method protocols and whether they can do it 

### 21 efficiently. 

### 22 The second primary area of focus is the 

### 23 performance of the EDSP Tier 1 battery. As we break apart the 

### 24 Tier 1 assays and click it back together, is the battery 

### 25 performing as it was intended to? Does it give us information 

### 26 on thyroid, androgen and estrogenic pathway? Can we do the same

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1 job with less assays; and if so what would we be missing when we 

### 2 remove some of the critical assays which we move forward? 

### 3 And this was, again, at the impetus of the 

### 4 SAB/SAP panel back in 1999 that was pretty clairvoyant in 

### 5 suggesting that we convene a panel of independent experts to 

### 6 review all the screening data for approximately 50 to 100 

### 7 compounds but, more specifically, with an eye towards revising 

### 8 the process and eliminating those methods that just don't work. 

### 9 This SAP, of course, is not a revalidation of 

### 10 the Tier 1 assays, but it's intended to look at the Tier 1 

### 11 assays with real-world chemicals. As exemplified in all SAPs, 

### 12 this is an open and transparent public process intended to give 

### 13 the Agency insights from an independent, external scientific 

### 14 peer review. And based on their findings in 2008, the current 

### 15 state of science at that time indicate that the Tier 1 assays 

### 16 and batteries were indeed capable of identifying potential 

### 17 estrogenic, androgen and thyroid endocrine-active compounds. 

### 18 And more importantly, it's an appropriate 

### 19 starting point to detect potential endocrine disruptors, 

### 20 acknowledging that the state of the science was advancing very, 

### 21 very quickly and that we could supplement the battery with 

### 22 additional test methods. 

### 23 But the Tier 1 battery was, in fact, pointed 

### 24 out to be very, very informative because it include multiple 

### 25 taxa and different modes of action endpoints and provide a broad 

### 26 range of metabolism and needed corroboration so that we can

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1 control for any false negative and false positive results as we 

### 2 move forwards. 

### 3 Today we are starting with the initial list of 

### 4 EDSP chemicals that was submitted for Tier 1 screening back in 

### 5 2009. That data has started rolling in, submitted to the Agency 

### 6 beginning 2011, and we received the last set in 2013. We hand 

### 7 selected among the 52 pesticide chemicals 21 unique chemicals 

### 8 for the reasons stated here, that they really represent a range 

### 9 of physicochemical properties inclusive of volatility, a broad 

### 10 range of log Kow values as well as solubility. 

### 11 We also looked to establish a subsample that 

### 12 had a broad range of biological activity and inclusive of 

### 13 various classes of pesticide chemical. So includes chemical 

### 14 classes representative of emides, azols, organophosphates and 

### 15 pyrethroids, et cetera. These pesticide chemicals are designed 

### 16 with clear biological activity, and that's why as an Agency we 

### 17 felt these selected chemical would be the way to test the Tier 1 

### 18 assays more robustly. 

### 19 So the Tier 1 assays from our initial review 

### 20 has demonstrated again, as 2008 had concluded, to be useful 

### 21 information to evaluate the potential interactions of E, A and T 

### 22 signaling pathways. There were no major problems identified in 

### 23 Tier 1 assays, and again, we demonstrated here in our evaluation 

### 24 that laboratories as a whole were able to execute each of the 

### 25 assay protocols in accordance with the respected test 

### 26 guidelines, and they were able to achieve specific performance

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1 criteria noting that there were some minor deviations from the 

### 2 performance criteria. Those differences were not considered 

### 3 substantial. 

### 4 The second half of our evaluation was to look 

### 5 and focus on the battery, whether the battery performed as 

### 6 intended and whether there were opportunities to reduce the 

### 7 battery and not sacrifice any sensitivity. We looked at the 

### 8 battery in terms of the specific pathways that was intended to 

### 9 be informative for. So starting at the top for thyroid, what we 

### 10 found was that the amphibian metamorphosis assay and the rat 

### 11 pubertal assays were indeed able to give us different types of 

### 12 information across the taxa and reinforce the importance of 

### 13 having both taxa in the screening battery to reduce the 

### 14 potential for false negatives. 

### 15 For the androgen pathway, we found that the in 

### 16 vitro assays weren't able to consistently exhibit significant 

### 17 effects when effects were observed in the in vivo assays, and 

### 18 this could be due to potentials in differences in metabolism and 

### 19 different whole animal systems that are not able to be 

### 20 demonstrated in in vitro assays. These possible redundancies 

### 21 across in vivo assays in terms of the Hershberger, male pubertal 

### 22 and the fish assay, were observed, albeit there were noted some 

### 23 overt toxicity. 

### 24 For the estrogen pathway, we found again that 

### 25 the female pubertal and fish assays are indeed very important in 

### 26 evaluating the estrogenic pathway across taxa. The uterotrophic

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1 here we note was consistently negative for all chemicals, but 

### 2 that's not surprising because there was no representative 

### 3 estrogen agonist in our subsample. I want to again confirm that 

### 4 this estrogen pathway and all of the Tier 1 assays associated 

### 5 with it had been validated in 2008. And it's particularly 

### 6 important to note that although the uterotrophic assay was 

### 7 consistently negative, it was never positive while the in vivo 

### 8 system assays were negative. 

### 9 So the fundamental charge to the panel today 

### 10 is really to receive your overall comment on the assay, the 

### 11 laboratory performance and advice on how to address some of the 

### 12 technical deviations and other issues encountered with specific 

### 13 assays. We noted in our White Paper examples of overt toxicity, 

### 14 cytotoxicity and difficulties in solubility of different 

### 15 compounds. 

### 16 The second area is to receive your comment on 

### 17 Tier 1 battery performance and whether there are unnecessary 

### 18 redundancies among the 11 assays and whether we can do the job 

### 19 of answering the question of potential interaction with fewer 

### 20 assays. 

### 21 I leave you with this quote from Aristotle 

### 22 which to me punctuates the review that we have before us, and 

### 23 that is, "The whole is greater than the sum of its parts." 

### 24 Certainly the Tier 1 eleven assays are indeed very informative 

### 25 and they're a good tool. But it's intended to provide us an 

### 26 answer to the question and the question is whether the chemical

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1 has potential to interact with the endocrine system. If we can 

### 2 do that job with less assays, it's important to understand what 

### 3 information we would be missing if we were to reduce the 

### 4 battery. 

### 5 And again, I want to acknowledge the OCSPP 

### 6 team that worked on this evaluation. They're listed here, Jess 

### 7 Rowland, Greg Akerman, John Liccione, Amy Blankinship, Catherine 

### 8 Aubee, Tom Steeger, Vicki Dellarco, Patience Browne, Don 

### 9 Bergfelt and Calvin Powell, all just very important contributors 

### 10 to the scientific evaluations and some who will be speaking 

### 11 during the course of today. 

### 12 Here I leave you with this last slide, and 

### 13 this is just the order of the presentations for today. 

### 14 Following me is Dr. Les Touart who will be providing some 

### 15 background on the current validated Tier 1 screening assays and 

### 16 going into the history a little bit more on the conceptual 

### 17 framework. 

### 18 Dr. Greg Akerman will be going over the in 

### 19 vitro assays, Dr. Liccione the in vivo mammalian assays. Amy 

### 20 Blankinship will be going over the fish assay, Catherine Aubee 

### 21 the frog assay, and Tom Steeger, Catherine and Amy will 

### 22 collectively go over the battery performance, and then we'll 

### 23 close out with an overall summary provided by Dr. Tom Steeger. 

### 24 Thank you very much, and I welcome any 

### 25 questions. 

### 26 **DR. DANIEL SCHLENK:**Thank you, Mary.

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1 Any general questions regarding the overview? 

### 2 Yes, Dr. Ehrich. 

### 3 **DR. MARION EHRICH:**Could I ask you to put up 

### 4 that slide again that had all 11 batteries just to take another 

### 5 look at? Thank you. 

### 6 **DR. DANIEL SCHLENK:**Did you have a question? 

### 7 No? Okay. 

### 8 Dr. Klaine. 

### 9 **DR. STEPHEN KLAINE:**Thanks for the 

### 10 presentation. I'm sure this is a lot of work in terms of 

### 11 analyzing this. I'm still puzzled as to the choice of the 21 

### 12 chemicals out of the 52, and was the strategy to minimize or, 

### 13 you know, to use your effort wisely in terms of analyzing the 

### 14 data? In other words, why didn't you just do all 52? 

### 15 **DR. MARY MANIBUSAN:**So that's a very good 

### 16 question, and what we've written into the White Paper is our 

### 17 selection process. We were very specific in choosing these 21 

### 18 chemicals for the reasons articulated, that they represent a 

### 19 broad range of pesticide chemicals within that 52-chemical 

### 20 category. 

### 21 It also had very clearly a broad range of 

### 22 physicochemical properties that we again wanted to demonstrate 

### 23 that the Tier 1 assays were able to perform for. And it had a 

### 24 broad range of biological activities so inclusive of herbicides, 

### 25 rodenticides and fungicides; it even includes an inert compound. 

### 26 This subsample is really representative of the

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1 remaining chemicals within that 52 class, but as you said, we 

### 2 would like to receive the panel's recommendation and advice 

### 3 before completing and dedicating resources to completing all of 

### 4 the remaining 52 chemical assessments. 

### 5 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 6 **DR. GEOFFREY SCOTT:**Just a follow-up question 

### 7 to Dr. Klaine's question. Of those compounds that were 

### 8 screened, what proportion of total pesticide use do you think 

### 9 those represent? It may not be a precise number, but is it a 

### 10 major proportion of pesticide use or is it in the middle or is 

### 11 it the smaller portion? I guess what I'm saying, out of about 

### 12 1,000 plus compounds, what percentage of total use does that 

### 13 possibly represent? 

### 14 **DR. MARY MANIBUSAN:**Maybe I can start and 

### 15 pass it over to Dr. Bradbury. 

### 16 So these initial lists of pesticide chemicals 

### 17 were prioritized based on exposure. So it had to meet four 

### 18 particular exposure pathways in order to be moved into the list. 

### 19 So it had to be used in a residential setting, occupational, be 

### 20 used for food and water. So because of that, this was 

### 21 prioritized in a higher category for the initial list to be used 

### 22 for screening. 

### 23 Let me pass it to Dr. Bradbury for additional 

### 24 information. 

### 25 **DR. STEVEN BRADBURY:**As Mary indicated, it 

### 26 was back 2007'ish, I think, when we put out for public comment a

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1 process to get that first 50 to 100 compounds. And we laid out 

### 2 that we would try and get a good cross-section of 

### 3 active-ingredient pesticides as well as a cross-section of 

### 4 high-production volume inert ingredients. 

### 5 And so the process that we put out for public 

### 6 comment wasn't to get as quantitative, as you've implied in your 

### 7 question, Dr. Scott, but we've qualitatively capture chemicals 

### 8 that have a higher potential for exposure. So we looked at 

### 9 whether or not a compound could be used, as Mary said, in a 

### 10 residential setting. Was it detected in water? Was it a 

### 11 food-use pesticide which is an indirect measure that could be in 

### 12 the human food chain, those kinds of parameters, and we made 

### 13 sure that every chemical in that list of 61 was in at least 

### 14 three of the four exposure pathways, so all of these compounds 

### 15 have at least three of the four exposure pathways. 

### 16 Having said that, the compounds themselves, if 

### 17 you can see in the public, the list of 67, many of them 

### 18 represent, for example, insecticides and herbicides are very 

### 19 commonly used. 

### 20 The other point of sort of how we're moving 

### 21 forward is in the one graph that Mary put up which showed sort 

### 22 of the pesticide process of how we're going to go through an 

### 23 evaluation and the non-pesticides. So for the pesticide active 

### 24 ingredients, we're using our registration review program which 

### 25 is our 15-year cycle for reevaluating pesticides. 

### 26 And so once we kind of know how all the

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1 machinery is going to work, we'll use that process to 

### 2 systematically go through the active ingredients, although we 

### 3 may use some read-across and other techniques not to have to 

### 4 test every single one or screen every single one, so a 

### 5 qualitative approach to the exposure potential; and as you'll 

### 6 see the list, many of them are pretty common. 

### 7 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 8 **DR. GEOFFREY SCOTT:**Thank you all for that 

### 9 clarification. What you're telling us is it's a good 

### 10 representative group, and that was really the intent of the 

### 11 question. It wasn't clear before exactly without following all 

### 12 the details, so thank you for that explanation. 

### 13 **DR. STEVEN BRADBURY:**And if I could, to 

### 14 follow up on Dr. Klaine's question which was related and Mary's 

### 15 response, is that yeah, we did take a look, you know, at peak 

### 16 log Kows, some very soluble compounds, some, if you look at the 

### 17 list of 67 there are plenty of pyrethroids on there, and those 

### 18 of us who have done experiments with pyrethroids know what that 

### 19 entails. 

### 20 So we knew we had a pretty good cross-section 

### 21 to add some other P Chem properties and then also taking a look 

### 22 at a fairly representative cross-section of insecticidal mode of 

### 23 action, herbicidal mode of action. Again, as Mary pointed out, 

### 24 part of the challenge of this group is how to get enough dose, 

### 25 just enough, knowing that if you go too high that insecticidal 

### 26 and herbicidal mode of action will start to kick in and so we

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1 figured that would be a pretty robust group, and to get some 

### 2 feedback partway through the effort. 

### 3 So, Dr. Klaine, it was a bit of a balancing 

### 4 act. One option would have been to do all fifty-some and then 

### 5 if you come back and go well, you all really got it wrong, that 

### 6 would be an option. But we felt a more prudent option would be 

### 7 to take a good cross-section and get some initial feedback from 

### 8 you, because you may have some suggestions to go back and 

### 9 rethink a few things on these twenty one and we can also use 

### 10 that knowledge as we go forward with the remaining 

### 11 thirty-something. And so it's kind of balancing getting 

### 12 feedback and using our resources efficiently. 

### 13 **DR. DANIEL SCHLENK:**Dr. Piersma. 

### 14 **DR. ALDERT PIERSMA:**Just to follow up on 

### 15 these questions, one more detail is from the report I understand 

### 16 that the Tier 1 data for 50 compounds were submitted, and after 

### 17 that the 21 were selected. Was the Tier 1 data in any way 

### 18 influencing the selection of the 21 compounds? 

### 19 **DR. MARY MANIBUSAN:**No. In fact, we 

### 20 understood which 52 were going to be submitted to the Agency and 

### 21 we specifically chose those 21 chemicals, because they again 

### 22 represented that broad range of physicochemical properties and 

### 23 pesticidal mode of action. 

### 24 **DR. DANIEL SCHLENK:**Dr. Patino. 

### 25 **DR. REYNALDO PATINO:**I may be missing 

### 26 something but why not all 52? I mean the more information the

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1 better. Or is it that it may be an effort-related problem here 

### 2 that you didn't have time to do all 52? I mean in my opinion, 

### 3 the more information the better. 

### 4 **DR. STEVEN BRADBURY:**It isn't more is better 

### 5 -- not to say more isn't better -- but it's also balancing time 

### 6 and time in terms of when to get some feedback as we go forward. 

### 7 From a scientific standpoint it's get back to (Q) SAR and 

### 8 read-across and chemical classes. Do we have a sense of that 

### 9 universe of fifty-some chemicals with the 21 and our conclusion 

### 10 was yes. 

### 11 It's reasonably representative of the 

### 12 insecticidal mode of action in the group of 67. It's reasonably 

### 13 representative of the herbicidal mode of action in the group of 

### 14 67, the fungicides in the 67, the log Kow range. If somebody 

### 15 was going to ask me are the 52 representative of all 1,500 

### 16 active ingredients, the answer would be no. Inherently it can't 

### 17 be. 

### 18 The SAP said get 50 to 100 and see how it's 

### 19 going. So what we decided to do to balance resource and good 

### 20 timing to get input from peer-review panel, let's take a look at 

### 21 21 that are reasonable, very reasonable cross-section of 

### 22 fifty-some, and get some feedback. Because part of your advice 

### 23 may be, I hope, yeah, the assays are generally executed 

### 24 reasonably well, but here's a couple of things to think about as 

### 25 you look at this assay or that assay. 

### 26 Okay, good. We can go back and touch up the

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1 21, and we'll know that going into the remaining 30 as we take 

### 2 your advice and move forward. 

### 3 **DR. DANIEL SCHLENK:**Dr. Klaine. 

### 4 **DR. STEPHEN KLAINE:**I think you answered 

### 5 this, but just to kind of follow up. So in the relatively near 

### 6 future we could expect the rest of the analysis of the other 

### 7 compounds? Okay. 

### 8 **DR. STEVEN BRADBURY:**We're nodding our head 

### 9 yes. 

### 10 **DR. DANIEL SCHLENK:**Thanks. Dr. Nelson. 

### 11 **DR. NELSON HORSEMAN:**This sort of touches a 

### 12 similar question, but assume from this list that the EDSP has 

### 13 10,000 compounds for which this set of assays is being applied. 

### 14 And understanding that it's not this committee's purview or 

### 15 responsibility, those non-FIFRA compounds, is there useful 

### 16 information about the performance of these individual or battery 

### 17 tests in the rest of EDSP that this panel could be informed by? 

### 18 **DR. MARY MANIBUSAN:**So again, we're going 

### 19 back to the issue of how representative is the subsample of 21 

### 20 chemicals that we're bringing before this panel for review? And 

### 21 again, the focus is on the performance of the assays and battery 

### 22 and not on the chemical risk assessments. So how they perform 

### 23 for the chemical, what decisions we make is not subject to this 

### 24 review. This review is just primarily looking at methodology. 

### 25 And that's the reason why the subsample is so important. 

### 26 Among that 21 subsample is an inert ingredient

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1 that would be in parallel with the Safe Drinking Water Act, 

### 2 would be consistent and similar to those chemicals. But in 

### 3 terms of the broad range of SDWA chemicals, again, the initial 

### 4 list was focusing on pesticide chemicals as a start. But we 

### 5 feel that that is a robust, you know, list of chemicals upon 

### 6 which to challenge the Tier 1 assays and the battery. 

### 7 **DR. DAVID DIX:**If I could follow up. I think 

### 8 as we move forward in the SAP processes, the upcoming weight of 

### 9 evidence SAP will bring in some additional data that I think 

### 10 will satisfy some of these questions and comments. And then 

### 11 moving forward as we expand the program and take a more 

### 12 integrated computational approach to analysis, I think that will 

### 13 address these issues even more so. 

### 14 We see that as the future, and just to 

### 15 emphasize, I think that getting this intermediate feedback sort 

### 16 of midway through this dataset in the first round of Tier 1 

### 17 testing will be extremely valuable to us, and that's why we set 

### 18 up the schedule the way it is. 

### 19 **DR. DANIEL SCHLENK:**Thanks. Okay, we ready 

### 20 to move on for background presentation? 

### 21 Okay. Presenter is Leslie Touart who is with 

### 22 the Exposure Assessment Coordination and Policy Division, Office 

### 23 of Science Coordination and Policy. Dr. Touart. 

### 24 **DR. LESLIE TOUART:**Thank you, Dr. Schlenk, 

### 25 and I thank the panel for being here and good morning. 

### 26 My purpose here is to talk a little bit about

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1 the background of the Tier 1 battery, to discuss a little bit 

### 2 about how the assays address elements within the pathways that 

### 3 are utilized. 

### 4 Going back and just remember the endocrine 

### 5 system, recognizing that it's an integrated system of organs and 

### 6 tissues, chemical messengers, to maintain homeostasis within an 

### 7 organism primarily through a system of negative feedback 

### 8 signaling. The estrogen, androgen and thyroid subpart of these 

### 9 endocrine axes is really just a subpart, it's only part of that. 

### 10 The focus of the endocrine screening program is on these 

### 11 hormones because they were considered to be primary and suitable 

### 12 for the available science at the time to develop assays to look 

### 13 at these. And so this is what was targeted and developed. 

### 14 The endocrine system as we know can be 

### 15 perturbed at multiple sites and by multiple mechanisms. So we 

### 16 wanted to develop a battery of assays that we would be able to 

### 17 pick up and screen for all of the known mechanisms at the time. 

### 18 The considerations that were given were that 

### 19 there's complex interactive nature of the endocrine system, and 

### 20 it's clear that these should be screened for apical activity in 

### 21 addition to some of the more molecular or in vitro kinds of 

### 22 things. Since the main indications in terms of reproduction, 

### 23 growth, development, you know, these are the key things that the 

### 24 Agency is concerned about in terms of its risk assessment. So 

### 25 we want to be able to be sure that we're looking and screening 

### 26 for those kinds of activities because these are going to be more

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1 comprehensive and inclusive. 

### 2 So the battery itself was designed to ensure 

### 3 detection of estrogen, androgen, thyroid hormonal system 

### 4 interactions. It fulfills the EDSTAC recommendations that a 

### 5 range of taxonomic groups be included, and it includes 

### 6 sufficient diversity of endpoints to maximize sensitivity and 

### 7 minimize false negatives. 

### 8 Again, we wanted to emphasize the need for 

### 9 apical assays to provide a more comprehensive assessment in the 

### 10 context of what was being done for the screening. So in general 

### 11 it meets the overall objective of detecting potential for 

### 12 endocrine-mediated effects regardless of the particular 

### 13 mechanism of action. 

### 14 Again, as already have been mentioned, it's a 

### 15 two-tiered approach that we're utilizing and what was 

### 16 recommended by the EDSTAC, with Tier 1 designed to detect 

### 17 potential to interact with the endocrine system and the pathways 

### 18 we're focused on are estrogen, androgen and thyroid. 

### 19 This Tier 1 includes both in vitro and in vivo 

### 20 assays, and if the context is a positive then you would consider 

### 21 need for Tier 2. 

### 22 Tier 2 then is the higher-level design to 

### 23 corroborate interactions that may have been seen in Tier 1, to 

### 24 characterize adverse effects if these occur, and provide 

### 25 dose-response information from these. 

### 26 In general, the Tier 2 tests consist of

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1 lifecycle and multigenerational-level studies and cover a broad 

### 2 range of taxa including the mammals, birds, frogs, fish, and 

### 3 even including invertebrates for the connection that they might 

### 4 have in terms of actions that may be seen. 

### 5 The assays that are included in Tier 1 are 

### 6 basically these, and we've seen the similar slides before, but 

### 7 the in vitro assays include steroidogenesis, the estrogen 

### 8 receptor binding and a transcriptional activation assay, an 

### 9 androgen receptor binding and aromatase. 

### 10 There are a couple of in vivo assays that have 

### 11 the HPG axis interrupted, and these are uterotrophic and 

### 12 Hershberger. Then there are the whole-animal assays which are 

### 13 pubertal female, pubertal male and amphibian metamorphosis and 

### 14 then a fish short-term reproduction. 

### 15 These assays are designed to work together in 

### 16 an integrative fashion to maximize sensitivity and minimize 

### 17 false negatives. The in vitro assays as a group provide 

### 18 sensitivity and mechanistic clues in terms of what may be going 

### 19 on or what an initiating event may be. The in vivo assays 

### 20 provide for more integrative responses and include metabolism 

### 21 and distribution considerations. 

### 22 The battery itself relies on similarities of 

### 23 endocrine systems across taxa and includes redundancies in these 

### 24 assays as well as some assay-unique endpoints in terms of being 

### 25 able to cover and interpret what may be going on to allow for 

### 26 conclusions that there is indeed potential interactions with the

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1 hormone or endocrine system. 

### 2 And it's interpreted by a weight of evidence 

### 3 of the battery in addition to other relevant information that 

### 4 may exist, other toxicologic information, literature information 

### 5 that may be available. And it's this weight of evidence rather 

### 6 than the sum of, you know, any positive or negatives in terms of 

### 7 responses of the assays themselves. 

### 8 This is another familiar slide in terms to 

### 9 identify across the assays the coverage with the various 

### 10 hormonal pathways in terms of which assays are reacting to 

### 11 elements of perturbations along the pathway for estrogens, the 

### 12 androgens. In the steroid synthesis, you know, the androgen 

### 13 steroid and estrogen steroid syntheses themselves and really 

### 14 towards the right we have the HPG, HPT axes, and these are 

### 15 really going to be comprehensive in terms of interactions and 

### 16 include both the mammalian as well as fish and amphibian. 

### 17 To get into a little bit more specifics for 

### 18 the individual pathways and the variety of assays that relate or 

### 19 speak to these, for the estrogen pathway first, we have ER 

### 20 binding which detects chemicals that bind to the estrogen 

### 21 receptor. This is critical since that would be an initiating 

### 22 activity for what would ultimately become the outcome within 

### 23 that particular pathway. 

### 24 There's the estrogen receptor, the 

### 25 transcriptional activation assay which detects the receptor 

### 26 interaction. And with this particular assay, we're able to

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1 differentiate ER agonist from antagonist. The uterotrophic is 

### 2 designed to detect chemicals that act through the ER. It has 

### 3 the HPG interrupted so will be using ovariectomized or immature 

### 4 individuals, and it incorporates metabolism as well. But it 

### 5 would be able to indicate any activity that would be determined 

### 6 by an estrogen signal independent of the hypothalamus and 

### 7 pituitary interaction. 

### 8 The pubertal female is designed to detect 

### 9 chemicals that act on the mammalian estrogen system and there 

### 10 are key endpoints within this particular assay to respond to 

### 11 that from an integrated or comprehensive assessment. And the 

### 12 same with the fish; it's designed to detect chemicals that act 

### 13 with the fish estrogen system. 

### 14 The one key with the fish is it's fish during 

### 15 the reproductive portion of the lifecycle. So for compounds 

### 16 like estrogen as well as the androgen, which are reproductive 

### 17 hormones, we have the ability to detect what might be going on. 

### 18 And although reproduction is somewhat nonselective in terms of 

### 19 being endocrine-specific, it does pick out endocrine activities 

### 20 and with combinations of other endpoints and other information 

### 21 within the battery, we're able to determine if it's primarily 

### 22 through endocrine mechanism or not. At least that's the 

### 23 consideration that we have. 

### 24 Within the androgen pathway, a similar set 

### 25 where we've got in vitro assay with the AR binding which is 

### 26 designed to detect compounds that bind to the androgen receptor.

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1 There is a Hershberger which is an in vivo assay which detects 

### 2 compounds that act through the androgen receptor. It can 

### 3 distinguish between agonist and antagonist, you know, the aspect 

### 4 of this particular assay. It incorporates metabolism and likely 

### 5 uterotrophic. It is independent of the hypothalamus and 

### 6 pituitary interaction because these would be castrated 

### 7 individual. 

### 8 The pubertal male is another in vivo assay 

### 9 designed to detect chemicals that act on the mammalian androgen 

### 10 system through a variety of pathways or tissues that would be 

### 11 responsive to this and developmental cues so the indications 

### 12 would be from a kind of comprehensive interaction. And fish, 

### 13 you know, likewise, in vivo detecting chemicals that act through 

### 14 the fish androgen system and specifically with endpoints like 

### 15 secondary sex characteristics in reproduction. There would 

### 16 indication of activity that would be prevalent and able to 

### 17 compare that with information gleaned from some of the other 

### 18 assays within the pathway in terms of the action overall. 

### 19 Thyroid pathway is limited to the three 

### 20 assays, all in vivo. We weren't able to identify or at least 

### 21 have recommended the in vitro thyroid assays. That's still an 

### 22 area of active research. But for now we're limited to in vivo 

### 23 activities which would be comprehensive. 

### 24 With both the pubertal female and pubertal 

### 25 male, these are designed to detect hormonal changes through 

### 26 measures of things like T4, TSH and then thyroid histology. For

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1 the amphibian metamorphosis, this detects changes in thyroid 

### 2 histology but also developmental changes that would occur 

### 3 through the metamorphosis which is a good indicator and a 

### 4 general indicator for vertebrate thyroid controls. 

### 5 Steroidogenesis is another aspect that's 

### 6 considered within the battery. We have aromatase assays 

### 7 specific to detecting chemicals that would inhibit aromatase 

### 8 action in an in vitro system. There is steroidogenesis which is 

### 9 a little bit more comprehensive in terms of any interactions or 

### 10 perturbances in steroid metabolism that could inhibit enzymes in 

### 11 the ultimate steroid synthesis. 

### 12 The in vivo assays of the pubertal male, 

### 13 female as well as the fish screen, because these are 

### 14 comprehensive in terms of the estrogen/androgen pathways, would 

### 15 detect any interferences within the steroid metabolism as well. 

### 16 So in trying to consider the real strength of 

### 17 the battery overall in terms of the various kinds of activities 

### 18 and the number of assays that speak to these that either 

### 19 compliment or potentially provide a little redundancy in terms 

### 20 of action, you know, this table kind of compares that. 

### 21 For estrogenic activity, it's generally done 

### 22 fairly well of the five assays that are looking at various 

### 23 components across the pathway with ER binding, transcriptional 

### 24 activation, the uterotrophic, the female pubertal and fish. 

### 25 And going down the line for things like 

### 26 anti-estrogenic, there are three assays that would be able to

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1 detect along those lines. There is the androgenic, we've got 

### 2 four assays; and anti-androgenic, the same four assays. 

### 3 In compounds that modulate steroidogenesis, 

### 4 we've got five assays with two in vitros as well as the three 

### 5 key in vivos that deal with the estrogens and thyroids. 

### 6 For aromatase, we're back with really only 

### 7 four because the male pubertal doesn't provide, you know, really 

### 8 good information in terms of aromatase interactions in terms of 

### 9 the kinds of endpoints that are going to be more androgen 

### 10 specific. 

### 11 For hypothalamic pituitary function in terms 

### 12 of the HPG axis overall, we have to deal with the integrated in 

### 13 vivo assays where we have the pubertal, the fish and the 

### 14 amphibian on the HPT axes. Anti-thyroid activity, male, female 

### 15 pubertals are directed at this as well as amphibian 

### 16 metamorphosis primarily through the histopathology as well as 

### 17 the hormone measures that would be made in the rat pubertal. 

### 18 For thyromimetic activity, we're limited to 

### 19 the amphibian metamorphosis. And this is done through the kind 

### 20 of development, either accelerated development or things like 

### 21 asynchronous development that would seen, would be indicators 

### 22 for this particular type of activity. 

### 23 In terms of looking at battery overall and 

### 24 trying to interpret, you know, we do this through weight of 

### 25 evidence review. That uses professional judgment. Some 

### 26 endpoints are more diagnostic and specific than others. Effects

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1 seen in multiple endpoints and across multiple assays will carry 

### 2 the most weight. 

### 3 You know, we will be considering weight of 

### 4 evidence review on a forthcoming SAP later this summer, and so 

### 5 we'll focus here just in terms of how the battery performs in 

### 6 providing information that would be utilized within kind of a 

### 7 weight of evidence context. 

### 8 Really we anticipate two possible outcomes. 

### 9 You know, either there is sufficient potential to identify 

### 10 within the review of the assays in terms of E, A or T acuity 

### 11 which could warrant additional analysis in Tier 2 testing. The 

### 12 other outcome would be the potential to consider either low or 

### 13 nonexistent. 

### 14 We went back and looking at previous 

### 15 SAP in 2008 in terms of its review of the Tier 

### 16 1 screening battery, we had a charge question at that time, are 

### 17 there any unnecessary redundancies for MoAs across the battery? 

### 18 19 The panel assessed that, considered each assay 

### 20 in reference to different modalities for the EAT pathways, 

### 21 examined degrees of complimentarity or concordance across the 

### 22 assays and examined any degree of redundancy or concordance. 

### 23 And their conclusion was that in vitro assays convey specificity 

### 24 and sensitivity but provide no complementarity or redundancy 

### 25 within the battery for these in vitro assays alike. But the in 

### 26 vivo assays provide both complementary and redundancy within the

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1 battery. 

### 2 The overall recommendations were based on the 

### 3 current state of science. This proposed set of Tier 1 assays 

### 4 are an appropriate starting point, but EPA should consider this 

### 5 as a work in progress and expects to continue to develop, refine 

### 6 and review. That brings us here in terms of providing feedback 

### 7 on some of the initial outcomes of the battery performance in 

### 8 terms of how well these assays are working and to what degree we 

### 9 see redundancies or complimentarity in terms of how the assays 

### 10 function. 

### 11 So kind of to summarize, multiple assays are 

### 12 required to comprehensively screen E, A and T hormone systems. 

### 13 You know, there is not a one size fits all. In vitro assays are 

### 14 good for well understood mechanisms such as receptor binding. 

### 15 The in vivo assays with intact HPG or HPT axes are good for 

### 16 efficiently screening complex processes to make sure we are 

### 17 identifying outcomes that may be acting through mechanisms that 

### 18 we might not be as secure about. 

### 19 Multiple taxa and mode of action endpoints 

### 20 provide a range of metabolism and needed corroboration. And 

### 21 then the complete battery is needed to support a weight of 

### 22 evidence finding of low or no potential for EAT activity. 

### 23 So with that, I thank the panel for your 

### 24 attention and can respond to any specific questions or 

### 25 clarification on the battery itself. 

### 26 **DR. DANIEL SCHLENK:**Any questions for Dr.

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1 Touart? Yes, Dr. Delclos. 

### 2 **DR. BARRY DELCLOS:**One question. Before your 

### 3 grand summary slide, is there an anti-estrogenic component in 

### 4 the guideline for the uterotrophic assay? Because in the White 

### 5 Paper I think it said that there is, but nobody did it. 

### 6 **DR. LESLIE TOUART:**For the uterotrophic assay 

### 7 as part of the validation effort, there was a component part 

### 8 that dealt with the antagonism in terms of what was used. But 

### 9 that didn't perform so well, so it wasn't included in the final 

### 10 guideline. So it's something that could still, you know, be 

### 11 developed, but it didn't perform well enough for us to consider 

### 12 using it at the time. 

### 13 **DR. DANIEL SCHLENK:**Any other questions? 

### 14 Yes, Dr. McManaman. 

### 15 **DR. JAMES McMANAMAN:**In your steroidogenesis 

### 16 assays, are those gene assays, or are they based on actual 

### 17 measurements of products in the adrenal? 

### 18 **DR. LESLIE TOUART:**Yeah, for the 

### 19 steroidogenesis as well as the aromatase, these are in vitro 

### 20 assays. They're not gene-based. They are actual 

### 21 component-based to look at actual products in terms of 

### 22 productions of the particular hormones like testosterone or 

### 23 estradiol. So they are measured in terms of that context. 

### 24 And I should mention that the assays 

### 25 themselves, that there is discussion in later presentations so 

### 26 there will be a little bit more detail in terms of the actual

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1 assays and some of the functional elements of those. 

### 2 **DR. DANIEL SCHLENK:**Yes, Dr. Piersma. 

### 3 **DR. ALDERT PIERSMA:**I have a general question 

### 4 about the level of depth of the presentation of the data. 

### 5 Basically the way you present the data is either it gives us a 

### 6 response or not in the various assays. And in order to 

### 7 appreciate the results, it might even help for you to go into a 

### 8 little more depth, but this has not been done purposely on 

### 9 things like directionality, potency within the different assays 

### 10 and how they compare among the assays. 

### 11 What particular endpoint within 

### 12 multiple-endpoint assays that was affected that drove the either 

### 13 plus or minus within the different tests, that kind of level? 

### 14 Have you thought about presenting it at that level and why 

### 15 haven't you done it that way? 

### 16 **DR. MARY MANIBUSAN:**So in terms of the degree 

### 17 of detail inclusive of directionality and endpoint analysis 

### 18 across the different assays, that will be the composition of the 

### 19 July SAP. So we've purposely refrained from moving in that 

### 20 direction because we wanted to hold the methodology standardized 

### 21 so that we can look at just the performance of the assay and 

### 22 battery and not draw any conclusions for chemical assessments 

### 23 and its ability to initiate particular endocrine activity. That 

### 24 will be the subject of the July meeting. 

### 25 **DR. DANIEL SCHLENK:**Dr. Dix to add? 

### 26 **DR. DAVID DIX:**Yeah, just to add to Mary's

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1 response, I think, Dr. Piersma, your comments could very well 

### 2 lead to some very useful suggestions from this panel in how we 

### 3 might revise, not just our analysis but our presentation of 

### 4 those analyses and communication. 

### 5 **DR. DANIEL SCHLENK:**Thanks. Dr. Green? 

### 6 **DR. SHERRIL GREEN:**I have another general 

### 7 question about the metamorphosis assays that I'll hear more 

### 8 about later in Xenopus, but I was wondering, you know, the 

### 9 Xenopus FETAX test already exists; and are there not elements of 

### 10 that very standardized, very well-used, documented test that 

### 11 would be applicable to the Tier 1 assay here and be cheaper, 

### 12 faster? Just maybe some elaboration on that would help me. 

### 13 **DR. LESLIE TOUART:**In determining the context 

### 14 of how the amphibian metamorphosis assay was selected, again, 

### 15 the context was that the Xenopus developmental model was 

### 16 considered to be well understood and could be adapted in terms 

### 17 of a thyroid activity screen fairly easily. The initial context 

### 18 was perhaps a tail resorption assay, but that was felt, well 

### 19 that's usually coming a little bit after the big peak within the 

### 20 thyroid level. 

### 21 The FETAX was considered, but FETAX doesn't 

### 22 really provide us good information in terms of thyroid activity 

### 23 to the context of the histopathology that's done on the gland 

### 24 from what we can see. And then the developmental cues that are 

### 25 identified in terms of accelerated development and, you know, 

### 26 things like asynchronous development that come later than the

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1 FETAX window would allow for. 

### 2 So FETAX was discounted, you know, kind of 

### 3 early in the considerations, and then the effort was moved to 

### 4 develop metamorphosis which went through the validation process 

### 5 and for the thyroid active compounds that we used to challenge 

### 6 it were determined to be effective on those. 

### 7 **DR. DANIEL SCHLENK:**Any other questions for 

### 8 Dr. Touart? 

### 9 Okay. Thank you. Let's go ahead and take a 

### 10 first break. And I'm going to go by this clock on the wall 

### 11 here, so let's be back at 20 'til 11. 

### 12 (Whereas a break was taken) 

### 13 **DR. DANIEL SCHLENK:**Our next presentation on 

### 14 in vitro assays is by Gregory Akerman who is with the Health 

### 15 Effects Division, Office of Pesticide Programs. 

### 16 **DR. GREGORY AKERMAN:**Good morning. My name 

### 17 is Greg Akerman. I'm from the Health Effects Division, Office 

### 18 of Pesticide Programs. I would like to first begin by 

### 19 presenting an overview of the EDSP assay performance 

### 20 presentations. 

### 21 As been previously mentioned, this slide shows 

### 22 the order of the presentations. I will begin by presenting the 

### 23 results of the Tier 1 in vitro assays. John Liccione will 

### 24 present the results of the in vivo mammalian assays. Amy 

### 25 Blankinship will follow with presentation of fish short-term 

### 26 reproduction assay, and Catherine Aubee will complete the assay

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1 performance session with a presentation of the amphibian 

### 2 metamorphosis assay. 

### 3 The goals of the assay performance are listed 

### 4 on this slide. The primary goal for this analysis is to 

### 5 evaluate how well the Tier 1 assays can be executed by the 

### 6 testing laboratories based on defined performance criteria and 

### 7 the test guidelines. The second goal of this exercise to 

### 8 identify any issues associated with the interpretation of 

### 9 responses within each of the assays. 

### 10 As mentioned previously, there are a total of 

### 11 21 chemicals that were selected as case studies for this 

### 12 analysis, and these chemicals represent chemical space of 52 

### 13 pesticide actives that were included in the first group of 

### 14 chemicals to receive EDSP Tier 1 test order. This subset of 

### 15 chemicals represented a broader range of physical-chemical 

### 16 properties, chemical classes and pesticide activities. 

### 17 Please note as the presenters discuss the 

### 18 results of the assays that the number of chemicals evaluated for 

### 19 each of the assays may differ as some of the assays were 

### 20 satisfied by other scientifically relevant information commonly 

### 21 referred to as O-S-R-I or OSRI. OSRI is defined as the existing 

### 22 data used to satisfy guideline requirements. 

### 23 This table shows the number of case study 

### 24 chemicals evaluated for each of the 11 Tier 1 assays. As 

### 25 previously mentioned, some of the study requirements are 

### 26 satisfied by OSRI. Please also keep in mind that although some

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1 of the assays have the same number of case studies, for example, 

### 2 AR binding, uterotrophic, Hershberger and pubertal female all 

### 3 have 17 case-study chemicals. This does not necessarily mean 

### 4 that they are the same 17 chemicals for all four assays. Fish 

### 5 short-term reproduction assay is all 21 chemicals evaluated for 

### 6 that assay. 

### 7 This slide outlines the data review process 

### 8 that was followed for evaluating the Tier 1 assays. The Agency 

### 9 has developed a standard set of standard evaluation procedures, 

### 10 or SEPs, for each of the 11 Tier 1 assays. These SEPs were used 

### 11 in conjunction with the appropriate EDSP Test Guidelines to 

### 12 review the studies. 

### 13 The Agency also developed data evaluation 

### 14 records or DERs. The DERs capture the data to determine how 

### 15 well the assay conforms to the guideline. It also documents 

### 16 information on materials and methods, study results, made strong 

### 17 conclusions as well as the Agency's conclusions. 

### 18 The Agency used a multi-tiered approach to 

### 19 review the Tier 1 data. Primary reviews were performed either 

### 20 by the Agency or contract scientists. Secondary reviews of the 

### 21 DERs were performed by Agency scientists. And then finally, an 

### 22 additional level of review was conducted by an interdisciplinary 

### 23 committee to ensure consistency in the interpretation of the 

### 24 assay results across all sets of chemicals. 

### 25 Before discussing individual assays' results, 

### 26 I'd like to also mention two topics that will be discussed

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1 throughout the talks today. One is performance criteria. Most 

### 2 of the Tier 1 in vitro assays and some of the in vivo assays 

### 3 have performance criteria established for controlled chemical 

### 4 performance and/or specific assay parameters or conditions. And 

### 5 some of the performance criteria are related to the second 

### 6 topic, overt toxicity. 

### 7 Overt toxicity was observed in some of the in 

### 8 vivo assays, and some examples of overt toxicity including 

### 9 bodyweight loss in rats and possibly in fish studies, clinical 

### 10 signs or erratic behavior in swimming or aquatic studies. For 

### 11 the purpose of this analysis, we note where overt toxicity was 

### 12 seen, and we indicate if potential endocrine-related effects 

### 13 were seen in the presence of overt toxicity. 

### 14 Next we're going to move into the discussion 

### 15 of the in vitro assays. One this slide are the five in vitro 

### 16 assays that we've mentioned previously, the ER binding assay, 

### 17 the ER transcriptional activation assay or ERTA, AR binding 

### 18 assay, steroidogenesis assay and aromatase assay. 

### 19 This slide shows an overview of how we'll 

### 20 present the five in vitro assays. First, I will provide 

### 21 background information on each assay. Then I will discuss the 

### 22 evaluation of the study including performance criteria, general 

### 23 observations from the selected studies and the results. 

### 24 Finally, I will provide concluding statements 

### 25 of how well the assay was executed. 

### 26 Starting with the ER binding assay, this

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1 assay, as Dr. Touart had mentioned, measures the ability of a 

### 2 test chemical to interact with ER. Rat uterine cytosols is the 

### 3 source for the estrogen receptor and both ER alpha and ER beta 

### 4 are expressed in this tissue. The assay cannot distinguish 

### 5 between ER agonism and ER antagonism. 

### 6 A total of 18 chemicals were evaluated for the 

### 7 ER binding assay preliminary analysis. ER binding assay has two 

### 8 components, the saturation binding experiment and the 

### 9 competitive binding experiment. I will talk about each one 

### 10 individually. 

### 11 Starting with saturation binding, the purpose 

### 12 of the saturation binding experiment is to determine if the ER 

### 13 binds estradiol correctly as indicated by a dissociation 

### 14 constant, or Kd value, and are the ER estrogen receptors present 

### 15 in reasonable numbers in the cytosol preparation as indicated by 

### 16 the Bmax. Saturation binding experiment is performed by using a 

### 17 constant concentration of receptor with increasing 

### 18 concentrations of the radiolabled estradiol. The amount of 

### 19 radioactivity bound to the receptor is measured. 

### 20 In the competitive binding experiment, the 

### 21 objective is to determine whether the test chemical interacts 

### 22 with the ER. In this case, the concentration of the radiolabled 

### 23 ligand, estradiol and the estrogen receptors are held constant 

### 24 and incubated with increasing concentrations of the test 

### 25 chemical. If the test chemical does interact with the estrogen 

### 26 receptor, increasing concentrations of the competitor will

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1 result in a descending concentration response curve. 

### 2 The next slide, I show an example of a 

### 3 competitive binding curve. The competitive binding assay is 

### 4 composed of three independent runs. Each run includes a strong 

### 5 positive control which is unlabeled estradiol, a weak positive 

### 6 control norethindrone as all the studies we evaluated used 19 

### 7 norethindrone as the weak positive control. And it also 

### 8 includes a negative control. The test chemical is included and 

### 9 tested over a range from 10 to -10 to 10 to -3 molar or up to 

### 10 the limit of solubility. In this example, the test chemical did 

### 11 not interact with the estrogen receptor. 

### 12 Binding classifications are listed on this 

### 13 slide. The classifications of a chemical is based on the result 

### 14 of three independent binding runs. The three classification 

### 15 categories are interactive, not interactive and equivocal. 

### 16 A run is classified as interactive if more 

### 17 than 50 percent of the radiolabeled estradiol has been displaced 

### 18 from the receptor. The run is classified as not interactive if 

### 19 less than 25 percent of the radiolabeled ligand has been 

### 20 displaced. And a run is considered equivocal if it does not 

### 21 meet the criteria for interactive or non-interactive. 

### 22 Shown here are the performance criteria for ER 

### 23 saturation binding. The parameters include Bmax, Kd and 

### 24 nonspecific binding. For the 18 chemicals evaluated, all 

### 25 saturation binding performance criteria were met. I'd also like 

### 26 to point out that the 18 chemicals evaluated represents the

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1 results from six different testing laboratories. It was common 

### 2 for some laboratories to evaluate multiple test chemicals used 

### 3 in the same cytosol prep. 

### 4 This slide shows performance criteria for 

### 5 estradiol for the competitive binding experiment. The left 

### 6 column lists the parameters for the response curve, and the 

### 7 middle columns show the expected values for each criteria. The 

### 8 column on the right show the actual ranges of values observed 

### 9 across the 18 chemicals evaluated. 

### 10 This graph summarizes the performance criteria 

### 11 for the positive control 17 beta estradiol. The dark gray boxes 

### 12 indicate the performance criterion was met. Although it may be 

### 13 difficult to distinguish the shades of gray, the important point 

### 14 emphasized here is that the large majority of the chemicals 

### 15 performance criterion were met. 

### 16 This slide shows the binding classifications 

### 17 of the 18 chemicals evaluated. Please note, this table was 

### 18 incorrectly labeled on page 27 of the White Paper. That's Table 

### 19 4. The table on this slide shows the correct result of the 18 

### 20 chemicals. None of the 18 chemicals classified as interacted 

### 21 with the ER. Two were classified equivocal, and 16 were 

### 22 classified as not interactive. 

### 23 For the ER binding assay, we conclude that 

### 24 although the number of the studies evaluated for this analysis 

### 25 did not meet all of the performance criteria, in general, when 

### 26 the performance criteria were not met, the values fell slightly

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1 outside the expected ranges. Overall, the laboratory 

### 2 performance of the ER binding assay was generally acceptable. 

### 3 Next I'd like to move to the ER 

### 4 transcriptional activation assay. This system evaluates 

### 5 potential of a chemical to bind and activate the ER using a 

### 6 luciferase reporter gene system. 

### 7 Some general background information is 

### 8 provided on this slide. The assay uses the HeLa-derived cell 

### 9 line containing human ER alpha expression construct and an 

### 10 estrogen response luciferase construct. 

### 11 Test chemical binds as an ER agonist, 

### 12 luciferase enzyme is produced. When the substrate luciferin is 

### 13 added, it caused the light-emitting reaction that we measured. 

### 14 This assay measures ER alpha agonist activity only. Nineteen 

### 15 chemicals were evaluated in this analysis. 

### 16 A chemical is considered positive in this 

### 17 assay if inducive response is at least 10 percent of the maximum 

### 18 response of the positive control which in this assay is one 

### 19 animal estradiol. The positive response must be observed in at 

### 20 least two of the three experimental runs. 

### 21 Test guide recommends that concurrent 

### 22 reference chemicals be included in each experiment to ensure 

### 23 that the test system is running properly. Listed here are the 

### 24 four reference chemicals with the expected ranges of values for 

### 25 each parameter. 17 beta estradiol is used as a strong positive 

### 26 control. 17 alpha estradiol is included as a weak positive

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1 control. Methyltestosterone is included as a very weak positive 

### 2 control, and corticosterone serves as a negative control count. 

### 3 For 17 beta estradiol, all performance 

### 4 criteria were met for 10 of the 19 chemicals. 17 alpha 

### 5 estradiol, all parameters were met for 5 of the 19 chemicals. 

### 6 Testing facilities did appear to have difficulty meeting the 

### 7 guideline performance criteria for 17 alpha methyltestosterone. 

### 8 However, in most cases for the referenced chemicals, the values 

### 9 that did not meet the criteria fell slightly outside of the 

### 10 expected ranges. 

### 11 This table shows the classification of the 19 

### 12 chemicals evaluated with ERTA. There were 5 positives, 13 

### 13 negatives, and one study was considered inconclusive based on 

### 14 cytotoxicity that was observed in the reference chemicals that 

### 15 compounded interpretation of the study. 

### 16 In conclusion, the ERTA, all the performance 

### 17 criteria for the referenced chemicals were not met for the 

### 18 majority of the chemicals. However, in most cases, the values 

### 19 were close to the guideline ranges and did not affect the 

### 20 interpretation of the assay. Most chemicals had difficulty 

### 21 meeting performance criteria for the very weak agonist, 17 alpha 

### 22 methyltestosterone. And all but one chemical was classified as 

### 23 either negative or positive in this assay. 

### 24 Next I'll move to the AR binding assay. I 

### 25 will not spend a lot of time going into the background 

### 26 information since it's the same principle as seen in the ER

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1 binding assay. This assay detects the ability of the chemical 

### 2 to interact with the androgen receptor. 

### 3 Seventeen chemicals were evaluated for this 

### 4 assay. As with the ER binding assay, there were two components 

### 5 to the assay, saturation binding and competitive binding. The 

### 6 AR saturation binding experiment is similar to ER binding with 

### 7 the exception that AR binding assay uses the synthetic androgen 

### 8 R1881 as a reference androgen ligand and rat prostate cytosol as 

### 9 a source of the AR. 

### 10 The objective interpretation of this assay is 

### 11 similar to the ER binding assay. The assay uses constant 

### 12 concentration of the radiolabeled ligand, constant concentration 

### 13 of receptor with increasing concentrations of the test chemical. 

### 14 If the test chemical competes with the ligand, a descending 

### 15 concentration curve is produced as shown in the next slide. 

### 16 Here we see an example of an AR competitive 

### 17 binding curve. The assay is composed of three independent runs 

### 18 with each run containing unlabeled R1881 as the strong positive 

### 19 control, dexamethasone as a weak positive control, and the test 

### 20 chemical tested in the range from 10 to -10 to 10 to -3 molar or 

### 21 up to the limit of solubility. 

### 22 In this example, the test chemical showed a 

### 23 positive response producing a descending concentration response 

### 24 curve. The binding classifications have shown, on this slide, 

### 25 for classification purposes the term "binder" and "non-binder" 

### 26 are equivalent to the terms that are used in the ER binding

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1 assay which are interactive and not interactive. 

### 2 This slide shows performance criteria of the 

### 3 Kd and nonspecific bindings for the AR binding assay. 

### 4 And here, we see the results of the saturation 

### 5 binding performance criteria. The Kd values were not met for 4 

### 6 of the cytosol preparations that were used for a total of 8 

### 7 chemicals. All laboratories met the nonspecific binding 

### 8 criteria. 

### 9 Performance criteria for the competitive 

### 10 binding experiments is shown here with the curved parameters 

### 11 from the bottom, top plateau levels and the hill slope. The 

### 12 expected values of strong positive and weak positive controls 

### 13 are shown. 

### 14 This graph shows how well the assays met the 

### 15 competitive binding performance criteria for R1881. In nearly 

### 16 all cases, the performance criteria were met for the strong 

### 17 positive control. And for the weak positive control, all 

### 18 performance criteria were met for all chemicals. 

### 19 This table shows the classifications for the 

### 20 17 chemicals evaluated in the AR binding assay. Three chemicals 

### 21 were classified as binder. Ten were classified as non-binder. 

### 22 Three were classified as equivocal, and one was considered 

### 23 untestable because it was not soluble above concentrations of 10 

### 24 to 6 molar in the assay system. 

### 25 In conclusion for the AR binding assay, 

### 26 although the performance criteria were not met for all

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1 chemicals, particularly with regards to saturation binding; 

### 2 however, when the cytosols were used in the competitive binding 

### 3 criteria, nearly all performance criteria were met. Overall, 

### 4 the laboratory performance of the AR binding assay was generally 

### 5 acceptable. 

### 6 Next I'll move to the steroidogenesis assay. 

### 7 This assay uses the adrenal corticocarcinoma cell on H295R. The 

### 8 cell line expresses all enzymes needed to synthesize 

### 9 testosterone and estradiol. Eighteen chemicals were evaluated 

### 10 in this analysis. 

### 11 This diagram shows the basics of how the assay 

### 12 is performed. The assay is generally conducted in 24 well 

### 13 plates, and the endpoints measured are estradiol and 

### 14 testosterone production. Three independent runs are conducted, 

### 15 and each run includes the positive control for inducer and 

### 16 inhibitor of hormone production. 

### 17 This slide shows a test chemical that resulted 

### 18 in estradiol inhibition on the left panel, and the graph on the 

### 19 right shows an example of test chemicals that inhibit 

### 20 testosterone production. Positive responses are indicated by a 

### 21 statistically significant increase or decrease of estradiol/ 

### 22 testosterone production. 

### 23 Performance criteria for basal-level 

### 24 production of testosterone, estradiol and the cell system are 

### 25 shown here as well as the criteria for the minimum level of 

### 26 hormone induction inhibition for the positive control chemicals

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1 for both estradiol and testosterone. 

### 2 The graph on this slide shows how well the 18 

### 3 chemicals performs in this assay. Although it may be difficult 

### 4 again to read all the shades of gray, the overall message is 

### 5 that most of the performance criteria are met for most of the 

### 6 parameters. Again, in cases where the performance criteria were 

### 7 not met, they were typically close to the expected values and 

### 8 did not impact the interpretation of the study. 

### 9 This table shows the results of the 18 

### 10 chemicals. Six chemicals were a negative in this assay, and ten 

### 11 chemicals showed some effect on either estradiol and/or 

### 12 testosterone production. Two chemicals were classified as 

### 13 equivocal. 

### 14 In conclusion for this steroidogenesis assay, 

### 15 laboratory performance in the steroidogenesis assay was 

### 16 generally consistent across the 18 chemicals evaluated. 

### 17 Although not all performance criteria were met for every 

### 18 chemical, the experimental values were close to the expected 

### 19 values, and the deviations did not affect the interpretation of 

### 20 the study. 

### 21 Finally, the last in vitro assay I'll discuss 

### 22 is the aromatase assay. This assay is used to determine if a 

### 23 test chemical can inhibit Step 19 aromatase enzyme activity 

### 24 which is the enzyme that converts androgens to estrogens. The 

### 25 components of the reaction are shown here on this slide. A 

### 26 radiolabeled estrogen dione substrate is added to microsomal

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1 complex containing the aromatase plus the test chemical, 

### 2 concentrations of .1nM - 1.0mM. The endpoint measurement in 

### 3 this assay is the production of tritiated water. 

### 4 Some additional background information on this 

### 5 assay measures the rate at which tritiated water is produced 

### 6 with increasing concentrations of test chemical. Three 

### 7 independent runs are conducted. Each run has a full activity 

### 8 control, background activity control, and each run also includes 

### 9 eight concentrations of the positive control and eight 

### 10 concentrations of the test chemical. Eighteen chemicals were 

### 11 evaluated for this analysis, and all 18 studies evaluated used 

### 12 human recombitant microsomals as the source for aromatase. 

### 13 Classification categories for this assay are 

### 14 shown here. A chemical is classified as an inhibitor if there's 

### 15 a 50 percent or more reduction in aromatase activity. Chemicals 

### 16 that allow 50 to 75 percent activity are classified as 

### 17 equivocal, and non-inhibitors do not fit the inhibition model or 

### 18 they do not reduce activity by 25 percent. 

### 19 Performance criteria for this assay are shown 

### 20 here. Top half of the table show the criteria for experimental 

### 21 parameters, and the bottom half show the performance criteria 

### 22 for the positive controlled response curves. 

### 23 This slide shows the performance criteria 

### 24 results. The performing laboratories were able to meet most of 

### 25 the criteria for most of the chemicals. In the case of the 

### 26 criteria not met, values predominantly fell slightly outside

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1 expected ranges and generally did not impact the overall 

### 2 interpretation of the study. 

### 3 For the 18 chemicals evaluated in this assay, 

### 4 two chemicals were classified as inhibitors, five chemicals were 

### 5 classified as equivocal with those chemicals showing some 

### 6 limited inhibition at the highest test concentration. Eleven 

### 7 chemicals were classified as non-inhibitors aromatase activity. 

### 8 The laboratory performance aromatase assay was 

### 9 generally consistent across the test chemicals evaluated. As 

### 10 with the other in vitro assays, most of the performance criteria 

### 11 were met, and in cases where they were not met, the results 

### 12 slightly exceeded or fell outside the expected range of the 

### 13 performance criteria. 

### 14 Finally, I'd like to briefly discuss a few 

### 15 additional observations in this initial set of chemicals. One 

### 16 is proficiency testing. Proficiency testing for the 

### 17 laboratories was not consistently reported across chemicals and 

### 18 in some cases proficiency data were not included in the study 

### 19 reports, or in other cases, proficiency testing was not 

### 20 mentioned in the study report. 

### 21 The second observation has to do with 

### 22 solubility and cytotoxicity. Test chemical and solubility and 

### 23 cytotoxicity was observed with many of the chemicals at the 

### 24 highest test concentrations. When test chemical solubility 

### 25 issues were encountered some, but not all laboratories followed 

### 26 EPA guidance by adding additional half-log concentrations as

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1 recommended in many of the in vitro test guidelines. 

### 2 These additional half-log concentrations 

### 3 helped to better define a lower portion of the response curve 

### 4 and aided the reviewers in interpreting the results. We 

### 5 encourage the use of half-log concentrations when appropriate. 

### 6 And the last observation was for saturation 

### 7 binding data, the saturation binding data from the binding 

### 8 assays, the ER and AR binding assays. These data were not 

### 9 always submitted within the study report, although they were 

### 10 submitted subsequently upon request by the Agency. We 

### 11 encouraged laboratories to include those data within the study 

### 12 reports for reviewing purposes. 

### 13 Thank you for your attention. 

### 14 **DR. DANIEL SCHLENK:**Any questions for Dr. 

### 15 Akerman? 

### 16 Dr. Scott. 

### 17 **DR. GEOFFREY SCOTT:**Across the different 

### 18 assays, the word "slight" is used. Is that a consistent 

### 19 quantitative deviation from the norm across the assays? 

### 20 **DR. GREGORY AKERMAN:**We use that word just to 

### 21 characterize when the values fell barely outside that we didn't 

### 22 really use the same criteria. Of course, there were some 

### 23 instances where the values fell considerably outside the 

### 24 obvious. But for the most part, if they were very close to the 

### 25 expected ranges we used the word slightly. 

### 26 **DR. GEOFFREY SCOTT:**I mean is it a percentile

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1 or, you know, is it within 10 percent, 5 percent? You know, I 

### 2 think that was the problem I had in reading through this 

### 3 section, is not having that quantitative. 

### 4 I understand when a value is pretty close and 

### 5 we want to help you with this. So I think one of the things 

### 6 that would help us in helping you with defining this is, you 

### 7 know, I happen to kind of agree with most instances if 

### 8 something's just slightly outside of the norm, you make a value 

### 9 judgment as to whether or not that's affecting the assay or not. 

### 10 11 So I think the panel will probably want to 

### 12 deliberate and comment on that. So any information you can 

### 13 provide us on that would be helpful. 

### 14 **DR. DANIEL SCHLENK:**Okay. Dr. Schultz. 

### 15 **DR. TERRY SCHULTZ:**Would it be safe to say 

### 16 that the Agency has more experience with the ER-related tests 

### 17 than the AR-related tests? And I only refer to that because 

### 18 there has been a couple of Scientific Advisory Panels that have 

### 19 kind of focused on that area within the prescreening scheme. 

### 20 **DR. MARY MANIBUSAN:**As the statute is focused 

### 21 on estrogenic pathway, that has been our primary focus with the 

### 22 extension to the androgen and thyroid. 

### 23 In terms of our experience, I don't believe 

### 24 our experience is that different across ER and AR. It's a 

### 25 matter of bringing before the panel our experience with this 

### 26 initial list and how this initial list performs for the Tier 1

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1 assays as a whole. 

### 2 **DR. DANIEL SCHLENK:**Dr. Schultz, follow? 

### 3 **DR. TERRY SCHULTZ:**Yes, as a follow-up. Has 

### 4 the Agency looked at revising the performance guidelines so that 

### 5 they can do a better job of capturing what is going to be the 

### 6 inherent inter-laboratory rare-ability in some of these test? 

### 7 I, like Dr. Scott, am concerned about using the word "slight" 

### 8 and things like that. That doesn't sound very scientific and it 

### 9 does lead to perception that there's something going on that is 

### 10 varied in the report or the information and the data. 

### 11 **DR. DANIEL SCHLENK:**Dr. Touart, going to 

### 12 respond to that? 

### 13 **DR. LESLIE TOUART:**The context with the 

### 14 various assays in terms of the performance criteria that were 

### 15 imposed were part of the validation program originally. And so 

### 16 those fell within the context of what we perceived performance 

### 17 was being achieved in the number of iterations that we have. 

### 18 I think the context here in a broader, you 

### 19 know, test of the assays in context in terms of how these are 

### 20 done through, you know, routine laboratory testing, you know, 

### 21 there is the indication that in data they were seeing some fall 

### 22 short of these criteria. And so the consideration is well, are 

### 23 we still able to make determinations, you know, in terms of 

### 24 determining that the assay was conducted satisfactorily, and we 

### 25 can form a conclusion, in which case the performance criteria 

### 26 could, you know, conceivably be modified to accommodate that.

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1 In those cases where it leads to more ambiguities or concerns. 

### 2 When we set performance criteria, these things 

### 3 are also more or less recommendations, they are targets in terms 

### 4 of what we're trying to achieve to ensure quality, you know, 

### 5 studies to be performed. So they're not established as bright 

### 6 lines in terms of, you know, kind of a pass/fail situation. 

### 7 But there would be situations where you fall 

### 8 short enough in enough of the criteria that you might deem the 

### 9 test unsatisfactory and would need to be repeating. So I think 

### 10 there would be some room for going back and revisiting these 

### 11 based upon this broader context to determine if we need to 

### 12 modify or at least loosen some of these criteria to allow better 

### 13 interpretation and at least more successes in terms of 

### 14 laboratory conduct of these assays. 

### 15 **DR. DANIEL SCHLENK:**Okay. Dr. Maclatchy. 

### 16 **DR. DEBORAH MACLATCHY:**Similarly along those 

### 17 same lines, there were a number of places in the White Paper 

### 18 where I've seen that there was missing information coming from 

### 19 the lab. And one of the questions I have is how iterative is 

### 20 EPA when they're getting the information from the labs as far as 

### 21 whether or not the labs might have done things, and they didn't 

### 22 report them so the data's available or whether or not they 

### 23 didn't actually do that aspect and the data's not available? 

### 24 **DR. GREGORY AKERMAN:**Yeah, most cases the 

### 25 data was available; we just had to go back and ask them and then 

### 26 they sent it us. It wasn't like the data wasn't available.

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1 It's just not included within the study report for some reason. 

### 2 **DR. DEBORAH MACLATCHY:**Okay. So when it's 

### 3 not available, it clearly is not available? 

### 4 **DR. GREGORY AKERMAN:**Correct. 

### 5 **DR. DEBORAH MACLATCHY:**Okay. And I guess as 

### 6 a supplement to that, in some cases it looks like the criteria 

### 7 really hadn't been met. And did the labs redo the assay saying 

### 8 okay, this batch didn't work, and so I'm going to test the 

### 9 chemical again and then send in other data, or how did that 

### 10 process happen within the lab? 

### 11 **DR. GREGORY AKERMAN:**No. I mean there were 

### 12 instances, and you can tell from the study report that they had 

### 13 failed runs and they didn't submit and they had to repeat it and 

### 14 explain why there was failed runs for a particular experiment. 

### 15 But the data was submitted and they can come back in and submit 

### 16 additional data later if their criteria were not met. 

### 17 **DR. DANIEL SCHLENK:**Any other questions for 

### 18 the panel? 

### 19 Yes, Dr. Tillitt. 

### 20 **DR. DON TILLITT:**You know, aside from the 

### 21 performance criteria or really probably performance guidelines, 

### 22 you report that proficiency testing was not consistently 

### 23 reported. How comfortable are you with not getting all of that 

### 24 information and evaluating that performance criteria without 

### 25 having that proficiency testing? 

### 26 **DR. GREGORY AKERMAN:**Many of these labs have

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1 tested multiple chemicals now so I think they've shown that they 

### 2 are proficient in testing using these assays. There's only a 

### 3 few cases where the proficiency testing information is not 

### 4 reported. Other times they would make statements saying that 

### 5 the proficiency testing results were what they expected. They 

### 6 just didn't submit the data necessarily, but they showed the 

### 7 report and describe in the report that they were able to conduct 

### 8 the assay. 

### 9 They had performed the proficiency testing, 

### 10 but they just didn't include the actual data in the study 

### 11 report. 

### 12 **DR. DANIEL SCHLENK:**Could you state your 

### 13 name, please? 

### 14 **DR. JOHN LICCIONE:**John Liccione from HED. 

### 15 To give you an example, the steroidogenesis assay, when the 

### 16 proficiency test wasn't included, we carefully looked at the 

### 17 quality control plate that were run concurrently, that gave us 

### 18 another way of looking at proficiency. 

### 19 **DR. DANIEL SCHLENK:**Any other questions? 

### 20 (No audible response) 

### 21 **DR. DANIEL SCHLENK:**Okay. Let's go ahead and 

### 22 move on for in vivo mammalian assays. That will be Dr. 

### 23 Liccione. 

### 24 **DR. JOHN LICCIONE:**Thank you. Welcome 

### 25 everyone. In this presentation, I'm going to discuss the 

### 26 available in vivo rodent endocrine screening assays. And I will

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1 start with a brief description of each individual assay 

### 2 including its purpose and study design, how well it adhered to 

### 3 performance criteria, any technical issues that had been 

### 4 identified, and finally, a conclusion for each assay. 

### 5 You've already seen these assays in a number 

### 6 of slides, but just quickly to go over them one more time, we 

### 7 have four mammalian in vivo assays, the uterotrophic, 

### 8 Hershberger, male pubertal and the female pubertal assays. 

### 9 To begin with, we'll start with the 

### 10 uterotrophic assay. This assay evaluates the ability of a 

### 11 chemical to elicit an agonist estrogen response, and the 

### 12 important metric is an increase in uterine weight. It is a 

### 13 rapid screen and it incorporates first pass metabolism if the 

### 14 oral route is used. It has a mechanistic sensitivity and 

### 15 specificity, and there are two models that are available, the 

### 16 immature female and the adult ovariectomized female rat models. 

### 17 In the immature model, postnatal day 18 rats 

### 18 are treated for three days with dosing by either the oral or 

### 19 subcutaneous routes, and then the rats are subjected to a 

### 20 necropsy. In the adult ovariectomized model, which is the 

### 21 preferred model, adult females, ages six to eight weeks, are 

### 22 dosed for at least three days and then the rats are necropsied. 

### 23 Endpoints that are pertinent for both models 

### 24 include uterine weights, both wet and blotted, and other things 

### 25 such as bodyweight, food consumption, clinical signs and 

### 26 mortality. Optional endpoints include histology of the uterus,

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1 cervix and the vagina. 

### 2 A positive estrogenic response is defined as a 

### 3 significant increase in uterine weight. A positive control, 17 

### 4 alpha ethynyl estradiol is included in the assay to verify 

### 5 sensitivity in performance of the assay. 

### 6 From a baseline study with the positive 

### 7 control, an effective dose of 70 to 80 is selected for use in 

### 8 the assay. The performance criteria for this assay is that the 

### 9 blotted uterine weights should be .09 percent of the final 

### 10 bodyweight for the immature model and .04 percent of the final 

### 11 bodyweight for the adult ovariectomized model. 

### 12 In regard to the summary of the performance of 

### 13 this assay, all 17 chemicals were negative for the estrogenic 

### 14 component that was measured. And the positive control did 

### 15 increase uterine weight as expected. There were no major 

### 16 technical issues with the assay. Baseline control studies were 

### 17 not reported for two test chemicals and overt toxicity was noted 

### 18 at the highest dose tested for one chemical and this included 

### 19 decreased bodyweight and some clinical signs. 

### 20 In conclusion, the assay performance was 

### 21 consistent across all 17 test chemicals. The assay performed as 

### 22 expected, and dosing was considered adequate. 

### 23 Moving on to the Hershberger assay, the 

### 24 Hershberger assay detects androgenic agonists, antagonists as 

### 25 well as 5 alpha-reductase inhibitors. It is also a rapid screen 

### 26 and incorporates first pass metabolism if an oral route is used.

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1 It has mechanistic specificity and sensitivity. In this assay, 

### 2 postnatal day of rats from 42 to about 48 male rats that had 

### 3 been castrated are dosed for 10 days. Neither the oral or the 

### 4 subcutaneous route can be used, and the high dose tested is 

### 5 selected at or below a maximum tolerated dose not to exceed a 

### 6 limit dose. 

### 7 The primary endocrine-related endpoints that 

### 8 are measured are based on the weights of five known 

### 9 androgen-dependent tissues in the castrated male rats. These 

### 10 include the ventral prostate, seminal vesicles, the levator ani 

### 11 and bulbocavernosus muscles -- herein I will just refer to it as 

### 12 LABC -- Cowper's glands and the glans penis. Other endpoints 

### 13 include clinical signs, bodyweight mortality, weights of the 

### 14 liver, adrenal and the kidney. Optional endpoints could include 

### 15 testosterone, luteinizing hormone as well as follicle 

### 16 stimulating hormone. 

### 17 A positive androgenic response is defined as 

### 18 an increase in two or more of the androgen-dependent weights. A 

### 19 positive anti-androgenic response is identified as a decrease in 

### 20 two or more androgen-dependent weights. This assay includes 

### 21 performance criteria for the organ weights, specifically the 

### 22 maximum recommended percent coefficient of variations for the 

### 23 sex accessory tissues. 

### 24 For the summary of the performance of this 

### 25 assay, none of the 17 chemicals showed an androgenic response. 

### 26 Seven did show an anti-androgenic response. Four of the seven

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1 were in the presence of overt toxicity. Effects included a 

### 2 decreased bodyweight, food consumptions and clinical signs. 

### 3 There were no major issues identified in the assay. The percent 

### 4 of coefficient variation in excess of performance criteria was 

### 5 noted in 14 of the 17 studies, but this did not have a major 

### 6 impact on study interpretation. 

### 7 In conclusion, the assays conformed to the 

### 8 guidelines. The assay performance was consistent across the 17 

### 9 chemicals tested, and dosing was considered adequate based on 

### 10 the studies that were submitted. 

### 11 Moving on to the female rat pubertal assay, 

### 12 the purpose of the female rat pubertal assay is to identify 

### 13 pubertal development and thyroid function in the intact female 

### 14 juvenile rats. It has the ability to detect antithyroid, 

### 15 estrogenic, anti-estrogenic chemicals or alterations in the 

### 16 hypothalamic pituitary gonadal axis. 

### 17 In the study design, female rats are treated 

### 18 orally daily from postnatal days 22 to 42. The weights and age 

### 19 of attainment of vaginal opening are recorded. Statistical 

### 20 analysis recommended include an ANCOVA an analysis of covariance 

### 21 with bodyweight at weaning as covariate for the age and 

### 22 bodyweight at vaginal opening as well as for organ weights. 

### 23 Endpoints measured include the 

### 24 endocrine-related endpoints, specifically age at vaginal 

### 25 opening, first estrus, changes in estrus cyclicity, weights and 

### 26 histology of the ovary, uterus, thyroid and levels of T4 and

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1 TSH. Other endpoints include clinical signs, mortality, 

### 2 bodyweight and clinical chemistry, weights of the liver, 

### 3 pituitary, adrenal and the kidney. 

### 4 For dose selection, the high dose is selected 

### 5 at or just below the maximum tolerated dose but not to exceed 

### 6 the limit dose. The toxicity of the profile of the chemical is 

### 7 also considered. The second dose level is spaced to produce a 

### 8 lesser degree of toxicity relative to the high dose tested. 

### 9 With regards to some strength and limitations 

### 10 of the assay, this assay involves a sensitive period of 

### 11 development, the pubertal period. It has a well-established 

### 12 knowledge base of estrogen and thyroid-dependent endpoints. 

### 13 There is variability of organ weights associated with the 

### 14 ovarian cycle. And the assay has an inability to identify 

### 15 specific mechanisms of action. 

### 16 In assessing the results in terms of what a 

### 17 positive response is, the direction and magnitude of the changes 

### 18 in the following endpoints are considered, particularly any 

### 19 alterations in the vaginal estrus, vaginal opening, first 

### 20 estrus, estrus cyclicity, weights and histology of the ovaries 

### 21 and the uterus, changes in thyroid weight and histology and the 

### 22 levels of T4 and TSH. 

### 23 Performance criteria for the vehicle-control 

### 24 includes mean coefficient variation and range of values for a 

### 25 number of endpoints including the weights of uterus, ovaries, 

### 26 thyroid, adrenal, kidneys and pituitary, T4 levels, final

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1 bodyweight and age and weight of vaginal opening. However, 

### 2 performance criteria are available only for the Sprague-Dawley 

### 3 rat. 

### 4 For the summary of the performance of this 

### 5 assay, 6 of the 17 chemicals displayed a response in at least 

### 6 one estrogen-dependent endpoint. Four of the six were in the 

### 7 presence of overt toxicity the effect were generally decreased 

### 8 bodyweight, food consumption and clinical signs. 

### 9 Eleven of the seventeen chemicals showed a 

### 10 response in the thyroid-related endpoints, and four of the 

### 11 eleven were in the presence of overt toxicity. 

### 12 There were no major issues that compromised 

### 13 the conduct and/or interpretation of the study. There were some 

### 14 minor deviations in percent coefficient outside the limits of 

### 15 the performance criteria. But this was considered not to have 

### 16 any major impact on the interpretation of the study. 

### 17 In conclusion, the assays were conducted in 

### 18 accordance with the guidelines, and the assay performance was 

### 19 consistent across all of the chemicals tested, and dosing was 

### 20 considered adequate. 

### 21 Moving on to our last assay, the male rat 

### 22 pubertal assay, the purpose of the male pubertal assay is to 

### 23 identify effects on pubertal development and thyroid function in 

### 24 the intact male juvenile rat. It detects chemicals with 

### 25 antithyroid, androgenic, anti-androgenic activities for any 

### 26 perturbations in the HPG axis.

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1 In terms of the study design, male rats are 

### 2 treated orally daily from postnatal day 23 to 53. Rats are 

### 3 examined for preputial separation beginning on postnatal day 30. 

### 4 Statistical analysis also include an ANCOVA, a bodyweight at 

### 5 weaning as to covariate, for age and body weight preputial 

### 6 separation and also organ weights. 

### 7 Endpoints again are measured including the 

### 8 endocrine-related endpoints, age and weight at preputial 

### 9 separation, the weights of ventral prostate, dorsal prostate, 

### 10 LABC, seminal vesicle, epididymides, testes, thyroid, histology 

### 11 of the epididymides, testes, thyroid, T4 and TSH and 

### 12 testosterone levels. 

### 13 Other endpoints include the clinical signs, 

### 14 mortality, bodyweight, weights of liver, kidneys, pituitary and 

### 15 adrenal and also histology of the kidney and some clinical 

### 16 chemistry measurements. 

### 17 For dose selection, very similar to the female 

### 18 rat pubertal assay, the high dose is selected at or just below 

### 19 the maximum tolerated dose but not to exceed a limit dose. The 

### 20 toxicity profile is also considered in the dose selection. The 

### 21 second dose level is spaced to produce a lesser degree of 

### 22 toxicity relative to the high dose tested. 

### 23 With respect to strengths and limitations, the 

### 24 assay involves a sensitive pubertal period of development and 

### 25 includes metabolism, and it has a well-established knowledge 

### 26 base of androgen- and thyroid-dependent endpoints and has an

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1 inability to identify specific mechanisms of action. 

### 2 And similarly to the female rat pubertal assay 

### 3 in terms of how do we assess a positive response, we look at the 

### 4 direction and magnitude of the changes and the important 

### 5 endpoints that would include alterations in preputial 

### 6 separation, changes in the androgen-sensitive organ weights, 

### 7 testosterone levels, thyroid weight and histology with T4 and 

### 8 TSH levels. 

### 9 In regard to performance criteria, the 

### 10 vehicle-control also includes criteria for the mean coefficient 

### 11 and variation and range of values for various endpoints, the 

### 12 important endpoints relating to accessory tissues, epididymides, 

### 13 testes, thyroid, adrenal, kidneys, liver, pituitary as well as 

### 14 testosterone levels, age at preputial separation, the levels of 

### 15 T4 and TSH, weaning weight and final weight. 

### 16 For summary of performance, 9 of the 16 

### 17 chemicals showed a response in at least one androgen-dependent 

### 18 endpoint. Two of the nine were in the presence of overt 

### 19 toxicity. Once again, decreases in bodyweight, food 

### 20 consumption, clinical signs. 

### 21 Seven of the six chemical showed a response in 

### 22 thyroid-related endpoints. Two of the seven were in the 

### 23 presence of overt toxicity. There were no major deficiencies 

### 24 that compromised the conduct or interpretation of the studies. 

### 25 And in conclusion, the assay was conducted in 

### 26 accordance with the guidelines. Assay performance was

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1 consistent across all 16 chemicals, and dosing was considered 

### 2 adequate. 

### 3 **DR. DANIEL SCHLENK:**Okay. Any questions for 

### 4 Dr. Liccione? 

### 5 Dr. Selvage. 

### 6 **DR. DANIEL SELVAGE:**Hi. Dan Selvage. Other 

### 7 than overt toxicity signs and adrenal weight, I may know the 

### 8 answer to this, but I just want to ask; did you guys measure any 

### 9 HPA axis parameters, like corticosterone or anything like that? 

### 10 **DR. JOHN LICCIONE:**Oh, no the stress 

### 11 hormones? 

### 12 **DR. DANIEL SELVAGE:**Yeah. 

### 13 **DR. JOHN LICCIONE:**Stress is obviously one of 

### 14 the concerns that they can affect and that's why one of the 

### 15 covariants are generally included in these assays. We know that 

### 16 that could -- but none of these assays measured in general 

### 17 corticosterone. 

### 18 **DR. DANIEL SELVAGE:**Okay. So no ACTH or 

### 19 anything like that? 

### 20 DR. JOHN LICCIONE: No. 

### 21 **DR. DANIEL SCHLENK:**Dr. Liccione, can I just 

### 22 ask you to speak up in the mic a little bit closer? Thanks. 

### 23 **DR. DANIEL SCHLENK:**Yeah. Dr. Green. 

### 24 **DR. SHERRIL GREEN:**I wanted to know what were 

### 25 the strains, the genetic backgrounds, of both the male and the 

### 26 female rats, and was that consistent across all these studies?

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1 **DR. JOHN LICCIONE:**The Sprague-Dawley rat was 

### 2 used in the male and female pubertals except for one chemical 

### 3 that used the Wistar rat. 

### 4 **DR. SHERILL GREEN:**And male and female 

### 5 Sprague-Dawley? 

### 6 **DR. JOHN LICCIONE:**Right. Sprague-Dawley, 

### 7 Wistar. 

### 8 **DR. SHERRIL GREEN:**And I have a second 

### 9 question, if that's all right, related to the housing and the 

### 10 standardization across the study. Were they all in the same 

### 11 kind of cages, similar or same food, bedding, water? Just to 

### 12 rule out the possibility of androgenic, estrogenic effect? 

### 13 **DR. JOHN LICCIONE:**Right, exactly. Right. 

### 14 And they adhered to the guidelines in terms of randomizing the 

### 15 bodyweight they grew -- for the female pubertal assay obviously 

### 16 it's very important to present the final estrogen content of the 

### 17 diet. They all conformed and we didn't see any major problems 

### 18 with housing in range of the bodyweight and content of the diets 

### 19 in the female pubertal were critical. 

### 20 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 21 **DR. JACK LEONARD:**Was there any comparison 

### 22 made between the route of administration of the test chemicals? 

### 23 I noticed sub-q was suggested as well as oral gavage. Was a 

### 24 comparison made between those two routes of administration? 

### 25 **DR. JOHN LICCIONE:**In terms of the 

### 26 guidelines?

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1 **DR. JACK LEONARD:**In terms of testing the 

### 2 efficacy or the impact of the compound on any of the in vivo 

### 3 assays you were talking about. 

### 4 **DR. JOHN LICCIONE:**Oh, okay. They came from 

### 5 the validation studies, the recommendation for the uterotrophic 

### 6 assay to use the subcutaneous, more came based on the validation 

### 7 as a screening assay to enhance the response. And for the 

### 8 Hershberger, both oral or subcutaneous can be used, and I think 

### 9 in most cases they used oral. All cases for Hershberger used 

### 10 the oral. I think there was one subcutaneous for the 

### 11 uterotrophic. Most elected to use the oral which is okay with 

### 12 the guidelines, the profile of the chemicals considered. Of 

### 13 course, for the pubertals they were all oral. 

### 14 **DR. DANIEL SCHLENK:**Dr. Delclos. 

### 15 **DR. BARRY DELCLOS:**Since you mentioned 

### 16 phytoestrogen, in the validation studies, I know that the OACD 

### 17 in the uterotrophic assay didn't test the phytoestrogen levels. 

### 18 Did they do that in the pubertal assays as well? Was there any 

### 19 effect on the thyroid hormones? 

### 20 **DR. JOHN LICCIONE:**Oh, you mean in terms of 

### 21 validating it? 

### 22 **DR. BARRY DELCLOS:**When it was validated, did 

### 23 they do varied phytoestrogen contents in the pubertal assays as 

### 24 well? 

### 25 **DR. LESLIE TOUART:**This is Les Touart 

### 26 responding. During the validation programs, as part of the

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1 development of the assays, that was something that was looked at 

### 2 in terms of phytoestrogens, and even looking at 

### 3 phytoestrogen-free type diets, and that's difficult because 

### 4 within the rats they still need some sterile components within 

### 5 the diet itself. 

### 6 But in conduct I think that the 

### 7 recommendations that were made were to kind of limit the amounts 

### 8 to the extent and try to use some standardized diets, you know, 

### 9 to do that. And as long as the diets were, you know, kind of 

### 10 uniform or standard, I think that was considered to be 

### 11 sufficient. But more details would be available in the actual 

### 12 validation reports for those assays as they were developed. 

### 13 **DR. BARRY DELCLOS:**I was just wondering, I 

### 14 mean the range is quite large, up to 350 parts per million, I 

### 15 think. I'm familiar with the uterotrophic assays. I don't 

### 16 remember much about the other assays or whether that was tested 

### 17 and if thyroid hormone levels would be something. I would be 

### 18 interested in knowing if they were varied across. That's okay. 

### 19 I understand. 

### 20 **DR. DANIEL SCHLENK:**Yes, Dr. Piersma. 

### 21 **DR. ALDERT PIERSMA:**In those cases where you 

### 22 had a specific effect on the endpoint in the assay in the 

### 23 presence of overt toxicity, did you always consider the specific 

### 24 effect as the primary effect or the primary mechanism affected? 

### 25 Or were there cases where you would consider the overt toxicity 

### 26 mediating the other endpoint? I mean have you made those

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1 assessments in each specific case? 

### 2 **DR. JOHN LICCIONE:**Yeah. We did consider the 

### 3 overt toxicity as whether that's driving the endocrine-related 

### 4 effect. So that's where we noted those cases where 

### 5 endocrine-related endpoints that were changed were in the 

### 6 presence of overt toxicity in cases where they were not at the 

### 7 lower doses. We didn't conclude mechanistically but we did know 

### 8 that that's a concern. 

### 9 **DR. ALDERT PIERSMA:**Okay. So to be 

### 10 absolutely clear, where you stated that there was overt toxicity 

### 11 also present, it is not clear whether the compound had a 

### 12 specific effect on the specific endpoint? 

### 13 **DR. JOHN LICCIONE:**Exactly. Exactly. And 

### 14 actually, one of the charge questions related to the overt 

### 15 toxicity. 

### 16 **DR. ALDERT PIERSMA:**Thank you. 

### 17 **DR. DANIEL SCHLENK:**Dr. Horseman? 

### 18 **DR. NELSON HORSEMAN:**Well, back in Dr. 

### 19 Touart's talk he presented a very nice slide showing the 

### 20 construction of the feedback loops and such, which there's a lot 

### 21 of information inherent in that. I noticed the only assay in 

### 22 which the LH and FSH variables may be optional is in the 

### 23 Hershberger assay. Is there information in that LH and FSH data 

### 24 that -- was that done so that's optional, and is there 

### 25 information in there that would help to increase the information 

### 26 content?

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1 **DR. JOHN LICCIONE:**Are you referring to 

### 2 during the validation of the assay the LH and FSH? 

### 3 **DR. NELSON HORSEMAN:**I'm mostly referring to 

### 4 the conduct of this Tier 1 screening. 

### 5 **DR. JOHN LICCIONE:**Oh, no, no. 

### 6 **DR. NELSON HORSEMAN:**None of those were? 

### 7 **DR. JOHN LICCIONE:**The FSH or LH was not 

### 8 included. 

### 9 **DR. DANIEL SCHLENK:**Okay. Any other 

### 10 questions? 

### 11 Dr. Scott. 

### 12 **DR. GEOFFREY SCOTT:**Geoff Scott. Just a 

### 13 little clarification on the selection of the second dose. Could 

### 14 you just go back and re-amplify how you do that again, and in 

### 15 terms of design? I understood the top dose and how you did 

### 16 that. 

### 17 **DR. JOHN LICCIONE:**Okay. What the guidelines 

### 18 specify is you have a high dose that should not exceed the 

### 19 maximum tolerated dose. And for the female and male pubertals 

### 20 it's half that. 

### 21 **DR. GEOFFREY SCOTT:**Oh, so 50 percent? 

### 22 **DR. JOHN LICCIONE:**But keep in mind the 

### 23 toxicity profile of the chemicals considered in all of these 

### 24 labs did provide dose selection rationale for selection of the 

### 25 doses, which we found adequate in terms of the range findings 

### 26 and sometimes other, actually in Hershbergers and uterotrophic

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1 assays, the results of dose to inform on these apical assays. 

### 2 **DR. GEOFFREY SCOTT:**You answered my -- what I 

### 3 really want to know is there are specific guidelines? 

### 4 **DR. JOHN LICCIONE:**Yeah. That's correct. 

### 5 **DR. GEOFFREY SCOTT:**Okay. Thank you. 

### 6 **DR. DANIEL SCHLENK:**Dr. Klaine. 

### 7 **DR. STEPHEN KLAINE:**Just to follow up on 

### 8 that, so in every case when you did find overt toxicity at the 

### 9 higher dose, and you reported that, you saw no effects at the 

### 10 lower dose? 

### 11 **DR. JOHN LICCIONE:**No. In some cases, there 

### 12 were endocrine-related effects that were in the absence of overt 

### 13 toxicity. We wanted to dissect it out clearly without making 

### 14 any conclusions about the overt toxicity or mechanism knowing 

### 15 that there were some effects at the lower doses. 

### 16 **DR. STEPHEN KLAINE:**Okay. And they were 

### 17 scored positively? 

### 18 **DR. JOHN LICCIONE:**Right. Correct. It's 

### 19 based on the performance criteria and guidelines. 

### 20 **DR. DANIEL SCHLENK:**Any other questions, 

### 21 clarification? 

### 22 Yes, Dr. Roby. 

### 23 DR. KATHERINE ROBY: Just then to clarify, 

### 24 when there was toxicity at a higher dose, the guidance was not 

### 25 to repeat two lower doses but only one? 

### 26 **DR. JOHN LICCIONE:**Well, what the guidelines

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1 say is you can do a limit dosing if there's no toxicity or 

### 2 you're really not exceed the maximum tolerated dose, and then 

### 3 you'd go to a lower dose; in the pubertal assays it's halfway 

### 4 where you don't have any evidence of any adverse effects. 

### 5 DR. KATHERINE ROBY: So then to clarify, if 

### 6 there was toxicity at a higher dose, then only a single lower 

### 7 dose would be included in the analysis? 

### 8 **DR. JOHN LICCIONE:**Yes. Often there was only 

### 9 one. Occasionally, they did two, but most of the time was only 

### 10 one dose. 

### 11 **DR. DANIEL SCHLENK:**Okay. Dr. Horseman. 

### 12 **DR. NELSON HORSEMAN:**So this obviously is the 

### 13 favorite question about the toxicity. And it relates to the 

### 14 guidance to give the compound at or near the maximum-tolerated 

### 15 dose which seems to me that invites toxicity. Given that these 

### 16 assays depend upon a healthy system, are you comfortable with 

### 17 that piece of the guidance, to use the maximum tolerated dose as 

### 18 the place to start and then only use one lower dose? 

### 19 **DR. JOHN LICCIONE:**Yeah. Well, in terms of 

### 20 the screening assay, of course, you want to make sure you're 

### 21 testing high enough so that you can demonstrate -- this is in 

### 22 the screening assay so you want to give some kind of a 

### 23 challenging dose. But yet, at the same time, you want to try to 

### 24 get at that maximum -- not to exceed that maximum tolerated dose 

### 25 which sometimes is a challenge. 

### 26 And from that, you want to go to a lower dose

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1 where you know you can back off, or you can have a dose that 

### 2 will -- where if there's an endocrine-related effect, that 

### 3 you'll be able to pick it up. So that's the intent. 

### 4 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 5 **DR. GEOFFREY SCOTT:**Just to follow up on 

### 6 that, when you look at our exposure diagram at the maximum 

### 7 tolerated dose, what percent of the coefficient or variance are 

### 8 you seeing above that with the measurements of that? 

### 9 **DR. JOHN LICCIONE:**It depends on which 

### 10 parameter you're talking about, if I understand correctly. 

### 11 **DR. GEOFFREY SCOTT:**Well, what I'm trying to 

### 12 find out is if you're at the maximum tolerated dose, and 20 

### 13 percent of the time you're above it by your variance, you're 

### 14 above the maximum tolerance which is getting back to Dr. 

### 15 Horseman's question. I'm just trying to understand is that a 

### 16 slight deviation, going back to semantics here? 

### 17 I'm just trying to get my arms around this 

### 18 because it would seem like trying to go back to what Nelson 

### 19 pointed out. You want to be right at that -- I understand where 

### 20 you're coming from too, you want to be there where your 

### 21 challenging the animal -- 

### 22 **DR. JOHN LICCIONE:**Right. 

### 23 **DR. GEOFFREY SCOTT:**-- but you don't want to 

### 24 kick him over the edge. So I'm just trying to figure out are we 

### 25 kicking him over the edge a little bit of the time, you know, or 

### 26 what percentage of the time?

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1 **DR. JOHN LICCIONE:**We'll come back there. We 

### 2 didn't analyze it in that regard. In some cases, we noticed -- 

### 3 we were trying to get at the overt toxicity, but we can revisit 

### 4 that. 

### 5 **DR. GEOFFREY SCOTT:**If I can follow up, it 

### 6 would be interesting to look and see when you had those 

### 7 deviations, at what percentage of the time did you have to go to 

### 8 that second dose? That might be very helpful at least in seeing 

### 9 how it all fits. 

### 10 **DR. JOHN LICCIONE:**Okay. 

### 11 **DR. DANIEL SCHLENK:**Okay. And we can get 

### 12 that report tomorrow? Is that possible? Yeah? Okay. Great. 

### 13 Yes, Dr. Leonard. 

### 14 **DR. JACK LEONARD:**Were there any guidelines 

### 15 provided for dietary content of iodine? I know the thyroid is 

### 16 the key component we're talking about here and was wondering 

### 17 whether or not you had some guidelines as to what the iodine 

### 18 content of the commercial diet were? 

### 19 **DR. JOHN LICCIONE:**Not in the available 

### 20 guidelines that we had that I'm aware of. 

### 21 **DR. DANIEL SCHLENK:**Okay. Any other 

### 22 questions before we take our EAT activity? 

### 23 Sorry. I've been holding off on that all day. 

### 24 Okay. Let's go ahead and break for lunch, and let's reconvene 

### 25 at 1 p.m. Thanks. 

### 26 (Whereas a lunch break was taken)

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1 **DR. DANIEL SCHLENK:**Okay good afternoon 

### 2 everyone. We're ready to reconvene; just a couple of 

### 3 announcements. If you are a public commenter and would like to 

### 4 make your presentation today we do have time available on the 

### 5 agenda for that this afternoon. So if you have your materials 

### 6 if you could get them to the EPA staff before this afternoon's 

### 7 break that would be great. 

### 8 Right now I believe Dr. Dourson is going to be 

### 9 presenting today rather than tomorrow. We still will have the 

### 10 public commenting period tomorrow morning as well. 

### 11 But if you have your materials ready we can do 

### 12 that this afternoon just to sort of save a little bit of time 

### 13 there if you're up for that. Yeah we will have the full comment 

### 14 period tomorrow so that is not going away it's just if we could 

### 15 get some folks in this afternoon that would be great. Okay? 

### 16 So before we get started I believe we had some 

### 17 clarification comments from the Agency in regards to some of the 

### 18 overt toxicity questions. And could you just state your name? 

### 19 Thanks. 

### 20 **JESS ROWLAND:**Thank you. I'm Jess Rowland. 

### 21 I want to give some context to the dose selection for the in 

### 22 vivo assays. For most of the studies they ran a range finding 

### 23 studies with the three or four dose levels. 

### 24 And based on the results of those studies they 

### 25 selected the high dose and the lower dose. If they didn't run a 

### 26 range finding study they used the existing knowledge from the

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1 other standard toxicology studies to see where the NOAEL and 

### 2 LOAEL fell in the 14-day studies and the 28-day studies and used 

### 3 that. 

### 4 So for other chemicals that we felt that the 

### 5 high dose selected were appropriate, except for like one out of 

### 6 17 in the uterotrophic had a kind of a short overt toxicity, 3 

### 7 out of the 17 from the Hershberger, five out of the 17 for the 

### 8 female pubertal, and three out of 16 for the male pubertal. 

### 9 For a few studies that the high dose showed 

### 10 some overt toxicity, and even though toxicity was very -- we 

### 11 usually consider the criteria and decrease of more than ten 

### 12 percent body weight, clinical signs for acceleration in some 

### 13 cases decrease in food consumption, so those doses were run 

### 14 concurrently. And for the Hershberger or the androgenic phase 

### 15 they tested two doses, for the antiandrogenic they used three 

### 16 doses. 

### 17 On some of the pubertal we had two doses for 

### 18 some clinicals and three for the others. So the overall picture 

### 19 of the 67 studies may be right on target because most of the 

### 20 studies were selecting the high dose and the lower dose. 

### 21 **DR. DANIEL SCHLENK:**Okay, any questions, 

### 22 clarifications of panel members regarding that? Okay thank you. 

### 23 Let's move ahead and start talking about our fish assays and 

### 24 presentation there will be from Amy Blankinship from 

### 25 Environmental Fate and Effects Division, Office of Pesticide 

### 26 Programs.

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1 **AMY BLANKINSHIP:**Good morning. I will be 

### 2 discussing the fish short-term reproduction assay, which is 

### 3 referred to hereafter as the FSTRA. 

### 4 Shown in the photo taken from the Agency's 

### 5 webpage on the FSTRA is a test species fathead minnow. I would 

### 6 like to start by giving an overview of the presentation today. 

### 7 First I will provide a brief background on the assay. Second I 

### 8 will discuss the evaluation of the available studies as it 

### 9 relates to the execution of the assay and performance criteria 

### 10 as well as some general observations on the conduct and results 

### 11 of the studies. And finally some concluding remarks. 

### 12 The FSTRA is intended to be a screening assay 

### 13 which provides information on reproductive fitness and the 

### 14 potential to interact with the endocrine system. It can be used 

### 15 to detect potential estrogen and androgen agonist and antagonist 

### 16 as well as chemicals that affect steroidogenesis and other 

### 17 interactions along the HPG axis. 

### 18 The assay contains endpoints that are 

### 19 reflective of changes associated with interactions with the HPG 

### 20 axis such as plasma vitellogenin regimens in male fish. The 

### 21 FSTRA is conducted using an aqueous exposure and the test 

### 22 species is a fathead minnow. The fish are distributed into 

### 23 breeding groups of two males and four females. 

### 24 The breeding groups are monitored for 14 days 

### 25 prior to exposure to evaluate reproductive output, which will 

### 26 allow for a systematic method of distributing the breeding

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1 groups to the control and treatment groups, after which the fish 

### 2 are exposed to the chemical for 21 days. 

### 3 Typically there are three test concentrations 

### 4 in a negative control. And if a solvent carrier is used, a 

### 5 solvent control, there are four replicates for treatment. This 

### 6 assay examines many endpoints both during the study and at test 

### 7 termination. 

### 8 Shown here are the data endpoints or 

### 9 parameters that are evaluated in the FSTRA which are recorded in 

### 10 the data evaluation record or DER. The individual parameters 

### 11 are listed in the left hand column and the type of information 

### 12 recorded for each endpoint is listed in the right hand column. 

### 13 The endpoints include parameters such as 

### 14 survival and growth along with other reproductive endpoints such 

### 15 as daily measurements, fecundity and fertilization success. 

### 16 Other endpoints indicative of reproductive fitness are measured 

### 17 at test termination for each sex and include measurement of 

### 18 secondary sex characteristics including nuptial tubercles, 

### 19 gonadal somatic index, or GSI, and gonadal staging and 

### 20 histopathology. 

### 21 The biochemical marker of plasma vitellogenin 

### 22 or VTG is included as well. Optional endpoints include 

### 23 measurement of sex steroids including testosterone and 

### 24 estradiol. It is noted that alterations in some endpoints such 

### 25 as vitellogenin may provide more diagnostic utility in 

### 26 evaluating a potential endocrine interaction. Whereas

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1 disruption in other endpoints, such as a decrease in fecundity, 

### 2 may be due to nonendocrine or nonspecific mechanism. 

### 3 There are six performance criteria identified 

### 4 in the FSTRA guideline. These criteria are referred to control 

### 5 performance for the endpoints of percent fertility, percent 

### 6 survival and fecundity rate. 

### 7 The three other criteria are reflective of 

### 8 test chemical exposure and environmental conditions including 

### 9 the maximum variability and chemical exposure for a treatment 

### 10 group as well as the minimum percent dissolved oxygen 

### 11 concentration and range of water temperature in the test 

### 12 chambers. 

### 13 As with any of the identified criterion it is 

### 14 anticipated that slight deviations will occur and the conduct 

### 15 and result of each study are evaluated as a whole to determine 

### 16 the degree of confidence in the ability to discern the potential 

### 17 for endocrine interaction. 

### 18 Twenty-one FSTRA studies with unknown 

### 19 endocrine potential were evaluated for their ability to adhere 

### 20 to the six identified performance criteria. 

### 21 This figure summarizes the number of studies 

### 22 out of the 21 evaluated that met each of the six performance 

### 23 criteria. The number of studies that met each criterion is 

### 24 identified by the green bars and the studies that did not are in 

### 25 orange. 

### 26 As illustrated 75 percent or more of the

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1 studies met each criterion except for the test concentration 

### 2 variance, which may be anticipated with some of the more 

### 3 difficult to test substances due to their physical chemical 

### 4 properties such as low water solubility and absorption 

### 5 properties. 

### 6 All of the studies were able to maintain water 

### 7 temperature within the guideline range and all but one study met 

### 8 the control survival criterion. 

### 9 Shown here is another summary figure, but this 

### 10 figure summarizes how many of the six criteria each study met. 

### 11 Four of the 21 studies met all of the six criteria with another 

### 12 11 studies meeting all but one. For the 11 studies that met all 

### 13 but one, the table at the bottom of the slide identifies which 

### 14 one of the six criteria the study did not achieve. 

### 15 Most of the 11 studies did not achieve either 

### 16 test concentration variance or the control fecundity followed by 

### 17 the dissolved oxygen, survival and fertility. If the study did 

### 18 not meet two or more of the six criteria typically the unmet 

### 19 ones were test concentration variation along with the control 

### 20 fecundity and/or dissolved oxygen concentration. 

### 21 The first of the six criteria that I will 

### 22 discuss in greater detail is the measured test concentration 

### 23 coefficient variance or CV. A CV for each test concentration 

### 24 was calculated using the 21-day treatment mean or time weighted 

### 25 average. Twelve out of the 21 studies were able to maintain 

### 26 test concentrations within the recommended 20 percent variance

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1 with another four CVs ranging up to 30 percent. 

### 2 For some of the 21 evaluated chemicals their 

### 3 physical chemical properties could have made maintaining test 

### 4 concentrations in the water column more difficult. In an effort 

### 5 to maintain test concentrations some laboratories increased the 

### 6 test solution to an overt rate in the test system. 

### 7 Additionally some laboratories did utilize the 

### 8 solvent carrier to help facilitate dosing. Furthermore in an 

### 9 effort to better characterize exposure some laboratories took 

### 10 additional measurements during the 21 days. 

### 11 The second criterion is control fecundity. 

### 12 The fecundity is monitored in both the pre-exposure and exposure 

### 13 phase. During the pre-exposure phase fecundity is measured to 

### 14 ensure adequate spawning in a breeding group prior to exposure 

### 15 and to allow for distribution of the breeding group for the test 

### 16 concentration using a randomized block design. 

### 17 While not a performance criteria, the 

### 18 guideline has a recommended fecundity rate during the 

### 19 pre-exposure phase and 20 of the 21 studies met this 

### 20 recommendation. During the exposure phase the criterion for 

### 21 control fecundity is that each of the four replicates in a 

### 22 control should achieve either 15 eggs per female per 

### 23 reproductive day or spawn at least every four days. 

### 24 If the study used a solvent carrier then these 

### 25 criteria were applied to the solvent control replicates as well. 

### 26 All control replicates in 15 of the 21 studies met one of these

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1 two conditions. However, if evaluating fecundity using an 

### 2 average rate, in 17 of the 21 studies had mean control fecundity 

### 3 rate of 15 eggs per female per day or greater. 

### 4 The third criterion is controlled 

### 5 fertilization success. As in fecundity fertilization success is 

### 6 assessed daily. The guideline states that on average 95 percent 

### 7 or more of the eggs produced in the controls should be 

### 8 fertilized. Seventeen of the 21 studies met this and the mean 

### 9 fertility in the other four studies range from 90 to 94 percent. 

### 10 11 For the fourth criterion of control survival 

### 12 90 percent or greater survival of controlled fish over the 

### 13 21-day is desired as there are 16 females and eight males in a 

### 14 control for a total of 24 fish, this allows for two mortalities. 

### 15 16 All but one study was able to maintain 

### 17 survival at 90 percent or greater during the test duration. The 

### 18 control survival in the remaining study was 83 percent. The 

### 19 study authors in that one study stated that mortality during the 

### 20 pre-exposure phase was within the allowable US EPA guideline 

### 21 mortality for an acclimation period and that all other control 

### 22 performance criteria in the study were met. 

### 23 The remaining two criteria referred to 

### 24 environmental conditions during the test. The guidelines states 

### 25 that dissolved oxygen or DO should be maintained at 60 percent 

### 26 saturation or greater during the 21-day exposure. Per the

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1 guideline, DO should be measured in each test replicate at least 

### 2 weekly. Fifteen of the 21 studies were able to maintain DO at 

### 3 60 percent or greater. In the other six studies at least one DO 

### 4 measurement was below 60 percent with recorded minimums of 45 to 

### 5 58 percent. 

### 6 Given the reported frequency of measurement of 

### 7 DO, it was not always possible to know the duration under which 

### 8 the DO may have been less than 60 percent. However, once a 

### 9 decrease in dissolved oxygen below 60 percent was measured, the 

### 10 laboratories added aeration or in some cases increased aeration 

### 11 if it was already present. 

### 12 Decreases in DO were observed in studies that 

### 13 used a solvent carrier as well as studies that did not. For 

### 14 water temperature all 21 studies were within the guideline 

### 15 criterion of 25+/- 1 degree Celsius. 

### 16 In addition to DO in water temperature Table 1 

### 17 of the guideline has reported maximum limits on other water 

### 18 quality parameters such as total organic carbon and residual 

### 19 chlorine. 

### 20 While not all of the 21 studies recorded 

### 21 measurements for these parameters, based on historical water 

### 22 quality information for these laboratories the agency deemed 

### 23 that the dilution water used in these studies was adequate to 

### 24 support survival, growth and reproduction of fathead minnows. 

### 25 Overall the control performance for the 21 

### 26 evaluated studies was generally within or close to the guideline

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1 criteria with four of the studies meeting all six criteria and 

### 2 another 11 meeting all but one. In the cases of the dissolved 

### 3 oxygen and test concentration variation, two of the most missed 

### 4 criteria laboratories appear to be responsive to these criteria 

### 5 by taking measures to increase dissolved oxygen and/or reducing 

### 6 variation in chemical exposure. 

### 7 Now I'd like to discuss some general 

### 8 observations in a FSTRA after evaluating a 21 study, with one of 

### 9 these being the occurrence of incorrectly sexed fish from the 

### 10 FSTRA. In this assay fish are distributed to test chambers by 

### 11 two males and four females. This sorting occurs at the 

### 12 beginning of the pre-exposure phase. As the study is generally 

### 13 initiated using newly mature males and females, the guideline 

### 14 recognizes potentials for misidentifying the sex of a fish 

### 15 during this distribution process and recommends adding 

### 16 additional tanks during the pre-exposure phase. 

### 17 As the thought is that once a fish starts to 

### 18 actively reproduce during the pre-exposure phase it will become 

### 19 apparent that a fish was misidentified and this breeding group 

### 20 can be excluded. However, upon evaluation of the 21 studies, 6 

### 21 studies or 30 percent had at least one replicate with an 

### 22 incorrectly sexed fish. In all cases it was a male mistaken for 

### 23 a female, which was typically not realized until test 

### 24 termination. 

### 25 The figure in this slide represents how many 

### 26 replicates contained an incorrectly sexed fish for each of those

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1 six studies. Half of the six studies had only one replicate 

### 2 with an incorrect sex ratio whereas one study or 25 percent had 

### 3 an incorrect ratio. For the studies that recorded the missed 

### 4 sexed fish, the study author suggested the fecundity value of 

### 5 that replicate to account for the additional male. 

### 6 However, for the other gender specific 

### 7 endpoint such as GSI and vitellogenin some laboratories excluded 

### 8 the missed sexed male while others did not. The Agency thinks 

### 9 it is appropriate to remove the missed sexed fish from all 

### 10 endpoint analyses. As laboratories gain experience with this 

### 11 assay, the occurrence of missed sexed fish may diminish. 

### 12 The second topic I would like to discuss is a 

### 13 selection of test concentrations in the FSTRA and the appearance 

### 14 of potential overt toxicity in the reported results. The 

### 15 guidelines suggest that the highest test concentration in the 

### 16 study should not result in increased overt toxicity compared to 

### 17 the control. The guidelines provide some suggestions for 

### 18 studying the highest test concentration which include using 1/3 

### 19 of the 96 hour LC50. The use of the limit of solubility or a 

### 20 maximum concentration of 100 parts per million may also be used. 

### 21 Based on the table in this slide, about half 

### 22 of the 21 studies used, at least in part, 1/3 have an acute 

### 23 LC50, but the few other studies using a limited solubility or 

### 24 the necessary maximum concentration as the high dose. 

### 25 This slide discusses the observation of 

### 26 potential overt toxicity in the FSTRA. Potential overt toxicity

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1 in this evaluation is defined as (1) a reduction in survival, 

### 2 (2) the reporting of clinical signs of toxicity in a treatment 

### 3 group such as abnormal swimming, lethargy or decreased feeding 

### 4 and these affects were only evaluated qualitatively and in some 

### 5 cases the number of fish affected was not always reported for 

### 6 clinical signs of toxicity; and (3) a statistically significant 

### 7 reduction in body weight compared to the control. 

### 8 For reductions in body weight it is noted that 

### 9 reductions may or may not be due to potential endocrine 

### 10 interactions, and that changes in body weight could be affected 

### 11 by alterations in other endpoints such as fecundity. The top 

### 12 figure shows the number of studies that recorded an effect one 

### 13 or more of the three main categories of overt toxicity. 

### 14 Please keep in mind that a study may have 

### 15 reported more than one effect which is why the three categories 

### 16 add up to more than 21. Thirteen studies reported effects on 

### 17 one or more of the three categories with another four studies 

### 18 reporting a decrease in body weight only. 

### 19 Generally the observed overt toxicity was 

### 20 reported only in the high concentration. This is illustrated in 

### 21 the bottom figure in which 70 percent of the studies that 

### 22 reported a potential overt toxicity only reported it in the 

### 23 highest treatment group. 

### 24 There are seven main endpoints evaluated in 

### 25 the FSTRA with four of them being gender specific. These 

### 26 endpoints are presented in the figure on the slide. Changes

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1 and/or alterations in these endpoints compared to the control 

### 2 were evaluated for the 21 studies. However, only three of the 

### 3 21 studies measured sex steroids. 

### 4 An effect was reported at least once for all 

### 5 seven endpoints. However, an effect in one of these endpoints 

### 6 is often reported at the same concentration where the presence 

### 7 of potential overt toxicity was also reported. In this figure 

### 8 the red bars represents where an effect was observed only in the 

### 9 presence of potential overt toxicity. 

### 10 The orange bars represent where an effect was 

### 11 observed both with and without accompanying overt toxicity. For 

### 12 example, an effect on fecundity was observed at both the high 

### 13 and mid-test concentration but abnormal swimming was only 

### 14 reported at the highest test concentration. 

### 15 And finally the green bars indicate that an 

### 16 effect was reported without any associated overt toxicity. 

### 17 From the graph, changes or alterations in 

### 18 fecundity in female gonadal histopathology were reported most 

### 19 often. Other general observations observed in these 21 studies 

### 20 were that for control male or female fish, the GSI values were 

### 21 within the guidelines reported range. 

### 22 Additionally, male control fish were reported 

### 23 to have the appropriate secondary sex characteristics of nuptial 

### 24 tubercles. Whereas female control fish did not, which is 

### 25 normal. These observations further indicate that the fish used 

### 26 were sufficiently appropriate for use in this study.

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1 For the endpoint in vitellogenin the 

### 2 appearance of potential outliers was observed in several 

### 3 studies, primarily for male fish. As normal male fish do not 

### 4 express vitellogenin, and the measurement is often at or around 

### 5 background levels, variation of individual male fish may be 

### 6 expected. The next slide will further illustrate this. 

### 7 And finally while an optional endpoint, as 

### 8 stated, sex steroids were only measured in three of the 21 

### 9 studies. Inclusion of these measurements could potentially be 

### 10 quite useful in interpreting results when evaluating for a 

### 11 potential endocrine interactions. 

### 12 As indicated this slide shows two examples of 

### 13 a measurement of male plasma vitellogenin for two different 

### 14 chemicals. For the figure on the left, Chemical L, while the 

### 15 range of values for the negative control or NC and the lowest 

### 16 treatment group, or L1, are close together, there was one value 

### 17 for both groups that is must greater than the other values as 

### 18 noted by the red circles. 

### 19 For the highest treatment group, or L3, it is 

### 20 statistically different from the control. However this may be 

### 21 due to the two values near the top of the box plot whiskers. 

### 22 For the figure on the right, or Chemical A, 

### 23 none of the treatment groups are statistically different from 

### 24 the control and for the mid-treatment, or L2, one value with 

### 25 orders of magnitude greater than the other values in this study, 

### 26 which may be hard to see, but it definitely is.

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1 In these examples these outliers were visually 

### 2 observed with no statistical test performed. In the 21 studies 

### 3 some laboratories removed perceived outliers while other 

### 4 laboratories included all data. In this evaluation the Agency 

### 5 included all reported data unless it was clear that the reported 

### 6 value was due to a technician or instrument error. However 

### 7 plotting the data like as shown here can be quite useful when 

### 8 evaluating whether a chemical is truly affecting vitellogenin in 

### 9 fish for a study. 

### 10 Overall for the FSTRA the assay is genially 

### 11 being conducted as expected with a majority of the evaluated 

### 12 studies meeting all or most of the performance criteria. 

### 13 Observations of decreased survival, body weight and/or other 

### 14 clinical signs of toxicity were observed in many of the 

### 15 evaluated studies. 

### 16 Therefore revisiting or additional guidance on 

### 17 dose selection may be warranted. However, the observation of 

### 18 overt toxicity was generally isolated to the highest treatment 

### 19 group which would allow for evaluation potential endocrine 

### 20 interaction in the two remaining test concentrations. However, 

### 21 having three concentrations available for evaluation would be 

### 22 optimal. 

### 23 Additionally the Agency recommends excluding 

### 24 fish from the data analysis that were incorrectly sexed. And 

### 25 finally although the measurement of plasma sex steroids is an 

### 26 optional endpoint, the endpoints are endocrine specific and are

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1 anticipated to provide a very useful line of evidence and 

### 2 whether a chemical is potentially impacting the HPG axis. 

### 3 Therefore the Agency emphasizes the importance 

### 4 of measuring sex steroids when the blood sample volume is 

### 5 sufficient. And I'll take any questions. 

### 6 **DR. DANIEL SCHLENK:**Any questions from the 

### 7 panel? Yes Dr. Patino. 

### 8 **DR. REYNALDO PATINO:**I'm curious to know a 

### 9 more specific rationale for your recommendation to exclude the 

### 10 incorrectly sexed males from analysis of endpoint sex with 

### 11 vitellogenin and nuptial tubercles. What is your rationale for 

### 12 excluding that one fish? 

### 13 **AMY BLANKINSHIP:**The thought would be that 

### 14 prior to pre-exposure those fish were identified by the 

### 15 technician as a female. So they probably weren't expressing 

### 16 normal male secondary sex characteristics and so they may not be 

### 17 acting as normal male fish. And so having them carry through, 

### 18 through all the evaluation, may not be appropriate if they 

### 19 weren't necessarily acting normally or exhibiting normal 

### 20 secondary sex characteristics prior to any chemical exposure. 

### 21 **DR. DANIEL SCHLENK:**Okay Dr. Leonard. Oh do 

### 22 you have follow up? 

### 23 **DR. REYNALDO PATINO:**Follow up. Yeah I can 

### 24 see the rationale for nuptial tubercles but how about 

### 25 vitellogenin where all the levels should be close to being on 

### 26 the table?

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1 **AMY BLANKINSHIP:**Yes and so that's a good 

### 2 point and so I recommend that anything like that you put in the 

### 3 record for consideration, but that is a good point. 

### 4 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 5 **DR. JACK LEONARD:**So I want to make sure I 

### 6 follow up a little and understand so that when a fish with 

### 7 missed sex that was discovered at the end of the study? 

### 8 **DR. AMY BLANKINSHIP:**Yes, typically. 

### 9 **DR. JACK LEONARD:**So you had a dataset for 

### 10 that fish up until the end of the study. 

### 11 **DR. AMY BLANKINSHIP:**So that fish, if it was 

### 12 caught at test termination, should have a full suite of 

### 13 endpoints. 

### 14 **DR. JACK LEONARD:**Right. If you evaluated 

### 15 those endpoints in that missed sex fish, did they differ 

### 16 significantly from the correctly sexed male? 

### 17 **DR. AMY BLANKINSHIP:**In the evaluations that 

### 18 I looked at we did try; because like I said some laboratories 

### 19 excluded them and some did not. So we removed them. Sometimes 

### 20 it did not make a difference in the overall endpoint, especially 

### 21 for vitellogenin where there can be some variation; so generally 

### 22 we're starting with an end of 8 if we have all 8 male fish 

### 23 available. 

### 24 So the thought of with the end of that value 

### 25 each fish could contribute significantly to that mean value and 

### 26 statistical analysis. But in some of the cases I looked at it

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1 did not appear to change the overall sort of effect level. 

### 2 **DR. DANIEL SCHLENK:**Dr. Horseman. 

### 3 Dr. nelson horseman: A point was made about 

### 4 the coefficient variation and measured test concentrations, were 

### 5 those variances normally distributed or typically skewed? And 

### 6 if not normal how were they skewed? 

### 7 **DR. AMY BLANKINSHIP:**I did not look at that 

### 8 in that detail. I do know that the coefficience of variance for 

### 9 each treatment group did vary quite a bit. 

### 10 In this assay there is a 10x spread between 

### 11 concentration; so if you're approaching near the limit of 

### 12 solubility, or a chemical that has an absorption properties, a 

### 13 10x factor between concentrations could result in significant 

### 14 differences between how the chemicals are measured compared to 

### 15 the nominal concentrations for that treatment group. So there 

### 16 was quite a range of CVs even within a study for each of the 

### 17 three treatment group. 

### 18 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 19 **DR. GEOFFREY SCOTT:**Along those same lines on 

### 20 the coefficient of variance of dose, you talked about the 

### 21 variance relative to dose in terms of the range of doses. What 

### 22 about the effect of time in the assays as conditions change in 

### 23 the test chambers and the size of organisms get bigger over 

### 24 time; have you looked at the time variable on variance, whether 

### 25 the coefficient of variance increases over time? Is that part 

### 26 of the problem? I'm just trying to get to the source of the

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1 variance. 

### 2 Amy Blankinship. Sure. Yeah as expected they 

### 3 can change over time so in some cases the measured 

### 4 concentrations were time-weighted averages just to sort of help 

### 5 account for that, especially if they took additional 

### 6 measurements over time. But generally in terms of my 

### 7 understanding it the coefficient variance just was sort of based 

### 8 on all the measurements that were taken over 21 days for each 

### 9 individual treatment growth. 

### 10 **DR. GEOFFREY SCOTT:**Follow up. The 

### 11 guidelines do allow for adjusting, you said several of labs did 

### 12 adjust flow rate I believe? 

### 13 Amy Blankinship: Correct. 

### 14 **DR. GEOFFREY SCOTT:**The guidelines do allow 

### 15 for that? 

### 16 Amy Blankinship: Maybe not directly but the 

### 17 goal is to help try and maintain test concentrations. So in 

### 18 terms of common practice for aquatic toxicity testing it would 

### 19 be common to increase the turnover rates in the system to help 

### 20 maintain test concentrations. 

### 21 **DR. GEOFFREY SCOTT:**And what would be the 

### 22 allowances of increases. Is that a ten percent increase? What 

### 23 rates are in the guidance? 

### 24 **AMY BLANKINSHIP:**There are no particular 

### 25 defined rates in the guidance; generally it's sort of dictated. 

### 26 Sometimes if you don't use a solvent in terms of how much of

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1 your test dosing solution you can get through the system in 

### 2 terms of your solubility of your highest test concentration 

### 3 stock solution. But this study asks for a minimum of five 

### 4 turnovers. So I wouldn't suspect that you can have turnovers of 

### 5 at least maybe 2/3 of that or greater. To me that would still 

### 6 think that the fish would still sort of operate normally and 

### 7 still behave properly. 

### 8 **DR. GEOFFREY SCOTT:**One last comment. Had 

### 9 any of the studies -- I assume the studies were measuring the 

### 10 exposure in aquaria with fish. 

### 11 **AMY BLANKINSHIP:**Right. 

### 12 **DR. GEOFFREY SCOTT:**Have you had any blank 

### 13 determinations where you have an empty chamber to look at the 

### 14 delivered dose so that you know whether or not the variance is a 

### 15 function of the dose or is a variance of what is going on in the 

### 16 tank? Because that is getting to your source of variance, it 

### 17 helps your endpoint. 

### 18 So I just ask if there are considerations of 

### 19 those criteria; because if you know you're delivering the dose 

### 20 and it's just not there because of a lot of intrinsic factors in 

### 21 the bioassay itself, uptake absorption of organics in the 

### 22 system. I think at least you know the dose was there and I 

### 23 think that's the real key thing everybody is trying to lean in 

### 24 on. 

### 25 **AMY BLANKINSHIP:**Sure. So laboratories would 

### 26 do a sort of a pre-exposure to make sure that at least a test

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1 initiation of the dose is sort of there or where they're at. 

### 2 But there is not an abiotic tank in this test system, which is 

### 3 not common -- I'm going to say -- for regulatory aquatic 

### 4 toxicity testing for fish. 

### 5 **DR. GEOFFREY SCOTT:**I can only say a lot of 

### 6 the sub-legal work we've done, we've always done that just so we 

### 7 can be sure we're delivering the dose. And that way you know at 

### 8 least you're delivering the dose because everything else is 

### 9 subject the variance and you don't know what that source of 

### 10 variance is. If it's in the delivery then you need to adjust 

### 11 the delivery. 

### 12 If it's not then you need to think about what 

### 13 it is that you can do about it. 

### 14 **AMY BLANKINSHIP:**I will say further on that, 

### 15 some laboratories do measure their stocks at each sampling point 

### 16 so they will so -- I say there is not an abiotic, but they will 

### 17 necessarily sometimes measure their dosing solution to help 

### 18 characterize proper dosing at least. 

### 19 Dr. Jeff Scott: And I think that is a good 

### 20 QAQC requirement because they're probably making the volume 

### 21 measurements too. So if you know the volume and you know your 

### 22 dosing stock, that's an indirect way of measuring it without 

### 23 having to measure. 

### 24 **AMY BLANKINSHIP:**You're absolutely right they 

### 25 do monitor and check the delivery system generally twice a day 

### 26 and so that they have a weekly dosing of stock solutions each

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1 time. You're correct, that is a good way to check for that. 

### 2 **DR. GEOFFREY SCOTT:**Well that might be worth 

### 3 putting in the reporting as well to say that's a way of not 

### 4 having to measure but reporting what you already did. 

### 5 **DR. DANIEL SCHLENK:**Let's make those comments 

### 6 when we get to the question that would be great. Dr. Tillitt? 

### 7 Dr. Donald Tillitt: I'm still not exactly 

### 8 clear in terms of the high CV. Are those mainly in the high 

### 9 concentration or were they mainly in the low concentrations, do 

### 10 you know that? 

### 11 **AMY BLANKINSHIP:**I don't know that off the 

### 12 top of my head, but I will say that for some of them the high 

### 13 group was fine and then as we sort of went that 10x factor, 

### 14 especially if they had a high absorption property, those CVs 

### 15 tended to increase with the lower concentration just because 

### 16 they tended to absorb to surfaces. So I don't know off the top 

### 17 of my head sort of where the distribution amongst the low middle 

### 18 and high concentrations for the 21 assays. 

### 19 **DR. DONALD TILLITT:**Nor if it was close to 

### 20 the limit of protection for the assay for the chemical analysis? 

### 21 **AMY BLANKINSHIP:**Correct. I would say that 

### 22 probably the measurements of what the LOQs and LOD were probably 

### 23 appropriate on these assays. 

### 24 **DR. DONALD TILLITT:**I have one more question 

### 25 to follow up. Have you done covariate analysis at all in terms 

### 26 of the males that showed out of range for unexpected high

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1 vitellogenin? Have you done covariate analysis with the 

### 2 estrogen concentrations on the females from the same tank? 

### 3 **AMY BLANKINSHIP:**No we have not and I will 

### 4 say that I only have estrogen levels right now in maybe in those 

### 5 three of the 21 studies. So that's an endpoint that is optional 

### 6 and often not done. 

### 7 **DR. DONALD TILLITT:**Thank you. 

### 8 **DR. DANIEL SCHLENK:**Any other questions from 

### 9 the panel? Okay thanks. Let's go ahead and move on to our next 

### 10 presentation. This will be by Catherine Aubee, who is from 

### 11 Environmental Fate and Effects Division, Office of Pesticide 

### 12 Programs. 

### 13 And again let me remind the panel that we will 

### 14 have the Agency back up tomorrow just if you have any questions 

### 15 that you've been pondering over the night or when you're meeting 

### 16 together, to bring back up, you can do that tomorrow. 

### 17 **CATHERINE AUBEE:**So thank you Dr. Schlenk, 

### 18 thank you everybody. As Dr. Schlenk introduced, my name is 

### 19 Catherine Aubee I am a biologist in the Environmental Fate and 

### 20 Effects Division along with Amy and Dr. Steeger. 

### 21 Our division has the primary responsibility 

### 22 for reviewing pesticide studies submitted under both the FSTRA 

### 23 guideline and the amphibian metamorphosis assay guideline which 

### 24 I will be presenting this afternoon. 

### 25 My presentation follows the general structure 

### 26 of the AMA chapter in the white paper if you've had the pleasure

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1 of looking at that already. I'll first give a brief overview of 

### 2 the intended purpose of the AMA and its general experimental 

### 3 designs. 

### 4 I will introduce some of the quality control 

### 5 elements mentioned in the test guideline and discuss how well 

### 6 the studies we've reviewed so far have informed those criteria. 

### 7 I'll then discuss in a little bit more detail, 

### 8 specific observations from our first round of studies. You'll 

### 9 note that some of these issues have been identified in the 

### 10 charge questions to the panel, which we will discuss more I 

### 11 believe again tomorrow. 

### 12 The amphibian metamorphosis assay, as its name 

### 13 implies, uses frog metamorphosis as a model for evaluating 

### 14 potential affects with a hypothalamic-pituitary-thyroid or HPT 

### 15 axis and it's downstream thyroid hormonal pathway. 

### 16 It is one of three assays as discussed earlier 

### 17 within the two Tier 1 EDSP battery that looks at this potential 

### 18 for interaction with the HPT axis. The other two are the male 

### 19 and female pubertal rat assays, which was presented by Dr. 

### 20 Liccione. 

### 21 Like the fish short term reproduction assay 

### 22 the AMA uses a 21 day aqueous exposure method. The test species 

### 23 is the African quad frog or the Xenopus laevis which is a very 

### 24 well characterised model species, which is fully aquatic 

### 25 throughout its life cycle. 

### 26 That assay is initiated with tadpoles at

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1 Nieuwkoop-Faber or NF Stage 51. Depending on environmental 

### 2 conditions, this stage is usually reached within 17 days post 

### 3 fertilization. Tadpoles are assigned to test substance 

### 4 treatment levels at one of at least three treatment 

### 5 concentrations or to a concurrent control. A solvent control is 

### 6 also included if the solvent is used in the study. 

### 7 However, the guideline recommends against the 

### 8 use of a solvent whenever possible. Each treatment level 

### 9 includes at least four replicates containing at least 20 

### 10 tadpoles per rep. 

### 11 If you are unfamiliar with xenopus this is 

### 12 just an image taken from the test guideline which shows the hind 

### 13 limb bud of the tadpole at stage 51; so this is what it looks 

### 14 like when we begin the test. 

### 15 This is just to give you a sense of the 

### 16 morphological markers used in determining the NF stage of the 

### 17 tadpoles. Hind limb morphology is one of the key biomarkers 

### 18 during this period. Others include forelimb emergence and tail 

### 19 resorption. 

### 20 For the AMA, endpoints are evaluated at two 

### 21 time points, day 7 and day 21. At day 7, endpoints are expected 

### 22 to be particularly sensitive to potential thyroid agonism as 

### 23 background levels of thyroid hormone are essentially negligible 

### 24 or nonexistent at this stage of development. 

### 25 By day 21, at test termination, endpoints are 

### 26 expected to be more sensitive to thyroid antagonisms as thyroid

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1 hormone levels are peaking as the tadpoles approach metamorphic 

### 2 climax. Day 7 endpoints are evaluated in a subsample of 

### 3 tadpoles which are not returned to the test population. 

### 4 The same endpoints plus thyroid histopathology 

### 5 and individual observation with clinical signs of toxicity are 

### 6 evaluated in the remaining tadpoles at test termination. The 

### 7 key endpoints of those sampling points, which you see here 

### 8 include NF stage as we mentioned, hind limb length which is 

### 9 normalized to snout-vent length to account for growth. 

### 10 We analyzed snout-vent length separately and 

### 11 also body weight. Most test guidelines include quality control 

### 12 criteria which may be identified as validity elements, 

### 13 performance criteria or some combination of the two. The AMA 

### 14 test guideline includes both of these. 

### 15 In the AMA the validity elements address the 

### 16 minimum number of viable individuals, replicates and treatments 

### 17 needed to provide confidence in determining whether there is a 

### 18 treatment related affect in the test. 

### 19 This is generally associated with the 

### 20 statistical power of the test design. By viable in this case we 

### 21 are talking about units that are not compromised by mortality, 

### 22 other obvious overt toxicity or human or technical errors, and 

### 23 those do happen from time to time. 

### 24 In general a reviewer will not automatically 

### 25 discount the results of the study if one or more validity 

### 26 elements are not met, but will carefully assess all aspects of

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1 the study before reaching a conclusion as to its validity or 

### 2 usefulness. 

### 3 Performance criteria in the AMA are included 

### 4 more as an indicator of whether environmental and exposure 

### 5 conditions are adequate and consistent. These are intended to 

### 6 ensure the health of the test organisms and to promote 

### 7 successful performance of the study, or in other words to 

### 8 minimize potential confounding factors. 

### 9 These criteria relate to the consistency of 

### 10 exposure concentrations, either 21 day coefficient variation 

### 11 each treatment level, NF stage distribution at test termination 

### 12 and water quality characteristics. It is helpful to the 

### 13 reviewer when each of these criteria is addressed explicitly in 

### 14 the study report. 

### 15 This figure gives an overview of how many of 

### 16 the 18 studies included in the preliminary review of the AMA 

### 17 satisfied each of the performance criteria. The black bars 

### 18 which are largest all the way through here represent the studies 

### 19 which met the criteria. 

### 20 The white bars indicate studies which did not 

### 21 meet them and the grey bars -- which are not really grey on this 

### 22 slide, I apologize -- represent studies which did not provide 

### 23 enough information for the reviewer to determine whether or not 

### 24 the criteria were met. 

### 25 So for example the fourth bar down -- I'll 

### 26 attempt to use the pointer here, I'm not allowed to use the

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1 remote at home so bear with me. The fourth bar down represents 

### 2 the performance criteria for NF stage distribution and controls 

### 3 at test termination. The guideline recommends that this should 

### 4 include less than or equal to four stages between the tenth and 

### 5 90th percentiles. 

### 6 So most of the studies we looked at reported 

### 7 that they did meet this criterion. However, three studies only 

### 8 reported median stage values and did not report individual level 

### 9 data or report the stage distribution; so we weren't able to 

### 10 really determine if they met the criteria or not. 

### 11 Another example is with replicate mortality. 

### 12 In some cases mortalities were only reported on a per treatment 

### 13 basis. Individual replicates were not identified. With these 

### 14 studies it was unclear, and you can see those here on the bottom 

### 15 -- not quite the bottom but the next two rows up. In these 

### 16 studies it was unclear whether the mortalities were spread 

### 17 across replicates or occurred in a separate single replicate so 

### 18 it could not be determined whether a given replicate was 

### 19 compromised. 

### 20 The 21-day CV, this is right up here in the 

### 21 middle, was the performance criterion which was most likely to 

### 22 be missed. This was not surprising as Amy discussed earlier 

### 23 with a longer term test of aquatic exposure. 

### 24 The deviations from the recommended CVs were 

### 25 usually minor. In this case I'll say roughly within five 

### 26 percent of that 20 percent or occurred in only one treatment

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1 level leaving two treatments still for analysis. 

### 2 In such cases as long as measured 

### 3 concentrations for different treatment levels did not overlap 

### 4 time weighted averages could be employed to make more accurate 

### 5 conclusions relating exposure levels and any effects observed in 

### 6 the study. 

### 7 However, in one case from this sample the 21 

### 8 day CVs were extremely high, up to 60 percent, and were coupled 

### 9 with extremely poor recovery of the test substance despite the 

### 10 use of the solvent. 

### 11 Aside from the one major deviation in that 

### 12 case, overall study performance for the AMA was fairly 

### 13 consistent with the validity and performance criteria in the 

### 14 guidelines. 

### 15 So we discussed that a couple of the 

### 16 performance criteria are related to NF stage and controls at 

### 17 test termination. The guideline recommends that the minimum 

### 18 median stage at day 21 be NF stage 57, that's for control 

### 19 tadpoles. 

### 20 However, individuals which have reached NF 

### 21 stage 61 or greater are generally excluded from the analysis of 

### 22 continuous endpoints related to growth, such as snout-vent 

### 23 length and body weight, because morphometric changes during 

### 24 metamorphic climax would skew the distribution and might reduce 

### 25 the chance of detecting a treatment related affect. 

### 26 Therefore performing laboratories have to

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1 strike a careful balance between attaining the minimum 

### 2 recommended median stage and controls at test termination, but 

### 3 not having too many tadpoles which progressed beyond NF stage 

### 4 60. 

### 5 While no laboratories have reported problems 

### 6 in meeting the minimum recommended stage, some laboratories have 

### 7 reduced feeding during the end-life portion of the study to 

### 8 reduce the likelihood of control tadpoles progressing beyond NF 

### 9 Stage 60. 

### 10 Dietary considerations such as these are also 

### 11 of interest when reflecting on the incidence of spinal curvature 

### 12 in Xenopus Laevis tadpoles in these studies. Spinal curvature, 

### 13 which is also referred to variously as bent tail, crooked tail, 

### 14 scoliosis or I think tail flex in the comments that we'll hear 

### 15 tomorrow. This is a musculoskeletal deformity, which may 

### 16 resolve itself naturally in Xenopus through metamorphic 

### 17 progression. 

### 18 This table which you should see here and which 

### 19 follows on the next slide shows some of the factors which have 

### 20 been proposed as possibly being linked to the incidence of 

### 21 spinal curvature; it includes the quantity and type of feed and 

### 22 iodide availability. 

### 23 Genetic factors may also play a role, although 

### 24 the spinal curvature that we are discussing here should not be 

### 25 confused with the bent tail mutations, which is a recessive 

### 26 mutation described by Drouin (phonetic) in 1992. That is

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1 actually a lethal mutation associated with internal 

### 2 haemorrhaging, and so we don't want confusion on the terminology 

### 3 there. 

### 4 As discussed in the White paper experimental 

### 5 work by our colleagues in the Office of Research and Development 

### 6 suggests that the prevalence of spinal curvature, at least 

### 7 within their test populations of Xenopus Laevis and Xenopus 

### 8 Tropicalis, is strongly linked to the type and quantity of feed. 

### 9 I should make a couple of clarifying remarks here regarding 

### 10 statements in the Paper. 

### 11 The principal investigator for the Olmstead et 

### 12 al. Manuscript which is cited in the Paper has since left the 

### 13 Agency, but he clarified for us that none of the tadpoles in 

### 14 those studies were intentionally overfed, so that is 

### 15 mischaracterized perhaps in the paper. Although they do see 

### 16 associations with different types of feed and things perhaps as 

### 17 protein content. 

### 18 Also we cited the Paper as in prep because 

### 19 that's how it was cited on an EPA webpage, but that is no longer 

### 20 current. I don't think it's currently being reviewed by any 

### 21 journal so we just wanted to make those clarifying remarks. 

### 22 So back to this lovely busy table, I think 

### 23 there is an even busier version in the Paper that you have. You 

### 24 can see that we're tracking on several variables here. I'm 

### 25 going to skip ahead a little bit for time but you can see we 

### 26 looked at the different types of feed and they're only a few

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1 different kinds that are being used. 

### 2 Whether or not feed was reduced compared to 

### 3 the guideline recommendations and if it says yes that this means 

### 4 that the laboratory reported reducing feeding by 50 percent 

### 5 compared to the guideline. We also looked at the quantity of 

### 6 iodide in food, if that was reported or if it was reported by 

### 7 the manufacturer. 

### 8 Background levels of iodide in water. This is 

### 9 hugely based on periodic analysis just historical data from the 

### 10 lab and not necessarily during a given study. And then whether 

### 11 or not iodide supplementation was given during the study. 

### 12 So in only one case of the 18 was 

### 13 supplementation provided, and I think the guideline recommends 

### 14 that if it's below .5 micrograms per liter in the water that 

### 15 supplementation should be given. 

### 16 So you don't see a clear pattern here you also 

### 17 see very high variability in the incidence and prevalence of 

### 18 spinal curvature. It was reported in almost all of the studies. 

### 19 A few of the studies didn't really say if they saw it or not, 

### 20 but if they did describe it they usually provided numbers. 

### 21 And it could have occurred anywhere from a few 

### 22 tadpoles to as much on this side you see as much as 87 percent, 

### 23 this is in controls up to 92 percent in treatments. So there 

### 24 doesn't seem to be any indication that this is treatment related 

### 25 in this studies but it does occur sometimes at relatively high 

### 26 proportions of the test pathogens.

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1 At this time the initial industry data as we 

### 2 just described in the table do not suggest any single factor 

### 3 that appears to be predicted of the spinal prevalence of spinal 

### 4 curvature. However, again, given that the abnormality has been 

### 5 documented in up to 87 percent of control tadpoles we do seek 

### 6 advice on what may be done to reduce the occurrence of this 

### 7 abnormality and regarding whether, and at what point, the 

### 8 prevalence of spinal curvature maybe so great as to render the 

### 9 result of the study unreliable; at least for the purposes of 

### 10 screening for thyroid-related effects, which is what we are 

### 11 interested in here. 

### 12 So if everybody is tired of that table we'll 

### 13 switch gears a little bit and talk about thyroid histopathology 

### 14 in the AMA. This is the one endpoint that overlaps with the 

### 15 male and female pubertal rat assays. 

### 16 Because histological presentations are 

### 17 expected to change throughout metamorphosis the test guidelines 

### 18 recommends that the test specimen selected for thyroid 

### 19 histopathology at day 21 be stage matched to the median NF stag 

### 20 in controls. So in other words if the median stag in controls 

### 21 is 58 then you want your specimens from the other treatment 

### 22 levels selected for thyroid histopathology also to be stage 58 

### 23 if possible. 

### 24 And this way we control for effects on 

### 25 development and improve the sensitivity of the thyroid 

### 26 histopathology analysis. The test guideline identifies several

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1 diagnostic and additional thyroid histopathology criteria. In 

### 2 our review of the data we summarized the incidence and severity 

### 3 of these various diagnoses and incorporate any other relevant 

### 4 narrative descriptions such as cell shape for context. 

### 5 Based on the initial data submissions the 

### 6 thyroid histology analysis appears to be fairly straightforward. 

### 7 However in some cases the shift toward decreased incidence in 

### 8 severity of a particular diagnosis such as follicular cell 

### 9 hypertrophy were reported when treatments were compared to 

### 10 concurrent controls. 

### 11 This was only, however, identified as a 

### 12 potential treatment-related effect by one histologist even 

### 13 though it was seen in multiple studies. This histologist noted 

### 14 that it might suggest a potential treatment-induced 

### 15 developmental delay because thyroid follicular cells normally 

### 16 increase in height during tadpole development. 

### 17 This example of the data here on the slide 

### 18 shows how similar observations in different studies may lead to 

### 19 different conclusions by different histopathologists. Therefore 

### 20 that Agency seeks advice on general topic of interpreting 

### 21 histological indicators of mild developmental delay during 

### 22 metamorphosis and particularly any indicators which may present 

### 23 as decreases in incidence or severity when compared to controls. 

### 24 These may be counterintuitive to reviewers 

### 25 with extensive training and traditional toxicology or 

### 26 ecotoxicology, but perhaps not in endocrinology or

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1 histopathology. 

### 2 I mentioned before that the guideline 

### 3 recommends against the use of a solvent in test substance 

### 4 delivery whenever possible. However solvents continue to be 

### 5 frequently relied upon in aquatic toxicity tests and the AMA is 

### 6 no exception. Solvents may interfere with the test directly or 

### 7 indirectly by increasing microbial growth or affecting uptake of 

### 8 the test substance. 

### 9 When a solvent is used, concurrent solvent 

### 10 controls are required and are compared to the negative, which is 

### 11 solvent free or a dilution water only control, to determine 

### 12 whether the solvent is causing any effect. Solvent effects were 

### 13 noted in six of all the 18 AMA studies which we evaluated, and I 

### 14 believe ten of those used a solvent. 

### 15 Acetone was used in one case as the solvent 

### 16 and the remaining five of those six cases Dimethylformamide was 

### 17 used, or DMF. In all cases where a solvent was used the 

### 18 concentration was equal to or below the maximum limit of 0.1 

### 19 ml/l which was recommended in guidelines for standard aquatic 

### 20 toxicity tests. 

### 21 In one case where solvent effects were still 

### 22 observed the solvent concentration was .02 ml/l. This is 

### 23 consistent with the even lower maximum concentration suggested 

### 24 for studies of potential endocrine effects, which we cite in the 

### 25 standard evaluation procedures for both the FSTRA and the AMA 

### 26 test guidelines.

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1 Even in these cases where a relatively low 

### 2 solvent concentration is used, the presence of the solvent 

### 3 effect makes it challenging to determine whether and to what 

### 4 extent the test substance itself is affecting endpoints in the 

### 5 AMA. 

### 6 In this example statistically significant 

### 7 effects on day 21, normalize timeline length were observed in 

### 8 the solvent control and in all treatment levels we see this here 

### 9 in green and red if it shows up for you. 

### 10 You'll note that we identify the statistical 

### 11 test in these tables for each comparison -- that's just a side 

### 12 note -- consistent with the guideline if you see a monotonic 

### 13 trend just based on visual observation, and the results we'll 

### 14 use the Jonckheere-Terpstra test. Otherwise, we typically use 

### 15 Dunnet's test if the data satisfies assumptions for normality 

### 16 and homogeneity of variance. Or we'll use the nonparametric 

### 17 Mann-Whitney test. 

### 18 In the same study - moving to the next slide - 

### 19 a slight promotion of day 7 snout-vent length was observed in 

### 20 the solvent control in intermediate treatment level while a 

### 21 significant decrease was observed at the highest treatment 

### 22 level. 

### 23 With respect to body weight marginal solvent 

### 24 effects were also observed on day 21. Ultimately solvent 

### 25 effects on growth and other parameters complicate the 

### 26 interpretation of data and they in some cases reduce confidence

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1 in the studies ability to detect the treatment related effect. 

### 2 The studies can also be difficult to evaluate 

### 3 when they show nothing. In cases where there is confidence in 

### 4 actual exposure conditions based on the analytical chemistry 

### 5 throughout the test, the absence of an effect is just that, no 

### 6 effect. 

### 7 However the test substance that demonstrates a 

### 8 very poor recovery and has measured concentrations which 

### 9 fluctuate or decline considerably throughout the course of the 

### 10 study may not be amenable to an aqueous exposure method. In 

### 11 such a case the absence of an effect in the AMA may reflect an 

### 12 absence of exposure rather than providing confidence that the 

### 13 substance does not interact with the HPT axis in amphibians. 

### 14 The AMA test guideline points to an OEC 

### 15 guidance document which provides numerous techniques for testing 

### 16 successfully with difficult test substances, which is what they 

### 17 are termed very appropriately. However, there may be cases when 

### 18 a substance is so poorly soluble or highly lipophilic that an 

### 19 aqueous exposure is unrealistic and uninformative. 

### 20 In such cases the guidelines suggest 

### 21 considering a dietary exposure protocol as an alternative to the 

### 22 aqueous exposure in the AMA. However the Agency is currently 

### 23 unaware of any validated dietary exposure protocols for testing 

### 24 effects of the substance on frog metamorphosis. 

### 25 The figure you see here just represents the 

### 26 measured concentrations from the one case earlier that I

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1 mentioned where there was a major deviation from the 21 day 

### 2 exposure CVs for the study and you can see this is the high 

### 3 treatment level. 

### 4 It drops down so that the day 21 levels 

### 5 basically overlap with the other ones. This is the intermediate 

### 6 and the low treatment level. And the other thing just to note, 

### 7 other than the major drop at the high level, is that their 

### 8 actual achieved concentrations are about 1/3 of what their 

### 9 nominal concentrations were. So this was difficult on many 

### 10 levels. 

### 11 For the most part the challenges which I 

### 12 discussed here and which are discussed in more detail in the 

### 13 White Paper are the exception rather than the rule. Laboratory 

### 14 performance of the AMA has generally met expectations based on 

### 15 the general scientific soundness of the studies and the validity 

### 16 and performance criteria which are detailed in the test 

### 17 guidelines. 

### 18 The assay appears to be an integral and 

### 19 informative part of the Tier 1 battery in that it provides data 

### 20 regarding the affects of the test substance on frog 

### 21 metamorphosis, which is an HPT dependent process. 

### 22 The high quality of most studies reviewed thus 

### 23 far provides the assurance that the method is reproducible 

### 24 across laboratories as initially shown in the assay validation, 

### 25 and that the test guideline and other guidance materials 

### 26 generally provide sufficient information for a laboratory to

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1 successfully conduct the test. 

### 2 That said, as we continue to gain experience 

### 3 with these studies and these datasets there may be specific 

### 4 areas where additional targeted guidance could be useful to both 

### 5 laboratories and reviewers. Such areas may include additional 

### 6 guidance for difficult to test substances for example when or 

### 7 how to perform dietary exposure test, minimizing the background 

### 8 occurrence of spinal curvature in xenopus tadpoles and ensuring 

### 9 consistent interpretation of thyroid histopathology indicators, 

### 10 which may be diagnostic of mild developmental delay. 

### 11 And so that concludes my presentation and I 

### 12 believe all of the individual assay presentation. I'm happy to 

### 13 take any questions. 

### 14 **DR. DANIEL SCHLENK:**Any questions from the 

### 15 panel. Dr. Patino. 

### 16 **DR. REYNALDO PATINO:**I have a number of 

### 17 questions but I'll start with two. 

### 18 **CATHERINE AUBEE:**Okay. 

### 19 **DR. REYNALDO PATINO:**In regards to the bent 

### 20 tails and what that might mean in terms of the validity of the 

### 21 other endpoints or how that may affect, did you actually try to 

### 22 determine whether the individuals with bent tails were apart 

### 23 from the others in terms of the other influence of growth or 

### 24 other endpoints? 

### 25 **CATHERINE AUBEE:**No we haven't performed any 

### 26 analysis like that. The data are not presented to us in such a

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1 way that we could figure that out. In other words we don't 

### 2 usually get data that say for this individual snout-vent length 

### 3 was this and this individual also showed bent tail. 

### 4 **DR. REYNALDO PATINO:**It would be useful to 

### 5 have that information, it would be easy to determine. 

### 6 **CATHERINE AUBEE:**Right. 

### 7 **DR. REYNALDO PATINO:**The other question I 

### 8 have is one of the concerns or one of the questions you had is 

### 9 about the interpretation of the thyroid histopathology and what 

### 10 that means. 

### 11 **CATHERINE AUBEE:**Right. 

### 12 **DR. REYNALDO PATINO:**Also I guess your 

### 13 previous answer may have answered the second question I have. 

### 14 So you have that some compounds suggested that there was a 

### 15 reduced overall ranking of the thyroid condition. Was that 

### 16 associated also with reduced development in the same individual? 

### 17 **CATHERINE AUBEE:**What I showed was just 

### 18 intended to be an example. The type of question that you're 

### 19 asking is what we would anticipate then looking at when we're 

### 20 looking at the weight of evidence for an individual chemical. 

### 21 So those are the types of questions that we're thinking about. 

### 22 And this one case I don't think that that was the case. 

### 23 So in the one case that I showed you I think 

### 24 we would be able to sort out on our own looking at the other 

### 25 data and saying no it's not really consistent. 

### 26 However, our concern is more that we're seeing

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1 that sort of trend and those diagnoses across several studies 

### 2 and we just don't know in terms of the interpretation of that on 

### 3 its own. 

### 4 **DR. REYNALDO PATINO:**It's difficult to 

### 5 interpret on its own if you don't always develop the 

### 6 developmental stage. 

### 7 **CATHERINE AUBEE:**Right. We do have data on 

### 8 developmental stage usually for each organism. Although it is 

### 9 not required in the guideline, we do recommend that data be 

### 10 reported to us on an individual level with individual 

### 11 identifiers when possible, so that we can track the end-life 

### 12 measurements with the histopathology analysis, which is usually 

### 13 done separately and after the fact. But it's not something we 

### 14 get all the time. 

### 15 **DR. REYNALDO PATINO:**Alright, thank you. 

### 16 **DR. DANIEL SCHLENK:**Dr. Green? 

### 17 **DR. SHERRIL GREEN:**I have a couple of 

### 18 questions for you as well. Would it be possible for the panel 

### 19 to see images of the thyroid histopathology with examples of 

### 20 your grading system, which I think was zero to three, so we 

### 21 could have some perspective on what's being evaluated? 

### 22 **CATHERINE AUBEE:**Sure. 

### 23 **DR. SHERRIL GREEN:**Maybe tomorrow or later 

### 24 sometime. 

### 25 **CATHERINE AUBEE:**Okay, we don't routinely 

### 26 receive images for these studies. We ask that they keep them on

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1 file. That's something we would have to make a special request 

### 2 for from the company. What we do have and what we have 

### 3 referenced, I think, within the body of the charge question 

### 4 document, are the guidance documents which are available to 

### 5 industry and to reviewers which have some representative 

### 6 micrographs of those severity grading scales. 

### 7 **DR. SHERRIL GREEN:**Okay. Thanks, wonderful. 

### 8 **CATHERINE AUBEE:**So that may be useful. 

### 9 **DR. SHERRIL GREEN:**Along that line, how do 

### 10 you control between company reviewers or the contract labs for 

### 11 intra and inter observer agreement? Because this is fairly 

### 12 subjective and it's difficult if the changes are slight and you 

### 13 don't use a software system to measure. 

### 14 **CATHERINE AUBEE:**Right. I think we recognize 

### 15 that as a limitation. One of the things which Director 

### 16 Manibusan may be mentioning later is that a histopathology 

### 17 workshop or training session is anticipated sometime in the near 

### 18 future. 

### 19 **DR. SHERRIL GREEN:**Okay that's good. 

### 20 **CATHERINE AUBEE:**But other than that the 

### 21 guidance documents are sort of our work in progress and we'd 

### 22 like to build on that. 

### 23 **DR. SHERRIL GREEN:**Right. Would we be able 

### 24 to see for bent tail possibly? Do you have any images of the 

### 25 bent tail samples of what it looks like? 

### 26 **CATHERINE AUBEE:**I don't have any from the

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1 specific studies. 

### 2 **DR. SHERRIL GREEN:**Okay. 

### 3 **CATHERINE AUBEE:**I think we have a public 

### 4 commenter who again did some work while with the Agency and I 

### 5 don't know if he intends to present it or not. Dr. Touart did 

### 6 you want to say something? 

### 7 **DR. DANIEL SCHLENK:**Dr. Touart? 

### 8 **DR. LESLIE TOUART:**Just a follow up; in terms 

### 9 of the kinds of guidance that is provided in doing the 

### 10 histopathology, there is a histopathology guidance document that 

### 11 goes into a lot of detail with a lot of images and analysis to 

### 12 demonstrate to help insure kind of uniformity across 

### 13 pathologists that would be looking at these. 

### 14 **DR. SHERRIL GREEN:**Okay great. I have a 

### 15 couple of more. 

### 16 **DR. DANIEL SCHLENK:**Sure. 

### 17 **DR. SHERRIL GREEN:**With regards to the bent 

### 18 tail, you know, I'm aware that there is a mutation that is 

### 19 lethal, but there are also some naturally occurring mutations 

### 20 that are not lethal. And when labs breed and they use wild 

### 21 caught frogs, sometimes they introduce bent tail into their 

### 22 group from breeding. 

### 23 Have you attempted any mutagenesis screening 

### 24 on some of these sample cohorts so you could see if you have it 

### 25 in your family lines? 

### 26 **CATHERINE AUBEE:**We have not within our

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1 program. I cannot state for the Office of Research and 

### 2 Development. Unfortunately I don't know if they're on the line 

### 3 remotely with us right now. I know that they have looked at 

### 4 some of the other environmental and genetic factors. I don't 

### 5 know what type of screening they may have done, however. 

### 6 **DR. DANIEL SCHLENK:**Is it possible to contact 

### 7 them during the week so that we can get that information? 

### 8 **CATHERINE AUBEE:**Yes. Absolutely. We will 

### 9 ask for it. 

### 10 **DR. SHERRIL GREEN:**Another question related 

### 11 to the tails. One Slide 8 there was no comment on tail 

### 12 appearance or parameter as one of the things that you would 

### 13 test, that you would evaluate one of the criterion; I was 

### 14 wondering if you meant not to include that and why not? Or is 

### 15 this something that came up after this? 

### 16 **CATHERINE AUBEE:**In terms of the historical 

### 17 perspective on what is in the test guidelines, Dr. Touart may be 

### 18 able to talk about that a little bit more. However, this is 

### 19 something that we've recognized as an issue as people are 

### 20 starting to do the studies. 

### 21 We discussed it in a corrections and 

### 22 clarifications document that was published after the test 

### 23 guidelines came out and basically said keep an eye on this, make 

### 24 sure other performance criteria are being met, and we are 

### 25 definitely going to pay attention to whether or not it appears 

### 26 to be a treatment related effect.

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1 Dr. Touart, I don't know if you want to speak 

### 2 to the guideline a little bit more. 

### 3 **DR. LESLIE TOUART:**Just a follow up as 

### 4 Catherine said, the context is if we see some type of a 

### 5 concentration related, you know, response to that that would be 

### 6 reported, but the occurrence, because it is somewhat prevalent. 

### 7 8 One thing to note with the kind of tail 

### 9 curvature, it's really more of a muscular anomaly than it is a 

### 10 skeletal anomaly within the tail at that time. It appears that 

### 11 what you have is an asymmetry within the muscles of the tail in 

### 12 terms of how they grow. 

### 13 Recognize that within the metamorphosis of 

### 14 these tadpoles, the tail is growing but also being resorbed over 

### 15 the time course; so that the context of how the tail could 

### 16 presumably get (inaudible) and stuff is there, but there are 

### 17 these factors whether their mutations are possible to enter in. 

### 18 19 But the general observation for these tadpoles 

### 20 in terms of once the tails are absorbed or once they go through 

### 21 metamorphosis, there doesn't seem to be any correlation between 

### 22 the survivability of the individual beyond that. 

### 23 But other than that kind of general context 

### 24 the actual assay itself isn't looking at specific outcomes of 

### 25 that unless there is something that appears to be concentration. 

### 26 **DR. SHERRIL GREEN:**Okay that's all I have.

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1 **DR. DANIEL SCHLENK:**Okay. Dr. Schultz you 

### 2 had your hand up. 

### 3 **DR. TERRY SCHULTZ:**Because question five and 

### 4 six directly apply to two very specific components of your talk, 

### 5 don't feel that the committee or panel is picking on you but we 

### 6 kind of need this kind of information if we're going to provide 

### 7 the components back. So I know I'm not supposed to make 

### 8 comments just questions I'm going to list a series of questions 

### 9 and just get a yes or no and get a feel for this. 

### 10 The curvature of the spine is that a 

### 11 dorsal/ventral kinking? Does it have flexure that goes down or 

### 12 is it a lateral flexure? 

### 13 **DR. LESLIE TOUART:**Primarily lateral. 

### 14 **DR. TERRY SCHULTZ:**Does it curve primarily in 

### 15 the anterior region of the tail? 

### 16 **CATHERINE AUBEE:**Yes. 

### 17 **DR. TERRY SCHULTZ:**Does appearance tend to be 

### 18 related to a specific stage. In other words because you get to 

### 19 a certain stage you can start to see it and it might not be 

### 20 recorded there or not? 

### 21 **CATHERINE AUBEE:**Sure. I'll speak on that 

### 22 for a moment if that's all right. In most cases what we're 

### 23 seeing is it is not reported at test initiation with NF stage 

### 24 51. However at some point during the study it begins to appear 

### 25 and it generally seems to increase when we get that information; 

### 26 it appears that it increases over the duration of the study.

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1 So for example the study may report starting 

### 2 to see it in tadpoles around day seven or eight and then you get 

### 3 this really high prevalence by test termination. 

### 4 **DR. TERRY SCHULTZ:**Is there any anatomical 

### 5 pathology studies that you're aware of that's been associated 

### 6 with this lesion? 

### 7 **CATHERINE AUBEE:**Not that I know of but I 

### 8 don't know for certain. 

### 9 **DR. TERRY SCHULTZ:**Is there any biochemical 

### 10 or other information to suggest that it's associated with 

### 11 connective tissue defects? 

### 12 **CATHERINE AUBEE:**Not that I'm aware of. 

### 13 **DR. TERRY SCHULTZ:**Okay. 

### 14 **DR. DANIEL SCHLENK:**Piersma? 

### 15 **DR. ALDERT PIERSMA:**One very general question 

### 16 after all these details is what I noticed in the report is that 

### 17 you state that the reviews you've done of the data is of a 

### 18 preliminary nature. Both in the beginning and the end of the 

### 19 text I find that. What do you mean by that? Does it mean that 

### 20 this test has been analyzed so far in less steps than other 

### 21 tests, or do I need to weight these results differently from 

### 22 other tests? What do you want to say? 

### 23 **CATHERINE AUBEE:**What that means is that we 

### 24 are waiting for feedback from you before we finalize the 

### 25 reviews. As Dr. Bradbury mentioned earlier we're very 

### 26 interested in your feedback and if there is something that we

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1 should perhaps adapt or look at differently we would like to be 

### 2 sure to incorporate that before finalizing anything that then 

### 3 becomes a final public record for these chemicals. 

### 4 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 5 **DR. JACK LEONARD:**Has there been an attempt 

### 6 to make a relationship between the molecular cell height and the 

### 7 snout/tail limb in your data sets? 

### 8 **CATHERINE AUBEE:**To my knowledge no one has 

### 9 looked at that specifically although some of the 

### 10 histopathologists do appear to have had access to the actual 

### 11 study data. And then in some cases they will discuss that in 

### 12 more of a qualitative sense. 

### 13 **DR. DANIEL SCHLENK:**Dr. Scott? 

### 14 **DR. GEOFFREY SCOTT:**In the report itself you 

### 15 did not cover this in your remarks, but in Table 32 there is a 

### 16 mention of the suppliers. And I didn't hear you mention that in 

### 17 your talk. I'm sure you all probably did look at that and maybe 

### 18 you can comment on what your interpretation is in looking at the 

### 19 data on suppliers if you all had looked into that with greater 

### 20 detail. 

### 21 **CATHERINE AUBEE:**Thank you Dr. Scott. I was 

### 22 thinking of that also when Dr. Green asked her question earlier 

### 23 about screening for mutations and specific populations. 

### 24 I don't know if that's been done at the 

### 25 laboratory level either. Something that, as you pointed out, we 

### 26 took out of the slide just because it really didn't fit

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1 space-wise onto the slide itself, was that we are tracking on 

### 2 the supplier for test organisms when that is reported. It 

### 3 usually is. 

### 4 But again basically that is just keeping track 

### 5 of the name and does it seem like we see more bent tail in this 

### 6 case when it's all coming from the supplier. 

### 7 Generally, again this is a relatively small 

### 8 sample in that we have only a few labs that we're looking at 

### 9 with a few different suppliers; so it's hard to say 

### 10 qualitatively what is going on there. But you can tell, if you 

### 11 imagine that this supplier is pretty much aligned with what the 

### 12 lab here is, you can see there does seem to be at least visually 

### 13 some difference across labs and potentially suppliers. Does 

### 14 that get at your question? 

### 15 **DR. GEOFFREY SCOTT:**Yeah that's what I was 

### 16 asking. Thank you. 

### 17 **DR. DANIEL SCHLENK:**Dr. Patino? 

### 18 **DR. REYNALDO PATINO:**I have another question 

### 19 in concerns to the thyroid histology. And that is, what is the 

### 20 rationale for using a published guide of rankings versus using 

### 21 the internal controls for this study? 

### 22 **CATHERINE AUBEE:**Okay. Again, the published 

### 23 guide was actually created during the validation of the test 

### 24 itself. So it was sort of work that went hand in hand and 

### 25 because there wasn't really anything out there before looking at 

### 26 amphibian thyroids during metamorphosis. So they did a lot of

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1 work and they catalogued it as well as possible. And that is 

### 2 what is resulted then in the published guide. 

### 3 Now there are a couple of different ways to 

### 4 evaluate the histopathology criteria for the amphibian thyroid 

### 5 described in the guide. There are certain things, which you may 

### 6 know or not, that are diagnosed: We assigned the incidence, the 

### 7 number of observations, and severity based on a grading scale of 

### 8 0 not remarkable to 1, 2, 3 being the most severe. 

### 9 In some cases those are based on the normal 

### 10 micrographs which, for example, you might see in the guide that 

### 11 could be used as a reference case. For other specific regions 

### 12 it's intended to be based on what you see in the control 

### 13 specimen. 

### 14 However, what I should clarify is that even 

### 15 when something is diagnosed based on the control micrographs in 

### 16 the published guide, I think how it should be interpreted is 

### 17 then based on what you see in the controls for the study. 

### 18 So for example even if we go to the particular 

### 19 diagnosis that we were discussing here, you can see an incidence 

### 20 of ten individuals diagnosed as grade 1 follicular cell 

### 21 hypertrophy in the controls. 

### 22 So this is based on what the normal diagnosis 

### 23 or what the normal micrograph would be. But then what we're 

### 24 thinking is for the treatment levels we would see how these line 

### 25 up compared to the controls. So when interpreting a treatment 

### 26 related affect we are looking at the concurrent controls.

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1 **DR. REYNALDO PATINO:**Okay. 

### 2 **CATHERINE AUBEE:**Does that answer your 

### 3 question? It's very confusing. 

### 4 **DR. REYNALDO PATINO:**Somewhat. I do have a 

### 5 follow up. 

### 6 **CATHERINE AUBEE:**Okay. 

### 7 **DR. LESLIE TOUART:**Just to add a little bit 

### 8 more clarification with that. In context the controls, you're 

### 9 using the histopathology guidance document to read controls as 

### 10 well as treatments. So in the lesions and instance and severity 

### 11 grades that are used would be matched to what the guidance 

### 12 document in terms of criteria are listed. 

### 13 So if you have a severity grade of 2, you know 

### 14 that would be identified, it could be in control, it could be in 

### 15 treatment and those kinds of things. The pathologist himself 

### 16 would be going back and kind of comparing it. Once they've 

### 17 graded the variety of slides and stuff to try to determine okay 

### 18 is there some type of a concentration related response, are they 

### 19 able to sort the slides in some capacity in that context. 

### 20 But in general it's not that you're comparing 

### 21 an effect, you know, in the atlas versus what your actual 

### 22 control will be because that's being compared within the same 

### 23 context. 

### 24 **DR. REYNALDO PATINO:**I understand. You are 

### 25 comparing and indirectly the control is compared against an 

### 26 external standard so to speak.

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1 **DR. LESLIE TOUART:**Correct. It's just a 

### 2 matter of once everything has been read and you've got all your 

### 3 gradings then you can go back and compare, okay did the actual 

### 4 instances or follicular hikes that have been measured, is that 

### 5 different from control to the treatments in that sense. But in 

### 6 terms of the actual grading, that's based upon kind of the 

### 7 standard that would be provided within the guidance documents. 

### 8 **DR. REYNALDO PATINO:**So a follow up 

### 9 concerning thyroid histopathology. This measures are kind of 

### 10 semi quantitative, qualitative your ranking the severity of the 

### 11 lesion or whatever you call it. Have you considered using a 

### 12 more continuous variable such as in this case would be thyroid 

### 13 side height? 

### 14 Have you any thoughts about doing that and 

### 15 having a continuous variable for the thyroid histopathology? 

### 16 Currently with the guideline we don't, it's all ranking. 

### 17 **DR. LESLIE TOUART:**I think the context, I 

### 18 mean, we did have a histopathology workshop, whatever they 

### 19 consider that in terms of considerations of how these things 

### 20 might be considered, but one aspect given that this is a screen, 

### 21 we move more towards keeping it somewhat qualitative. 

### 22 I think if one moves into evaluations that 

### 23 would be more say in the realm of Tier 2, definitive type 

### 24 testing, the need for evaluating a little bit more qualitatively 

### 25 in context, using continuous measures and using your statistical 

### 26 elements in terms of determination, would be perhaps

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1 appropriate. But in this context it was decided to try to keep 

### 2 it simple because histopathology is one of the more expensive 

### 3 aspects of these assays and so we want to keep it as concise and 

### 4 simple as is practical in the context. 

### 5 So that was the, I think, the determining 

### 6 factors in terms of what went into the guidance. 

### 7 **DR. REYNALDO PATINO:**So this would be 

### 8 something for Tier 2? Thyroid side height? 

### 9 **DR. LESLIE TOUART:**Conceivably, and we look 

### 10 forward to further recommendations that you may have on this. 

### 11 **DR. REYNALDO PATINO:**Follow up question. 

### 12 Concerning controls, what is your rationale -- not just for this 

### 13 but the other endpoints in the AMA. What is your rationale for 

### 14 comparing against the native control when there is a solvent as 

### 15 opposed to the solvent control? 

### 16 **CATHERINE AUBEE:**I'm being looked at because 

### 17 this is a policy of our division. This is just the standard 

### 18 practice for evaluating eco-toxicity data within our division. 

### 19 I believe it ties back to what is in the regulations, the 40 CFR 

### 20 which actually have a statement on the laboratory practices that 

### 21 say if there is solvent interference then the study really 

### 22 should be thrown out and invalidated. 

### 23 And so based on that our practice in the past 

### 24 has been to compare to the negative control but to test to see 

### 25 if there is a solvent effect using a t-test or something like 

### 26 that, and if there is then we look at things a little bit more

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1 carefully and make some decisions as to the overall validity of 

### 2 the study. 

### 3 Now this is a new paradigm for us and, of 

### 4 course, we recognize that it is quite common practice in many 

### 5 cases to compare it to the solvent control. However, for the 

### 6 reasons that we've discussed earlier it does sometimes -- even 

### 7 if we do that -- it does raise some questions. In some cases 

### 8 it's just a matter of how much uncertainty you're willing to 

### 9 deal with. How much a solvent may be masking an effect in the 

### 10 study. 

### 11 **DR. REYNALDO PATINO:**Okay. 

### 12 **DR. DANIEL SCHLENK:**Okay Dr. Horseman. 

### 13 **DR. NELSON HORSEMAN:**Back to the thyroid 

### 14 histology. I understand the discussion of the comparison made 

### 15 to controls on this. Is there an effort to determine whether 

### 16 the thyroid histology is concordant internally with the 

### 17 metamorphic stage of those animals? 

### 18 It seems that the more informative question is 

### 19 that, whether thyroid histology corresponds to what one would 

### 20 expect with that metamorphic staging. 

### 21 **CATHERINE AUBEE:**I think we do that perhaps 

### 22 indirectly by recommending that at least the specimen selected 

### 23 the stage matched. Ideally we would be looking at individuals 

### 24 which all have the same developmental stage, and although then 

### 25 we don't necessarily look to see okay are these characteristics 

### 26 of the thyroid, are they representative of what we would see at

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1 that stage, we operate under the assumption that if they are all 

### 2 at the same stage then they should all have similar thyroid 

### 3 characteristics; and if there is a difference then that could be 

### 4 a potential effect. 

### 5 **DR. NELSON HORSEMAN:**Just a follow up. If 

### 6 there is then a treatment effect on stage how does that affect 

### 7 your selection of histopathology specimens? 

### 8 **CATHERINE AUBEE:**This sort of bridges over to 

### 9 what I will show in another presentation where we're showing the 

### 10 decision logic for the AMA test guideline, but it is in the test 

### 11 guideline. 

### 12 The first thing I would say is if we see an 

### 13 effect on developmental stage that it appears to be advancing 

### 14 developmental stage according to the test guidelines that could 

### 15 be a hit for the screens. So we might consider that a positive 

### 16 finding for the screen and so even if there is uncertainty as to 

### 17 the thyroid diagnoses because there is variability there we 

### 18 already know that there is some sort of effect. 

### 19 Now where it gets a little bit more 

### 20 complicated is, as we mentioned, if we see a developmental delay 

### 21 and then we are unable to stage match those specimens. The 

### 22 guidance recommends that you get as close as possible. So for 

### 23 example if there is a developmental delay and you can't get the 

### 24 same stage you would select the next closest one down. 

### 25 So we try to get it close but it does 

### 26 introduce some more uncertainty, I think, into the analysis.

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1 **DR. DANIEL SCHLENK:**Dr. Green. 

### 2 **DR. SHERRIL GREEN:**I have a different line of 

### 3 question. I was looking on slide number nine, the test 

### 4 concentration CV = 20% over 21 days and in the report there it 

### 5 looks like only 12 out of the 18 studies met that performance 

### 6 criteria. 

### 7 And I listened very carefully to your 

### 8 explanation but just for my own edification, could you reiterate 

### 9 that one more time? I'm struck by how many of the studies 

### 10 didn't meet that performance and it seems to me like test 

### 11 concentration is so critical to this entire body of work. 

### 12 So if you could explain one more time the 

### 13 discrepancy there between the only six labs having met that 

### 14 criteria and the others not. Thank you. 

### 15 **CATHERINE AUBEE:**Sure. As you pointed out I 

### 16 think it was about maybe 10 studies. I can't tell exactly on 

### 17 here and I don't remember the exact number, but I think that ten 

### 18 studies met the criteria and something like six to eight did not 

### 19 meet the criteria for 21 day CVs. 

### 20 In most cases the criterion is = 20% so those 

### 21 cases may have been where at the high treatment level where it's 

### 22 really close to the limit of solubility they got slightly 

### 23 outside that. Or conversely, as the case was brought up before, 

### 24 we did see the opposite also where at very low concentrations 

### 25 close to the limit of detection it was slightly outside that. 

### 26 The point that I was making was that except

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1 for the one case where I showed you the different figure -- and 

### 2 I'll bring that up again here. This was pretty drastic, but this 

### 3 is uncharacteristic for most of the chemicals at least in the 

### 4 subset that we analyzed. 

### 5 So we knew based on the physical chemical 

### 6 characteristics of this chemical that it was going to have 

### 7 issues. But in most cases even if they did not meet that 

### 8 criterion -- so say for the other five chemicals that missed it, 

### 9 it was a relatively minor deviation, so maybe 5 percent or less, 

### 10 that's a rough approximation. Or it was at only one treatment 

### 11 level and so one of the other criteria within the guideline is 

### 12 that you have to have two uncompromised treatment levels. You 

### 13 start with three, so if you have less confidence in one because 

### 14 of something weird like this you may still be able to get enough 

### 15 information out of that study based on the remaining two. 

### 16 **DR. SHERRIL GREEN:**Just so I recall 

### 17 correctly, tissue levels of the test substance were not measured 

### 18 in the tadpoles themselves, only in the water. 

### 19 **CATHERINE AUBEE:**Correct. 

### 20 **DR. SHERRIL GREEN:**Okay thank you. 

### 21 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 22 **DR. GEOFFREY SCOTT:**Yeah I think this figure 

### 23 is really illustrative of the problem with getting to this 

### 24 dosing conformance because if you look in this data that you 

### 25 have posted here, at least in the lower two doses, at the start 

### 26 of the test and well in beyond the doses seem pretty

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1 consistently at I think you said about 30 percent of the 

### 2 nominals. 

### 3 So the real question is what is causing that, 

### 4 where is that other 70 percent? And I think that's why this 

### 5 kind of information is real useful and I guess I assume you all 

### 6 are looking at those kinds of differences. I think this is a 

### 7 good example of having this kind of data to be able to look at, 

### 8 to sort out where the variance may be. 

### 9 **DR. DANIEL SCHLENK:**Okay. All right one 

### 10 more. We're about ready to break. Yeah Dr. Patino: 

### 11 **DR. REYNALDO PATINO:**Okay last question. So 

### 12 in the report you mentioned that you normalized hind limb length 

### 13 by snout-vent length. And I think tomorrow in the public 

### 14 comments there may be a question about that procedure. But my 

### 15 question for you is have you determined that the relationship 

### 16 between these two variables is proportional or not? And that 

### 17 would probably address questions about the validity of doing 

### 18 that. 

### 19 **CATHERINE AUBEE:**We do not test to see if 

### 20 there is a relationship between snout-vent length and hind limb 

### 21 length prior to doing the analysis where it's simply operating 

### 22 under the recommendations of what was put into the guideline. I 

### 23 had the chance to look back at the validation reports that came 

### 24 out in three phases and they did look at this specifically. 

### 25 They did different types of normalizations. 

### 26 They also tried normalizing to body weight and they decided at

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1 least based on the subset of reference chemicals that they used, 

### 2 that normalizing the hind limb length was the most appropriate. 

### 3 They didn't necessarily recommend doing any sort of other tests 

### 4 for relationships between that and snout-vent length beforehand. 

### 5 I will say that in our first set of chemicals 

### 6 when we do our analysis it's sort of a batch process for the 

### 7 statistics, so we run all the data through; so non-normalized 

### 8 hind limb length is something that is analyzed although it's not 

### 9 one of the recommended endpoints. And then pretty much all of 

### 10 the cases where we saw an effect on normalized hind limb length, 

### 11 it was also significant for the non-normalized. We just didn't 

### 12 then report that because it wasn't a guideline. 

### 13 **DR. REYNALDO PATINO:**May I follow up? Is 

### 14 there any way you can provide the rationale on the development 

### 15 of the guideline? Is there some document where we can see why 

### 16 they recommended that? I was not involved in that so I don't 

### 17 know and I'd like to know. 

### 18 **CATHERINE AUBEE:**Sure. I can pull the 

### 19 relevant sections of the earlier reports if that is helpful and 

### 20 I don't know if Dr. Touart wants to speak to that. 

### 21 **DR. REYNALDO PATINO:**Thank you. 

### 22 **DR. LESLIE TOUART:**There was some discussion 

### 23 within the integrated summary report that was developed for the 

### 24 AMA that went for the peer review and the validation activity to 

### 25 consider that. 

### 26 One thing to remember though is that the hind

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1 limb length is measured at day seven and at day seven there is 

### 2 not a lot of other measures that are being made since the test 

### 3 is being carried throughout. 

### 4 So the snout-vent length is an indicator of 

### 5 size so it can normalize to a size that would seem to be 

### 6 appropriate for context. So that is a situation for providing 

### 7 it in that particular case because weight or other indications 

### 8 are not as effective. 

### 9 **DR. DANIEL SCHLENK:**Okay. If it's okay with 

### 10 Dr. Steeger we're going to take a break now and then we can 

### 11 combine your presentations at the end of that is okay. And 

### 12 there will be ample time because this will be a combined 

### 13 presentation. If you have other questions that come up during 

### 14 the break you can ask them at that time as well. So let's go 

### 15 ahead and take a 15 minute break and be back at 2:45. 

### 16 (Whereas a break was taken) 

### 17 Okay. I have another announcement again for 

### 18 public presenters. If you have a presentation please get that 

### 19 to Fred either after the meeting today or before 8:30 tomorrow 

### 20 morning if you're up for tomorrow morning. Again, public 

### 21 commenters if you have a presentation please get that to Fred 

### 22 Jenkins after the meeting today or before 8:30 in the morning. 

### 23 Okay. Let's go ahead and continue with our presentations and 

### 24 Dr. Steeger if you're up on the battery performance. 

### 25 **DR. TOM STEEGER:**All right thank you very 

### 26 much.

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1 Before I move into the battery performance 

### 2 section I just want to make one comment and that is that over 

### 3 the past several presentations talking about the individual 

### 4 assay performances, the agencies highlighting issues that have 

### 5 been associated with some of the performance of the assays 

### 6 potentially high CVs in some of the assays where measured 

### 7 concentrations may not have been close to nominal. 

### 8 I think that the important thing to keep in 

### 9 mind here is that while those might occur in a test they did not 

### 10 necessarily occur across all treatments within the test. 

### 11 So the Agency considers the performance 

### 12 criteria as guidelines; they aren't meant to be hard and fast 

### 13 rules; that if you exceed the criteria then it automatically 

### 14 discounts the utility of the study. Most of these studies as 

### 15 has been indicated in the presentations have met the performance 

### 16 standards while some replicates within the treatment groups may 

### 17 not have. 

### 18 In general, I think that what we're seeing is 

### 19 that across the assays and across the different measurement 

### 20 endpoints, including the chemical/physical and environmental 

### 21 conditions of the tanks themselves, there tended to be 

### 22 relatively good consistency across the different chemicals that 

### 23 were tested. 

### 24 With that said there are some chemicals that 

### 25 are very difficult to test, and those were used as actual 

### 26 examples where solubility and high organic carbon partition

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1 co-efficient really do challenge any type of aquatic testing 

### 2 with those chemicals. 

### 3 So moving into battery performance in the 

### 4 following presentations; battery performance will be discussed 

### 5 with respect to each of the endocrine modalities. 

### 6 Catherine Aubee will be discussing battery 

### 7 assays associated with the thyroid pathway. Amy Blankinship 

### 8 will be providing an overview of the assays associated with the 

### 9 androgen pathways and Greg Ackerman will provide an overview of 

### 10 the assays associated with the estrogen pathways. 

### 11 In evaluating the performance of assays and 

### 12 the battery, the goal of the analysis was to evaluate the extent 

### 13 of complementarity of responses within individual assays and the 

### 14 extent of concordance of responses across assays. That is the 

### 15 concept of redundancy for each of the three endocrine pathways. 

### 16 In considering the extent to which there is 

### 17 redundancy across assays, the analysis also examines whether or 

### 18 not it may be possible to reduce the number of assays and still 

### 19 adequately identify potential interactions with the E A or T 

### 20 hormonal pathways. 

### 21 As discussed during the presentation by Dr. 

### 22 Touart, in 2008 the SAP concluded that the EDSP Tier 1 assay 

### 23 served as an appropriate screen for disrupters of the E A and T 

### 24 axis and that the ability of the assays to identify endocrine 

### 25 active substances is enhanced through what was considered by the 

### 26 SAP to be adequate complementarity within assays and redundancy

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1 across assays to provide safeguards against false negatives or 

### 2 false positives. 

### 3 This analysis used the 2008 FIFRA SAP report 

### 4 is a guide in terms of evaluating endpoints within assays for 

### 5 complementarity and the consistency of responses across assays 

### 6 as indicators of potential effects on specific endocrine 

### 7 pathways. 

### 8 The concept of complementarity and redundancy 

### 9 is used in this document, in the White Paper as defined by the 

### 10 2008 SAP, complementarity describes the concordance of endpoints 

### 11 within assays and redundancy describes the concordance of 

### 12 endpoints across assays that are intended to evaluate an 

### 13 interaction in a particular E A or T pathway. 

### 14 As will be discussed in the following 

### 15 presentations for each of the hormone pathways evaluated, 

### 16 redundancy is evaluated across assays that are intended to 

### 17 provide evidence that a particular modality is potentially 

### 18 affected. 

### 19 For example six Tier 1 assays are considered 

### 20 capable of detecting chemicals with androgenic or antiandrogenic 

### 21 like activity that may disrupt the androgen hormonal signaling 

### 22 pathway, while three Tier 1 assays are capable of detecting 

### 23 chemicals that may disrupt the thyroidal hormone signaling 

### 24 pathway, and seven Tier 1 assays are capable of detecting 

### 25 chemicals with estrogenic or antiestrogenic like activity that 

### 26 may disrupt the estrogen hormonal signaling pathway.

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1 Within and across assays complementarity and 

### 2 redundancy are evaluated in the context of different modalities 

### 3 that span multiple levels of biological organizations, including 

### 4 molecular interactions and cellular organ and whole organs and 

### 5 responses for each hormonal class that is the E A or T hormonal 

### 6 systems that the battery was expected to assess. 

### 7 The 2008 SAP defined these biological levels 

### 8 or organizations consisting of receptor/ targeted binding, cell 

### 9 responses, enzyme activity, organ responses, male and female 

### 10 system integrity, developmental such as thyroid and comparative 

### 11 system integrity. As such, the battery of assays provides a 

### 12 means of integrating information across multiple levels of 

### 13 biological organization and across multiple taxa. 

### 14 As noted previously the first set of Tier 1 

### 15 test orders was issued for 67 chemicals by EPA in 2009. Fifteen 

### 16 chemicals were voluntarily cancelled or the manufacturer or 

### 17 importer agreed to cease all sales and distribution of chemicals 

### 18 in the pesticide market. 

### 19 Tier 1 assay data were actually submitted on a 

### 20 total of 52 chemicals. To facilitate timely feedback, a 

### 21 subsample consisting of 21 chemicals, which were considered to 

### 22 have adequately representative chemical space of the 52 

### 23 chemicals, were selected for this analysis. 

### 24 Of these, 18 case study chemicals were used in 

### 25 the preliminary analysis of the performance of the battery for 

### 26 the HPG estrogen hormonal pathway screening. Seventeen case

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1 study chemicals were used in the preliminary analysis of the 

### 2 performance of the battery for the HPG androgen hormonal pathway 

### 3 screening. 

### 4 Sixteen case study chemicals were used in the 

### 5 preliminary analysis performance battery of the hpt thyroid 

### 6 hormonal pathway screening. 

### 7 When positive Tier 1 findings are observed in 

### 8 the presence of overt toxicity the agency is aware there is 

### 9 uncertainty as to whether or not the responses are mediated 

### 10 through an endocrine pathway. 

### 11 The Agency, however, will evaluate the degree 

### 12 of confidence for endocrine responses that co-occur with 

### 13 systemic toxicity in future weight of evidence determination, 

### 14 and plan to do so on a case by case basis to maintain 

### 15 transparency. Cases where overt toxicity was observed are 

### 16 clearly identified in the section reviewing each assay and are 

### 17 noted in all tables including the battery review section. 

### 18 So we're ready to move into each of the 

### 19 individual data reach. 

### 20 **DR. DANIEL SCHLENK:**Okay we'll just hold 

### 21 questions at the end for everybody if that's okay. 

### 22 **CATHERINE AUBEE:**Hello again everybody. I 

### 23 will be presenting a very high level overview of the performance 

### 24 of the Tier 1 battery with respect to the detection of potential 

### 25 interactions with the HPT axis or downstream thyroid hormonal 

### 26 pathway.

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1 We tend to use those terms interchangeably 

### 2 within this presentation for better or worse. The important 

### 3 thing to note is that we currently lack truly mechanistic assays 

### 4 for identifying potential thyroid interaction in the Tier 1 

### 5 battery. 

### 6 You have seen this slide or some version of 

### 7 this table several times today -- we just love it's on the 

### 8 website. As Dr. Steeger mentioned multiple in vitro and in vivo 

### 9 assays provide more extensive complementarity and redundancy for 

### 10 detecting affects on the estrogen and androgen hormonal 

### 11 pathways, but the current battery provides much less 

### 12 complementarity and redundancy for detecting potential thyroid 

### 13 effects. 

### 14 What we do have are three fairly comprehensive 

### 15 in vivo assays which we have discussed in detail here today; 

### 16 with male and female pubertal rats and with metamorphosing 

### 17 amphibians. These work together to cast a wide net to detect 

### 18 chemical-induced perturbations which could be thyroid related. 

### 19 Within each assay multiple endpoints are use 

### 20 to screen for affects. The thyroid hormone levels including 

### 21 thyrotropin or thyroid stimulating hormones, TSH, and thyroxine, 

### 22 T4, are measured in the male and female pubertal rats. 

### 23 The 2008 FIFRA SAP recommended, "A significant 

### 24 affect of test substance on serum T4 must be considered a 

### 25 positive finding whether or not serum TSH is affected and 

### 26 whether or not the AMA is positive." That is from page 31 of

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1 their report. 

### 2 Therefore in this preliminary analysis of Tier 

### 3 1 battery performance, the agency considers a treatment related 

### 4 affect on TSH, T4 or thyroid weight or histology in the male or 

### 5 female pubertal rat to indicate a potential interaction with the 

### 6 thyroid hormonal pathway in those assays. 

### 7 The amphibian metamorphosis assay evaluates 

### 8 downstream affects on developmental biomarkers such as 

### 9 normalized hind limb length. 

### 10 These two endpoints are analyzed separately 

### 11 but are intended to be complimentary to one another within the 

### 12 AMA. They are grouped together in this presentation. 

### 13 The AMA overlaps with the pubertal rat assays 

### 14 in that it also evaluates thyroid histopathology and gross 

### 15 pathology. Although in the AMA thyroid weights are usually no 

### 16 reported. 

### 17 Whereas the Agency does not currently assign 

### 18 any particular relative weight to the thyroid related endpoints 

### 19 in the male and female pubertal rat assays, as compared to one 

### 20 another, the AMA test guidelines provides a methodical framework 

### 21 for evaluating the potential significance of responses in 

### 22 metamorphosis in frogs. 

### 23 This figure is from the test guideline and was 

### 24 developed during the international validation at the AMA. The 

### 25 decision framework is not a hard and fast rule, but it is 

### 26 proving very useful for reviewers who are evaluating data from

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1 test substances already known to be bioactive, in our case 

### 2 mostly pesticides, but with unknown potential for endocrine 

### 3 activity. 

### 4 Following this logic, significant indications 

### 5 of advanced development or the presence of a synchronous 

### 6 development are considered more diagnostic of potential thyroid 

### 7 interaction; whereas developmental delay may be considered an 

### 8 affect of generalized toxicity unless corroborated by thyroid 

### 9 histological findings. 

### 10 So what I'm going to show you now in this 

### 11 table is a very high level overview of the overall responses in 

### 12 each of the three thyroid relevant assays by case study 

### 13 chemical. You should note that at this phase of the review, 

### 14 where we are just broadly considering performance, we are not 

### 15 presenting detailed information on the direction and magnitude 

### 16 of affect of the different endpoint responses; or whether these 

### 17 responses when taken together tell a consistent story about what 

### 18 type of interaction may be occurring. 

### 19 We will definitely be considering and 

### 20 addressing those issues when we move into the more interpretive 

### 21 weight of the evidence evaluation for test chemicals. That 

### 22 again is the subject of a future meeting. 

### 23 Here we are simply showing whether there was a 

### 24 response and whether we saw responses in multiple endpoints or 

### 25 assays. That relates to the concept of complementarity and 

### 26 redundancy, which Dr. Steeger introduced earlier.

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1 In this table an X indicates a significant 

### 2 effect on one or more thyroid related endpoints in that assays. 

### 3 A blank cell indicates no effect. A footnoted "a" as you see 

### 4 here indicates that thyroid relevant effects occurred only in 

### 5 the presence of overt toxicity. 

### 6 For the amphibian metamorphosis assay where 

### 7 you see a footnote of "b" that indicates there was potential 

### 8 solvent interference in this study, and in this case this was 

### 9 the case we saw where there was a question as to whether the 

### 10 exposure levels in the study were adequate because the test 

### 11 substance appeared to be unstable. 

### 12 It's important to note that the number of Xs 

### 13 in this table should not be interpreted as the number of 

### 14 chemicals which may be thyroid active. We need to refine our 

### 15 analysis through the weight of evidence process before making 

### 16 any such determinations. There is no single clear pattern of 

### 17 responses in the assays which suggested that any one assay or 

### 18 endpoint was consistently more or less sensitive than in others. 

### 19 In the next slides I'm going to show you a 

### 20 handful of examples of the different combinations of responses 

### 21 that were seen. 

### 22 Something to note here is that all of these 

### 23 chemicals, again, have unknown endocrine potential. So we were 

### 24 not necessarily predicting any specific pattern of responses 

### 25 within the case study chemicals. 

### 26 In the first example which you see here, three

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1 of the 16 case study chemicals showed at least one endpoint 

### 2 response in all three assays. For these three chemicals, T4 was 

### 3 affected in all three cases in both male and female pubertal 

### 4 rats. 

### 5 TSH was also affected in only one case in the 

### 6 female rat. Amphibian development was affected in all three 

### 7 cases and was corroborated by effects on thyroid histology in 

### 8 two of those cases. However, effects on rat thyroid histology 

### 9 were observed only in one case in females and in the presence of 

### 10 overt toxicity. 

### 11 In the second example, three chemicals 

### 12 elicited a response in both male and female pubertal rats, but 

### 13 did not cause an effect in the frog. In one case here the 

### 14 affect in rats was observed only in the presence, again, of 

### 15 overt toxicity noted by an "a". 

### 16 And the other two endpoint responses were 

### 17 fairly consistent between male and female rats in once case, but 

### 18 differed -- in other words different endpoints were affected 

### 19 between sexes in the other case. Detectible differences in TSH 

### 20 were accompanied by changes in thyroid weight two out of three 

### 21 times in this study. 

### 22 In the third example the affects were seen in 

### 23 the frog, but not in the rat. Again this type of response was 

### 24 seen in three of 16 cases. Potential solvent interference was 

### 25 noted in two of these three cases. In one case where solvent 

### 26 affects were noted however the developmental affect was

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1 corroborated by affects on thyroid histology which were not 

### 2 present in the solvent controls. 

### 3 Here is example number four. No affects were 

### 4 seen in any of the guideline assays in these studies. For one 

### 5 of these three case study chemicals the footnote however, 

### 6 indicates that the test order requirements were satisfied by 

### 7 other scientifically relevant information. 

### 8 Interestingly in one of these submissions a 

### 9 significant effect was seen on T3, which was not a guideline 

### 10 required endpoint, however, it was not corroborated by any 

### 11 affects on T4 or TSH. 

### 12 The next example -- this is the last one, this 

### 13 is just a catchall for all the remaining four of the 16 case 

### 14 study chemicals and which responses varied across endpoints and 

### 15 assays. 

### 16 Here we have affects on T4 in female rats in 

### 17 two cases, corresponding to effects on thyroid histology on both 

### 18 female rats and in the frog in one case. 

### 19 We see effects on male TSH also corresponding 

### 20 to effects on thyroid histology in one case. And we have one 

### 21 chemical, which affected female rat thyroid weight but only in 

### 22 the presence of overt toxicity. No other effects were seen but 

### 23 there is a question as to whether exposure levels were adequate 

### 24 in this last AMA study that you see at the bottom. 

### 25 So what does all this suggests with respect to 

### 26 performance of the battery? We know that there is less

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1 complementarity and less redundancy built into the battery for 

### 2 the thyroid pathway as compared to the estrogen and androgen 

### 3 pathways. With that in mind we did not go into this review 

### 4 anticipating a certain pattern of responses from chemicals with 

### 5 unknown potential for interaction and we didn't get one. 

### 6 If anything the broad types of responses were 

### 7 distributed remarkably evenly across possible combinations. 

### 8 Some of this is expected because of inherent difference between 

### 9 the assays, the rat and amphibian assays focus on different 

### 10 portions of the thyroid pathways and there maybe be sex specific 

### 11 differences between male and female rats. 

### 12 The rat and amphibian assays also use 

### 13 different exposure methods. Tissue responses in the rats are 

### 14 more likely to incorporate effects due to first-pass metabolism. 

### 15 On the other hand tissue responses in the amphibian may reflect 

### 16 effects on peripheral tissue diodinations. 

### 17 Individual study requirements as described 

### 18 earlier in the review of Tier 1 assays also plays an important 

### 19 role in study interpretation. Given the widespread in responses 

### 20 and the general lack of complementarity and redundancy built 

### 21 into the battery for the thyroid pathway, the Agency concludes 

### 22 that it is premature to consider dropping any one of these three 

### 23 assays from the Tier 1 screen at this time. 

### 24 This conclusion is consistent with that of the 

### 25 2008 SAP which advised that all three assays, and particularly 

### 26 the amphibian metamorphosis assay, were critical to effectively

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1 screen for thyroid interaction. And that concludes my 

### 2 presentation and Amy Blankinship will be up next. 

### 3 **AMY BLANKINSHIP:**Hello again good afternoon 

### 4 and I will be discussing the evaluation of the androgen pathway 

### 5 for the EDSP Tier 1 battery performance. Again I would like to 

### 6 start by giving an overview of the presentation today. 

### 7 First I will provide a brief background on the 

### 8 assays that inform of the androgen pathways. Second I will 

### 9 discuss the concept of complementarity responses within an assay 

### 10 as well as redundancy across assays. Third I will present some 

### 11 examples of the different types of results across the assays and 

### 12 finally some concluding remarks. 

### 13 There are six assays that inform the androgen 

### 14 pathway. Three in vitro assays and three in vivo assays. The 

### 15 information on the slide is also found in table 38 of the White 

### 16 Paper. 

### 17 Seventeen chemicals were used to evaluate the 

### 18 androgen pathways Tier 1 battery performance. In regards to the 

### 19 inclusion of the in vitro aromatase and steroidogenesis assays 

### 20 and the androgen pathway evaluation, the 2008 SAP panel stated 

### 21 that androgens could also influence enzyme activities in these 

### 22 two assays and as such were including in this evaluation. 

### 23 The endpoints listed in this table encompass 

### 24 several different levels of biological organizations as well as 

### 25 the nine modalities presented by the 2008 SAP panel. 

### 26 For example the AR binding assay represents

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1 receptor or target binding while the aromatase and 

### 2 steroidogenesis assays represent enzyme activities. A change in 

### 3 the age at PPS or VO in the male and female rat pubertal assays 

### 4 represent a change at the organism level and correspond to the 

### 5 modalities of the male and female system integrity respectfully. 

### 6 Similarly an alteration in fecundity in female 

### 7 fish or nuptial tubercles in male fish would represent a 

### 8 potential alteration in female or male system integrity 

### 9 respectfully. The criteria that constitutes a response to each 

### 10 of these six assays for the androgen Tier 1 battery evaluation 

### 11 varies depending on the type of assays and what guidance is 

### 12 provided in the Agency guidelines. 

### 13 For the three in vitro assays, AR binding, 

### 14 aromatase and steroidogenesis, there is one endpoint for each of 

### 15 those assays that are used to inform the androgen pathway. In 

### 16 the analysis of the Tier 1 battery for the AR binding and 

### 17 aromatase assays a positive response is elicited when the 

### 18 chemical's activities surpasses a thresholds that is established 

### 19 in the guidelines. 

### 20 For the steroidogenesis assays, a positive 

### 21 response is obtained when the chemical's response is 

### 22 statistically different from the control. 

### 23 Additionally there are three in vivo assays 

### 24 for the androgen pathway. A Hershberger rat assay, the male rat 

### 25 pubertal assay and the fish reproduction assay. 

### 26 For the Hershberger assay, the guideline

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1 defines a response when any two of the five measured androgen 

### 2 dependent weights in the treated animals are different from the 

### 3 control. The response can be an increase or decrease in the 

### 4 tissue weights. 

### 5 In the male pubertal assay, a change in any 

### 6 one of the four endpoint categories listed in this table could 

### 7 be considered as indicative of a response for the androgen 

### 8 pathway for the purposes of the battery evaluation discussed 

### 9 here. 

### 10 And finally for the FSTRA, if a statistically 

### 11 significant difference from the control was found for one of the 

### 12 seven endpoints listed in the table, it could also be considered 

### 13 to be indicative of a potential response for the androgen 

### 14 pathway. 

### 15 As shown in the evaluations for thyroid, again 

### 16 this table shows the assay results for all 17 chemicals used to 

### 17 evaluate the androgen pathways, which is also presented at table 

### 18 39 in the White Paper. 

### 19 The Xs denotes that a response, as defined in 

### 20 the previous slide, was reported for that assay. 

### 21 Blank spaces indicate that no response was 

### 22 reported. In a few cases, equivocal results for the in vitro 

### 23 assays were also reported. An equivocal result is obtained when 

### 24 the chemicals response does not reach threshold established for 

### 25 a positive result, but is greater than the maximum allowable 

### 26 limit used to define a negative response.

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1 For the AR binding assay for Chemical C, the 

### 2 classification of untestable is defined by the guidelines as the 

### 3 test chemical not being soluble at the minimum concentration 

### 4 tested in the assay. 

### 5 For chemical T results from several Tier 1 

### 6 assays were not available as other scientific data were used to 

### 7 satisfy those assays. 

### 8 For the FSTRA for Chemical T, while there were 

### 9 not statistically significant endpoint alterations reported, 

### 10 decreasing trends were observed for several endpoints and as 

### 11 such at this time this study is classified as inconclusive. 

### 12 Additionally two other FSTRA assays results 

### 13 were deemed questionable due to potential solvent interferences 

### 14 or overall study conduct. 

### 15 In several cases for the in vivo assays a 

### 16 response was only observed in the presence of overt toxicity as 

### 17 identified in this table with an "a" or "b" superscript. 

### 18 Also please note in this table that if a 

### 19 response was observed in the Hershberger assay it was 

### 20 accompanied by a response in either the male pubertal or the 

### 21 FSTRA assays. 

### 22 Now I'd like to discuss the concept of 

### 23 complementary responses within an assay. As defined by the 2008 

### 24 SAP reports, complementarity is a concordance of endpoints 

### 25 within an assay. 

### 26 As there is only one endpoint evaluated for

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1 each of the three in vitro assays, evaluation of this concept to 

### 2 those assays is not appropriate. However, in the male pubertal 

### 3 assay there are four defined androgen responsive endpoints in 

### 4 that assay. Age at PPS, weight of androgen dependent 

### 5 reproductive tissue, histopathology of those tissues and plasma 

### 6 testosterone levels. 

### 7 As presented in the top table in the slide, 

### 8 none of the 17 chemicals elicited a response in all four 

### 9 endpoints. Additionally no alterations in the four defined 

### 10 endpoints were reported for seven chemicals. 

### 11 In the remaining chemicals two reported 

### 12 alterations in three of the four endpoints and four in three 

### 13 studies reported alterations in two or one of the four 

### 14 endpoints. 

### 15 For example, Chemical E resulted in changes in 

### 16 testosterone levels and androgen dependent organ weight for a 

### 17 total of 2 complimentary responses. 

### 18 For the Hershberger assay the endpoint is a 

### 19 change in two of the defined androgen dependent tissue weights, 

### 20 for which seven chemicals reported such a change. As a response 

### 21 for this assay is gauged by the change in tissue weights only, 

### 22 the ability to evaluate complementary responses is limited. 

### 23 In the FSTRA there are seven defined 

### 24 endpoints. Fecundity, fertilization success, secondary sex 

### 25 characteristics including nuptial tubercles, GSI, histopathology 

### 26 of gonadal tissues, plasma vitellogenin and the optional

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1 endpoint of plasma sex steroid concentration. 

### 2 None of the 17 chemicals elicited a response 

### 3 in all seven endpoints. Only one study reported no response 

### 4 whereas another study was inconclusive. Eight of the 17 studies 

### 5 reported altering three of the seven defined endpoints whereas 

### 6 additional four studies each reported alterations to five or two 

### 7 of the seven endpoints. For example, Chemical J reported 

### 8 alterations in five complimentary responses, which are listed on 

### 9 the slide. 

### 10 Now I'd like to discuss the concept of 

### 11 redundancy in modalities across the assays. Again, as defined 

### 12 by the 2008 SAP reports, redundancy is the concordance of 

### 13 endpoints across the assays within a modality that are intended 

### 14 to evaluate an interaction with a particular pathway, in this 

### 15 case the androgen pathway. 

### 16 Since the in vitro assays are specific in 

### 17 their endpoints and have different mechanisms of action for each 

### 18 assay, they have limited redundancy between them. However, 

### 19 while the AR binding assay is different from the aromatase and 

### 20 steroidogenesis assay, both the aromatase and steroidogenesis 

### 21 assays evaluate enzyme activity such as interactions with 

### 22 estradiol or testosterone, although via slightly different 

### 23 mechanisms. 

### 24 The table on this slide illustrates how for 

### 25 different chemicals modalities are conserved across the 

### 26 different assays. For some chemicals a redundancy was reported

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1 for one of the nine modalities as is shown for chemical E and G 

### 2 for alterations in organ response. While redundancies in three 

### 3 modalities were observed for Chemical S and include enzyme 

### 4 activity, organ response and male system integrity. 

### 5 Again, please be aware that some of the 

### 6 responses were only observed in the presence of potential overt 

### 7 toxicity, which are flagged here in the table. However, for the 

### 8 purposes of evaluating the battery performance, they are 

### 9 retained in this analysis. 

### 10 For chemical G, redundancy and organ response 

### 11 was reported for all three in vivo assays, which is 

### 12 characterised by a change in the androgen dependent tissue 

### 13 weights for both the Hershberger and the male pubertal assays, 

### 14 and as a change in vitellogenin in fish. For chemical J, a 

### 15 response in enzyme activity was reported for the male pubertal 

### 16 and the fish assay as well as the aromatase and steroidogenesis 

### 17 assays, which represents an alteration in sex steroid for all 

### 18 four assays. 

### 19 I'm sorry if the table is a little hard to 

### 20 read. 

### 21 Now I'm going to highlight some examples of 

### 22 the type of results reported across the six assays for the 

### 23 androgen pathway, regardless of whether their responses were 

### 24 redundant within a defined modality or that concordance was 

### 25 observed among endpoints regarding things such as direction of 

### 26 effect for a particular alteration for androgen pathway.

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1 Rather this table merely shows that some type 

### 2 of response, as defined by the table earlier, was reported for 

### 3 an assay. These results are from the larger overview table 

### 4 presented near the beginning of this talk and the footnotes are 

### 5 the same as in that table. 

### 6 In example one, these chemicals represent 

### 7 cases where there were multiple in vitro assays which were 

### 8 reported as a positive or a binder. There are three chemicals 

### 9 listed here. This list does not include chemicals where the in 

### 10 vitro results were reported only as equivocal or negative. 

### 11 In example 2 these chemicals represent cases 

### 12 where there were responses in multiple in vivo assays, but no 

### 13 positive or binding responses in the in vitro assays. 

### 14 The lack of in vitro response may provide 

### 15 insight into whether the response observed in the in vivo assays 

### 16 was mediated via an endocrine interaction or if it occurred 

### 17 along a different part of the pathway or if metabolism was 

### 18 involved. 

### 19 In both of these examples please notice that 

### 20 if a response was reported for the Hershberger assay a response 

### 21 was also reported for either the male pubertal or the FSTRA. 

### 22 In the final example, these chemicals 

### 23 represent cases where there were responses in both the in vitro 

### 24 assays and the in vivo assays. Please note that for some of the 

### 25 in vitro assays equivocal results were reported. 

### 26 In conclusion, complementary responses were

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1 observed within the in vivo assays as well as redundancy across 

### 2 the different modalities when evaluating across assays. 

### 3 However, the observation of overt toxicity 

### 4 needs to be considered when evaluating for potential endocrine 

### 5 effect. 

### 6 And finally it was observed that when a 

### 7 response was reported for the Hershberger assay it was 

### 8 accompanied by a response in the male pubertal and/or the fish 

### 9 assays. And I think we'll go to Greg Akerman. 

### 10 **DR. GREGORY AKERMAN:**Good afternoon this is 

### 11 Greg Akerman again. I will be discussing the evaluation of the 

### 12 estrogen pathway which is the final pathway in our EDSP Tier 1 

### 13 battery analysis. 

### 14 Following a similar overview is what Amy just 

### 15 presented; I will be following that similar format she used to 

### 16 describe the androgen pathway. I will first provide an overview 

### 17 of the assays and then the endpoints that were used to evaluate 

### 18 the estrogen pathway. Next I will describe the complementary 

### 19 responses within an assay and redundancy of responses across the 

### 20 assay to the E pathway. 

### 21 I will then summarize our observations by 

### 22 presenting examples of results across the assays and then 

### 23 providing a concluding statement for the E pathway battery 

### 24 performance. 

### 25 This slide shows the seven assays that are 

### 26 designed to identify potential interactions with the estrogen

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1 pathway. They're comprised of four in vitro assays, the ER 

### 2 binding, ERTA, aromatase and steroidogenesis assays. And three 

### 3 in vivo assays, uterotrophic, female pubertal and fish 

### 4 reproduction. 

### 5 Eighteen chemicals were evaluated for this 

### 6 preliminary analysis for the battery performance for the E 

### 7 pathway. The endpoints shown for each of the assays are 

### 8 categorized based on the nine modalities of the levels of 

### 9 organization as identified by the 2008 SAP. 

### 10 For example, ERTA represents cell signaling 

### 11 modality, aromatase and steroidogenesis represent enzyme 

### 12 activity modality. 

### 13 Whereas endpoints such as a change in age of 

### 14 vaginal opening in the female pubertal assay both fecundity and 

### 15 the fish reproduction assay represent a change at the organism 

### 16 level and corresponds to the modality of female integrity 

### 17 system. 

### 18 The criteria for determining a positive 

### 19 response for a particular endpoint is specific for each assay 

### 20 according to the Agency's test guidelines. The four in vitro 

### 21 assays in the uterotrophic assay each represented a single 

### 22 endpoint to inform the estrogen pathway. The ER binding, ERTA, 

### 23 aromatase assays positive response is based on the chemical's 

### 24 percent activities surpassing the specified threshold as 

### 25 established in the test guidelines. 

### 26 In the steroidogenesis assay specifically

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1 significant alterations in estradiol levels indicate a positive 

### 2 response. 

### 3 In uterotrophic assays a response is indicated 

### 4 by a specifically significant increase in uterine weight 

### 5 representing an organ response modality. 

### 6 For the female pubertal assay a change in any 

### 7 of the three endpoint categories shown in the slides indicate a 

### 8 positive response for the estrogen pathway. And finally for the 

### 9 fish a statistically significant difference in the control for 

### 10 any of the seven endpoints listed could be considered a response 

### 11 indicative of an estrogen pathway interaction. 

### 12 This slide show an overview of the results 

### 13 from the 18 chemicals evaluated from the seven assays that 

### 14 informed the estrogen pathway. This table is also presented in 

### 15 the White Paper on page 151. The Xs in the table indicate a 

### 16 response was observed as defined in the test guidelines. In 

### 17 some cases an equivocal response was reported as indicated by an 

### 18 "E". 

### 19 In other cases a response was observed only in 

### 20 the presence of overt toxicity. These responses are denoted 

### 21 with an X followed by the superscript "a" or "b" which 

### 22 corresponds to footnote at the bottom of the slide to indicate 

### 23 the type of overt toxicity that was observed. 

### 24 The superscript "c" in the fish assay for 

### 25 chemical B indicates the results were deemed questionable due to 

### 26 potential solvent interference in footnote "d" for the fish

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1 assay. 

### 2 Chemical G indicates that the study's conduct 

### 3 was questionable and the data may not be reliable. 

### 4 As recognized by the 2008 SAP, the in vitro 

### 5 assays in general have few or no complementary endpoints. 

### 6 Therefore the analysis for complementary responses within assays 

### 7 will focus on the three in vivo assays for the estrogens 

### 8 pathways. 

### 9 As described in the previous presentation, 

### 10 complementarity is defined as a concordance of endpoints within 

### 11 an assay. In the upper panel this slide shows the number of 

### 12 reported alterations in three endpoint categories; sexual 

### 13 development characteristics, weight of estrogen dependent 

### 14 reproductive organs and histopathology of the estrogen dependent 

### 15 reproductive tissues for the female pubertal assay. 

### 16 As shown here no chemicals show complimentary 

### 17 responses in all three endpoint categories in the female 

### 18 pubertal assay. Three chemicals reported complementary 

### 19 responses in two endpoint modalities and three chemicals showed 

### 20 response in a single modality. Of the 18 chemicals, 11 

### 21 chemicals showed no response. 

### 22 Note the table at the bottom of the slide that 

### 23 no chemical induced a response in the uterotrophic assay. 

### 24 This table shows the complementary responses 

### 25 observed in the fish assays. There are seven identified 

### 26 estrogen response endpoints for this assay; fecundity,

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1 fertilization success, secondary sex characteristics, 

### 2 gonado-somatic index, gonadal histopathology and plasma 

### 3 vitellogenin and the optional endpoint of plasma steroid 

### 4 concentrations. 

### 5 None of the 18 chemicals evaluated for the 

### 6 estrogen pathway showed a response at all seven endpoints. And 

### 7 one study was considered inconclusive. 

### 8 As shown in this table a majority of the 

### 9 chemicals showed a response in three or more of the estrogen 

### 10 responsive endpoints in this assay. 

### 11 In the example provided on the screen Chemical 

### 12 V showed three complementary responses for the estrogen pathway, 

### 13 those responses included fertility, secondary sex 

### 14 characteristics and fecundity. However, these effects were seen 

### 15 in the presence of potential overt toxicity in the form of 

### 16 decreased body weight. 

### 17 Next we looked at redundancy and modalities 

### 18 across the assay. As stated in previous presentations, 

### 19 redundancy is the concordance of endpoints across the assays 

### 20 within a modality that are intended to evaluate potential 

### 21 interactions with specific pathways. 

### 22 Here we look at the redundancy of responses 

### 23 across assays with the estrogen pathways. The in vitro assays 

### 24 are designed to be more mechanistic in nature and therefore have 

### 25 limited redundancy built in across the assays. 

### 26 Chemical S shows that organ response in the

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1 female pubertal assay and fish assay as well the equivocal 

### 2 response from aromatase inhibition. Chemicals M and Q show 

### 3 response in two modalities, enzyme activity and female system 

### 4 integrity in both the female pubertal and the fish assay. 

### 5 Chemical K showed a response in three 

### 6 modalities, enzyme activity in aromatase assay and both cell 

### 7 response and female system integrity responses in the female 

### 8 pubertal and fish assays. 

### 9 Next we looked at chemicals that showed 

### 10 multiple responses across the seven assays for the estrogen 

### 11 pathway. This includes results regardless of whether or not 

### 12 there was redundancy within a defined modality. These results 

### 13 are taken from the larger overview table that was presented 

### 14 earlier and did not include chemicals where only equivocal 

### 15 results were observed. 

### 16 In example 1 the top of the panel in the 

### 17 slide, there were seven cases where responses were seen in 

### 18 multiple in vitro assays. Please note that in the three cases 

### 19 where there were positive responses in the estrogen receptor 

### 20 transcriptional activation assay, there were no responses in the 

### 21 ER binding or the uterotrophic assays. All seven cases showed a 

### 22 response in the steroidogenesis and fish assays. 

### 23 Example 2, Chemical G and Q represent two 

### 24 cases where there were multiple responses in the in vivo assays 

### 25 which showed no corresponding responses in the in vitro assay. 

### 26 Note that the ERTA was considered inconclusive for Chemical Q.

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1 In Example 3 we show ten cases where the 

### 2 response was observed in in vitro and in vivo assays. None of 

### 3 these responses were in the steroidogenesis and fish assays. 

### 4 In conclusion complementary responses were 

### 5 observed within the in vivo assays and redundancy observed 

### 6 across the Tier 1 assays. The observation of overt toxicity 

### 7 needs to be considered when evaluating potential endocrine 

### 8 interactions. Finally for all chemicals evaluated for this 

### 9 analysis, there were no positive responses in the uterotrophic 

### 10 or the ER binding assays. 

### 11 **DR. TOM STEEGER:**Hi. This is Tom Steeger and 

### 12 I am going to be summarizing the information that has been 

### 13 provided throughout the day. 

### 14 The intent of this effort was to evaluate 

### 15 whether laboratories were able to consistently execute EDSP Tier 

### 16 1 battery of 11 assays, and to highlight issues that were 

### 17 identified in the conduct of those assays, particularly those 

### 18 that may impact the interpretation of the study. 

### 19 Additionally this effort aimed to evaluate 

### 20 whether there was a concordance of information that is 

### 21 complementary within assays and to evaluate whether there was a 

### 22 concordance of information that is redundancy across assays. 

### 23 The Agency is committed to reducing the number 

### 24 of animals involved in testing and toward lowering the costs 

### 25 associated with testing; recognizing that the battery of Tier 1 

### 26 tests in combination with testing already required to support

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1 chemical registrations is high in terms of both the number of 

### 2 animals used and the costs of conducting and reviewing such 

### 3 studies. 

### 4 Therefore the review of the data from the EDSP 

### 5 Tier 1 battery of assays also considered whether or not a 

### 6 modified number of assays within the battery could adequately 

### 7 ensure the identification of potential interactions with the 

### 8 estrogen, androgen or thyroid hormonal pathways. 

### 9 The Tier 1 tests required data on 52 chemicals 

### 10 and a subset of these were selected for this analysis. The 

### 11 subset of chemicals represents a range of chemical classes as 

### 12 well as a range of biological activities in terms of pesticidal 

### 13 modes of action including an inert chemical. 

### 14 As has been discussed in the preceding 

### 15 presentations with respect to the ability of laboratories to 

### 16 consistently conduct the Tier 1 battery of assays, performance 

### 17 was typically consistent across the assays in performing 

### 18 laboratories. While some performance standards were not met, 

### 19 the deviations were not considered significant and it did not 

### 20 impact the interpretation or reliability of the data. 

### 21 In general the Agency's review of the studies 

### 22 using the subset of 21 chemicals indicated that the assays were 

### 23 providing useful information for evaluating potential 

### 24 interactions with the E A and T signaling pathways. 

### 25 Based on the information provided in the 

### 26 preceding series of presentations regarding assay and battery

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1 performance, some of the issues identified in the performance of 

### 2 the in vivo assays included dose selection and overt toxicity. 

### 3 With respect to the question of whether 

### 4 individual assays provided complementary results within the 

### 5 assays and whether there was a concordance of information across 

### 6 assays, the individual assays measuring multiple parameters did 

### 7 demonstrate complementarity; that is a concordance of measuring 

### 8 endpoints within an assay. And the battery of assays identified 

### 9 for a particular pathway demonstrated a concordance of 

### 10 measurement endpoints across assays that is redundancy. 

### 11 As discussed in the preceding slide 

### 12 presentations for the estrogen and androgen pathways there were 

### 13 complementary responses for two or more endpoints within the in 

### 14 vivo pubertal and FSTRA assays. Additionally, a few of the case 

### 15 studies exhibited complementary responses in the in vivo male 

### 16 and female pubertal and/or FSTRA along with the relevant in 

### 17 vitro assays for the estrogen and androgen pathways. 

### 18 However, there were some cases where 

### 19 concordance was not observed between the in vitro and the in 

### 20 vivo assays. These situations emphasize the importance of the 

### 21 multiparameter in vivo assays and the potential differences 

### 22 between these pathways across taxa. 

### 23 With respect to the thyroid pathway the 

### 24 available assays were designed with less complementarity with a 

### 25 redundancy across assays. This emphasizes the importance of the 

### 26 in vivo amphibian and the rat pubertal assays in measuring the

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1 potential effects of the thyroid pathways. 

### 2 Overall based on review of the EDSP Tier 1 

### 3 assays and test results using the subset of chemicals, 

### 4 complementarity was demonstrated within assays and redundancy 

### 5 across assays consistent with their intended design for 

### 6 evaluating potential interactions with the E A or T signaling 

### 7 pathways across taxa. 

### 8 With respect to the question as to whether 

### 9 there may be some redundancy that could be reduced in terms of 

### 10 the number of assays required as the Tier 1 battery, the 

### 11 preceding presentations indicate the chemicals which elicited a 

### 12 response in the Hershberger assay also elicited a response in 

### 13 the male pubertal assay and/or the fish short term reproduction 

### 14 assay. 

### 15 Also, the uterotrophic assay did not show a 

### 16 response with any of the chemical subset used to evaluate the 

### 17 battery performance. The SAP is being asked to provide input 

### 18 based on the White Paper and information presented during the 

### 19 preceding presentations whether there may be an opportunity to 

### 20 reduce the number of assays while still ensuring adequate 

### 21 complementarity and redundancy. 

### 22 This concludes the presentations on the 

### 23 overall analysis of the assay and battery performance and the 

### 24 extent to which there was a concordance of information within 

### 25 assays that is complementarity and across assays, redundancy. 

### 26 Several assays within the Tier 1 battery may represent

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1 unnecessary redundancies. SAP input is being sought on the 

### 2 analysis and the extent of necessary redundancy. 

### 3 In closing I would like to emphasize what was 

### 4 stated by Dr. Manibusan during her opening presentation and that 

### 5 is that this SAP is not focused on the weight of evidence 

### 6 analysis that will be used in evaluating whether chemicals 

### 7 transition to higher tier testing. 

### 8 The Agency is aware that the magnitude and 

### 9 direction of an effect, the consistency of responses within and 

### 10 across assays, the route of exposure, bioactivation and 

### 11 metabolism, existing effects and other scientifically relevant 

### 12 information, or OSRI, all have a role in determining the extent 

### 13 to which the studies discussed during this SAP will be used in 

### 14 decision making. 

### 15 However, these aspects of the decision making 

### 16 process will be discussed during subsequent SAPs. The focus of 

### 17 this SAP is on assay and battery performance. Thank you and 

### 18 we're open for questions. 

### 19 **DR. DANIEL SCHLENK:**Any questions from the 

### 20 panel? Let me just remind the Agency and panel to direct your 

### 21 question to the specific person and then if that person can just 

### 22 state your name when the question comes in that would be great 

### 23 thanks. Dr. Green? 

### 24 **DR. SHERRIL GREEN:**I have a question for Dr. 

### 25 Steeger. This is a very broad general question but of those 21 

### 26 different chemicals that were selected from that set of the 52,

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1 were any of those chemicals already known to have effects that 

### 2 are well documented on any of the EAT pathways? 

### 3 **DR. TOM STEEGER:**The Agency has, as a policy, 

### 4 determined that prior to this point to the development of the 

### 5 EDSP testing battery that we did not have the capacity to 

### 6 identify chemicals that were acting on endocrine related 

### 7 processes. 

### 8 With that said there are certainly open 

### 9 literature studies and there are lines of evidence that will be 

### 10 used in evaluating which studies move to higher tier testing 

### 11 among the chemical domain that was used in this subset that 

### 12 could be used to support the conclusion that they may be acting 

### 13 on endocrine mediated processes. 

### 14 That may sound like a roundabout answer, but 

### 15 I'm reflecting the Agency's policies that up until this point we 

### 16 didn't have the capacity for making that determination. We are 

### 17 aware of studies and there are endpoints in our other existing 

### 18 158 studies that could be useful in making such determinations, 

### 19 though. 

### 20 **DR. SHERRIL GREEN:**I have a follow up 

### 21 question to that. So I guess right to the heart of my question 

### 22 would be how would you validate that this Tier 1 collection of 

### 23 three assays actually does predict what you think it would 

### 24 predict, and indicate that a compound should go on to Tier 2 

### 25 testing? 

### 26 **DR. TOM STEEGER:**I think that the capacity of

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1 the battery to serve as a means for detecting whether a chemical 

### 2 is acting on E A or T pathways has already been established 

### 3 through the use of known standards in prior SAPs and through 

### 4 ring testing of the battery. So the capability has already been 

### 5 established. 

### 6 The difficulty that I think that the panel is 

### 7 faced with here and one that the Agency, those of us that are 

### 8 seated here, are faced with is making sense of what the 

### 9 responses from unknown chemicals are, and patterns that you 

### 10 might expect given the 11 assays in terms of complementarity 

### 11 within an assay or redundancy across assays, do you see those 

### 12 patterns starting to be illuminated. 

### 13 We believe that based on the information that 

### 14 we've presented that indeed the individual assays are working 

### 15 together as expected, however, we're not talking about standards 

### 16 here, we're talking about unknowns. But there seems to be a 

### 17 concordance of information that will ultimately have to be 

### 18 weighed in light of other lines of evidence and that would be 

### 19 part of the weight of evidence analysis. 

### 20 **DR. DANIEL SCHLENK:**Questions? Dr. Schultz. 

### 21 **DR. TERRY SCHULTZ:**Were any of the 52 

### 22 chemicals run through any of the pre-screening components in 

### 23 your program? 

### 24 **DR. TOM STEEGER:**The answer to that is yes. 

### 25 **DR. DANIEL SCHLENK:**Dr. Horseman. 

### 26 **DR. NELSON HORSEMAN:**I believe this is

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1 probably for Dr. Steeger, maybe if somebody else wants to chime 

### 2 in. 

### 3 Am I correct in assuming that all 21 of these 

### 4 example compounds, or for that matter all 52 of the ones in the 

### 5 larger set, are chemicals that have been toxicity tested 

### 6 previously in ordinary toxicology screens, and for which there 

### 7 are safe levels defined or virtue that non-ED set of modalities. 

### 8 **DR. TOM STEEGER:**As we indicated during the 

### 9 presentation the chemicals that were selected as part of the 

### 10 subset and the 52 that were identified, our pesticide mode of 

### 11 action, one of which is an inert, and to support the pesticide 

### 12 registrations they are required under FIFRA to have a testing 

### 13 that is conducted both human health and ecological tests, a full 

### 14 suite of chemicals -- both exposure and effects data are 

### 15 required to support the registration of pesticides. 

### 16 So yes there are a battery of 158 studies that 

### 17 are required to support the registration of pesticides. 

### 18 **DR. NELSON HORSEMAN:**A follow up. Excuse me 

### 19 go ahead. 

### 20 **DR. DANIEL SCHLENK:**Actually why don't you 

### 21 follow up first and then we'll go. 

### 22 **DR. NELSON HORSEMAN:**Well the follow up to 

### 23 that would then be, given the inherent theory behind the 

### 24 endocrine disrupter mode of action, would you be interested in 

### 25 seeing tests presumably at this level rather than Tier 2 of 

### 26 these compounds at something like 2X the safety levels defined

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1 by these other toxicological tests as opposed to maximum 

### 2 tolerated dose and these sort of doses that we are seeing here, 

### 3 as a more relevant insight into their possible endocrine 

### 4 disrupter modality as opposed to the normal toxicological 

### 5 modalities. 

### 6 **DR. TOM STEEGER:**The EDSP battery of tests as 

### 7 was indicated by previous presenters, the intent is to try and 

### 8 develop the highest dose that goes up to a point where you know 

### 9 that you're starting to stress the animal, or test the animal, 

### 10 relative to the chemical itself and then back off that dose so 

### 11 that you have suitable understanding that you've tested a large 

### 12 enough concentration, but a range of doses that we feel that if 

### 13 it passes the screen we have covered both sides if we tested up 

### 14 high enough and we've tested down low enough. 

### 15 So I think that that is the general rationale 

### 16 that Dr. Touart can add to that if he wishes. 

### 17 **DR. DANIEL SCHLENK:**I believe he had some 

### 18 comments for something else as well right? 

### 19 **DR. LESLIE TOUART:**Yeah a couple of 

### 20 observations I mean just to go back a little bit. The battery 

### 21 of assays was selected based on the validation principles. And 

### 22 we were required to develop a validated set of assays to 

### 23 evaluate for endocrine modalities, so it's that context that is 

### 24 used to try to identify. Shouldn't the findings or other 

### 25 information that is within the literature be somewhat suspect 

### 26 until they are considered in light of the validated assays in

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1 context? 

### 2 They will be considered as part of weight of 

### 3 evidence considerations in terms, but in terms of how existing 

### 4 data may inform, I think that's the purpose of why we were 

### 5 mandated by Congress to establish an endocrine screening 

### 6 program, was concern that existing studies that are based on the 

### 7 toxicological endpoints and stuff we are looking at may not 

### 8 actually cover all particular endocrine activities and concerns. 

### 9 Methodologies like on the mammalian side, but 

### 10 the rat (inaudible) had been recognized early on and endocrine 

### 11 relevant endpoints added to that, and that's why that particular 

### 12 assay is already considered acceptable. We are evaluating on 

### 13 the ecological side, new methodologies that would be able to 

### 14 focus on that. 

### 15 So those are all aspects of consideration and 

### 16 with the 52 compounds that are part of this initial battery, 

### 17 they were selected based upon exposure criteria not on existing 

### 18 toxicological information, although for some of these there is 

### 19 literature that may reflect and that will be incorporated or 

### 20 considered as part of weight of evidence when the battery of 

### 21 assays are considered altogether. 

### 22 **DR. DANIEL SCHLENK:**Thank you. Any other 

### 23 questions? Dr. McManaman? 

### 24 **DR. JAMES MCMANAMAN:**So did you look at the 

### 25 concordance between your assays? Let me get specific here to 

### 26 help out the question. Is that on the thyroid assay it shows

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1 Compound A, Chemical Code A had a female pubertal effect, but on 

### 2 none of the other assays did it have a female pubertal effect. 

### 3 So if it's having an effect on one why isn't 

### 4 it having an effect on the others? So just to see how closely 

### 5 -- what we are looking at -- is Compound A the same for each of 

### 6 these assays or did you mix them up in terms of the codes on 

### 7 them? 

### 8 It just seems kind of -- if it's having an 

### 9 effect in one assay it should be having the same effect in 

### 10 another assay. 

### 11 **DR. TOM STEEGER:**The codes are not mixed up 

### 12 across these assays. The chemical codes are consistent across 

### 13 all the assays. The information that we have and that the panel 

### 14 does not have is the magnitude and direction of an effect that 

### 15 is being measured in any one of the assays. And I think that 

### 16 that needs to be considered in looking at the fact that you 

### 17 might have an effect on one in vivo assay and not on another. 

### 18 It could also represent that there might be 

### 19 differences between taxa that is one of the reasons that 

### 20 redundancy has been incorporated into the assays; if Catherine 

### 21 would like to add more on her understanding of the thyroidal 

### 22 assays? 

### 23 **CATHERINE AUBEE:**I have nothing to add. 

### 24 Thank you. 

### 25 **DR. JAMES MCMANAMAN:**So it's presumably the 

### 26 same taxa so that wouldn't be the explanation because it's the

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1 rat assay right or the pubertal? 

### 2 **DR. TOM STEEGER:**That's correct, but there 

### 3 can be differences between sex. 

### 4 **DR. JAMES MCMANAMAN:**But if it was a female 

### 5 pubertal assay, though, so the sex wouldn't be the explanation 

### 6 there either. I guess. 

### 7 **CATHERINE AUBEE:**Could you please clarify and 

### 8 can you restate your specific question about Chemical A. I'm 

### 9 not sure I understood it. 

### 10 **DR. JAMES MCMANAMAN:**Yes, so the female 

### 11 pubertal assay -- I'm asking about the female pubertal assay; it 

### 12 shows a positive result in the T but negative results for the E. 

### 13 **CATHERINE AUBEE:**So across pathways is what 

### 14 you're asking? Okay. 

### 15 **DR. JAMES MCMANAMAN:**Yes, right, yeah. 

### 16 **CATHERINE AUBEE:**Okay, well I think that Amy 

### 17 said she would like to speak to that. 

### 18 **AMY BLANKINSHIP:**If I understand your 

### 19 question correctly it's because the thyroid for the female 

### 20 pubertal has very specific endpoints like in terms of thyroid 

### 21 histopathology, TSH, T4. Whereas looking for the E or the A, 

### 22 while it's the same assay we only evaluated for like the 

### 23 estrogen sort of a change in VO or estrogen dependent weights or 

### 24 androgen dependent weights. So they won't necessarily overlap 

### 25 in terms of looking at a response for the E, the A and the T 

### 26 because they have two different endpoints they're looking for

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1 those pathways. 

### 2 **DR. JAMES MCMANAMAN:**So is it possible to try 

### 3 to identify some concordance across assays? I mean because like 

### 4 you said they're using one specific assay for E, for instance, 

### 5 and another one for the G. Is there a way to combine those two 

### 6 so that you could get a full spectrum or full view of what's 

### 7 going on with a particular compound? 

### 8 **AMY BLANKINSHIP:**They are the same single 

### 9 assay so within that single assay they are looking at VO 

### 10 estrogen dependent weights and the thyroid endpoints in that; 

### 11 they've just been broken up for this analysis of the different 

### 12 pathway. 

### 13 So as I understand it for the weight of 

### 14 evidence we would look at the totality of all the endpoints 

### 15 affected in that single female pubertal assay for that chemical 

### 16 and how it fits into each of the E the A and the T pathways for 

### 17 an interaction for that single chemical. 

### 18 **DR. JAMES MCMANAMAN:**So in other words the 

### 19 question should be held until you get the weight of evidence 

### 20 component of it? Okay. 

### 21 **DR. DANIEL SCHLENK:**Dr. Bradbury you have 

### 22 something to add? 

### 23 **DR. STEVE BRADBURY:**Just for clarification 

### 24 just in case because I was getting a little confused too. 

### 25 So in a pubertal assay, say a female rat and 

### 26 female pubertal assay, that same rat is giving information on E

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1 and T from the same animal. So it's not like three separate 

### 2 pubertal assays. It's one pubertal assay. But those rats are 

### 3 giving us male information on A and information on T; it's 

### 4 female information on E and information on T. So you could see 

### 5 a response for T because TSH changed and T4 changed, but none of 

### 6 the E related endpoints did anything. So show a negative for E 

### 7 but positive for the T. But it's the same information content 

### 8 for that rat that is giving us information on two pathways. 

### 9 **DR. JAMES MCMANAMAN:**I have a second 

### 10 question. It looks for the estrogen binding assay that none of 

### 11 the assays appear to be positive. And the same thing for the 

### 12 uterotrophic assay. So this relates to the question of whether 

### 13 you can reduce the number of animals. 

### 14 Is it your contention that none of these 

### 15 compounds would have been positive for these assays because it 

### 16 seems odd that there was no positive result in any of these 

### 17 compounds for any of these basic binding or the uterotrophic 

### 18 assays. Or are these assays just so insensitive that they would 

### 19 not have picked up? Do you feel that there is a possibility 

### 20 that these assay are so insensitive it wouldn't have picked up a 

### 21 positive response? 

### 22 **DR. TOM STEEGER:**The assays have been 

### 23 validated against known standards. So we know that they are 

### 24 capable of detecting a response if it is actually binding to the 

### 25 receptor. So I don't think that that's in question. Whether 

### 26 these chemicals are actually binding to the receptor, the data

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1 suggests that based on the assay responses that they are not. 

### 2 So the fact that you have within the domain of 

### 3 the chemical space that was used in this subsample, nothing 

### 4 that's binding to that particular -- or positive in those 

### 5 particular assays does not speak to the utility of the assay. 

### 6 It just tells us something about the chemical space in which 

### 7 we're looking. 

### 8 **DR. DANIEL SCHLENK:**Dr. McManaman? 

### 9 **DR. JAMES MCMANAMAN:**So in the universe of 

### 10 the chemicals you think you just happened to select 19 chemicals 

### 11 or 21 chemicals that just didn't have any estrogenic activity in 

### 12 terms of being able to bind to the estrogen receptor or activate 

### 13 the uterotrophic response? 

### 14 **DR. TOM STEEGER:**I think that based on the 

### 15 preliminary analysis of the assay results by themselves, without 

### 16 taking into account other scientifically relevant information 

### 17 that would be suggested of what we have. 

### 18 **DR. DANIEL SCHLENK:**Dr. Horseman? 

### 19 **DR. NELSON HORSEMAN:**With regard to this 

### 20 question on concordance -- and I believe it does relate to this 

### 21 discussion -- have you applied any sort of a logic tree to these 

### 22 concordances? In other words, for example, something showing 

### 23 agonist activity in the ERTA assay ought to be predicted to have 

### 24 a positive effect in the uterotrophic assay, absent some oddball 

### 25 metabolic circumstance or something. 

### 26 Therefore that particular concordance ought

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1 maybe to have a weight different than, you know, happenstance of 

### 2 something being positive, aromatase and ER binding or something. 

### 3 **DR. DANIEL SCHLENK:**I think we're going to 

### 4 get in that in July perhaps. 

### 5 **DR. NELSON HORSEMAN:**We probably are. So 

### 6 maybe that's just a warning shot. 

### 7 **DR. DANIEL SCHLENK:**Just not to jump the gun 

### 8 a little bit there. 

### 9 **DR. NELSON HORSEMAN:**Maybe that's a warning 

### 10 shot. 

### 11 **DR. DANIEL SCHLENK:**Let's just kind of focus 

### 12 on these particular assays here. Yeah. Any other questions? 

### 13 Dr. McManaman. 

### 14 **DR. JAMES MCMANAMAN:**Sorry I have one other 

### 15 clarification question. So for the ER binding assay, the 

### 16 equivocal assays; were they equivocal because they truly gave 

### 17 equivocal results or were they equivocal -- and I may have 

### 18 missed it when you explain it -- but were they equivocal because 

### 19 some other component of the assay failed in terms of that they 

### 20 didn't meet standards; you know, certain numbers of the samples 

### 21 didn't or the controls didn't meet the standards that you set 

### 22 for them. 

### 23 So was it a true equivocality or was it some 

### 24 other technical reason why they were given an E-value? 

### 25 **DR. TOM STEEGER:**The definition of equivocal 

### 26 for that assay is very well defined. And I will let my

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1 colleague here who is biting his lips speak. 

### 2 **DR. GREGORY AKERMAN:**They were true 

### 3 equivocal based on the response curve for those chemicals, so it 

### 4 had nothing to do with the assay. They're actually classified 

### 5 by the test value classifications. 

### 6 **DR. DANIEL SCHLENK:**Dr. Shultz. 

### 7 **DR. TERRY SCHULTZ:**I have a question to the 

### 8 chair. We can't move forward to weight of evidence, can we move 

### 9 backwards to pre-screening? 

### 10 **DR. DANIEL SCHLENK:**Well since those minutes 

### 11 have just gone out I believe I guess it's if we have enough time 

### 12 you can elaborate if you like if you have a question. I'll 

### 13 leave that up the Agency if they wish to answer that. 

### 14 **DR. TERRY SCHULTZ:**This will be a question to 

### 15 the Agency, but it comes from the fact that a number of the 

### 16 panel members have been surprised that a number of the 

### 17 chemicals, of the 51 or 21 chemicals, so few of them are ER 

### 18 binders when we feel we know a great deal about ER binding 

### 19 domain. 

### 20 And so when you start to ask questions is 

### 21 there redundancy and assays and these other things, the question 

### 22 that seems to be leading from the panel is, were the 51 

### 23 chemicals evaluated in your ER binding pre-screen so that there 

### 24 weren't any of those in there, because that would explain why 

### 25 you don't have any ER binding at all or was it just luck of the 

### 26 draw.

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1 **DR. TOM STEEGER:**The selection of the 52 

### 2 chemicals was based on exposure and not on activity. 

### 3 **DR. DANIEL SCHLENK:**Dr. Bradbury did you want 

### 4 to add something? 

### 5 **DR. STEVE BRADBURY:**I'm glad Dr. Schultz 

### 6 brought it up because I was thinking about his earlier questions 

### 7 and I'm glad we did this. 

### 8 As Dr. Steeger said, the first 67 actually 

### 9 that got test orders was based purely on an exposure metric. 

### 10 And because in part the Tier 1 assay were still under 

### 11 development, in part the expert system and related technologies 

### 12 done in previous SAPs are still a work in progress, it really, 

### 13 at that point in time back in 2005/2006 we didn't really have an 

### 14 effective base toolbox to help inform us. 

### 15 I think it's a fair point, especially as we to 

### 16 into weight of evidence, taking a look at for example would be 

### 17 what do we know about receptor binding domain, how many 

### 18 (inaudible) between two pockets, what's the Log Kow of these 

### 19 things? And I think that kind of information would be helpful 

### 20 to bring in to bear as we take a look at the in vivo responses 

### 21 and in vitro responses. 

### 22 And as we discussed in the January SAP, 

### 23 clearly as we look at the non pesticidal compounds and the inert 

### 24 ingredients, we're going to be using the physical chemical 

### 25 properties for exclusion. We're going to be looking at 

### 26 computational toxicology or expert systems to help hone in on

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1 which ones that go through testing and bringing exposure ideas 

### 2 as you all reported out in the panel. 

### 3 And even with the active ingredients, although 

### 4 we didn't go into great detail, we had that box about read 

### 5 across, which was meant as a placeholder to use other kinds of 

### 6 information to help move forward. 

### 7 Clearly the July SAP is an important step 

### 8 along that process to give us advice on how to use other kinds 

### 9 of information along with Tier 1 data. It could be 

### 10 pre-screening data; it could be post -- Tier 2-ish data to help 

### 11 inform that. 

### 12 **DR. DANIEL SCHLENK:**So it sounds like the 

### 13 entire slide will be present for our July meeting then it sounds 

### 14 like. Dr. Roby. 

### 15 DR. KATHERINE ROBY: Kind of continuing on the 

### 16 same idea, given that apparently none of the test chemicals 

### 17 under review were estrogenic and given the history behind the 

### 18 uterotrophic assay and understanding the sensitivity of that 

### 19 assay, what's the rationale behind questioning that assay within 

### 20 the battery? 

### 21 **DR. MARY MANIBUSAN:**I'm going to take a stab 

### 22 at answering your question. The question that we're posing to 

### 23 the panel is not only that of battery performance as performance 

### 24 against this subsample of chemicals that we've chosen among the 

### 25 52. It's asking a question of whether there is unnecessary 

### 26 redundancies within the battery.

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1 And with that I only want to share a point 

### 2 made in the White Paper; and that is for the uterotrophic, 

### 3 certainly they were all negative for the chemicals that we've 

### 4 chosen to evaluate. But they were never a case where the 

### 5 uterotrophic was positive and in vivos were negative. 

### 6 And so the question to the panel is, if we 

### 7 remove the uterotrophic assay what valuable information would we 

### 8 be missing? What type of information would we be missing to 

### 9 introduce additional uncertainty that we could not accept in 

### 10 order for us to answer the question of whether the chemical has 

### 11 the potential to interact with E, A and T? Understanding again, 

### 12 as we've all said, the Tier 1 battery of assays have been 

### 13 already validated against referenced compounds, both very strong 

### 14 agonist, very strong antagonist, negative and weak binders. 

### 15 So we know that they work. The question to 

### 16 the panel is for today bringing forward real world chemicals 

### 17 that have not been preselected because of their activity, but 

### 18 preselected based on exposure, based on biologic activity and 

### 19 representative of the 52 chemicals that we have, there are 

### 20 pesticide chemicals. Were challenging the Tier 1 assays and 

### 21 asking that question. Can we do better; can we do the same with 

### 22 fewer assays. 

### 23 **DR. DANIEL SCHLENK:**Any other questions? Dr. 

### 24 Horseman. 

### 25 **DR. NELSON HORSEMAN:**Just a follow up on 

### 26 this; there is the issue of savings or eliminating unnecessary

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1 redundancies of FSTRA. There is also the issue of you making 

### 2 sure that this level provides adequate screening to avoid moving 

### 3 things in the second level unnecessarily. 

### 4 So could maybe a couple of people, maybe you 

### 5 Mary, primarily, comment on the balance between those two values 

### 6 in making this particular piece of advice that we're asked to 

### 7 make. 

### 8 **DR. MARY MANIBUSAN:**I'll start. I think what 

### 9 we've been doing over the course of today is looking back at the 

### 10 foundation placed down by the EDSTAC founding members in terms 

### 11 of the purpose of the Tier 1 assays and its intention to serve 

### 12 as a screen. 

### 13 And having said that they recognize even back 

### 14 in 1998 when they had written their report, that to serve as an 

### 15 adequate screen you need to maximize sensitivity. Certainly 

### 16 we're more accepting of false positive as a screen than false 

### 17 negatives. But it also goes on to state very clearly that we 

### 18 need to balance the false positives in order for it to serve as 

### 19 an appropriate screen. 

### 20 We as an agency have always acknowledged the 

### 21 cost and price of all the required studies that we've asked for. 

### 22 I believe that was the intention behind the EDSTAC conceptual 

### 23 framework, that we not move chemicals forward for higher 

### 24 definitive, highly costly long-term studies without ensuring 

### 25 that that chemical even has a potential to interact with the 

### 26 endocrine system.

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1 So the task before this panel is not an easy 

### 2 one and that's why we're bringing it before you for 

### 3 consideration and brining this clear representative subsample 

### 4 that is intended to be robust enough to challenge the Tier 1 

### 5 assays, and demonstrate again whether or not it can perform for 

### 6 the intention of answering the question that we have before us. 

### 7 We're not answering the question of does it 

### 8 interact and what dose does it do it so we can integrate that 

### 9 into our risk assessment; because certainly we understand that 

### 10 the level of uncertainty there is a lot lower. We're looking at 

### 11 the Tier 1 again as a screen within that conceptual framework 

### 12 with the original intent of the EDSTAC members at that time, 

### 13 which I believe is still relevant. But it's not an easy task 

### 14 and we acknowledge that. 

### 15 **DR. DANIEL SCHLENK:**Dr. Green. 

### 16 **DR. SHERRIL GREEN:**This is for Mary. I think 

### 17 I have it straight now. So my earlier question about validation 

### 18 retrospectively on chemicals that are known; so as I understand 

### 19 it now each of these assays have been validated individually 

### 20 with specific chemicals that are known and that's well 

### 21 documented. 

### 22 The task for this panel is to, if we can, 

### 23 determine if we can streamline these tests so that maybe some 

### 24 would not be necessary or reasons why some must be included. 

### 25 But the July -- the next session -- would go on to develop that 

### 26 algorithm and say what if, then this.

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1 So that maybe some of the say AMA, the 

### 2 amphibian metamorphosis tests, would not be germane and wouldn't 

### 3 be part of the Tier 3 unless there is some criteria that was 

### 4 identified by the other two. Is that the sort of direction? 

### 5 **DR. STEVE BRADBURY:**I'll give it shot and 

### 6 then you can ask me to clarify again. Just to back up, each of 

### 7 the assays went through their individual assay validation other 

### 8 folks have described. And then in 2008 we came to the SAP not 

### 9 so much to ask them to redo their validation exercise that 

### 10 already had been done through OECD or other peer review 

### 11 processes. But we did ask just overall did we kind of do it 

### 12 right, assay by assay, and the answer was yes. 

### 13 The big part of that 2008 SAP was to start to 

### 14 get some feedback on this issue of how to think about redundancy 

### 15 across assays and how to look for complementary responses within 

### 16 an assay if it has multiple endpoints. And that was an 

### 17 important -- in our opinion -- an important framework that the 

### 18 SAP in 2008 provided us. And you see variations on that table 

### 19 from the 2008 SAP that was getting at, in 2008 what do you all 

### 20 think? Do you think this 11 is reasonable in terms of E, A and 

### 21 T and redundancy across assays that would look at E, look at A 

### 22 and look at T? Do you think it's reasonable in terms of assays 

### 23 that have multiple endpoints or some coherency or some 

### 24 concordance we should expect? 

### 25 And the SAP said yes. Eleven assays seem to 

### 26 be the right way to get started in terms of E, A and T, looking

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1 at what goes on within an assay and looking at what goes across 

### 2 an assay. But at that point that approach was based on 

### 3 chemicals that weren't necessarily the same across all those 

### 4 validation exercise. 

### 5 So now we've got 20 something chemicals that 

### 6 we brought to sort of test out the rationale that the SAP had in 

### 7 terms of, for this assay -- for this SAP -- when you take a look 

### 8 at assays looking E, assays looking at A and assays looking at 

### 9 T, are we seeing patterns that the 2008 SAP and we all talked 

### 10 about in 2008 and sort of seeing it play out the way it seemed 

### 11 it should play out; realizing there would be some noise in 

### 12 there, but is it sort of playing out the way you would expect. 

### 13 And what we're chewing up today is some 

### 14 initial input from you all, at least, in terms of -- you have 

### 15 the 2008 SAP that said you need 11 to get enough redundancy and 

### 16 complementariness type of word -- I'll blame you guys for that. 

### 17 18 Is it playing out the way the 2008 SAP figured 

### 19 it would, and/or is there some indication that we may be able to 

### 20 say there is more redundancy than you might need? And that's 

### 21 reflected in the charge questions we're asking you in terms of 

### 22 here is what we knew in 2008 based on the data we had in 2008, 

### 23 but it all came from chemicals designed to validate the assays. 

### 24 25 Now we're looking at 21 assays handpicked for 

### 26 any hypothesis ahead of time is the E, A or T potential. Here

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1 is what we're seeing, is the pattern of redundancy and 

### 2 complementariness playing out like in 2008? Steady as she goes 

### 3 or maybe there is some indication that we may have some 

### 4 additional redundancy in there that you could pull away but 

### 5 still not erode your ability to look at potential for E, A and 

### 6 T. That's the step we're at. 

### 7 **DR. DANIEL SCHLENK:**Mary? 

### 8 **DR. MARY MANIBUSAN:**And just to follow up on 

### 9 Dr. Bradbury's response to Dr. Green's question, the other half 

### 10 of your question deals with what we're forecasting for the July 

### 11 SAP, the weight of evidence. 

### 12 So that weight of evidence will be bringing 

### 13 forward the Tier 1 assay evaluation and integrating it with 

### 14 other scientifically relevant information so that we can answer 

### 15 the question of whether that chemical has no potential to 

### 16 interact with the endocrine system -- we can put those aside 

### 17 --or whether we need to advance that chemical because there is 

### 18 clear indication of potential interaction with the endocrine 

### 19 system, that it needs to move forward for Tier 2 testing. 

### 20 That's the critical assessment that we'll be 

### 21 bringing forward to the July SAP. It will not be revamping any 

### 22 tiered screening or testing program but more brining the 

### 23 information to bear so that we can look at it in an integrated 

### 24 fashion in a weight of evidence approach and make a decision of 

### 25 whether that chemical has an interaction with E, A or T. 

### 26 **DR. STEVE BRADBURY:**And part of the reason of

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1 breaking it out into a couple of SAPs would be, you know, if we 

### 2 had concluded the assay didn't work or looking at the assays 

### 3 together, just throwing our hands up and there is no rhyme or 

### 4 reason of what's going on. It's going on as we told you, but 

### 5 getting that kind of feedback rather than try to roll it all up 

### 6 into one period with you all. Because if we were seeing things 

### 7 that were strange with the assays, it would be kind of good to 

### 8 get some feedback from you at this stage and then carry, you 

### 9 know, insights into the weight of evidence methodology that 

### 10 we're putting together and getting some feedback on that 

### 11 methodology. 

### 12 **DR. DANIEL SCHLENK:**Thank you Dr. Bradbury. 

### 13 Yes Dr. Patino. 

### 14 **DR. REYNALDO PATINO:**I have a related 

### 15 question but going back a little bit. I think this is a 

### 16 question for Dr. Ackerman and it relates specifically for the 

### 17 binding assay. You said -- so each assay had a weak interacter 

### 18 as a reference control right? And my understanding is that 

### 19 maybe sometimes that weak compound may not perform as within the 

### 20 guidelines. Would that have affected your designation in that 

### 21 assay, the compound of interest, either a designation as 

### 22 equivocal or negative, if in the assay itself a weak binder 

### 23 cannot bind as expected. 

### 24 **DR. GREGORY AKERMAN:**Certainly it would 

### 25 have given us less confidence in those results if we saw a weak 

### 26 binding controlled chemical that didn't respond properly. I

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1 don't think that was the case in any of these examples. Again, 

### 2 we saw no corresponding response in the uterotrophic assay to 

### 3 suggest that. 

### 4 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 5 **DR. GEOFFREY SCOTT:**Just one question from me 

### 6 and sometimes I'm a little slow. When you look at your results 

### 7 where you have assays that are based upon the percent 

### 8 displacement versus those that have comparisons with the control 

### 9 -- an inherent control. We always say the best control gives 

### 10 you the best ability to discriminate whereas inherent we really 

### 11 control those variables. 

### 12 And so one of the questions I have as you look 

### 13 through these data, have you all looked at that comparison of 

### 14 those assay regardless of which part of the endocrine system 

### 15 they're looking at in terms of their performance across the 

### 16 complementarity in all of these redundancies, in terms of 

### 17 whether or not those differences in the ability to discern an 

### 18 effect may be underlying there. I'm just curious; I think some 

### 19 of these assays I expected to perform -- particularly on the in 

### 20 vitro assays -- better. So I just wanted to ask if you all had 

### 21 a look at that. 

### 22 **DR. TOM STEEGER:**Again, the individual assays 

### 23 and the full battery has already been evaluated through ring 

### 24 testing and we know that they are capable of detecting the 

### 25 chemicals. For some of those assays, even today, you're running 

### 26 internal standards and you sort of have that assurance that they

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1 are functioning as expected. 

### 2 But you're right not all the assays are 

### 3 running standard. But we have to rely on the effort that has 

### 4 gone into preparing these batteries as part of the validation 

### 5 effort as having been successful and that these assays are 

### 6 performing as expected. 

### 7 **DR. GEOFFREY SCOTT:**I just remember in the 

### 8 '97 meeting when we were looking at this whole cascading among 

### 9 the pathway, looking at that chapter and helping write that 

### 10 chapter. I just remembered we were talking about how when an 

### 11 organism integrates that may give you a more reliable estimation 

### 12 and I'm really surprised with some of the results with the 

### 13 binding assays not predicting more. 

### 14 And I'm just trying to sort that out so I 

### 15 apologize for thinking out loud but I'm trying to help you get 

### 16 some more information. So anyway I just thought I would add 

### 17 this question. I know we had a lot of discussion a long time 

### 18 ago about how this would play out. So I thank you for your 

### 19 response. 

### 20 I recognize, in fact, when I look at the 

### 21 figures in the report on the assays and look at those positive 

### 22 controls, weak controls and the negatives, I mean every one of 

### 23 those assays is ready to get at. So I'm just trying to sort 

### 24 things out. 

### 25 **DR. TOM STEEGER:**Dr. Scott we appreciate your 

### 26 input and I think you raised valid concerns. I think that the

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1 assays you need to keep in mind that they don't stand alone but 

### 2 they will be considered in the context of other scientifically 

### 3 relevant information. And again what you see giving in these 

### 4 presentations is a very high level view of the responses; 

### 5 underlying that are the components of those assays and what 

### 6 direction each one is moving in and what kind of response did 

### 7 you have across the full range of doses or concentrations that 

### 8 were tested. 

### 9 So hopefully with knowing that the battery has 

### 10 been validated and then complimented with the idea that this 

### 11 information doesn't exist in and of itself, that it's considered 

### 12 in a weight of evidence context, that we will avoid making type 

### 13 2 errors. 

### 14 **DR. DANIEL SCHLENK:**Okay. Dr. Ehrich. 

### 15 **DR. MARION EHRICH:**A very simple question on 

### 16 the chemicals for table 3. Are they all high production 

### 17 chemicals widely used? 

### 18 **DR. TOM STEEGER:**For table 3? 

### 19 **DR. MARION EHRICH:**That's the 21 you chose 

### 20 based on tubercle chemical properties and so forth; were they 

### 21 also high production chemicals, highly likely for exposure? 

### 22 **DR. TOM STEEGER:**As indicated initially, the 

### 23 chemicals that were selected for testing in the first tier of 

### 24 test orders were based on the likelihood of exposure. So the 

### 25 tests that were part of the subsamples, the chemicals that were 

### 26 part of the subsample, indicated they represent a broad range of

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1 chemical domain and many of them are pesticides and one has 

### 2 actually an inert. 

### 3 So the inert would certainly be a high 

### 4 production volume chemical. 

### 5 **DR. DANIEL SCHLENK:**Okay any other questions? 

### 6 7 Okay I think what we are going to do is I 

### 8 think we're going to call it a day for today and Dr. Dourson 

### 9 will be so kind as to make his presentation tomorrow. I didn't 

### 10 want to throw you under the bus. Most people, I think, are 

### 11 pretty much overwhelmed at this point. 

### 12 So what I'd like to do is we'll conclude the 

### 13 Agency's presentations today, but if possible in the morning if 

### 14 we can have you back at the beginning and then have any final 

### 15 comments that you may have. And then we'll conclude your 

### 16 comments at that point and then begin the public commenting 

### 17 period after that. 

### 18 So with that let me just state to the panel we 

### 19 need to meet in the room over here after a quick break. We can 

### 20 meet over here after adjournment to discuss what you need to do 

### 21 tonight. And so Fred has a couple of comments to say. 

### 22 **DR. FRED JENKINS:**If I could have everybody's 

### 23 attention really quick. I have a few quick notes for the public 

### 24 commenters for tomorrow morning. 

### 25 First I want to note that the public comment 

### 26 period is a very important time and we want to make the most out

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1 of it for everyone. So I ask number one, for the public 

### 2 commenters if you are providing a power point presentation 

### 3 please see me either right after this meeting or first thing in 

### 4 the morning no later than 8:30 so that I can make sure that I 

### 5 have enough time to load your presentations on the computer. 

### 6 Also very important note, public commenters 

### 7 please it's important that you arrive on time. We don't want 

### 8 you to miss your opportunity to provide your public comments 

### 9 because once the public comment period is closed and the panel 

### 10 starts their deliberations on the charge questions, there will 

### 11 be no more opportunity to provide public comments. So please 

### 12 make sure you're here and available to provide your comment when 

### 13 it's your time to go. 

### 14 Also, very important as well I ask that each 

### 15 public commenter please stay within your time that was allotted 

### 16 for you, that you initially requested. If you asked for 10 

### 17 minutes please don't speak any more than 10 minutes. If you 

### 18 asked for an hour or so or an hour and ten minutes to be 

### 19 specific, please don't speak any more than that particular time. 

### 20 21 We only have a couple of days left and we want 

### 22 to make sure that we get everything done that needs to happen, 

### 23 including having all the public commenters get their time and 

### 24 also make sure that we have ample time for the panel to 

### 25 deliberate on the charge questions. 

### 26 So thank you everybody here for a very

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1 successful first day and we'll see you all early tomorrow 

### 2 morning. The meeting starts at 9 am. Thank you everybody good 

### 3 bye. 

### 4 (Whereas the meeting was adjourned for the 

### 5 day) 

### 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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1 **DR. DAN SCHLENK:**Good morning everyone. 

### 2 Today is day 2 of our panel. It's entitled Scientific Review of 

### 3 the Endocrine Disruptor Screening Program, Tier 1 Assay and 

### 4 Battery Performance. 

### 5 As is customary, we'll just go around the room 

### 6 real quick to introduce the panel. Just state your name and 

### 7 your affiliation. I'll begin. My name is Dan Schlenk. I'm a 

### 8 Professor in Environmental Toxicology at University of 

### 9 California, Riverside. 

### 10 **DR. STEPHEN KLAINE:**Good morning. I'm Steve 

### 11 Klaine and I'm a Professor of Ecotoxicology at Clemson 

### 12 University. 

### 13 **DR. MARION EHRICH:**Marion Ehrich, Professor 

### 14 of Pharmacology and Toxicology at Virginia Tech where I teach 

### 15 veterinary medical students and I'm a member of the Panel. 

### 16 **DR. JAMES MCMANAMAN:**Hi, Jim McManaman, 

### 17 Professor of Reproductive Biology, University of Colorado. 

### 18 **DR. BARRY DELCLOS:**Barry Delclos. I'm with 

### 19 the FDA National Center for Toxicological Research in Arkansas. 

### 20 **DR. GEOFFREY SCOTT:**Jeff Scott, National 

### 21 Oceanic & Atmospheric Administration, Center for Coastal 

### 22 Environmental Health & Biomolecular Research in Charleston. I'm 

### 23 an Aquatic Toxicologist. 

### 24 **DR. DANIEL SELVAGE:**Dan Selvage. I'm an 

### 25 Associate Professor of Pharmacology and Toxicology at the 

### 26 University of New England and I specialize in endocrine and

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1 neuroendocrine system. 

### 2 **DR. TERRY SCHULTZ:**Terry Schultz, Emeritus 

### 3 Professor, Department of Comparative Medicine, College of 

### 4 Veterinary Medicine, the University of Tennessee. 

### 5 **DR. DONALD TILLITT:**Don Tillitt. I'm an 

### 6 Environmental Toxicologist with the Department of Interior, U.S. 

### 7 Geological Survey, Columbia, Missouri. 

### 8 DR. KATHERINE ROBY: Kathy Roby, Reproductive 

### 9 Endocrinology, University of Kansas Medical Center. 

### 10 **DR. ALDERT PIERSMA:**Aldert Piersma, Professor 

### 11 of Reproductive Toxicology, Utrecht University, and National 

### 12 Institute of Health in the Netherlands. 

### 13 **DR. REYNALDO PATINO:**Reynaldo Patino. I am 

### 14 with the USGS Texas Cooperative Fish and Wildlife Research Unit 

### 15 at Texas State University and I specialize in endocrine 

### 16 disruption in fish and amphibians. 

### 17 **DR. DEBORAH MACLATCHY:**Deborah Maclatchy, 

### 18 Professor of Biology at Wilfrid Laurier University in Waterloo, 

### 19 Ontario, Canada, and I am an Comparative Endocrinologist and 

### 20 Ecotoxicologist. 

### 21 **DR. JACK LEONARD:**I'm Jack Leonard. I'm a 

### 22 Molecular Endocrinologist, Professor of Microbiology and 

### 23 Physiological Systems at UMASS Medical School in Worcester, 

### 24 Massachusetts. 

### 25 **DR. NELSON HORSEMAN:**Nelson Horseman at the 

### 26 University of Cincinnati, a Professor in Physiology.

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1 **DR. SHERRIL GREEN:**Sherril Green. I'm a 

### 2 Professor in Comparative Medicine at Stanford University School 

### 3 of Medicine. 

### 4 **DR. DANIEL SCHLENK:**Thank you everyone. As 

### 5 mentioned yesterday, you've had a chance to sort of dwell a bit 

### 6 upon some of your questions and some of the charge questions. I 

### 7 believe the Agency has some additional information to provide 

### 8 you as well, so at this particular time, let's go ahead and, if 

### 9 you guys want to make that presentation then hold your questions 

### 10 until after that presentation for final clarifying questions at 

### 11 that point. 

### 12 **DR. CATHERINE AUBEE:**Good morning everyone, 

### 13 this is Catherine Aubee. I'm going to be following up with a 

### 14 couple of clarifying remarks in response to specific questions 

### 15 from the panel yesterday regarding aspects of the amphibian 

### 16 metamorphosis assay, specifically regarding the occurrence of 

### 17 spinal curvature or bent tail, and then also briefly touching on 

### 18 hind limb length normalization. 

### 19 Some of you requested images of what spinal 

### 20 curvature looks like in these specimens. I think it may have 

### 21 been Dr. Green yesterday. 

### 22 These are images from the unpublished Olmstead 

### 23 et al. manuscript. This is actually in Xenopus tropicalis but 

### 24 the ORD labs work with both species and we believe they're 

### 25 seeing similar types of phenomenon in both species, so I think 

### 26 this is representative.

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1 And the top one here, A, this is considered a 

### 2 mild form or essentially absent. And B, it's posterior to the 

### 3 hind limbs and considered to be a moderate curvature in this 

### 4 manuscript. And C, it's anterior to the hind limbs and much 

### 5 more severe, could possibly be retained following metamorphosis. 

### 6 So that's just to give you a quick overview of what it looks 

### 7 like. 

### 8 I want to reemphasis that the initial industry 

### 9 data that we've looked at suggests that these abnormalities are 

### 10 not treatment-related, at least in these cases. If they were 

### 11 treatment-related, we would be more concerned about whether or 

### 12 not, specifically, it is a thyroid-related affect that we're 

### 13 seeing, whether or not the curvature resolves itself through 

### 14 metamorphosis and perhaps what implications that might have for 

### 15 different amphibian species, which may or may not resorb their 

### 16 tails. 

### 17 However, the generally similar prevalence of 

### 18 spinal curvature in control and treatment tadpoles in each given 

### 19 study that we've reviewed, that's shown in the table that you 

### 20 have in the White Paper, makes this more an issue of study 

### 21 performance and not treatment effects. 

### 22 Therefore, our interest is in whether or how 

### 23 much the incidents of spinal curvature may introduce uncertainty 

### 24 into the interpretation of the AMA, with respect to screening 

### 25 for potential thyroid related affects. We also seek advice on 

### 26 what may be done to reduce the occurrence of the abnormality.

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1 I wanted to briefly summarize some of what's 

### 2 in the Olmstead et al. manuscript because we just had one 

### 3 sentence about that. I don't know if Dr. Olmstead will be 

### 4 speaking more about this today in his public comments. 

### 5 But it does show correlations between several 

### 6 factors and the prevalence of spinal curvature and the Xenopus 

### 7 tropicalis specimens, which they studied. Those factors 

### 8 included the type of diet, body size and developmental stage, 

### 9 which ORD believes are especially important, and the strain of 

### 10 the tadpole used. 

### 11 The highest are squared observed, I believe, 

### 12 for any single one of these factors was 0.66. They did not 

### 13 identify any significant differences between spawns within a 

### 14 string. One of the diets tested included sera micron 

### 15 co-formulated with other elements such as Brine Shrimp. 

### 16 However, the study did not evaluate sera micron alone, which is 

### 17 the recommended diet in the AMA test guideline. 

### 18 Our ORD colleagues have indicated that they 

### 19 have reduced the prevalence of spinal curvature in their test 

### 20 populations to less than 10 percent by careful regulation of the 

### 21 diet with respect to both formulation and amount and generally 

### 22 avoiding overfeeding. 

### 23 They emphasize the importance of also 

### 24 considering flow rate in the tank, so this is study-specific and 

### 25 an absolute amount of feeding, therefore, cannot be given in the 

### 26 guideline. That needs to be considered by the labs with respect

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1 to flow-rate in the tanks. 

### 2 However, industry submissions in which the 

### 3 sera micron feeding regime was reduced by 50 percent did not 

### 4 show a lower prevalence of spinal curvature. Therefore, simply 

### 5 reducing the amount of feed does not appear to resolve this 

### 6 problem. 

### 7 The Agency is proposing a modified diet for 

### 8 the proposed Tier 2 frog assay, which will be brought before the 

### 9 SAP next month. That information may be used to help inform the 

### 10 Tier 1 test guideline in the future. 

### 11 Ultimately, the Agency does not believe that 

### 12 this phenomenon in itself compromises the utility of the AMA. 

### 13 However, it would be beneficial to identify any circumstances 

### 14 under which a high prevalence of spinal curvature might 

### 15 compromise the performance of the study or complicate the 

### 16 interpretation of results, in other words, how much is too much. 

### 17 18 It would also be helpful to identify other 

### 19 steps which could be taken to minimize the occurrence of spinal 

### 20 curvature in different test populations; therefore, the Agency 

### 21 seeks advice on these issues. 

### 22 One other question yesterday, I think it was 

### 23 from Dr. Schultz as to whether there was a particular 

### 24 developmental window which appeared to be more sensitive to the 

### 25 development of scoliosis. In conferring with our ORD 

### 26 colleagues, they indicated that NF stage -- and I'm sorry, we're

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1 not on this one yet -- NF stage 51 to 55 appeared to be the most 

### 2 sensitive stages. 

### 3 This slide here just shows an example of the 

### 4 different normalization approaches that were considered for 

### 5 evaluating the hind limb length measurements. This is in 

### 6 response to Dr. Patino's question yesterday. And this is from 

### 7 the OECD Phase 3 validation report for the AMA. They considered 

### 8 three different normalization approaches normalizing to body 

### 9 weight, to whole body length, which is HLL-W, which you see 

### 10 here, or to snout-vent length, which is the last column HLLS. 

### 11 Generally, they determine that normalizing to 

### 12 body weight was not appropriate. Based on these results, they 

### 13 didn't necessarily see a significant difference between -- I 

### 14 don't want to say statistically significance but they didn't 

### 15 determine that one approach, either whole body length or 

### 16 snout-vent length, was better than the other. But they did 

### 17 indicated that as tadpoles approach metamorphic climax because 

### 18 of the changes in the morphometric ratios, whole body length 

### 19 would probably not be as appropriate. Therefore, normalizing to 

### 20 snout-vent length was sort of more a safe bet. 

### 21 In this one example, you see the comparisons 

### 22 from the same datasets. This is one lab. This is data 

### 23 generated with the chemical BP2. And you can see that when 

### 24 evaluating day 21 hind limb length alone, not normalized, you 

### 25 get significant differences at two treatment levels compared to 

### 26 controls. You get it in only one treatment level when

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1 normalizing to whole body length and at no treatment levels when 

### 2 normalizing the snout-vent length. 

### 3 In another dataset, you see significant 

### 4 differences at the top two treatment levels non-normalized. 

### 5 When normalizing to whole body length, you see it only at the 

### 6 top treatment level. 

### 7 I think the most noteworthy thing here is that 

### 8 for day 7, hind limb length, which we believe is really the most 

### 9 useful time point for this measurement, you identify significant 

### 10 difference at four treatment levels, whereas you only identified 

### 11 them in two when using the non-normalized endpoint. However, at 

### 12 day 21, again, you only detect it at the top treatment level. 

### 13 The bottom line from the OECD Phase 3 

### 14 validation report was that they felt the snout-vent length 

### 15 normalization approach was probably the best way to go. They 

### 16 did believe it was appropriate to normalize for growth in some 

### 17 manner, but they also recognized that it would probably be 

### 18 useful to reevaluate that normalization approach once more data 

### 19 were available, and so this is just a little bit more 

### 20 information for you on that point. 

### 21 And that concludes my remarks. 

### 22 **DR. DANIEL SCHLENK:**Thank you. Any 

### 23 questions? Yes, Dr. Green? 

### 24 **DR. SHERRIL GREEN:**Thank you for the images. 

### 25 I have a question. Would the water quality analysis that I 

### 26 assume would be performed weekly from the various contract labs

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1 be available to the Agency and the Panel members? Meaning that, 

### 2 particularly, in the group with the 92 percent occurrence of 

### 3 bent tail, it would be really important to look at that water 

### 4 quality, everything from ammonia to temperature to pH. All 

### 5 those things can be impactful on the development of this kind of 

### 6 finding. 

### 7 **DR. CATHERINE AUBEE:**So those data are 

### 8 available to the Agency at this point and we have considered 

### 9 those in the review of the data. Generally, all of the studies 

### 10 met performance criteria for water quality so they were at least 

### 11 within those limits. 

### 12 Now, within that limit, we have not evaluated 

### 13 to see if there are potentially differences between studies 

### 14 related to the differences in the spinal curvature. Are there 

### 15 specific measurements from, for example, the highest prevalent 

### 16 study that would be useful to you? 

### 17 **DR. SHERRIL GREEN:**Most of what I know about 

### 18 in bent tail has been reported anecdotally. So as you can 

### 19 imagine, it would be the place I would start, with the group I 

### 20 would start because the number is so aberrant from any other 

### 21 population. 

### 22 And it might be other conditions related to 

### 23 water quality; the type of housing, the flow-thru, ambient 

### 24 temperature in the room or something that's used to clean things 

### 25 with. You know they are all other things that could impact 

### 26 that.

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1 So nothing specific, to answer your question, 

### 2 but if there was an outlier in a particular parameter, it would 

### 3 be useful to see that. And also understanding the methods in 

### 4 which the various components of the water chemistry were 

### 5 determined. Were they tank-side dip sticks or were they 

### 6 spectrophotometric? Because the accuracy and the difference 

### 7 there, depending on the test that you use, can be pretty wide 

### 8 and some are more accurate than others. 

### 9 So understanding how they measured what they 

### 10 measured would be useful too. 

### 11 **DR. DANIEL SCHLENK:**Any other questions on 

### 12 this particular point or any other final questions or 

### 13 clarification that you have for the Agency? 

### 14 Okay. So at this point, I guess we'll 

### 15 conclude the information presented by the Agency and move on to 

### 16 our public commenting period. 

### 17 And let me remind the public commenters and 

### 18 any speaker, for that matter, please to speak into the 

### 19 microphone so that we can hear you properly. 

### 20 Okay. According to my list, our first speaker 

### 21 is Michael Dourson from Toxicology Excellence for Risk 

### 22 Assessment. And I'll just remind the Panel that handouts for 

### 23 Dr. Dourson's presentation were provided yesterday, it's the 

### 24 blue one. It looks like this. 

### 25 **DR. MICHAEL DOURSON:**Top of the morning to 

### 26 you all. I thank Dr. Jenkins and Schlenk for the invitation to

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1 present before the esteemed Panel. My name is Mike Dourson and 

### 2 I am a Board Certified Toxicologist and an expert in risk 

### 3 assessment. I'm not an expert in your area and it would 

### 4 probably be apparent if you ask me questions. 

### 5 TERA is an NGO, 501(c)(3) neutral science 

### 6 group and our mission is to provide information so others can 

### 7 protect public health. 

### 8 Two-thirds of our work is government, 

### 9 one-third is industry and we like to build coalitions and TERA 

### 10 is supporting my trip. And the comments that you're going to 

### 11 get are from a workshop that we've done but there are TERA 

### 12 comments on it. 

### 13 About eight months ago, I was approached by 

### 14 Daland Juberg and Susan Borghoff -- Dr. Juberg was with Dow 

### 15 AgroSciences and Susan Borghoff with Integrated Lab Systems -- 

### 16 with the idea of putting together a workshop. And when we first 

### 17 heard about the workshop, we knew EPA was doing some effort in 

### 18 this area so we thought, well why do we need to do this? And 

### 19 what they wanted to do very much was to bring all this industry 

### 20 information and experience to bear into one spot so we can share 

### 21 experiences and then give that information to anybody who needed 

### 22 it, including the EPA. 

### 23 So we thought okay, that's fine. That makes 

### 24 sense. And they wanted to very much make it open, and so we 

### 25 agreed to the task and it was going to be lots of people 

### 26 invited. And we also asked them to consider working the task

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1 through the Alliance for Risk Assessment, which as some of you 

### 2 may know, it's a consortium of other nonprofits that put things 

### 3 together. 

### 4 And the operating thing here is through an 

### 5 open transparent multi-stakeholder approach. So if you go into 

### 6 the Alliance for Risk Assessment, you have to be open and 

### 7 transparent and everything is available to everybody else. And 

### 8 there is a group that looks at these incoming projects with that 

### 9 in mind. Is it going to be open and transparent and everybody 

### 10 can work together. And you can see the group up here, mostly 

### 11 government people, travel representative. 

### 12 And so this group doesn't actually do the work 

### 13 but they do past judgment and say, "Hey, this is worthy of going 

### 14 into the Alliance and the work can proceed along those lines." 

### 15 And so it did. It went to the Alliance and you'll be able to 

### 16 find anything that happened at this conference at the Alliance 

### 17 website. 

### 18 Okay, now into the technical information, here 

### 19 is the organizing committee. One of the things to be obvious 

### 20 with this organizing committee is that it's a multi group. We 

### 21 have some nonprofits in here, we have some industry, and we have 

### 22 some government. And the intent, again, is to bring as many 

### 23 parties to the table as possible that wants to participate. 

### 24 And a couple of things that were obvious, when 

### 25 I finally understood this area a little bit more, was that the 

### 26 focus is giving back lessons learned at the workshop. And part

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1 of the reason for this is that the Tier 1 studies are costly. 

### 2 You know, if you run all of them, the rough cost is somewhere at 

### 3 a million dollars. I mean the people that actually do them will 

### 4 know. I don't. And it's costly in terms of animals, and so 

### 5 people wanted to not step forward in an unintelligent way. 

### 6 But there was another thing behind it. It was 

### 7 a lot of experience gained here and the scientists that actually 

### 8 did the studies, wanted to bring that and share it with people. 

### 9 And quite independent of their own group, they wanted to be able 

### 10 to say, hey, we did this and here is the successes we had, or 

### 11 not. And of course, challenges and lessons learned, this is 

### 12 what you're group's all about. It does this Tier 1 information 

### 13 give EPA what it needs. 

### 14 So workshop objectives, I mean this is 

### 15 probably to be true of any workshop; provide knowledge, identify 

### 16 best practices, discuss insights, interpreting data, explore 

### 17 biological mechanisms, engage perspectives. So that was the 

### 18 workshop objectives. 

### 19 And the workshop was designed into four 

### 20 blocks. And the first block was on performance and that's a lot 

### 21 of what you folks are talking about now, and the EPA has 

### 22 summarized nicely some data that's come from this industry and 

### 23 others' experiences. EPA summarized data on 21 or so chemicals, 

### 24 22 chemicals, and of course the industry brought information in 

### 25 for all of their chemicals and other people, not just industry. 

### 26

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1 And not only were these presentations given, 

### 2 but there were 20 abstracts given where there's a lot of 

### 3 detailed information at the workshop. And you can see that 

### 4 three people spoke at the workshop. We actually have two or 

### 5 maybe all three of these people here today, so again, if there 

### 6 are questions that are detailed, I'll just probably defer those 

### 7 to one of you. 

### 8 I think that was important from my perspective 

### 9 is some of the perspectives. And some of the perspectives are, 

### 10 first of all, Tier 1 is not designed for dose response. As Dr. 

### 11 Manibusan said yesterday, it really is hazard only. 

### 12 There were several technical difficulties 

### 13 identified in the in vitro assays. Meeting reference chemical 

### 14 performance was one of the highlighted ones. Laboratory 

### 15 constraints for ER and AR competitive binding assays was another 

### 16 technical difficulty with some of these in vitro assays. 

### 17 Meeting performance criteria for steroidogenesis was another 

### 18 technical difficulty that several people mentioned. 

### 19 On the in vivo assays, many of the people that 

### 20 were doing the tests had to do additional pilot studies, and 

### 21 therefore additional animals, in order to maximum the 

### 22 sensitivity as required under the criteria. So there were more 

### 23 animals used than intended and that's because of this idea of 

### 24 maximizing sensitivity. 

### 25 And a theme that came up routinely -- now 

### 26 realize we're only talking hazard identification -- but this

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1 idea of testing at such high concentrations; why do this? If 

### 2 the exposures are low and the potency is low such that the 

### 3 margin of exposure at the risk term is ten thousand, why are we 

### 4 doing these studies? Now again, we were doing it for hazard 

### 5 identification, but this kept coming back, you know somehow get 

### 6 in the idea of exposure and potency. 

### 7 Session two, practical applications. Again, 

### 8 you can see the cast of characters here. Dr. Gray is not here, 

### 9 I don't believe, but Dr. Marty and Dr. Borgert are here and they 

### 10 actually may be speaking later today. I actually don't know 

### 11 that, but in any event, those are the speakers. 

### 12 Here are some perspectives. Development of 

### 13 critical systematic evaluation; again, Tier 2. Tier 2 now is 

### 14 even more expensive than Tier 1, especially in terms of animals 

### 15 and money. There was a couple of posters that showed where -- 

### 16 there was some angst in this meeting. Again, when you bring all 

### 17 these people together, there are differences of opinion. 

### 18 We had some posters where it showed lots of 

### 19 testing data for these 11 bioassays and no effects. And the 

### 20 poster presenter said, "Well we told EPA to expect this. Why? 

### 21 Because we have tested this chemical with multiple million 

### 22 dollars of studies and we know there are no endocrine effects. 

### 23 But we did this anyhow and see, you know, we found what we told 

### 24 them we'd find." So there was that tension in the room. So the 

### 25 idea of going to Tier 2 where there is even more money and 

### 26 animals spent, needs to be done in a systematic way.

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1 One of the interesting ideas that came out was 

### 2 a Tier 1.5 screening strategy. Well what's Tier 1.5? Well it's 

### 3 some additional tests that you do before Tier 2, and you do that 

### 4 because the current scheme strives to avoid false negatives, 

### 5 which of course maximizes false positives. So do you want to 

### 6 really launch into Tier 2 with false positives? The answer is, 

### 7 well, maybe Tier 1.5. 

### 8 Another thing that came up was this two 

### 9 gatekeeper bioassays, Tier 0.5. It was kind of a nice 

### 10 presentation. Actually I was quite taken by it. This is Dr. 

### 11 Earl Gray his colleagues' presentation. 

### 12 Basically, you'd say two gatekeeper bioassays, 

### 13 the fish short-term reproduction study, the male rat pubertal. 

### 14 You do these two studies, and if they're both negative, you put 

### 15 the chemical in a hold testing box. You don't stop it. You 

### 16 just say, "Well we're going to put this in a hold and go to 

### 17 something else." 

### 18 And then finally the assessment application of 

### 19 alternate data, highly relevant, it replaced the Tier 1 assays. 

### 20 Now part of this is this high-throughput screening data. And 

### 21 the fact that Dr. Dix is now part of the OSCP is remarkable. I 

### 22 mean I didn't know about this but he comes from leading that 

### 23 group, this computational toxicology group, so I think that's 

### 24 going to be very important. 

### 25 Session three was the future, and we had all 

### 26 sorts of perspectives here, including David Dix, he gave a

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1 presentation. Mel Anderson and some of these people you know. 

### 2 We have Catherine Willett. She's in the audience as well, so 

### 3 again we have one or two of these people here today. So if we 

### 4 have questions about that that would be great. 

### 5 The perspectives here -- well again, the 

### 6 high-throughput screenings and compu-toxicology and In silico 

### 7 assays don't have the in vivo characteristics, we all know that. 

### 8 9 So if it doesn't have metabolism, cell-to-cell 

### 10 communication or tissue interactions; so that we have to think 

### 11 about validating these things before we actually use them. This 

### 12 idea of considering human and ecological exposure and especially 

### 13 this idea of exposure is valuable in prioritizing -- I actually 

### 14 didn't know until Dr. Manibusan's presentation yesterday, that 

### 15 EPA has already done this exposure cut. That's really 

### 16 important. 

### 17 Health Canada did their domestic substances 

### 18 list a couple of years back where they did 23,000 chemicals. 

### 19 They screened them on exposure. And then after they screened 

### 20 them on exposure, then they went to hazard. So it looks like 

### 21 EPA is doing something similar, and we need to emphasis that, 

### 22 you know, screen on exposure first. 

### 23 This idea of current collaborations focused on 

### 24 improving -- you know, we have human and ecological 

### 25 biomonitoring now. And if we can biomonitor the chemical and 

### 26 compare it to the critical effects point of departure -- that's

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1 another risk term. So the critical affect is a sentinel adverse 

### 2 effect and its point of departure. 

### 3 If we can match those two then we can start to 

### 4 winnow out on the basis of exposure. We don't have to do that 

### 5 chemical. Why? Because we biomonitored it in the human 

### 6 population and you can't even hardly even find it. And its 

### 7 point of departure -- it's not very potent so its point of 

### 8 departure is very, very high, so maybe that's a way to screen. 

### 9 And then this idea came up more than once, 

### 10 this idea of collaboration, coordination, communication amongst 

### 11 regulatory community. Well this is one reason TERA is here, we 

### 12 want to communicate the results of this. 

### 13 And several times people said, "You know we 

### 14 need to pause and reflect on current findings." Well of course, 

### 15 I mean we need to do this and this is what you folks are doing. 

### 16 We're pausing and we're reflecting on current findings. And I'm 

### 17 glad to be able to bring this to you folks as part of your 

### 18 deliberations along that line. 

### 19 We had a final session which was like "Hey, 

### 20 anybody can grab the microphone and add or subtract from what 

### 21 they heard." And a number of things came up; this idea of 

### 22 critical review and update to the guidelines for the conduct of 

### 23 individual assays. Boy, there was a lot said there, and again, 

### 24 your input on these various assays are important. 

### 25 Biomonitoring and exposure-based 

### 26 prioritization. If we know the existing human eco-exposures, we

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1 need to use that information. We test chemicals that have small 

### 2 margins of exposure and we do that first. You maybe even 

### 3 prioritize it. If you could, small margins of exposure, we're 

### 4 on it. Large margins of exposures, to the back of the line. 

### 5 Reduction in the number of animals in testing 

### 6 used; redesign the studies to avoid pilot studies. If we could 

### 7 do that, that would be great. Consider the gatekeeper bioassays 

### 8 that Dr. Earl Gray and his colleagues suggested. Hey, that 

### 9 might work, that's just one way. 

### 10 This idea of robust weight of evidence 

### 11 approach using potency exposure; again, the potency and exposure 

### 12 comes up -- I don't need to tell you folks this but dihydrogen 

### 13 monoxide is a neurotoxin, and yet its exposure, in order to 

### 14 cause neurotoxin, you need a lot of water. So therefore, why 

### 15 worry about that. And that's what this whole idea of potency 

### 16 exposure is, it keeps coming back. Again, it's this 

### 17 hazard-based system so we have to integrate that desire with the 

### 18 fact that it's hazard-based. 

### 19 And finally, I think we have -- this is my own 

### 20 perspective. I really was taken with this two-assay gatekeeper 

### 21 approach, maybe in part because Dr. Gray is such an entertaining 

### 22 speaker. But it makes a lot of sense to look at these two 

### 23 studies first, and your thoughts on that would be highly 

### 24 valuable to EPA as they struggle with this. 

### 25 From my point of view, from a risk 

### 26 perspective, it seem to benefit all stakeholders that were in

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1 the audience at this workshop, and those who were not. We did 

### 2 not have many environmental NGO colleagues there. But my sense 

### 3 is the gatekeeper approach would be valuable to them as well. 

### 4 Okay. So the summary was insight of 

### 5 practitioners and labs, so it was a lot of good information 

### 6 there, multiple stakeholders. And I think the most important 

### 7 thing here is all the presentations and workshop materials are 

### 8 available at the workshop website. 

### 9 And the final slide is the sponsors we had, a 

### 10 bunch of sponsors here including nine NGOs. 

### 11 So with that, I will thank you very much for 

### 12 your time and attention and maybe take one or two questions and 

### 13 we also have a four-page summary of the workshop that just came 

### 14 out a couple of days ago, so you guys get to see it first. 

### 15 Thank you. 

### 16 **DR. DANIEL SCHLENK:**Thank you. Any questions 

### 17 from the Panel? Okay. 

### 18 **DR. MICHAEL DOURSON:**All right, very good. 

### 19 **DR. DANIEL SCHLENK:**Okay. Our next 

### 20 presentation will be a series of presentations which will be 

### 21 from the Endocrine Policy Forum. And according to my list here, 

### 22 our first speaker is going to be Ellen Mihaich. And you have a 

### 23 host of materials, I believe, that I believe are in order of the 

### 24 speakers, or they're not? 

### 25 **DR. ELLEN MIHAICH:**They should be in order. 

### 26 Good morning. I'm Ellen Mihaich and I just have one quick

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1 slide. I'll be back later, but I just wanted to let you know 

### 2 what the next little bit is about. So these next presentations, 

### 3 nine or so, are from the Endocrine Policy Forum. The Endocrine 

### 4 Policy Forum is a consortium of more than 95 percent of the Tier 

### 5 1 test order holders. So we're the ones that gave you all the 

### 6 data after EPA did it for you, but we're the ones that generated 

### 7 this data. 

### 8 This group is the test order recipients and 

### 9 other interested stakeholders. And we, as I said, are 

### 10 responsible for all of the testing of the majority of the List 1 

### 11 chemicals. 

### 12 So I just wanted to set the stage so that you 

### 13 knew we're going to be going through each one of the screens and 

### 14 then have some overarching issues. And this is the culmination 

### 15 of 50 plus million dollars and a whole lot of years of 

### 16 experience and work. And as Mike Dourson just talked about, we 

### 17 pulled this together about a month ago and you're going to get 

### 18 it in an hour and 10 minutes, so good luck. 

### 19 **DR. DANIEL SCHLENK:**Thanks for the warning. 

### 20 Okay. Our first speaker is Steve Levine. 

### 21 **DR. STEVE LEVINE:**Good morning everybody. 

### 22 I'd also like to thank Mr. Jenkins and Dan for allowing me to 

### 23 provide comments this morning. I'm going to provide comments on 

### 24 three of the in vitro assays, the ER binding and the AR binding 

### 25 and the estrogen receptor transcriptional activation assay and 

### 26 I'll call it the ERTA assay as it was referred to yesterday.

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1 Again, I'm Steve Levine. I'm a Senior Science 

### 2 Fellow at Monsanto and I lead our ecotoxicology function. This 

### 3 is an area that I've been following for about a decade. I've 

### 4 had the opportunity to participate on several review committees 

### 5 of these in vitro assays. I've participated on the EDMVAC, 

### 6 which you heard about yesterday, between 2004 and 2006. I've 

### 7 also had the opportunity to participate in OECD expert groups 

### 8 and validation review committees, and most recently on an NIH 

### 9 review committee, looked at the BG1 ERTA assay. 

### 10 This is a quick overview of what I'm going to 

### 11 cover in the next five minutes or so; comments on AR and ER, 

### 12 approaches and challenges to the ERTA HeLa-assay, advantages of 

### 13 the new BG1 assay, which is functionally equivalent to the 

### 14 HeLa-assay but the methodology has several advantages, and the 

### 15 EPA is to be commended for allowing us to use that improved 

### 16 methodology for the ERTA assay; talk about ERTA assay 

### 17 performance in the context of the White Paper as well as the 

### 18 battery, comments on cell line assays, specifically with impacts 

### 19 of cytotoxicity and then finish up with a few closing remarks. 

### 20 So here are some general comments on the ER 

### 21 and the AR binding assays. Currently the assays are very large 

### 22 and cumbersome because they require many test-tubes with 

### 23 cytosolic preparations which require a large number of animals 

### 24 to be sacrificed. And currently we have advanced recombinant 

### 25 technology which can be used to replace this in a 

### 26 high-throughput format, and those are advances we should look

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1 for in the future. 

### 2 Some of the issues with these assays center 

### 3 around solubility issues at high concentrations. And in my 

### 4 opinion, the assay should be limited to a maximum concentration 

### 5 of 100 micromolar rather than 1000 micromolar. And in fact, 

### 6 many of the validations only went up to 100 micromolar, not a 

### 7 1000 micromolar. Those were added later during test guideline 

### 8 preparation. 

### 9 There is a desire across the testing 

### 10 facilities to harmonize both the AR and ER assays from a 

### 11 methodology standpoint as well as from a test guideline 

### 12 standpoint. 

### 13 The AR guideline seems more like an SOP and it 

### 14 really reflects Bill Kelsis (phonetic) SOP from the ER binding 

### 15 assay. The ER is more consistent with the modern test guideline 

### 16 providing guidance and is not as prescriptive. 

### 17 The screens generally appear to be 

### 18 cyto-specific but some compounds can result in false positives, 

### 19 and this is again because of the high concentrations that are 

### 20 required at the top end of the dose range where the assays and 

### 21 some compounds seem to crash. 

### 22 These assays could eventually be replaced with 

### 23 reliable and validated transcriptional activation assays. Those 

### 24 are functionally equivalent and have the advantage of telling 

### 25 the difference between agonist as well as antagonist. However, 

### 26 there's some watch-outs with those assays which we'll talk more

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1 about on the coming slides. 

### 2 And finally, we heard of EPA's SAR models back 

### 3 in January. And a SAR model was brought to the SAP back in 

### 4 2009, which is very predictive for potential estrogenic 

### 5 activity, and that should be used as a Tier zero screen as well 

### 6 as OSRI or Other Scientifically Relevant Information. 

### 7 This is the ERTA model and I'm not going to go 

### 8 into this, but I just showed this. Again, this is looking at 

### 9 more than just receptor ligand binding. Again, you can look at 

### 10 agonism versus antagonism. 

### 11 A brief slide on ERTA methodology. One of the 

### 12 issues with methodology in the HeLa-test guideline is the cells 

### 13 are treated only three hours after plating, and that's not a 

### 14 common practice. In the BG1, they're allowed to go overnight 

### 15 before treatment. This shows up as the inability of the cell 

### 16 line only three hours after plating to tolerate levels of DMSO 

### 17 beyond .1 percent. If they go overnight and they're allowed to 

### 18 recover, you can achieve higher DMSO levels up to one percent 

### 19 and that allows you to reach some of those higher test 

### 20 concentrations. 

### 21 Activity and cytotoxicity are assessed after 

### 22 24 hours and I'll have some more comments on cytotoxicity. 

### 23 The other key issue with the assay is the 

### 24 criterion for positive, which is the PC10 value, and we heard 

### 25 about that yesterday in the presentation, what the PC10 value 

### 26 is.

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1 This is a look at some of the validation data. 

### 2 These are the E2 curves from the validation and we can see 

### 3 that we're only able to achieve approximately 4X to 16X at very 

### 4 high concentrations of E2, the highest concentration being one 

### 5 nanomolar. 

### 6 The PC10 represents 10 percent of the maximal 

### 7 induction level. And even at the highest induction level at 

### 8 16X, that's only 1.6X. And typically with assays with these 

### 9 levels of inherent variability, experience in many labs, they 

### 10 don't see these as biologically significant until you're over a 

### 11 2-fold response and, you know, this improvement could be 

### 12 achieved by reaching higher levels of induction. 

### 13 The OECD validation review Panel had several 

### 14 reservations about the reliability of the PC10 value as a 

### 15 criterion for a positive. And in fact, in the OECD validation 

### 16 review, it was stated that if the PC10 value were to be used as 

### 17 an indicator of a positive response, it may lead to 

### 18 identification, a lot of false positives when testing a broad 

### 19 array of environmental compounds. And this was born out in the 

### 20 White Paper, and I'll speak more about that in a moment. 

### 21 ICCVAM, which made a publication in the late 

### 22 90s, in Appendix B7 it stated "An efficacy of 25 percent of the 

### 23 positive control should be considered as a positive response for 

### 24 the test chemical in that assay." So they're really looking at 

### 25 about a PC25. The V2 is the positive response. 

### 26 Consistent with that ICCVAM recommendation,

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1 the BG1 ERTA assay settled on a PC20 as the endpoint for a 

### 2 positive response. That was based on empirical information and 

### 3 that was what led to a reliable response and limited the number 

### 4 of false positives that could potentially come out of this 

### 5 screening assay. 

### 6 Assay Performance; overall, in my opinion 

### 7 there was poor concordance between the ER binding and the ERTA 

### 8 results using the PC10 as a criterion for a positive. In the 

### 9 ERTA assay there were five positives out of the 19 compounds 

### 10 evaluated. However, there were zero for the ER binding assay 

### 11 and also zero for the uterotrophic assay, and those are all 

### 12 mechanistically functionally equivalent assays. However, with 

### 13 the nanomole model you do get metabolism or transformation. 

### 14 In the BG1 assay, concordance between the ER 

### 15 binding assay and the ERTA assay was greater than 95 percent in 

### 16 their evaluation. And there was also very high concordance 

### 17 between receptor binding assay, the ERTA assay and the 

### 18 uterotrophic, and we would expect that for most compounds. 

### 19 And in fact, if you pull out some of the ones 

### 20 that were positive in the ERTA assay between the PC10 and the 

### 21 PC20, it gets you very close to 95 percent concordance, which is 

### 22 what you predict based on these earlier analysis, for the HeLa 

### 23 validation and the BG1 validation. 

### 24 Some comments on cytotoxicity, ICCVAM also 

### 25 made a recommendation on cytotox and it shouldn't be greater 

### 26 than 10 percent. And this is very important for all cell line

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1 assays, not only the ERTA assay. This test guideline permits up 

### 2 to 20 percent cytotoxicity, which is high, particularly if the 

### 3 assay is used to assess antagonism. We're not using it for that 

### 4 now but it certainly could be used for that in the future. 

### 5 And the reason for that is, if you have up to 

### 6 20 percent cytotoxicity you could have 20 percent inhibition of 

### 7 a response just due to cell death and that could flag as a 

### 8 positive. And the guidelines right now don't allow for 

### 9 correction of cytotoxicity, and they should. 

### 10 Cytotoxicity assessment should be quantitative 

### 11 rather than just purely visual ratings; that's an option in the 

### 12 BG1 just to do a categorical rating of cytotox. But in my 

### 13 opinion it really needs to be quantitative so you can correct 

### 14 for cytotox, particular if it's pushing near 20 percent. 

### 15 So a few closing remarks; a retrospective 

### 16 analysis is needed for studies using the PC10 value rather than 

### 17 the PC20 value in the weight of evidence evaluations for 

### 18 compounds judged to be positive with a value greater than a PC10 

### 19 but less than a PC20. 

### 20 And three of those five values, as I said, hit 

### 21 in that range. And one of those five is also considered to be 

### 22 likely a false positive. That gets you down to one out of 20, 

### 23 95 percent concordance. That gets you back to what has been 

### 24 looked at in the validations under HeLa and BG1. 

### 25 Further consideration needs to be given to 

### 26 cytotox and shouldn't be greater than 10 percent, in my opinion.

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1 And in any case, there should be a correction allowed for 

### 2 cytotoxicity, particularly when you're looking at an antagonism, 

### 3 and this will come up again in the steroidogenesis assay, H295R 

### 4 cell line assay. 

### 5 So with that, I'll close my comments. And if 

### 6 there's any questions, please. Thank you. 

### 7 **DR. DANIEL SCHLENK:**Thanks. Any questions 

### 8 for Dr. Levine? 

### 9 Okay, thanks. Moving along to our next 

### 10 presenter, this will be Sue Yi. 

### 11 **DR. SUE YI:**Good morning. My name is Sue Yi 

### 12 and I'm a Senior Toxicologist at Syngenta Crop Protection 

### 13 speaking on behalf of the EPF. I'd like to take this 

### 14 opportunity to thank you for the opportunity to speak, and what 

### 15 I'll do is just go through the Steroidogenesis and the Aromatase 

### 16 Assay. 

### 17 So just a quick overview. So mainly what I 

### 18 will focus on is the cytotoxicity issue, issues with solubility 

### 19 of the test compounds at high concentrations and the suitability 

### 20 of the assays themselves. 

### 21 So in terms of the steroidogenesis assay, I 

### 22 won't go into the details of the protocol, but one of the issues 

### 23 is the performance criteria, and that was discussed by EPA quite 

### 24 a bit. 

### 25 In these cell lines, the H295R cells, there's 

### 26 actually quite a bit of variability in cell behavior, and that

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1 is the hormone production at basal level. 

### 2 So in many of the cases, assays had to be 

### 3 repeated to get to or get close to the performance criteria, so 

### 4 there's quite a bit of variability. So in that respect, this 

### 5 decreases the usefulness of this assay. Not only does it have 

### 6 to be repeated many times to meet the criteria, so it's no 

### 7 longer rapid, nor is it actually cost-effective because it has 

### 8 to be repeated, but most importantly, this decreases the 

### 9 confidence of the data that's generated. 

### 10 There is also a 24-hour treatment period which 

### 11 is a bit arbitrary time point. As we've shown in several 

### 12 different publications that this is really an assumption that 

### 13 there is an increase or a decrease in the hormones that are 

### 14 being tested that that will be sustained. What we have is that 

### 15 there could be a time course effect in which maximal induction 

### 16 or inhibition could occur at 48 or 72 hours, but then decrease 

### 17 back to basal level. So it could be an adaptive or a quick 

### 18 response that returns to normal. 

### 19 And the other aspect that confounds the 

### 20 performance criteria is the method of analysis for the estrogen 

### 21 and the testosterone. So the gold standard for these analysis 

### 22 actually are LC/MS/MS method. RIAs are the second method of 

### 23 choice, and then ELISAs or EIAs, and that's because they're 

### 24 actually not quite as reliable. 

### 25 The reason being is that the different assay 

### 26 kits from different companies and within companies can be highly

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1 variable. These antibody assays are actually, especially at low 

### 2 concentrations of estradiol and testosterone, are plagued with 

### 3 non-specific binding cross-reactivity and matrix interference. 

### 4 So to increase the confidence if you're using 

### 5 an EIA immunoassay approach, the estradiol or the testosterone 

### 6 should be separated by the chromatography before running these 

### 7 analyses. And there's some general references to the 

### 8 validations and the experiments that were done for the 

### 9 cross-comparison of these assays. 

### 10 So I'll go back to the cytotoxicity issue. 

### 11 This actually has a major effect; it can have a major effect on 

### 12 hormone production. What we know is that estrogens have been 

### 13 demonstrated to be up-regulated to protect the cells in cases of 

### 14 cytotoxicity, especially oxidative stress mediated cytotoxicity. 

### 15 The solubility of test chemicals as well as 

### 16 the hydroxycholesterol which is used to bump up the basal 

### 17 production of E2 and testosterone, have cytotoxic and solubility 

### 18 issues. 

### 19 And a visual assessment of solubility is not 

### 20 quite scientific because it can actually be missed. Test 

### 21 compounds could precipitate out at actually very low 

### 22 concentrations even at about one micromolar that you can miss 

### 23 because of the volume at which you're using these assays. 

### 24 So I'll now move on to the aromatase assay. 

### 25 And some general comments about the aromatase assays is that the 

### 26 aromatase assay is only just looking at one half of the

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1 aromatase activity, so inhibition is the only assessment that is 

### 2 being made so induction is not even considered in this assay. 

### 3 It is a measure of tritiated water rather than 

### 4 estrone or estradiol production, so you could have nonspecific 

### 5 affects at high concentrations. 

### 6 The inhibition of aromatase assay can be 

### 7 actually assessed in something like the steroidogenic assay or 

### 8 other cell-based assays which can also detect induction of this 

### 9 enzyme. There is an issue of some of the solvent control and 

### 10 this sort of relates to the solubility. And it's 

### 11 test-tube-based so therefore it's actually quite labor intensive 

### 12 and probably can be miniaturized dramatically to a multi-well 

### 13 format and up to about 96 well. 

### 14 The aromatase assay uses the recombinant 

### 15 variant; a single recombinant variant of the CYP19 gene, the 

### 16 aromatase gene. So there's actually high variability of CYP19 

### 17 across tissue and across species. 

### 18 The result is that the aromatase activity that 

### 19 is being assessed may not be a true reflection of what happens 

### 20 in vivo. And in addition, because it is a single variant, it 

### 21 does not address individual or population variances. 

### 22 So there are issues in terms of the high 

### 23 concentration insolubility. There are issues with the enzyme 

### 24 denaturation that leads to false positives. There is also the 

### 25 lack of xenobiotic metabolizing capability, and so you are not 

### 26 assessing for what happens when these compounds are actually

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1 metabolized in vivo. 

### 2 There is some question about the longevity of 

### 3 the activity of the enzyme preparation because specific activity 

### 4 of those enzymes actually decrease with time and dependent on 

### 5 temperature, as well. So in this assay, protein concentration 

### 6 is the assessment, as opposed to the specific activity of the 

### 7 enzyme itself. And I've already touched upon the tritiated 

### 8 water being the measured endpoint rather than the estrone 

### 9 itself. 

### 10 So just as a wrap-up, what I'd like for you to 

### 11 take away from this presentation really is the key issues which 

### 12 are the cytotoxicity issues. So this confounds the 

### 13 interpretation of the data. And in vivo we know that 

### 14 cytotoxicity or some type of an insult to the system that could 

### 15 lead to cell death will actually induce aromatase or increase 

### 16 production of estradiol because estradiol is involved in 

### 17 protection of cells, especially in systems like the neurons 

### 18 and/or the cardiomyocytes. 

### 19 The solubility of the test compound at high 

### 20 concentrations, again, this is really important because we're 

### 21 using a solvent to go beyond the limit of solubility. And this 

### 22 is something that was touched upon by the EPA when discussing 

### 23 the AMA or the FSTRA. But it's interesting that in vitro and/or 

### 24 in vivo that we don't talk about the limit of solubility. And 

### 25 then the necessity of these assays, whether the aromatase assay 

### 26 is actually absolutely necessary or can we actually fold that

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1 into the steroidogenesis assay. Thank you. 

### 2 **DR. DANIEL SCHLENK:**Any questions? Dr. 

### 3 Schultz got one over there. 

### 4 **DR. TERRY SCHULTZ:**This is the second 

### 5 presentation we have heard the issue of cytotoxicity, yet one of 

### 6 the criticisms that we've had at earlier SAPs was the fact that 

### 7 we weren't taking the concentrations high enough. 

### 8 How do you rectify the issue of covering the 

### 9 issue of water solubility for a particular chemical and the 

### 10 tradeoff that you end up having in the fact that you had the 

### 11 potential of becoming near or inducing the cytotoxicity issue? 

### 12 **DR. SUE YI:**Right. So there are concerns 

### 13 about going up to extremely high concentrations in order to 

### 14 determine an effect of lets say estrogen binding or 

### 15 steroidogenesis. 

### 16 One of the things that do not get discussed 

### 17 actually is the solubility and the exposure at which the 

### 18 population, whether it's eco or human population get exposed to. 

### 19 And so it makes sense actually to take a look at the exposure, 

### 20 the limit of solubility, in order to determine what the highest 

### 21 concentration should be tested for that particular compound. 

### 22 **DR. DANIEL SCHLENK:**Okay, thanks. Any other 

### 23 questions? Okay, thank you. Our next presentation will be by 

### 24 Barbara Neal. 

### 25 **MS. BARBARA NEAL:**Good morning. My name is 

### 26 Barbara Neal. I'm a Toxicologist with ExPonent and I'm pleased

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1 to present comments on behalf of the EPF, which is the Endocrine 

### 2 Policy Forum. 

### 3 I want to briefly provide comments on the 

### 4 technical performance and reliability of the Uterotrophic and 

### 5 Hershberger assays and also comment on how these assays may add 

### 6 to the Tier 1 test battery either as a primary element or as 

### 7 secondary phased in elements. 

### 8 I think it's very clear from the literature 

### 9 that the Uterotrophic assay is well validated. It's 

### 10 scientifically sound. It's easy to interpret, the agonist 

### 11 option in particular reliably detects estrogenic agents, and 

### 12 both the ovariectomized mode and immature model appears 

### 13 reliable. 

### 14 There are a few difficulties with the assay. 

### 15 Range-finding studies are generally required for dose selection. 

### 16 And because the assay has a very short exposure duration, it's 

### 17 difficult to determine -- in any case, sometimes the MTD 

### 18 criteria are hard to meet clearly. 

### 19 Other issues; sometimes the external suppliers 

### 20 don't provide age of animals and the dates for ovariectomy. And 

### 21 in some cases we encountered incomplete ovariectomies. 

### 22 With immature model the primary limitation is 

### 23 the animal must be pre-pubertal. But EPA in its review 

### 24 identified no difficulties that were considered a compromise, 

### 25 the assay interpretation of reliability. 

### 26 Positive control data met the criteria in all

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1 cases, and I think the primary question stemmed on the doses of 

### 2 the 17 alpha-ethinylestradiol, or EE, where excessive doses 

### 3 could lead to non-dose related responses. 

### 4 Essentially, though, it is a gold standard 

### 5 assay, I think it's premature before establishing a weight of 

### 6 evidence strategy to remove it from Tier 1 and make that 

### 7 decision at this point. And I think that it can reliably help 

### 8 distinguish compounds that interact via the estrogen receptor 

### 9 from compounds that may cause sporadic or non-specific findings, 

### 10 for example fecundity in the fish study or in published 

### 11 literature in OSRI, in which cases group sizes may be small. 

### 12 And in either case, I think the utility may be greatest when 

### 13 there is consideration that the testing may have been done at 

### 14 overtly toxic doses. 

### 15 And I think the main animal welfare 

### 16 consideration is if the data are available some reproductive 

### 17 toxicity studies incorporate weanling uterine weights. And 

### 18 assuming the necropsies are done prior to puberty, these can be 

### 19 used in lieu of the uterotrophic assay and mimic the immature 

### 20 model. 

### 21 The Hershberger assay is, again, a reliable 

### 22 assay. I think it can be confounded by overt toxicity, as will 

### 23 be discussed for the pubertal assay. Marked decreases in body 

### 24 weights can lead to changes in some of the accessory sex organ 

### 25 weights. And it can also be confounded by hepatic enzyme 

### 26 inducers in Phase 2 of the assay, and that's where exogenous

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1 testosterone is added and you've got an increased clearance with 

### 2 enzyme induction. 

### 3 I think the assay guideline is complex as 

### 4 written. I work with several sophisticated contract labs and 

### 5 two of the three had real problems interpreting the protocol or 

### 6 developing a protocol based on the guideline. 

### 7 Again, the dose range-finding study is 

### 8 generally necessary. And even though hormonal analysis is an 

### 9 option in this assay, I think the group size of six per group is 

### 10 too small to reliably develop hormone data. 

### 11 Again, it's the subject of artifacts from very 

### 12 high doses and multivariate analysis which is recommended in the 

### 13 guideline may lead to an increased number of false positives, 

### 14 and that's based on the analyses of the validation studies. 

### 15 That assay is a gold standard assay. It's 

### 16 relatively specific for identifying androgenic, anti-androgenic 

### 17 or the five alpha hydroxide reductase mechanisms. And again, it 

### 18 provides additional information on activity and mode of action 

### 19 for compounds that show sporadic or less specific indications, 

### 20 and it may be particularly useful again in cases where there's 

### 21 high dose suggestions in the applicable assays. 

### 22 So I think it's premature, again, before 

### 23 developing the weight of evidence strategy and analyzing all of 

### 24 the data available to say we should remove the assay. I do 

### 25 think we could consider removing the androgenic phase or Tiering 

### 26 that later if there's an interaction with AR binding, since

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1 androgenic compounds seems to be very rare. And that concludes 

### 2 my comments. 

### 3 **DR. DANIEL SCHLENK:**Thank you. Any questions 

### 4 from the Panel? Okay, thanks a lot. Our next presenter will be 

### 5 Sue Marty. 

### 6 **DR. SUE MARTY:**Good morning. My name is Sue 

### 7 Marty. I'm a Toxicologist with the Dow Chemical Company. What 

### 8 I'd like to do is present some observations as a practitioner of 

### 9 the Tier 1 EDSP mammalian assays specifically related to the 

### 10 male and female pubertal assays. 

### 11 First I'd like to talk about the performance 

### 12 criteria. I understand and perfectly agree with the Agency's 

### 13 decision to include performance criteria on the pubertal assays. 

### 14 They're multimodal assays so they're designed to detect a 

### 15 variety of endocrine activity. Because of that, it's difficult 

### 16 to select one positive control, so this gives you a way to look 

### 17 at assay performance. 

### 18 I will say though that when you're looking at 

### 19 performance criteria coefficient of variation or CVs, I think 

### 20 it's important to remember that there are two contributors. Not 

### 21 only variance in assay performance, what we call proficiency, 

### 22 but also inherent biological variability. 

### 23 And based upon the Tier 1 data that were 

### 24 submitted, I think there's an opportunity to adjust some of the 

### 25 performance criteria that are listed in the test guidelines. 

### 26 So for instance, in the female pubertal assay,

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1 what we noted -- you can see some of the performance criteria 

### 2 that were commonly missed. What we noticed was that terminal 

### 3 body weight had CVs above the acceptable range. Now this is a 

### 4 body weight collected on an animal at a specific age. If we're 

### 5 missing performance criteria it suggests that maybe we didn't 

### 6 account for sufficiently biological variability. 

### 7 And we see the same thing when we look at the 

### 8 male pubertal assay. There's 32 performance criteria that are 

### 9 included in the assay. This list most commonly missed 

### 10 parameters for CVs outside the performance criteria on the left 

### 11 or means outside the performance criteria on the right. And 

### 12 again we see that we're missing CVs for things like final body 

### 13 weight. Again this suggests that maybe there is insufficiently 

### 14 accounted for the inherent biological variability, not that it's 

### 15 proficiency of a laboratory to collect the body weight. 

### 16 You'll note that there are footnotes for a 

### 17 number of the parameters in this table. These were parameters 

### 18 for which the performance criteria were missed in the 

### 19 inter-laboratory validation of the pubertal male assay. So this 

### 20 suggests that, in fact, over time, labs continue to miss the 

### 21 same performance criteria across labs. 

### 22 This is just a personal observation. I'm not 

### 23 sure about the accuracy of all the performance criteria that 

### 24 were reported in the white paper. I can only refer to the 

### 25 subset of data that came from my laboratory, but I noted that 

### 26 there were omissions in our datasets from the CVs for both the

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1 male and the female pubertal sections of the document. They're 

### 2 probably minor but, in fact, there were some omissions. 

### 3 Overall for lab performance criteria, I think 

### 4 there's an opportunity for the Agency to consider whether or not 

### 5 they should re-examine those criteria. And I think it would be 

### 6 great if they could give us some additional guidance on how 

### 7 those criteria are going to be used. 

### 8 This is just a quick slide on logistics for 

### 9 conducting the male and female pubertal assay related to the 

### 10 money, resources and time that are needed to do these assays. 

### 11 And I just wanted to call your attention to the second bullet 

### 12 point which is the animal use that's required for these assays. 

### 13 14 We're required to have 15 litters to conduct 

### 15 each the male and female pubertal assay. We try to conduct them 

### 16 together whenever possible. But the 15 litters are to control 

### 17 for litter effect, which is scientifically valid. However, what 

### 18 happens is that we actually are euthanizing more animals without 

### 19 any data collections, so 135 animals versus only 90 that we need 

### 20 to run the assay, so actually euthanizing more animals than 

### 21 we're using for data collection. 

### 22 So I was a little bit disappointed in the 

### 23 White Paper because I was hoping that it would discuss more of 

### 24 the issues that we had related to the pubertal assays. For 

### 25 instance, there's something on hormone measurement. The 

### 26 document acknowledges that changes in hormone measurements in

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1 the absence of any corresponding changes in other assay 

### 2 endpoints are meaningful, and this is a little bit problematic 

### 3 from the perspective that the hormone measurements are highly 

### 4 variable. They represent a value at a single point in time and 

### 5 they're subject to rapid stress-related changes. So these are 

### 6 sensitive assays. If hormone changes are sustained, I would 

### 7 expect to see changes in other endpoints over the 20- to 30-day 

### 8 dosing period. So it would've been helpful for me to see some 

### 9 more discussion about the Agency's thinking behind this point. 

### 10 For statistical analyses, we're required to 

### 11 analyze some data points three different ways, ANOVA, ANCOVA and 

### 12 linear trend test. This increases the potential for type 1 

### 13 error rate, so it would've been great if we had some 

### 14 understanding as to which of the statistical analyses are 

### 15 preferred and why. 

### 16 I would've liked to have seen some discussion 

### 17 on endpoint whose utility is unclear, and for this I'll highly 

### 18 the estrous cycle data. So estrous cycle can be highly variable 

### 19 in animals at its initiation. And we all have a limited 

### 20 monitoring period in the female pubertal assay for this 

### 21 endpoint, so it would be great if we had more description from 

### 22 the Agency on how they intend to use these data. 

### 23 In the pre-validation study, there was one 

### 24 pre-validation lab that failed to detect regular cycles in 12 

### 25 out of 14 control females in the pubertal assay. So with such a 

### 26 variable endpoint, it would be great to understand what the

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1 utility is in the eyes of the Agency. 

### 2 Someone asked yesterday whether or not diet 

### 3 can affect female pubertal assay endpoints, and it can. Dietary 

### 4 phytoestrogen requirement from a test guideline is actually 300 

### 5 micrograms per gram genistein equivalent. We're required to be 

### 6 below that level and most of our standard rodent diets are 

### 7 slightly above that level. 

### 8 So because of that most labs use low 

### 9 phytoestrogen diets. And in actuality, that means that the age 

### 10 at puberty onset vaginal opening in the females is actually 

### 11 about two days later than what we see in our multi gem studies, 

### 12 so it does have an impact. 

### 13 Complementarity across assays, this is the 

### 14 male and female pubertal assays. If you remove the studies for 

### 15 systemic toxicity, what we see is that approximately 50 percent 

### 16 of the assays are finding some indication of endocrine activity. 

### 17 Now the pubertal assays detect both endocrine and non-endocrine 

### 18 modes of action that are not seen in other Tier 1 assays, so if 

### 19 you'll offer differences in species, that could be meaningful, 

### 20 differences in gender, that may be meaningful. This can be very 

### 21 difficult to dismiss any positive findings in pubertal assays. 

### 22 I'm not going to go into this too much but 

### 23 this addresses a section in the report that talked about the 

### 24 aromatase assay contributing to understanding of androgen 

### 25 pathways. I would like to see a little bit more information on 

### 26 this because what we know is two of the mammalian in vivo assays

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1 that look at the androgen pathway, both the Hershberger and the 

### 2 pubertal male, they don't reliably detect aromatase inhibitors, 

### 3 so I would like to see a little more discussion on that. 

### 4 Our redundancy across assays, this is when you 

### 5 combine all the assay data and you're looking for concordance 

### 6 among the endpoints. Again, when you take out systemic 

### 7 toxicity, what we're seeing is 50 to 80 percent of the chemicals 

### 8 showing an indication of endocrine activity in either the 

### 9 estrogen, androgen or thyroid pathways. 

### 10 For me, the report lacked in sufficient detail 

### 11 for me to put any of this information into context. I can't 

### 12 tell whether or not these changes fit a pattern of responses 

### 13 that I would expect to see across assays. So for example, in 

### 14 the steroidogenesis assay, we had compounds that at higher 

### 15 concentrations would increase estrogen production in the H295R 

### 16 cells, but I never saw any indication of estrogenicity in any of 

### 17 the female pubertal assays. So for me, that kind of information 

### 18 is important to understand when I'm looking at assay 

### 19 performance. 

### 20 So in conclusion, the male and female pubertal 

### 21 assays are highly sensitive to detect potential endocrine 

### 22 activity. We see a high proportion of positives, but they're 

### 23 reliance on apical endpoints makes it difficult to determine the 

### 24 mode of action for endocrine related findings. The assays 

### 25 detect compounds that interact with estrogen or androgen 

### 26 receptor, affects steroidogenesis, alter the

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1 hypothalamic-pituitary-gonadal or thyroid axis, induce hepatic 

### 2 enzymes and enhance thyroxin or steroid hormone clearance, but 

### 3 they also detect nonspecific effects due to systemic toxicity 

### 4 and stress. Variability in some of the assay endpoints like 

### 5 hormone measurements and estrous cyclicity can complicate data 

### 6 interpretation. 

### 7 From my perspective, the specificity and 

### 8 toxicological relevance of these assays is somewhat unclear. 

### 9 And if gender and species differences are meaningful, it's going 

### 10 to be difficult to dismiss a Tier 1 pubertal assay positive 

### 11 because the assays detect different modes of action, both 

### 12 endocrine and non endocrine that are not looked at by other Tier 

### 13 1 assays. Thank you. 

### 14 **DR. DANIEL SCHLENK:**Any questions? Okay, 

### 15 thanks. The next presentation is by John Brausch. 

### 16 **DR. JOHN BRAUSCH:**Thank you very much. Good 

### 17 morning. My name is John Brausch. I'm an Aquatic 

### 18 Ecotoxicologist at the BASF Corporation. Today I'll be 

### 19 presenting the Amphibian Metamorphic Assay on behalf of the 

### 20 Endocrine Policy Forum. 

### 21 As we heard yesterday, there are a number of 

### 22 performance criteria with the amphibian metamorphic assay. One 

### 23 of the criterion that was most difficult to maintain was on test 

### 24 concentrations. Discussions with both the performing 

### 25 laboratories as well as in the EPA White Paper, this was common 

### 26 throughout in that only seven of 18 studies actually maintained

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1 the appropriate test concentration. 

### 2 It's important to note that these are 

### 3 mean-measured concentrations and the performance criteria 

### 4 indicate that you need to maintain less than 20 percent 

### 5 variation throughout the entire study. But it's important that 

### 6 the concentration is known throughout the study and is monitored 

### 7 throughout the entire 21-day assay. So what the organisms are 

### 8 actually being exposed to is in fact well known, it's just that 

### 9 it varies slightly or it varies from the nominal concentration. 

### 10 What the nominal concentration is set at is virtually 

### 11 irrelevant; it's what the organism is actually exposed to. 

### 12 The compounds that had the most difficulty in 

### 13 maintaining these test concentrations where the lipophilic or 

### 14 the highly biodegradable compounds. The tadpoles grow about 

### 15 10-fold during this 21-day study, so you have basically a 

### 16 10-fold increase in biomass from the beginning to the end, which 

### 17 really makes maintaining these concentrations very difficult. 

### 18 The next criteria that is difficult or had 

### 19 difficulty with the AMA is a synchrony among tadpoles. We heard 

### 20 yesterday that some tadpoles in the controls were exceeding 

### 21 stage 60. Stage 60 is important because this is where the 

### 22 tadpoles go from pro-metamorphosis to metamorphic climax. 

### 23 A number of things happened during metamorphic 

### 24 climax such as tail resorption and apoptosis, as well as major 

### 25 changes within the tadpole itself causing these indices to 

### 26 change very dramatically. One thing that happens is the

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1 tadpoles tend to start to lose weight after stage 60, therefore, 

### 2 making changes in comparisons between the controls after stage 

### 3 60 and the tadpoles that are before stage 60 very difficult. 

### 4 In 16 of 18 studies that were reviewed in the 

### 5 White Paper, controlled tadpoles exceeded stage 60, and actually 

### 6 four of these studies had greater than 20 percent of the 

### 7 controls exceeding the stage making additional statistical 

### 8 evaluation needed and very difficult. 

### 9 As we've heard, there's been a number of 

### 10 complications in dose setting. Dose setting was very difficult 

### 11 in the AMA mostly because there's a lack of historical data on 

### 12 amphibians for many of these compounds. 

### 13 The guidance indicated that a third of the 

### 14 LC50 or the maximum tolerated concentration should be used as 

### 15 its highest concentration. When using this concentration, 12 of 

### 16 18 studies showed overt toxicity at the highest concentration. 

### 17 One thing that was used in some studies was 

### 18 that a 10th of this LC50 was used as the highest concentration. 

### 19 This would still allow for thyroid affects to be observed, but 

### 20 also minimize the amount of overt toxicity. 

### 21 The AMA is actually the most animal-intense 

### 22 study in the 11-study battery. As many as 16,000 tadpoles were 

### 23 produced when only 320 tadpoles are actually needed for the 

### 24 study. This is additionally an expensive study costing upwards 

### 25 of $100,000, and it's a fairly long study for a screening assay. 

### 26

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1 So when you look at the whole battery, there's 

### 2 the three studies that can identify thyroid affects, the male 

### 3 and female pubertal as well as the AMA. 12 of the 16 studies 

### 4 reviewed showed and demonstrated thyroid activity, and seven of 

### 5 the 16 demonstrated thyroid activity in the AMA. 

### 6 There's only actually one instance where the 

### 7 AMA suggested thyroid activity that was not observed in the 

### 8 pubertal rat. Yesterday we heard there were three. And while 

### 9 three is true, the other two studies also had significant 

### 10 solvent affects. So without all the data, it's very hard to 

### 11 determine where this "thyroid activity" would have occurred, 

### 12 whether it be from a solvent or from the actual compound itself. 

### 13 This data is actually supported by a study 

### 14 from 2010 by Pickford et al. that was commissioned by the OECD 

### 15 in which he actually has in his abstract. There is little 

### 16 convincing evidence that an amphibian screening assay would 

### 17 detect significant numbers of thyroid active compounds that are 

### 18 not picked up in the mammalian assays. In fact, we saw five 

### 19 pubertal studies observe thyroid activity when the AMA did not, 

### 20 but this could also be due to nonspecific overt toxicity, but 

### 21 with the data that's provided, we are unsure. 

### 22 There are a number of strengths with the AMA. 

### 23 First it's important to know that histopathology was by far the 

### 24 most sensitive endpoint and very specific for thyroid activity. 

### 25 We saw the decision tree yesterday presented and it only 

### 26 indicates that the histopathology should be done for thyroid

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1 antagonist and not thyroid agonist. However, there is evidence 

### 2 that thyroid agonist can be observed in histopathology through 

### 3 what appears to be thyroid atrophy. 

### 4 Other strengths of the AMA is that it's the 

### 5 only assay that actually examines a developing thyroid versus a 

### 6 developed thyroid, and it examines in ecological receptor in the 

### 7 frog. It can also incorporate many different routes of exposure 

### 8 which is very important to try to get a full picture of 

### 9 potential thyroid activity. 

### 10 There are a number of limitations of the AMA. 

### 11 Most of these are actually going to be covered in the next 

### 12 couple of presentations you hear, so I will just gloss over 

### 13 those very quickly. 

### 14 Thyroid histopathology, although one of the 

### 15 benefits of the AMA, it also is currently a limitation as well 

### 16 because it is very important to have stage matched thyroids. 

### 17 The stage matched thyroids are actually very difficult to get 

### 18 because you have the lack of synchrony among the control as well 

### 19 as the exposed organisms. 

### 20 We've heard a bit about the normalize hind 

### 21 limb length, also this statistical analysis of the stage 60 

### 22 tadpoles; it makes statistical analysis extremely difficult. 

### 23 And then we've heard earlier today some more about the spinal 

### 24 curvature. 

### 25 There are a number of different alternatives 

### 26 to the AMA and we can talk about this for a long time. But if

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1 you refer to the public comments, there's about 18 pages for the 

### 2 amphibian metamorphic assay and all three of these are discussed 

### 3 much further. 

### 4 One alternative would be to use a stage fixed 

### 5 design, so basically saying that we would continue the assay 

### 6 until the tadpoles reach a certain stage, for example, stage 58 

### 7 versus this time fix. At 21 days, you terminate the assay 

### 8 regardless of where you're at. This is actually similar to the 

### 9 proposed Tier 2 LAGDA study in which is it more based on a stage 

### 10 versus a time. This would also assist in stage matching the 

### 11 thyroids and having much greater strength in the histopathology. 

### 12 Two other options would be to look at an 8-day 

### 13 amphibian assay, which was proposed in 2010, and this actually 

### 14 focuses primarily on biochemical endpoints, also a 14-day 

### 15 metamorphic assay similar to what EPA initially recommended 

### 16 could also solve some of the problems that we observed in the 

### 17 21-day assay. 

### 18 As currently designed, the AMA did not 

### 19 significantly add to the battery. The rat pubertal identified 

### 20 six or seven compounds that indicated thyroid activity. We say 

### 21 that it did not significantly add to the battery but it's as 

### 22 stated currently in the guidelines. A number of modifications 

### 23 could be made. It could really increase its utility. This 

### 24 could eliminate some of the growth issues that we've discussed 

### 25 or the statistical issues. It can also increase the power for 

### 26 the histology and have a little bit greater sensitivity and be

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1 more sure of what compounds actually do have thyroid activity. 

### 2 Thank you. 

### 3 **DR. DANIEL SCHLENK:**Any questions from the 

### 4 Panel? Yes, Dr. Green? 

### 5 **DR. SHERRIL GREEN:**I'd be interested in your 

### 6 opinion. Would a staged fixed design either result in an 

### 7 overall shorter assay time and/or the use of fewer animals? 

### 8 **DR. JOHN BRAUSCH:**It could definitely reduce 

### 9 the amount of time. Stage 58 would usually occur slightly 

### 10 before that 21-day that is currently established. I think that, 

### 11 even if it was the same length and you used the same amount of 

### 12 animals, there would be benefits in it because of the additional 

### 13 power that you would have in the histopathology. 

### 14 **DR. DANIEL SCHLENK:**Any other questions? 

### 15 Okay. Thanks Mr. Brausch. Our next presenter would be Allen 

### 16 Olmstead. 

### 17 **DR. ALLEN OLMSTEAD:**Good morning. My name is 

### 18 Allen Olmstead and I am an Ecotoxicologist at Bayer CropScience. 

### 19 My comments today are going to focus on the two charge 

### 20 questions that dealt specifically with the AMA. 

### 21 This abnormality, this condition that we've 

### 22 been discussing quite a bit, different people call it different 

### 23 names. You'll find it's called different things in the 

### 24 literature and people have sort of different definitions to it. 

### 25 So I think it's important -- I find these pictures really 

### 26 helpful in describing what it is that we're talking about and

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1 having a consistent identification so that people are onboard 

### 2 with what this is. 

### 3 This was already presented and described. It 

### 4 would like to add that if you look at the fore limbs and the 

### 5 hind limbs, you can tell that these tadpoles are at different 

### 6 stages. And in fact, the one that has the most severe 

### 7 abnormality is actually the least developed of these tadpoles. 

### 8 As was mentioned, you start to see these signs 

### 9 around stage 51, 54 although you can see them in older animals 

### 10 that didn't have them at that stage. And the severity of this 

### 11 condition tends to progress as the animals get larger and as 

### 12 they advance in the stages. 

### 13 So the middle panel there, at that stage you 

### 14 may have that level of abnormality, but at a further stage, that 

### 15 condition may progress and become worse. 

### 16 Another point I'd like to make is that this 

### 17 definition, sort of between the two different severities in 

### 18 terms of whether or not this curvature occurs anterior or 

### 19 posterior to hind limb lengths, I think it's important because 

### 20 when it occurs anterior to the hind limb lengths after the 

### 21 animal metamorphosis, you still sometimes see this condition in 

### 22 the adult frog. In addition, that severity of this condition 

### 23 may lead to inability of the tadpole to maintain itself in the 

### 24 water column which would impair its ability to feed as well as 

### 25 grow. 

### 26 Certainly, I think across the board we can say

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1 that any improvements in diet and husbandry for any animals that 

### 2 we work needs important considerations in things that we would 

### 3 want to improve; both we want thriving healthy animals in our 

### 4 research and for the applicable considerations there. 

### 5 One of the points that I think is important is 

### 6 that, does the development of this condition actually impair the 

### 7 ability of the assay to detect thyroid active compounds. And to 

### 8 my knowledge, we don't have any evidence that this would be the 

### 9 case. 

### 10 So when this research was done, to try and get 

### 11 a handle on this, the top panel was ascribed a score of zero, 

### 12 the middle one was ascribed a score of one and the last one was 

### 13 ascribed a score of two. So I bring that up to mention this 

### 14 figure, which is meant to address one of the questions from the 

### 15 Panel yesterday in terms of growth in relation to development of 

### 16 this abnormality. 

### 17 So these data points don't represent 

### 18 individual frogs, they represent means among the tanks. And 

### 19 there are two strains of -- this is Xenopus tropicalis 

### 20 illustrated here. You can see that one strain clearly develops 

### 21 this condition more than the other strain. That strain also 

### 22 actually grows larger than the other strain. 

### 23 And when this research was done, it wasn't 

### 24 done just to reduce this degree of abnormality, but also 

### 25 considerations were made to survivability as well as growth of 

### 26 the animal. So we really wanted to have large healthy animals

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1 but not have these conditions. And so one of the 

### 2 interpretations is over-feeding, sort of comes from this 

### 3 relationship. The more you push growth the more you tend to see 

### 4 this development. 

### 5 Personally, I feel that what we're looking at 

### 6 here is some sort of nutritional deficiency, and this is based 

### 7 on some other work that was done. Because we also looked at 

### 8 Rana Sylvatica and Rana Pipiens that were brought in from the 

### 9 field. 

### 10 With regard to the other charge question and 

### 11 thyroid histology, the important thing about where you make your 

### 12 baseline or where you put your baseline or your reference, is 

### 13 that you're always comparing to the control in these assays. 

### 14 So you can have an external baseline -- and 

### 15 this would be the Grim et al. guidance -- or you can have the 

### 16 internal baseline, which would be the controls themselves. But 

### 17 in the interpretation, it has to be made between the controls 

### 18 and the treatments. 

### 19 The last little cartoon here is that you want 

### 20 to know if the rabbit is finishing the race differently from the 

### 21 tortoise. It doesn't matter if you measure it from the start or 

### 22 the finish or some arbitrary point in between, you still come to 

### 23 the same conclusion. 

### 24 Thyroid activity in these animals is changing 

### 25 as you go through metamorphosis. This is a fixed time design, 

### 26 so at the end of it, we're getting a snapshot. Different assays

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1 will have different snapshots of what's going on, and so that's 

### 2 the important thing to consider in terms of interpretation, 

### 3 especially in terms of this idea or this observation of lower 

### 4 incidents, severity, hypertrophy compared to the controls. 

### 5 And so if you think of a thyroid antagonist 

### 6 activity, we would expect to see developmental delay along with 

### 7 increased hypertrophy compared to the controls. That's as the 

### 8 test is typically designed where your animals have not reached 

### 9 metamorphic climax. 

### 10 Certainly after you pass that metamorphic 

### 11 climax, then this indication of lower incidence severity of 

### 12 hypertrophy could be consistent with the developmental delay. 

### 13 Another explanation here is that the animals weren't properly 

### 14 stage-matched with the controls. 

### 15 And so, again, going back to my cartoon, I 

### 16 sort of think that if you don't have properly stage-matched 

### 17 controls then your point of reference is different for your 

### 18 controls and your treatments, and that's important. 

### 19 And another thing is that when you do have 

### 20 developmental delay it's important to realize that you are 

### 21 sub-sampling from these tanks from slightly different 

### 22 populations. So that's another consideration, even if you are 

### 23 stage-matching, there are other factors involved there. 

### 24 The stage-matching, the issues here with how 

### 25 the stage development of the animal impacts other endpoints is 

### 26 important considerations as well because the stage here is the

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1 confounding factor for statistical analysis of all the other 

### 2 endpoints, with the exception of developmental delay. And if we 

### 3 moved to a stage-fixed design, I believe that would alleviate a 

### 4 lot of the issues that we had. And with that, I thank you for 

### 5 your time. 

### 6 **DR. DANIEL SCHLENK:**Any questions? Dr. 

### 7 Green? 

### 8 **DR. SHERRIL GREEN:**So I just need some 

### 9 clarification here because your observation of bent tail in 

### 10 Xenopus laevis or Xenopus tropicalis -- lets start with that 

### 11 species -- you alluded to over-feeding. And I'm wondering if in 

### 12 your lab you've tested that hypothesis by purposefully 

### 13 over-feeding and inducing the condition in the Nigerian and the 

### 14 golden trouts. 

### 15 **DR. ALLEN OLMSTEAD:**The work that was done 

### 16 there was not focused on over-feeding. So none of the work in 

### 17 that Olmstead et al. manuscript actually dealt with different 

### 18 levels of feed. They were different diets, different components 

### 19 of the diet. In particular, one of the observations that was 

### 20 made was that when you included things that tend to have more 

### 21 protein than fat, we tended to see higher rates of scoliosis. 

### 22 You also tended to see increased birth rates. So I'm thinking 

### 23 of things like trout-starter, things that have, like, fishmeal. 

### 24 **DR. SHERRIL GREEN:**Right, which have been 

### 25 used for decades to feed those species in laboratory 

### 26 environments. These reports are relatively recent, including

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1 the one with the high frequency of bent tail, the 92 percent 

### 2 from one of the test laboratories. 

### 3 So it's really hard for me to get my head 

### 4 around this information when it hasn't been published yet and 

### 5 it's anecdotal. I see the pictures and I know that it's 

### 6 happening. But whether it's overfeeding or feeding too much 

### 7 protein, or it could be related to the changes in water quality 

### 8 as a result of the amount of excrement in the water on a 

### 9 particular change turnover system that isn't changed to 

### 10 accommodate the waste that's produced from that. You follow me? 

### 11 12 So there are other down-stream affects of 

### 13 overfeeding. So would you agree it's still a little murky to 

### 14 say -- no pun intended -- that overfeeding can consistently 

### 15 produce these findings? 

### 16 **DR. ALLEN OLMSTEAD:**I personally do not agree 

### 17 with that this as an overfeeding issue, but that is an 

### 18 interpretation. 

### 19 **DR. SHERRIL GREEN:**Okay, so let me interject 

### 20 here. All right. Thank you. Now I want to move to the second 

### 21 comment that you made about nutritional deficiencies. Okay. 

### 22 So that was on work that you did in your lab 

### 23 in wild-caught Rana Pipiens, which are not fully aquatic. They 

### 24 are semi-terrestrial. 

### 25 **DR. ALLEN OLMSTEAD:**It was based on similar 

### 26 work that was done mainly with Rana Sylvatica although a little

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1 bit of work was done with Rana Pipiens. And so that work was 

### 2 sort of done in collaboration with the University of Minnesota 

### 3 Duluth. We had mesocosms, typical mesocosms as they normally 

### 4 grow and we also tried to raise these in the labs. 

### 5 And we tried different types of diets and what 

### 6 we found is we had even worse severities of those conditions 

### 7 than what we saw with the Xenopus. 

### 8 Now one of the things in terms of husbandry of 

### 9 native amphibians is that when most people raise these in the 

### 10 lab, they get really tiny metamorphosis. There're like 10 

### 11 percent of what we actually were seeing in the lab. 

### 12 And so the animals that we raised in the lab, 

### 13 you know, they were smaller but they had high levels of this 

### 14 condition; whereas what we had in the mesocosms where it was a 

### 15 more natural type food, they grew to the size that we're seeing 

### 16 in the field but they did not have this condition. 

### 17 **DR. SHERRIL GREEN:**So what were you feeding 

### 18 them, Rana, in the lab when you were studying bent tail? 

### 19 **DR. ALLEN OLMSTEAD:**We tried sera micron, we 

### 20 tried trout starter. We tried fish plate food. We also had 

### 21 boiled lettuce, boiled spinach. It was a mix of diets that we 

### 22 looked at with Xenopus. 

### 23 **DR. SHERRIL GREEN:**Thank you. 

### 24 **DR. DANIEL SCHLENK:**Dr. Patino? 

### 25 **DR. REYNALDO PATINO:**I have a follow-up on 

### 26 the bent tail question. So just focusing on Xenopus and

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1 focusing on lab studies, do you have or do you know evidence 

### 2 that would show or suggest that the appearance or the incidents 

### 3 of bent tails is associated with or causes these problems in 

### 4 performance, such as growth or development? 

### 5 I mean is there evidence that would say, okay, 

### 6 this is a problem in terms of the other endpoints that are being 

### 7 measured in the assay that they will affect those animals? 

### 8 **DR. ALLEN OLMSTEAD:**Somewhat problematic in 

### 9 that the animals that tend to grow larger, the ones that tend to 

### 10 develop this, you know, you don't know if there is production in 

### 11 growth because of that; sort of putting the horse before the 

### 12 cart type of thing. 

### 13 The observations that I made was that when 

### 14 this condition gets really severe the animal is clearly not 

### 15 behaving normally. They have an impairment in how they move 

### 16 through the water column, and I would assume that that would 

### 17 impair their ability to feed. 

### 18 And so there are concerns with the compounding 

### 19 factor with the measurements there that are in the assay that -- 

### 20 I mean from what I observed, as long as the animals are able to 

### 21 move as an unaffected animal, I don't see how the endpoints that 

### 22 are measured in the assay would be impacted by that, which is 

### 23 why I would advocate -- I'm not just saying these animals have 

### 24 this condition, but do they have it anterior or posterior to the 

### 25 hind limbs. 

### 26 **DR. DANIEL SCHLENK:**Dr. Leonard?

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1 **DR. JACK LEONARD:**How tight was the 

### 2 population distribution in your tanks that showed bent tail? 

### 3 Did you find that the staging was broad, in terms of the 

### 4 distribution of the tadpole within a given tank? And was there 

### 5 relationship between the distribution and the incidence of bent 

### 6 tail? 

### 7 **DR. ALLEN OLMSTEAD:**So trying to rephrase 

### 8 your question; are you asking if those tadpoles that tended to 

### 9 reach metamorphosis earlier had higher incidences? 

### 10 **DR. JACK LEONARD:**No. I'm asking exactly how 

### 11 broad of an NF scale do you have in the tank when you have the 

### 12 bent tail. 

### 13 **DR. ALLEN OLMSTEAD:**The studies that were 

### 14 done with this were actually done two-stage as opposed to at any 

### 15 given time. 

### 16 So we went in and every time a tadpole entered 

### 17 stage 58 or 62, you pull it out and assess it then. 

### 18 Those particular studies were not done under 

### 19 the AMA guidelines. With the AMA guidelines, you have everybody 

### 20 that starts at stage 51 on a certain date, so you have a really 

### 21 tight variability there. These were done based on when they 

### 22 respond, so the variability there was much more spread out than 

### 23 what we would see in the AMA. 

### 24 **DR. DANIEL SCHLENK:**Dr. Horseman? 

### 25 **DR. NELSON HORSEMAN:**I'm going to get off the 

### 26 bent tail. So I want to ask about the logic. You make a strong

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1 argument for stage matching, and we heard this earlier from EPA, 

### 2 in the thyroid histology studies. 

### 3 I want to ask about the logic of that because 

### 4 just as a hypothetical, a treatment that accelerated the rate of 

### 5 metamorphosis, it seems to me if you stage matched those 

### 6 measurements of histology, you'd be selecting for animals that 

### 7 were less responsive to the testing factor. So I'm trying to 

### 8 figure out the logic in this thing. 

### 9 **DR. ALLEN OLMSTEAD:**I have to admit I didn't 

### 10 come up with this guideline and my experience was more dealing 

### 11 with Tier 2 test. And so I would be in favor of running this to 

### 12 a stage as opposed to a time. And I think what you're 

### 13 describing is what I refer to as when there is a developmental 

### 14 delay, we were basically sub-sampling for those animals that 

### 15 would least likely be affected. 

### 16 So again, this process of stage matching, we 

### 17 have to be aware of what sort of factors might be involved in 

### 18 there. I think the opposite or the other side to that is that 

### 19 when we look at validation exercises, when we look at those 

### 20 compounds that have anti-thyroid activity, what we see is we see 

### 21 the delays in development, but we see these thyroid changes at 

### 22 much lower concentrations as well. 

### 23 So it's sort of an adverse outcome pathway 

### 24 thing here. You see changes in the thyroid gland concentrations 

### 25 before you start seeing affects on development. So even though 

### 26 we are doing this sort of sub-sampling here, there is a question

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1 of thyroid histology is such a sensitive endpoint that, based on 

### 2 the validation work, I would argue that we should see evidence 

### 3 in changes in thyroid, even though we are purposely sampling 

### 4 here. 

### 5 **DR. NELSON HORSEMAN:**So you stick by your gun 

### 6 once it's stage-matched. 

### 7 **DR. ALLEN OLMSTEAD:**The other issues with the 

### 8 endpoints that were measured in the statistical analyses 

### 9 consistently, from looking at the AMA reports that I had, those 

### 10 were the big concerns, especially when you start dealing with 

### 11 overt toxicity; because that does have an impact on -- it could 

### 12 have an impact on development, it could have an impact on the 

### 13 size of the animal. 

### 14 The other nice thing about looking at time to 

### 15 a certain stage is that's a much more concrete measurement 

### 16 that's less abstract. When you say there's a stage difference 

### 17 between, you know, the treatment here is NF57 versus NF56, how 

### 18 you put that into ecological context? Whereas I think when you 

### 19 say the time to stage 58 was delayed by two days, I think that's 

### 20 more meaningful. 

### 21 **DR. DANIEL SCHLENK:**Dr. Patino? 

### 22 **DR. REYNALDO PATINO:**I have a question on 

### 23 stage matching and asking for your opinion here. What would you 

### 24 think of instead of matching the treatment to the subsample of 

### 25 control animals, matching the controls to what is most common in 

### 26 treatment, if the stages are there to match?

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1 **DR. ALLEN OLMSTEAD:**I think that's the other 

### 2 side of the coin if you are going to stage match. I think 

### 3 you'll still run into the same issues where you're still 

### 4 sub-sampling from these two different populations, and the 

### 5 possibility of having a compounding factor there still exist. 

### 6 **DR. DANIEL SCHLENK:**Dr. Scott? 

### 7 **DR. GEOFFREY SCOTT:**Just one question. Had 

### 8 there been any studies looking specifically at all of the 

### 9 ingredients in foods themselves that are used? Has anybody 

### 10 really done a detailed chemical analysis of the active 

### 11 ingredients in the food? Particularly, I'm thinking about the 

### 12 antioxidants that are used to stabilize the food. Has anybody 

### 13 done anything? 

### 14 **DR. ALLEN OLMSTEAD:**That sort of 

### 15 consideration was only done as sort of food fiber, ash protein 

### 16 that you typically get in these diets, but nothing to that 

### 17 equal. 

### 18 **DR. DANIEL SCHLENK:**Okay. Any other 

### 19 questions? All right, thanks. At this point, let's go ahead 

### 20 and take a break, if it's okay with Dr. Mihaich. We'll 

### 21 reconvene at 11:00. 

### 22 (Whereas a break was taken) 

### 23 **DR. DANIEL SCHLENK:**So I've been informed by 

### 24 our technical staff that the speakers need to sit as close as 

### 25 you can to that mic. Apparently we've been having some 

### 26 difficulties hearing in the back.

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1 Okay? All right, let's continue with our 

### 2 public commenting period by the Endocrine Policy Forum, and our 

### 3 next speaker is Ellen Mihaich. 

### 4 **DR. ELLEN MIHAICH:**Thank you. And I realize 

### 5 that other than giving my name, I didn't even tell you who I 

### 6 actually was earlier. I was just whisking in and whisking out. 

### 7 8 I am a consultant. I have my own consulting 

### 9 company and I am the Scientific Coordinator for the Endocrine 

### 10 Policy Forum, which is why I got to put all of this together for 

### 11 all of these people. I also have an adjunct appointment at Duke 

### 12 University teaching risk assessment, so I'm an environmental 

### 13 toxicologist and risk assessor. 

### 14 And right now I am going to be presenting on 

### 15 the FSTRA study. Dr. Katie Coady, with Dow Chemical, was not 

### 16 able to be here, but she put these slides together, so I will do 

### 17 my best to do her proud. 

### 18 It's a very similar setup to what you just 

### 19 heard from John Brausch on the amphibian metamorphosis study. 

### 20 There were difficulties with the performance criteria. Actually 

### 21 only four out of the 21 studies that were examined met all of 

### 22 the identified test validity criteria. It would be really great 

### 23 to actually have all 52 studies to understand what it was across 

### 24 the board. 

### 25 One of the biggest problems that we've had, 

### 26 and I think we can see that -- we saw that with the AMA, was

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1 this issue of mean measure test concentrations and the 

### 2 performance criteria for the aquatic. I mean these are 

### 3 difficult to test. A lot of these are difficult to test 

### 4 compounds. And as John mentioned, we do know at least what 

### 5 they're being exposed to because we do analytical confirmation 

### 6 of dose. 

### 7 But in the 21 studies that you have in your 

### 8 White Paper, nine of them did not meet this performance 

### 9 criteria, so 43 percent were not able to meet that performance 

### 10 criteria. 

### 11 The labs actually do a lot to try to meet 

### 12 this. It is a huge amount of up-front work that goes into this 

### 13 to try to get concentrations meeting the criteria that we have. 

### 14 They increase turn-over rates. They increase cleaning of the 

### 15 tanks; just all sorts of things in order to try to meet that 

### 16 criteria. 

### 17 In many cases or in some cases, we did have to 

### 18 use solvents. And then that came up yesterday, I believe, in 

### 19 some of the discussion that there is a bit of a lack of guidance 

### 20 on the issue for how to look at solvent controls versus food 

### 21 controls versus negative controls. And within the OECD, in 

### 22 fact, the standard practice within OECD is if your solvent and 

### 23 your negative controls don't match, you then compare it to your 

### 24 solvent control which is opposite of what is in the guideline; 

### 25 so I think having a little bit more understanding of why that 

### 26 is, we should probably take a better look at that too.

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1 We also had some difficulties in meeting the 

### 2 performance criteria for the fecundity endpoint. Of course 

### 3 fecundity is one of those apical endpoints not very specific to 

### 4 an endocrine mode of action, or not always specific to an 

### 5 endocrine mode of action. 

### 6 Six of the 21 examined studies in the White 

### 7 Paper did not achieve the 15 eggs per female per reproductive 

### 8 day, so it's 29 percent that didn't match it. Again, labs did a 

### 9 lot of work to try to meet this. In particular, they added 

### 10 collection trays underneath the spawning tubes so that they 

### 11 could try to catch all of the eggs, any eggs that might've 

### 12 fallen to try to up that to meet that performance criteria. 

### 13 I think we've also heard a lot about dose 

### 14 setting, and it's not just on the aquatic studies. It's done 

### 15 with the others, this overt toxicity. The guideline says that 

### 16 it should be a third of the acute LC50. That's one of the 

### 17 criteria and also based on solubility too, but this practice did 

### 18 actually lead to some overt toxicity. I mean think about it; 

### 19 you're doing a 96-hour LC50 typically to get that acute LC50 and 

### 20 you're taking that to a 21-day study. 

### 21 So it's really you're very much pushing the 

### 22 envelop as far as your maximum tolerated concentration. And out 

### 23 of the studies that were evaluated in the White Paper, 20 out of 

### 24 the 21 showed overt toxicity or some sort of sublethal affect. 

### 25 So I think that's something that we really do 

### 26 need to think about as far as, again, thinking about where we

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1 set our doses. Our recommendation is that 1050 acute LC50 seems 

### 2 to be a more appropriate place to put it. But also in trying 

### 3 to, again, meet this and trying not to have to repeat these 

### 4 studies over and over and over again to have doses that didn't 

### 5 have a lot of overt toxicity, a lot of registrants did prolonged 

### 6 range-finders, 7 to 14 days, before starting the study, and so 

### 7 that just adds more time and expenses to doing these. And we 

### 8 did also actually use the maximum dose separation in many of 

### 9 them so that you would at least have two treatments that didn't 

### 10 have the overt toxicity. 

### 11 Not quite as bad as the frog study but still 

### 12 pretty high in animal use, 400 to 500 fathead minnows that are 

### 13 usually put in here, but we also have range finders that could 

### 14 be an issue. And there is some trouble with meeting the weight 

### 15 requirements, and so you end up having to have a little more 

### 16 fish to make sure that you met the weight requirements. 

### 17 How this study is set up is you set up with 

### 18 extra tanks to go into this preexposure time to make sure that 

### 19 you have groups of fish that are reproducing well; so you over 

### 20 setup in order to be able to pick the ones to make sure that you 

### 21 have enough that are reproducing. 

### 22 So it's also a very expensive study. It's 

### 23 about $150,000, so it is quite labor-intensive and you need to 

### 24 know what you're doing. So it's high in animal use, high in 

### 25 cost, high in time, not really what I would consider to be a 

### 26 screen.

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1 There are some difficulties in endpoint 

### 2 interpretation, especially things like the apical endpoints in 

### 3 here that we always have to keep track on to make sure that 

### 4 things across the battery. I mean that's why this was setup as 

### 5 a battery and, you know, that was really smart that we can use 

### 6 all of the studies. 

### 7 But things like fecundity, it's sensitive but 

### 8 it's not really specific to an endocrine mode of action. 

### 9 Decreases in sex steroids, GSI fecundity, even vitellogenin. I 

### 10 mean vitellogenin is a great endpoint for an increase in male 

### 11 vitellogenin being exposed to an estrogen. But if you have, for 

### 12 example, a decrease in female vitellogenin, it could potentially 

### 13 be a path of toxicity; which has required us to all keep our 

### 14 fish just in case, depending on what you find in the end so 

### 15 that you might have to do some additional histopathology to try 

### 16 to understand what the affect is that you're seeing. 

### 17 We also have had troubles with bacterial 

### 18 infections, microsporidia, things like that, and that can in 

### 19 fact affect interpretation of the study. I just recently I had 

### 20 a study where I got about two weeks into the pre-exposure and 

### 21 they just stopped reproducing, and then you lose all those fish. 

### 22 While you can check them ahead of time, and that's good, 

### 23 sometimes it's not at the level to really see to know if it's 

### 24 going to cause a problem until you really get into it and start 

### 25 stressing through the reproduction. 

### 26 So there are some strengths of FSTRA. There

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1 are some very good endpoints, things like, again, increase in 

### 2 male vitellogenin, it's a great one. Presence of tubercles in 

### 3 females, another good one that make this study -- you can get 

### 4 some good information out of it. 

### 5 It is another one where you can see exposure 

### 6 with the aquatic route, so that's good. It gives us a little 

### 7 bit more information. It is useful I think in the battery to 

### 8 corroborate other findings, but you do, again, have to be very 

### 9 careful about the apical endpoints. It's an in vivo system with 

### 10 a species that we do know an awful lot about. And it actually 

### 11 is possibly a little superior than the pubertals in detecting 

### 12 steroids and synthesis inhibitors. 

### 13 But there are limitations, as we've seen with 

### 14 all of these studies that we talked about. But again, apical 

### 15 endpoints, lots of animals that we're using. There are just 

### 16 huge issues with getting material into water. It's just not one 

### 17 of the more fun things that we do. Long, expensive, and it is 

### 18 probably a little bit more limited than the male pubertal and 

### 19 the detection of anti-androgenic chemicals. 

### 20 So just some proposed alterations to the 

### 21 guideline, as you guys are thinking about this. Changing the 

### 22 dose setting to maybe a 10th of the acute LC50 could help us 

### 23 avoid some of these problems with overt toxicity. 

### 24 I think eliminating the fecundity and 

### 25 fertility endpoints, they're really not that helpful. And there 

### 26 is an OECD guideline study, the 230, that actually -- as we've

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1 done a lot of work through OECD and we ended up with two 

### 2 guidelines, the 229, which is the same as the FSTRA study here, 

### 3 the 890 guideline which has the fertility and fecundity 

### 4 endpoints, and then the 230 which does not; so we could consider 

### 5 maybe making some sort of a change towards that. 

### 6 I think, again, if we had all of the data and 

### 7 were able to do some sort of sensitivity analysis on some of 

### 8 these endpoints to see how much information they do bring to the 

### 9 table, it would be very helpful. And I think if we could have 

### 10 some clarity on statistical issues, maybe go back and revisit 

### 11 the issue of food controls versus comparing to the solvent 

### 12 control; that could be very helpful. 

### 13 And overall, it is a valuable assay in the 

### 14 battery. And again, because this is a battery, we can get some 

### 15 good information out of it. It has good sensitivity for 

### 16 estrogen and steroidogenesis inhibition. It is a waterborne 

### 17 exposure and it's an aquatic species, so that's another good 

### 18 plus for it. But certain modifications could really help us 

### 19 improve the conduct interpretation of the screen. I think 

### 20 that's it. 

### 21 **DR. DANIEL SCHLENK:**Okay, any questions? Dr. 

### 22 Scott? 

### 23 **DR. GEOFFREY SCOTT:**Ellen, we've heard from a 

### 24 number of folks this idea, attempted the LC50 with the frog and 

### 25 the fish, and one of the questions I had since y'all are 

### 26 conducting range-finding assays oftentimes with both of those

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1 assays; if you look at that attempt of the LC50, where does that 

### 2 put you in terms of the NOEC and LOEC if you were to calculate 

### 3 it in those range finders? Has anybody looked at that? 

### 4 **DR. ELLEN MIHAICH:**I haven't. 

### 5 **DR. GEOFFREY SCOTT:**Yeah, I know you're 

### 6 presenting but it just -- I should've asked it earlier, but 

### 7 since several folks have asked it and my brain cells have -- 

### 8 **DR. ELLEN MIHAICH:**I was just going to say, 

### 9 can I just turn to my friends? No? I think we'd have to get 

### 10 back to you on that. 

### 11 **DR. GEOFFREY SCOTT:**Well to me, that would be 

### 12 worth looking at. And it would give you some additional insight 

### 13 since we've heard this repeated recommendation. 

### 14 **DR. ELLEN MIHAICH:**Right. Thank you. 

### 15 **DR. DANIEL SCHLENK:**Any other questions? Don 

### 16 Tillitt? 

### 17 **DR. DONALD TILLITT:**Ellen, you recommend 

### 18 removing the fecundity. I'm somewhat surprised because that's 

### 19 the ecological relevant endpoint. I know it doesn't provide, 

### 20 you know, mechanistic, but it is an apical endpoint. 

### 21 **DR. ELLEN MIHAICH:**Yes, but you got to 

### 22 remember what the Tier 1 is. Tier 1 is a battery to look at the 

### 23 potential to interact with the endocrine system, it's not risk 

### 24 assessment. So there will be other studies, I'm sure. If there 

### 25 was some concern about an endocrine mode of action in this with 

### 26 good redundancy, good corroboration, and as long as the battery

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1 is working well, you would then go on to find that apical 

### 2 endpoint, which is the population-relevant endpoint. 

### 3 So you're not doing a risk-assessment on this. 

### 4 And because it's not mechanistic and can easily be perturbed by 

### 5 other modes or mechanisms of action, I mean think about it, you 

### 6 don't feel well, I think reproduction is going to go down, but 

### 7 it may have nothing to do with an endocrine mode of action, 

### 8 right? 

### 9 So I think that it just muddies the water from 

### 10 that perspective. Let's keep these things more mechanistic, 

### 11 more specific. Yes, it can provide some additional information, 

### 12 but I think if you were looking at the FSTRA study, what would 

### 13 be more important to me are things like male vitellogenin to 

### 14 indicate that there's some estrogenic component with the 

### 15 compound. 

### 16 **DR. DONALD TILLITT:**Even with the cost 

### 17 benefit being there? 

### 18 **DR. ELLEN MIHAICH:**Well the thing with the 

### 19 fecundity and fertility endpoints, it does add cost to the study 

### 20 because you have to do a lot of counting. So the study is a lot 

### 21 more resource-intensive and it is oftentimes -- when you can't 

### 22 meet that criteria, you take the study down and you start again 

### 23 because you don't want a study that's going to be rejected. 

### 24 So there are a lot of things that come in here 

### 25 and it may be that it has nothing to do with understanding 

### 26 whether or not there's a mechanism that is pertinent to the

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1 endocrine. 

### 2 **DR. DONALD TILLITT:**Along those same lines 

### 3 then would you recommend adding, as non-optional but required, 

### 4 the steroid analysis based on your same logic? 

### 5 **DR. ELLEN MIHAICH:**And I think Lisa is going 

### 6 to talk about that in just a minute. I know that there are 

### 7 issues and that's why it was not required. It's a blood issue, 

### 8 you know, small fish not enough. But I was just talking with 

### 9 someone else yesterday and there are methods that probably could 

### 10 look at that. I'm sure that there are some issues kind of 

### 11 similar to what Sue Marty just mentioned about a one-time point 

### 12 measuring a hormone and what that might mean; but I think we 

### 13 could probably get past that with some work. 

### 14 I think it's something to think about it, but 

### 15 you need to make sure that it was valid within the study. 

### 16 **DR. DONALD TILLITT:**Thank you. 

### 17 **DR. DANIEL SCHLENK:**Any other questions? 

### 18 Okay. I think you have another presentation, yes? Round two. 

### 19 **DR. ELLEN MIHAICH:**I do. You have to listen 

### 20 to me again. 

### 21 Okay. So as he's pulling that up, I just 

### 22 wanted to mention, I think you all got our 64 pages of comments. 

### 23 I meant to mention that when I first got up there. If you want 

### 24 additional information from what we're talking about here, I'm 

### 25 sure you can find it in there or catch any one of us after the 

### 26 fact.

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1 So the other presentation I just wanted to 

### 2 make was on the guidelines themselves. And I've been doing this 

### 3 endocrine stuff for a really long time. I was an EDSTAC 

### 4 groupie. I followed it around the world, it seems like. And 

### 5 I've also worked a lot through OECD to start working on some of 

### 6 these guidelines. 

### 7 And one of the problems that we've had, and I 

### 8 think you can probably get a sense to some extent from some of 

### 9 our discussion, was when these 890 series guidelines -- and 

### 10 please, if you haven't had a chance to page through them, you 

### 11 really should because you can see that there's some different 

### 12 quality to them. But at the time, there was no stakeholder 

### 13 review prior to their publication. And so what you see is some 

### 14 of the guidelines are done very well and others are really more 

### 15 like a protocol than a guideline. They're very prescriptive. 

### 16 If you've been involved with the SAP at all before, you probably 

### 17 have heard us talking about the corning hot plate. One of them 

### 18 says that you should have a Corning hot plate. You know, that's 

### 19 not really a guideline. 

### 20 Now, that being said, we have addressed some 

### 21 of that. But the problem is, is that the test orders that these 

### 22 registrants get is you have a requirement to follow the 

### 23 guideline. If they say you have to test the guideline, then you 

### 24 have to follow the guideline. So if you don't follow the 

### 25 guideline and don't use your Corning hot plate -- and I'm just 

### 26 being a little funny on that one. Like I said, that's not going

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1 to be a problem, I know that's not a problem, but there are 

### 2 other things in these guidelines like that, all right? 

### 3 You have to consult with the Agency. So for 

### 4 us, it's a bit of an efficiency issue, and for the Agency. And 

### 5 if you don't, then you probably have a protocol deviation in the 

### 6 study because, again, all of these studies are done under good 

### 7 laboratory practices and you have to be very cognizant of 

### 8 following guidelines. And if it says you're following the 

### 9 guideline, but you don't really follow it in your protocol, 

### 10 that's a protocol deviation. Okay? 

### 11 So the Endocrine Policy Forum -- again, 

### 12 because we are this consortia of all the test holders, we were 

### 13 able to get a lot of guidance on a lot of these points. So the 

### 14 Agency has worked very well with our questions to determine 

### 15 whether or not some of these things are necessary. And you will 

### 16 find on the endocrine website that there's a corrections and 

### 17 clarifications document, which is very helpful. So it's been 

### 18 very good to try to make sure that we have things like 

### 19 flexibility on software and lab equipment because, again, many 

### 20 of these guidelines really read just like a protocol. 

### 21 And there were also some errors in the 

### 22 guidelines that needed to be corrected. So some of the 

### 23 equations were wrong, some of the concentrations were wrong. 

### 24 You know, we've been working through these for the last few 

### 25 years and I think we're moving forward very well, but the 

### 26 guidelines haven't been formally updated. One of the things

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1 that I want to encourage you in your response back to the Agency 

### 2 is to say that it would be a really good idea to update these 

### 3 guidelines as you go forward. 

### 4 The other thing that we have that gets filled 

### 5 out -- and I think you had some -- someone mentioned from 

### 6 yesterday there are standard evaluation procedures and study 

### 7 profile templates that are also more or less called data 

### 8 evaluation records, DERs. And there are a number of 

### 9 inconsistencies that we have between the guidelines and those 

### 10 documents also; things like in the uterotrophic study and the 

### 11 standard evaluation procedure for the uterotrophic study, it 

### 12 talks about doing a study for both estrogen agonist and 

### 13 antagonist, but it's only the agonist portion that's supported 

### 14 in the guideline. So there is a disconnect between the 

### 15 evaluation procedure and the guideline. 

### 16 In the Hershberger Study there is inconsistent 

### 17 concentrations for the testosterone propionate between the test 

### 18 guideline, the SEP, and the DER. So I feel like we're at a 

### 19 point where we can start to try to make sure that all of these 

### 20 things come together. 

### 21 And it's important because, not only do 

### 22 contract research organizations and the laboratories doing this 

### 23 work need to know what the real answer is, you know, if there's 

### 24 a problem in an equation, but the Agency contractors and 

### 25 reviewers also need to know that too because they're filling out 

### 26 these study profile templates based on the study and it has that

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1 chance of having more of a question come up of whether or not 

### 2 we've met the guidelines. 

### 3 You have a number of charge questions on 

### 4 performance criteria. We've certainly talked about performance 

### 5 criteria and some of the challenges we've had. As Sue Marty 

### 6 said, there are 32 performance criteria in the male pubertal and 

### 7 all 15 studies that were evaluated in the White Paper had at 

### 8 least two out of acceptable range. In the AMA, seven out of 18 

### 9 were out of range. 

### 10 So from what John mentioned earlier, you're 

### 11 being asked to evaluate this. If you have changes in some of 

### 12 these performance criteria, now is, again, a great time to make 

### 13 sure that it gets put into the guidelines and the guidelines are 

### 14 updated. 

### 15 Again, you are only getting a chance to look 

### 16 at about 21 studies and a lot of the information is not 

### 17 completely transparent in trying to be able to understand what 

### 18 you're looking at. So here is a good opportunity to really try 

### 19 to get more information out of what the industry has already 

### 20 done. 

### 21 So I'm just making a recommendation that EPA 

### 22 should take the opportunity now to amend the guidelines and 

### 23 other related documents before issuing new test orders on which 

### 24 were likely to come around the corner pretty soon. And also, we 

### 25 would recommend that EPA allow for stakeholder comment of the 

### 26 Tier 2 guidelines which are going to be massive, and you'll get

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1 to see a month from now -- well not the guidelines, but at least 

### 2 the validation reports -- before requiring testing so that we 

### 3 can maybe circumvent some of these problems that we're bringing 

### 4 to the table right now. And with that, I'm done. 

### 5 **DR. DANIEL SCHLENK:**Any questions from the 

### 6 Panel? Dr. Green? 

### 7 **DR. SHERRIL GREEN:**Thank you for that 

### 8 presentation. I have a question. In your travels as a 

### 9 consultant and with the groups that you represent, would you say 

### 10 in your opinion that most of the contract labs are 1) AAALAC 

### 11 accredited where animals are used, and 2) GLP labs that are 

### 12 regularly inspected? 

### 13 **DR. ELLEN MIHAICH:**Yes to both of those 

### 14 things. These studies have to be done under GLP, so there's no 

### 15 option there. So the industry would only work with GLP labs for 

### 16 providing the data to EPA for this program. 

### 17 **DR. SHERRIL GREEN:**One of the topics that 

### 18 continually comes up in the discussion of bent tail in the 

### 19 metamorphic frogs and tadpoles, for example, is the leaning that 

### 20 there is a general idea that husbandry is a significant player 

### 21 in that result. And if these labs are abiding by the 

### 22 guidelines, you would think that that wouldn't be as prominent 

### 23 an occurrence as it appears to be in the reports that we've seen 

### 24 right now. Could you weigh in on your opinion about how that 

### 25 might happen? 

### 26 I've looked at the conditions in the husbandry

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1 guidelines. They're within a range that is generally practiced 

### 2 but I'm suspecting that perhaps there is a bigger deviation 

### 3 amongst labs. 

### 4 **DR. ELLEN MIHAICH:**Okay. So you have to 

### 5 think about this whole program. When this all started and 

### 6 EDSTAC came forward with the studies that you should be looking 

### 7 at, a lot of people, industry, EPA went off to start to really 

### 8 work on validating these assays. Many of them didn't exist and 

### 9 many of them changed greatly from what was suggested by EDSTAC. 

### 10 11 So you then start down this path of validation 

### 12 and I know the Agency gets a lot of grief for how long it took. 

### 13 It just takes time, you know? They had a lot of work to do. We 

### 14 had a lot of work to do to try to understand how best -- you 

### 15 know, husbandry things can take forever to really get at, and 

### 16 unfortunately a lot of times you don't have that luxury to 

### 17 really get at it. 

### 18 The other thing is, when validation is 

### 19 occurring, it's only being done in a couple of labs. So then 

### 20 when you get into the regulatory part of this, you're scaling up 

### 21 to have lots of these studies done in contract labs. 

### 22 Now I don't think the contract lab is doing 

### 23 something wrong. I think that it's just that we didn't see the 

### 24 effect as much during the validation as we're now seeing it when 

### 25 we're trying to push the system to get these done in a two-year 

### 26 timeframe, which was the requirement, although there was a lot

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1 of help from the Agency from the standpoint of offering 

### 2 extensions. 

### 3 I mean, this was all new for everybody and I 

### 4 think we're just learning. And I think that what that means is, 

### 5 this is what you guys are here to do, is to look at some of 

### 6 these problems and make some suggestions for maybe making some 

### 7 changes. But I think it would also be really important to look 

### 8 at all 52 studies. I mean that's what the SAP said in 2008, 50 

### 9 to 100 studies. You've got 52. 

### 10 It would really be a good opportunity to be 

### 11 able to look at all of the data and to start teasing apart some 

### 12 of these endpoints, sensitivity analyses, that sort of thing. 

### 13 You know, I think it's worth taking the time to stop and really 

### 14 put this program together well. 

### 15 **DR. DANIEL SCHLENK:**Okay. Dr. Schultz? 

### 16 **DR. TERRY SCHULTZ:**So the forum, would it 

### 17 prefer to look into changing the protocols now based on the 

### 18 information that's available on 21 or continue as was originally 

### 19 intended in the 2008 to do the 52 -- I think the 2008 said more 

### 20 than 50 but less than 100 chemicals. There were 67 and -- don't 

### 21 worry about the numbers as being exact, but start to change the 

### 22 protocols now based on the information you have on the 21 or 

### 23 wait till all the information is available on the 52 and then 

### 24 address that issue? 

### 25 **DR. ELLEN MIHAICH:**I think we would prefer to 

### 26 wait and do all 52. I mean, I think the best thing is you have

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1 a lot of data. But there are other things you can be doing at 

### 2 the same time. The EPA has a great program going right now on 

### 3 high-throughput. That was the January SAP. There are things 

### 4 that can continue to move forward there, be able to find useful 

### 5 information, perhaps. 

### 6 But I really think that it is very important 

### 7 to make use -- again, this is over $50 million just in testing 

### 8 cost, and that has nothing to do with other resources. And this 

### 9 is EPA's resources, this is industry's resources, so there's 

### 10 been a lot of time and money spent on this program and I think 

### 11 it really behooves us all to step back and look at the data that 

### 12 we have. 

### 13 **DR. DANIEL SCHLENK:**Okay. Any other 

### 14 questions? Okay. Our next presenter will be Lisa Ortego. 

### 15 **DR. LISA ORTEGO:**Hi, I'm Lisa Ortego. I work 

### 16 for Bayer CropScience. I'm an Ecotoxicologist there. I also 

### 17 Chair the Technical working group of the Endocrine Policy Forum. 

### 18 19 My comments are going to focus now on offering 

### 20 some considerations to the Panel and to EPA for ways we might 

### 21 reorganize the Tier 1 battery, the T1B to enhance its 

### 22 efficiency, to reduce the use of resources and perhaps the 

### 23 decision-making. 

### 24 So in EPA's white paper for this SAP, they 

### 25 proposed or at least suggested for the Panel to consider perhaps 

### 26 elimination of the Uterotrophic and the Hershberger assay. This

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1 consideration seems at least partly based on the fact that the 

### 2 pubertals are flagging more endpoints than the Uterotrophic and 

### 3 the Hershberger. But as you heard my colleague Sue Marty talk 

### 4 earlier, a number of the endpoints in the pubertals are 

### 5 susceptible to non-endocrine specific activity. And I think 

### 6 what the Uterotrophic and the Hershbergers bring, they inform on 

### 7 those endpoints as to whether or not we should suspect an 

### 8 endocrine mode of action or if there's maybe a non-endocrine 

### 9 specific mode of action at work. 

### 10 And as you heard Barb Neal discuss, the 

### 11 Uterotrophic and the Hershberger assays are among the more 

### 12 specific and extensively validated assays. They've undergone 

### 13 testing and regulatory use years before the rest of the T1B, so 

### 14 we have a lot of experience with them. We understand how they 

### 15 work. We understand their specificity and the information that 

### 16 they are telling us. 

### 17 So our opinion is that the recommendation to 

### 18 eliminate these assays from the T1B is probably not in the best 

### 19 interest in maintaining specificity and mechanistic information 

### 20 in the battery. 

### 21 However, there are some alternatives to 

### 22 consider for reorganizing how we do the Tier 1 battery. And the 

### 23 alternatives I'm going to present today, none of them recommend 

### 24 elimination of any of the tools in the battery. It's just a way 

### 25 to stage the battery in a different way such that we can 

### 26 facilitate a faster resolution for questions and also get at

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1 reduction of resources. 

### 2 So a couple of those alternatives, OECD and 

### 3 the European Center for Ecotoxicology and Toxicology of 

### 4 Chemicals both have framework. Both of these frameworks are 

### 5 heavily weighted toward evaluation of all existing data prior to 

### 6 beginning of any testing such that that information, whether 

### 7 it's industry generated or it's from the peer group literature 

### 8 can inform what test you might need to do. And those frameworks 

### 9 progress from tests in less organized levels of biology to 

### 10 higher levels of biological organization. So those are just a 

### 11 couple of examples. 

### 12 These alternatives are fully referenced in the 

### 13 comments that were submitted on behalf of Endocrine Policy 

### 14 Forum, so you can find more information about them there. 

### 15 In 2011, there was a paper that was published 

### 16 by Borgert et al. with some suggestions about how you might go 

### 17 about organizing the Tier 1 battery instead of just testing all 

### 18 11 at the beginning. Instead of a requirement, you just test 

### 19 all -- to use all 11 assays. Perhaps you start with the 

### 20 androgen receptor binding assay, Hershberger, the aromatase, 

### 21 steroidogenesis or female pubertal. And if you don't see any 

### 22 activity in any of those assays, maybe you don't need to go any 

### 23 further. 

### 24 And if we do an assessment like this and we 

### 25 supplement with other relevant information, so peer review 

### 26 literature, other industry data, then we can gather a lot of

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1 information about which of these tests are really necessary for 

### 2 us to run to understanding the effects that we may see. 

### 3 Another alternative -- and this is the 

### 4 proposal that you heard Mike Dourson talk about from the TERA 

### 5 workshop that Earl Gray presented there. It's similar in 

### 6 concept to the Borgert 2011 et al. paper. And in his paper, 

### 7 they proposed that the FSTRA and the male pubertal assay are 

### 8 sensitive and specific enough to stand alone as initial assays 

### 9 in the battery. And they based this on an analysis of 12 model 

### 10 compounds and they found that at least one fish and one rat 

### 11 assay indicated a positive finding with high concordance. 

### 12 So the FSTRA and the male pubertal could be 

### 13 performed first. If you have an effect in one of those assays, 

### 14 then you chose from the rest of the T1B what you need to 

### 15 characterize that affect and make a determination on whether you 

### 16 need, again, risk-assessment data from Tier 2 at the end of that 

### 17 evaluation. If no effects are observed in those two assays, 

### 18 then that chemical goes to a hold status essentially, and no 

### 19 further endocrine screening is necessary at that time. 

### 20 Now in their paper, they did not appear to use 

### 21 the fecundity endpoint. I haven't confirmed this with either 

### 22 Gary or Earl and we would need to do that, but it's not cited in 

### 23 the paper that the fecundity was a decision-maker. And if 

### 24 that's the case, that might be another way that we could reduce 

### 25 the amount of labor and cost and time associated with the FSTRA. 

### 26 However, the optional endpoints would need to be added to use

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1 the Ankley/Gray approach. 

### 2 So I think that if we're going to evaluate 

### 3 whether or not this is an appropriate way to go forward, we want 

### 4 to consider whether reproduction or fecundity is necessary and 

### 5 we'd want to consider that the optional steroid measurements may 

### 6 be a requirement instead of an option, going forward. 

### 7 Now in the Ankley/Gray paper, they focus on 

### 8 estrogen and androgen activity, but thyroid activity is also 

### 9 covered under this approach. The male pubertal assay includes 

### 10 thyroid evaluation or you can modify the Hershberger to get that 

### 11 information if somebody is more interested in pursuing the 

### 12 Hershberger as the male assay here. 

### 13 And I also wanted to say that the Pickford 

### 14 paper, which you heard John Brausch reference earlier, supports 

### 15 the idea that the thyroid data in the mammals is predictive of 

### 16 what's happening in the frogs. 

### 17 So they compared a number of chemicals, I 

### 18 think it was 20 to 30 chemicals across several thyroid modes of 

### 19 action and found very good concordance and that the mammal 

### 20 responses were predictive of the amphibian responses. 

### 21 In addition to this data, all pesticides have 

### 22 other studies in which thyroid evaluation are done. So this is 

### 23 not the only thyroid data for pesticides available, so you would 

### 24 have other mammal-based thyroid data to include. 

### 25 So just to conclude, wanted to say that there 

### 26 are a number of proposals that we could consider as an

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1 alternative to the elimination of the Uterotrophic and 

### 2 Hershberger. None of these approaches would propose eliminating 

### 3 any of the studies on the battery unless we just felt like they 

### 4 weren't working. 

### 5 Now the alternatives we think carry a high 

### 6 potential for reducing time and the resources, if you're looking 

### 7 at a gatekeeper type approach, you're talking about two assays 

### 8 and possibly done so that you can get chemicals through the 

### 9 process much faster. 

### 10 So we think that a staged approach like this 

### 11 transforms this from just a requirement to use all 11 assays 

### 12 into more a targeted assessment programs; so we're going to 

### 13 target what we need to do based on the data that we have and 

### 14 based on the new data that we don't. I'll be happy to take any 

### 15 questions that you have. 

### 16 **DR. DANIEL SCHLENK:**Questions from Dr. 

### 17 Patino. 

### 18 **DR. REYNALDO PATINO:**Do you know is there 

### 19 information on differences in the sensitivity -- you had various 

### 20 suggestions on all these stage approach to the Tier 1 assays to 

### 21 eliminate either option that you proposed, eliminate the 

### 22 amphibians metamorphosis assay. 

### 23 Do you know if the pubertal assay, I guess, is 

### 24 the male that you are proposing -- has anybody looked at the 

### 25 difference in sensitivity between the male pubertal assay when 

### 26 it comes to thyroid endpoints and the amphibian assay?

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1 **DR. LISA ORTEGO:**Well the only information 

### 2 that I have would be the information that was presented in the 

### 3 Pickford paper in which that -- I don't know if sensitivity per 

### 4 se was the exact endpoint they were using. But what they 

### 5 reported was when it was positive in the mammal, it was positive 

### 6 in the frog. There was one chemical that was an exception, it 

### 7 was the methoxychlor, was the exception. It was positive in the 

### 8 AMA and it didn't register in the rat data that they had. 

### 9 What I would suggest is there is a lot of data 

### 10 that's now been collected from the Tier 1 battery for these 

### 11 chemicals, for 50 chemicals, and that's the kind of evaluation 

### 12 that we would encourage to happen to make a determination on 

### 13 whether or not the AMA could be a second line to understand the 

### 14 response we saw based on the gatekeeper approach. I think the 

### 15 analysis needs to be done and I think we have the data to do it. 

### 16 **DR. DANIEL SCHLENK:**Dr. Horseman? 

### 17 **DR. NELSON HORSEMAN:**Let's see. This 

### 18 question is going to be asked from the perspective of having 

### 19 thought endocrinology to medical students for 20 years or 

### 20 something. 

### 21 In these pubertal assays, the tropic hormone 

### 22 assays or the LH/FSH/TSH in particular, I would also add ACTH to 

### 23 this. But those are generally optional, described as optional 

### 24 assays and apparently aren't done ordinarily. 

### 25 The reason I ask about this is that 

### 26 determining an endocrine mode of action ultimately relies on

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1 having that information. A patient comes in with delayed 

### 2 puberty, the only way you'd know whether that's an endocrine 

### 3 mode of action or endocrine cause of disease is by measuring the 

### 4 gonadotrophic hormones. It could be nutritional, genetic, could 

### 5 be lots of other things. 

### 6 So from that perspective, I'd like you to 

### 7 comment on the fact that that information isn't being captured 

### 8 in these assays. 

### 9 **DR. LISA ORTEGO:**Okay. I'm going to have to 

### 10 maybe defer to Sue Marty, but I believe thyroid hormone 

### 11 measurements are required for the pubertals. 

### 12 **DR. REYNALDO PATINO:**I'm specifically not 

### 13 asking about thyroid hormone. I'm asking about the tropic 

### 14 hormones. TSH could be the relevant ones here. 

### 15 **DR. LISA ORTEGO:**Yeah, okay. So I really 

### 16 don't have a response for you but Sue may want to. 

### 17 **DR. SUE MARTY:**Just a couple of things. TSH 

### 18 is a requirement, both for male and female pubertal, as is T4. 

### 19 Testosterone is measured in the males. We don't measure 

### 20 estradiol in the females because they're all cycling and we're 

### 21 not required to take in necropsy at specific stage in the cycle. 

### 22 As far as other tropic hormones, we don't 

### 23 measure them. One of the issues I think with LH and FSH is 

### 24 because of pulsatile release. It's really difficult to get a 

### 25 good handle on those hormone levels. 

### 26 **DR. LISA ORTEGO:**Thanks Sue.

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1 **DR. DANIEL SCHLENK:**Any other questions? 

### 2 Okay, thanks. So our last presenter for the Endocrine Policy 

### 3 Forum would be Chris Borgert. 

### 4 **DR. CHRIS BORGERT:**Thank you very much. I 

### 5 know it's been a long morning. I'll try to be brief. I am a 

### 6 consultant, as is Ellen Mihaich, and I serve as the Assistant or 

### 7 Associate Scientific Coordinator. We haven't really resolved 

### 8 that. I think the acronym is probably the same in either case. 

### 9 I want to take you to sort of a 50 thousand 

### 10 foot level now to think about some of what we've heard this 

### 11 morning to emphasize the need for a thorough review of the EDSP 

### 12 List 1 data. And I'll describe for you some work that the EPF 

### 13 is undertaking. I'll give you then a very specific example that 

### 14 I'm not really qualified to answer questions about because I'm a 

### 15 pharmacologist. I'm going to give you an ecotox example, from 

### 16 one that we've already heard probably more than you wanted to 

### 17 hear this morning, and then make some general points. 

### 18 Okay. So at this point, we now have data on 

### 19 the 50 to 100 chemicals that in 2008, the SAP recommended be 

### 20 reviewed to determine assay and battery performance. And so 

### 21 having that data available, I want to emphasize that EPA has all 

### 22 of the data. So far, what we have been able to collect, we're 

### 23 calling our control data project. 

### 24 And the number of datasets that we have for 

### 25 each assay is shown on the slide. So the number of datasets 

### 26 that we have is over here. It's probably similar to the numbers

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1 that EPA is showing you that have been analyzed, but this is far 

### 2 from the full dataset. We don't have the ability to issue test 

### 3 orders to our members, so we can only sort of beg and ask, and 

### 4 we have confidentiality issues. But we've been able to collect 

### 5 a substantial amount to this point in time and more is coming 

### 6 in. 

### 7 Now what we do with those data, and as I say, 

### 8 our goal here is to do a deeper dive because I think that's what 

### 9 the 2008 SAP recommended, and we've shared with you a number of 

### 10 concerns in assays and we think that requires then a deeper dive 

### 11 in many of these data. 

### 12 But for confidentiality reasons, we then code 

### 13 some of the parameters in the information that's submitted to 

### 14 us. And those that are listed here, we code them, we don't just 

### 15 get rid of them because many of these may be important 

### 16 parameters to evaluate in the performance of the assays, if you 

### 17 want to know something about inter and intra-lab variability, 

### 18 variability over time as well as a number of other parameters. 

### 19 So the next thing we do is make all the data 

### 20 uniform for ease of analysis by the statisticians. And these 

### 21 next couple of slides are just giving you a brief listing of 

### 22 some of the data information in addition to the simple data 

### 23 printouts of the control and test chemical responses. There is 

### 24 a lot of other information with these assays that may affect 

### 25 assay performance that we think needs to be taken into account. 

### 26 You've heard about some of those things already. I won't go

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1 through this list, but we've listed them for each assay. 

### 2 Now I want to turn quickly to an example 

### 3 you've already heard a lot about, and I'm just giving this as an 

### 4 example. I don't mean to imply that we've resolved any 

### 5 questions here, but just to give you an example. 

### 6 So for instance, in the amphibian 

### 7 metamorphosis assay, the test guideline talks about 

### 8 normalization of hind limb length with snout-vent length. 

### 9 Presumably those recommendations in the test guideline are based 

### 10 on what came out of 14 studies that were used in the validation 

### 11 in the OECD program. 

### 12 When data started coming into us, we noticed 

### 13 that there may be some questions around this procedure, so we 

### 14 tried to use our data collection project to do a more thorough 

### 15 evaluation. Here is the dataset for snout-vent versus hind limb 

### 16 length for -- I think this was just a subset of four that showed 

### 17 poor correlations. This is being used for normalization. It 

### 18 raises some questions. 

### 19 Just going to fly through the next slide or 

### 20 the next series of slides to give you a flavor of what we 

### 21 started to do. So going back then and looking at 18 studies 

### 22 we've collected, plus the 14 studies used in the OECD and 

### 23 plotting all those data. And I'm not the statistician who did 

### 24 that. I'm not a statistician. He's unable to attend the 

### 25 meeting today so I can't answer specific questions here. But 

### 26 just to go through and point out that when developmental stage

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1 was used as a covariate, at least at this level of analysis, 

### 2 there did appear to be a fairly tight relationship. So all the 

### 3 statistical parameters are listed here but this is only for a 

### 4 subset of control data. 

### 5 We don't think this is a necessarily answered 

### 6 question, and of course we haven't looked at test data. EPA has 

### 7 all of those data for the test article and the controls, and so 

### 8 this is the type of analysis that we think needs to be done in 

### 9 order to resolve these kinds of questions. So overall, we're 

### 10 continuing to cull through these data and trying to do a deep 

### 11 dive. We have a little bit of test article data. We have a lot 

### 12 of control data. Like I said, EPA has all the data. 

### 13 I think the lessons we're learning is that, 

### 14 first of all, potential problems are not going to be identified 

### 15 if you don't look for them. So doing some concordance analysis 

### 16 and showing that things lineup is nice, but we think there's a 

### 17 lot deeper dive that may need to be done. And now we have a 

### 18 broader dataset that can be used for that thorough analysis. 

### 19 You know, I actually served on EDSTAC. I was 

### 20 on the screening and testing advisory committee so I have to 

### 21 take some of the blame for the fact that we may have what we 

### 22 have. But then in 2008, 10 years after EDSTAC made its 

### 23 recommendations the SAP recommended this deeper dive after 

### 24 spending all of these resources. And remember that 50 million 

### 25 plus and the tens of thousands of hours is just since the test 

### 26 orders were issued. There was a decade of work that preceded

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1 that. 

### 2 So at this point, it really seems like it 

### 3 would be worth wild to step back and take a close thorough look 

### 4 at all of the data. For example, as I gave you, the test 

### 5 guideline for the AMA may need some revision to correct 

### 6 normalization procedures. But as we've also heard, that raises 

### 7 all kinds of confounding issues as well. I'm not expert enough 

### 8 to answer those questions, others are. But it certainly is 

### 9 going to take a more in-depth analysis of all the data, at least 

### 10 in my view. We need to do this kind of an analysis I think for 

### 11 all of the performance criteria. 

### 12 You've heard about how many performance 

### 13 criteria there are in the pubertal assays, for instance. And 

### 14 this kind of an analysis can help, first of all, understand what 

### 15 the overall endpoint variance is across all of the assays, and 

### 16 then may help tease out some of these issues related to the test 

### 17 article where you can get a response that may or may not be 

### 18 hormone-mediated. It may be a non-specific response. 

### 19 And I think it will also help in the 

### 20 interpretation issues for eventually getting into a weight of 

### 21 evidence because we'll know what the overall variance in these 

### 22 endpoints is across laboratories within laboratories. We'll get 

### 23 a better handle on the true biological variability in these 

### 24 endpoints so we can better discern which ones are giving us real 

### 25 information and which aren't. 

### 26 And so I think this has some real benefits to

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1 the Agency. This kind of an analysis, of course, is a lot of 

### 2 work. We understand that. But a lot of work has already been 

### 3 done. It would seem to be a shame, at least in my view, not to 

### 4 take advantage of that. That would help the Agency really 

### 5 assure people that assay performance and battery performance are 

### 6 there. It would help them then to improve the test guidelines 

### 7 that has been alluded to. It would help focus the refinement of 

### 8 this battery. 

### 9 You know, looking at the data on this in-depth 

### 10 sort of bases would help that you could actually then dry-run 

### 11 some of these alternative suggestions for staging the battery. 

### 12 You could actually use those test data to figure out then, first 

### 13 of all, which endpoints are really informative, and then dry-run 

### 14 some of these alternative approaches to see if they might 

### 15 actually be as or more useful and yet more efficient. 

### 16 So I'll conclude with that and just sort of 

### 17 pose a question for the Panel. How comfortable are you making a 

### 18 pronouncement on the performance of the assays themselves or on 

### 19 the battery as a whole with only a subset of the data that may 

### 20 represent only up to say 40 percent of the chemicals that have 

### 21 been tested? 

### 22 As a scientist, that would give me some pause, 

### 23 but I would encourage you to mull that question over and 

### 24 consider the comments that you've heard this morning. So thank 

### 25 you very much. 

### 26 **DR. DANIEL SCHLENK:**Okay. Any questions?

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1 You can stick around for questions if you want, if there are 

### 2 any. 

### 3 **DR. CHRIS BORGERT:**We've got Dr. Schultz over 

### 4 here. 

### 5 **DR. TERRY SCHULTZ:**So which would you 

### 6 recommend first; evaluating all 51 chemicals based on the 

### 7 protocols we have or starting to look at altering the protocols? 

### 8 You've offered both options in your discussions this morning. 

### 9 **DR. CHRIS BORGERT:**Well I may be 

### 10 misunderstanding. You know, I get confused easily so maybe I'm 

### 11 misunderstanding, but I don't see them as an either/or. 

### 12 It seems to me that you first have to look at 

### 13 endpoint performance, endpoint variance, across the assays, 

### 14 through laboratories, look at the effects, the possible effects 

### 15 of all these different conditions in the various assays to 

### 16 really understand what the performance of these endpoints is for 

### 17 determining the various hypotheses that are posed. 

### 18 You'd have to know something about that before 

### 19 you -- see, I think you have to have that baseline information, 

### 20 those baseline comparators before you start to then evaluate how 

### 21 you could streamline the battery. 

### 22 So I'm not saying it so much as an either/or. 

### 23 Maybe I'm naive, but it seems to me that it's first necessary to 

### 24 make those baseline comparisons with a deep dive on all the 

### 25 endpoints that we have, unless there are assays that people 

### 26 think that we can simply dispense with, a priori and not even

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1 bother evaluating those data, but I don't think that -- I 

### 2 haven't heard that suggestion yet, so I would think one, then 

### 3 the next. 

### 4 **DR. DANIEL SCHLENK:**Mr. Scott? 

### 5 **DR. GEOFFREY SCOTT:**In the analysis you 

### 6 presented, you all started looking at the control. And I guess 

### 7 one of the things I firmly believe that if you're going to use 

### 8 as a test or a battery test in this case, being intimately 

### 9 familiar with your controls is a starting point, and I assume 

### 10 that was some of the logic you all used in starting with that 

### 11 analysis, correct? 

### 12 **DR. CHRIS BORGERT:**That's correct. But to be 

### 13 candid and thorough in the answer, you know, there are 

### 14 confidentiality issues here too, so we thought it might be a 

### 15 much tougher sell to convince companies to release somewhat 

### 16 sensitive test article data. So we felt more confident that we 

### 17 could collect more of the control data but you're exactly right, 

### 18 we felt that that was the starting point, and so both factors 

### 19 played into it. 

### 20 **DR. GEOFFREY SCOTT:**Okay, thank you. 

### 21 **DR. CHRIS BORGERT:**As a scientist, I'd love 

### 22 to have all the control data and all the test data. I only know 

### 23 one group that has that, it's EPA. 

### 24 **DR. DANIEL SCHLENK:**Dr. Green? 

### 25 **DR. SHERRIL GREEN:**Just so I can have some 

### 26 perspective on how long it took you to do that; so you had 18

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1 studies. How long did it take you once you got the data from 

### 2 the various companies to go through each of them, pull up the 

### 3 control tables and enter those data points in? Because doing 

### 4 that at this point, of course, would require the Agency's 

### 5 additional resources and take more time. But just to know, how 

### 6 long, roughly, for those 18 studies did it take you to prepare 

### 7 those slides that you showed today? 

### 8 **DR. CHRIS BORGERT:**The statistician got them 

### 9 a little more than a week ago. 

### 10 **DR. SHERRIL GREEN:**Okay, thanks. 

### 11 **DR. CHRIS BORGERT:**Now what took us the most 

### 12 amount of time is collecting the data and standardizing the 

### 13 spreadsheets, but I think EPA has those data. They can tell you 

### 14 exactly what they have. But once you actually have the data -- 

### 15 and this was done by a couple of people, to be fair. You've 

### 16 already heard from Allen Olmstead. He was one of the people 

### 17 that was involved in this evaluation, and another statistician 

### 18 John Green from DuPont, but they were both busy with many other 

### 19 things while they were doing this so it wasn't even a solid week 

### 20 of time, so I think this is trackable. 

### 21 **DR. SHERRIL GREEN:**Thank you. 

### 22 **DR. DANIEL SCHLENK:**Mr. Patino? 

### 23 **DR. REYNALDO PATINO:**Just a quick question in 

### 24 the plot that you show of hind limb length and snout-vent 

### 25 length. Are those data just for the control or including all 

### 26 the tadpoles, treatments included?

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1 **DR. CHRIS BORGERT:**Those are only controls. 

### 2 Those are only controls. 

### 3 **DR. RENALDO PATINO:**Thank you. 

### 4 **DR. DANIEL SCHLENK:**Anyone else? Okay. 

### 5 Thank you, Dr. Borgert. 

### 6 **DR. CHRIS BORGERT:**All right, thank you. 

### 7 **DR. DANIEL SCHLENK:**At this point let's go 

### 8 ahead and take our lunch break and let's be back here at 1pm and 

### 9 we'll continue our public commenting period at that time. 

### 10 (Whereas a lunch break was taken) 

### 11 **DR. DANIEL SCHLENK:**Okay. As we continue our 

### 12 public comment period, our next presenter will be Clare Thorp 

### 13 who represents CropLife America. 

### 14 **DR. CLARE THORP:**Good afternoon. CropLife 

### 15 America appreciates being able to address the Panel and the time 

### 16 that you have given towards evaluating all the information that 

### 17 you have been provided with. 

### 18 My name is Clare Thorp. I'm the Senior 

### 19 Director of Human Health Policy within CropLife America and I 

### 20 administer all committees within CropLife America. I administer 

### 21 the Endocrine Policy Forum but they are not actually part of 

### 22 CropLife America, they are truly independent, as you've probably 

### 23 realized. So my job in that regard is more like providing 

### 24 administrative support and backup. 

### 25 I also administer the Human Health and 

### 26 Risk-Assessment Committee with draws from the toxicologist

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1 risk-assesses, epidemiologists, medical toxicologists, 

### 2 ecotoxicologists and scientists that reside with an industry. 

### 3 I'm represented on the committee that I administer, and it is 

### 4 their comments and those of the EPF that I am reflecting here. 

### 5 Just to give you a little bit about CropLife 

### 6 America, CropLIfe America is a not-for-profit national trade 

### 7 organization. So by way of background, CropLife America members 

### 8 have played a critical role in providing input into the Agency's 

### 9 development of the Endocrine Disruptive Screening Program. 

### 10 They have participated since EDSTAC was 

### 11 established in 1996, and specifically, it is our member 

### 12 companies who have been the recipients of the Tier 1 test orders 

### 13 and their products were chosen precisely because of the rich 

### 14 dataset that they had in the fact that they could therefore 

### 15 provide some comparisons between the high level testing and the 

### 16 screening test. 

### 17 But essentially it is CropLife America member 

### 18 companies which have been the laboratory facilities and the 

### 19 research arm of the EPA for the Endocrine Disruptive Screening 

### 20 Program. And the members have conducted this work at their 

### 21 expense and they were requested to test the 52 compounds. 

### 22 And this data will be used for further 

### 23 evaluation but these compounds are already data-rich. And it 

### 24 seems unfortunate that due to the time-constraints that we feel 

### 25 that there has been very short timeframe for the SAP to 

### 26 adequately consider the material that was placed on the Docket.

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1 And we are disappointed that all 52 compounds have not been 

### 2 reviewed. 

### 3 I want to specifically address the charge 

### 4 question, question 8, which is to look at the minimal number of 

### 5 screens and tests necessary to make sound decisions that by 

### 6 reducing the time needed to make the decisions around these 

### 7 screens, and to also establish whether or not the current 

### 8 battery of 11 assays can be modified to reduce animal usage or 

### 9 lower cost while adequately ensuring the EPA's ability to answer 

### 10 the question of whether a chemical has the potential to interact 

### 11 with the endocrine system. And I chose that word, interact, 

### 12 that is important here. 

### 13 And so from that regard, I want to discuss in 

### 14 my talk the testing that pesticides already go through, and so 

### 15 the data which is already available, the context as you would 

### 16 have it within which these Tier 1 tests can be evaluated. 

### 17 So on average, a new pesticide product takes 

### 18 about 10 years to develop. And regulatory testing, which is for 

### 19 the purposes for registration involves approximately 110 tests 

### 20 per compound to meet the data requirements necessary for 

### 21 registration, which is extremely extensive. And in addition to 

### 22 the test required for registration, the EPA can require further 

### 23 testing when more data is required. 

### 24 But there is also pre-market development 

### 25 testing which identifies potential hazards, and it also guides 

### 26 whether or not that project will ultimately be put forward for

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1 testing for registration purposes. So it's a little surprise to 

### 2 our member companies here, for example, that there were not 

### 3 positive hits on the uterotrophic assay because that would've 

### 4 already been pre-screened prior to even conducting the test 

### 5 required for registration. 

### 6 So when we look at what actually happens, 

### 7 pesticides come up for review, and then every 15 years they also 

### 8 come up for registration review, which can require further 

### 9 testing. And as noted in our submission to the Office of 

### 10 Management and Budget, which is required to look at the cost of 

### 11 all of these things, we calculated that it's approximately a 

### 12 million dollars per chemical to complete all the 11 assays. And 

### 13 it also takes about more than two years to actually complete 

### 14 these assays. And this needs to be taken into consideration in 

### 15 terms of not just financial costs but man-hours and the number 

### 16 of experimental animals that are involved. 

### 17 Again, this is just to show you what the data 

### 18 requirements already cover, and this speaks to the presentations 

### 19 that were made earlier by Ellen Mihaich and by Lisa Ortego and 

### 20 by Chris Borgert around the data that is already provided on 

### 21 these chemicals when the information is submitted for 

### 22 registration. 

### 23 So you have information on the product 

### 24 performance and the physical and chemical characteristics and on 

### 25 exposure. And you already have chemicals that are extensively 

### 26 tested in multiple mammalian and wildlife species for potential

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1 affects on development, reproduction, normal growth and 

### 2 including the structure and function of reproductive organs and 

### 3 all other vital organs controlled by hormones. 

### 4 So the question that we really want to pose is 

### 5 whether or not the Tier 1 and 2 tests are in addition to all the 

### 6 existing tests or whether or not we should be looking at an 

### 7 integrated and complementary testing approach when it comes to 

### 8 pesticide compounds. 

### 9 Again, current toxicological testing, and this 

### 10 addresses some of the comments that were made earlier, looks at 

### 11 both acute, sub-chronic and chronic exposure of very wide range 

### 12 of doses and a long duration, including multigenerational 

### 13 testing on reproduction and fertility. 

### 14 If we actually pull the mammalian toxicology 

### 15 data requirements and the global requirements, and these 

### 16 companies are multi-national, so generally speaking, if it's 

### 17 required in one jurisdiction, it's applicable across all 

### 18 jurisdictions. If they have to test for one country then it 

### 19 doesn't matter if another country doesn't need it, they still 

### 20 have to do that testing. And so this is the information that 

### 21 already currently exists. 

### 22 And for example, the two-generation 

### 23 reproduction test covers animals prior to conception, throughout 

### 24 pregnancy, the off-spring at birth. The off-spring are 

### 25 continued through the testing and then they're mated and then 

### 26 they're dosed throughout their pregnancy and then their

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1 off-spring from them, the second generation are also examined. 

### 2 So throughout this study, there is an 

### 3 opportunity to examine what's happening through production and 

### 4 development from pre-pregnancy, through pregnancy and throughout 

### 5 the development of the off-spring in two generations. There is 

### 6 a lot of data there. 

### 7 And so this test is also listed and one of the 

### 8 Tier 2 assays, and results from this test also cover some of the 

### 9 Tier 1 endpoints obtained from a pubertal assay. Now, okay, 

### 10 this test was mandated in 1998, but before that there was, 

### 11 nonetheless, reproductive data available on pesticide compounds. 

### 12 So we are concerned about the automatic 

### 13 testing of pesticides in the Tier 1 which could lead to 

### 14 excessive, unnecessary and wasteful practices that offer no 

### 15 additional benefits to the protection of human health, the 

### 16 environment or knowledge of these compounds from an ability to 

### 17 interact with the endocrine system already. 

### 18 This is the ecotox stage of requirements. 

### 19 Again, there's rather a lot of them. These are the global ones. 

### 20 So therefore, when evaluating the potential of pesticide 

### 21 products for their potential to interact with the endocrine 

### 22 system, CropLife America would like to encourage that the 

### 23 following approach be adopted moving forward in the future. 

### 24 The Tier 1 and possibly Tier 2 tests for 

### 25 registered products should only be required where existing test 

### 26 results combined with physical-chemical characteristics,

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1 absorption, distribution, metabolism, and elimination 

### 2 characteristics, and exposure data indicate data needs with 

### 3 respect to the potential estrogenic, androgenic and 

### 4 thyroidogenic activity. 

### 5 The requirement to conduct specific Tier 1 

### 6 assays should be tailored to fulfill only those areas where 

### 7 there are additional data needs. And we support the idea of 

### 8 gatekeeper assays for identifying potential endocrine activity 

### 9 as outlined in the paper by Ankley and Gray from the Office of 

### 10 Research and Development and the EPA, and using this approach to 

### 11 determine the necessity for further, if any, endocrine-specific 

### 12 testing is required. I actually have a copy of the paper here 

### 13 but I've only got one, so if you want to see it, I can make it 

### 14 available. 

### 15 And again, as already elaborated on by the 

### 16 previous speakers, we would like to see a move towards an 

### 17 integrated testing and evaluation program, one that doesn't 

### 18 require test to be conducted by rote or by requirement, and 

### 19 which references all the existing data, the OSRI that we 

### 20 referred to earlier to identify potential endocrine activity and 

### 21 so that we can guide the selection of the endocrine-specific 

### 22 test. 

### 23 And this means that the need for additional 

### 24 testing should be determined on a case-by-case basis, selecting 

### 25 from the available Tier 1 screening assays that will complement 

### 26 and fill the data needs in the existing information.

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1 And so on this basis, screening for endocrine 

### 2 potential of chemicals can be done effectively with a reduced 

### 3 number of assays. But as you can tell from the previous 

### 4 presentations, there are some issues with some of the assays and 

### 5 some of them fulfilled certain requirements and others do not, 

### 6 so again, it requires this sort of case-by-case approach where 

### 7 you look at what you need. 

### 8 And again, we also support the idea of staging 

### 9 so the decision can be made on specific assays that may not be 

### 10 required, and therefore, may be waived. That waiving would be 

### 11 based on, again, OSRI or Other Relevant Scientific Data, 

### 12 including, again, exposure and also physical chemical properties 

### 13 and so on and so forth. This was gone into in a lot more detail 

### 14 by the Endocrine Policy Forum. 

### 15 And our other comment relates to selecting the 

### 16 dose level. If this is to be an effective screen and we are to 

### 17 actually understand whether or not the compounds are actually 

### 18 interacting with the endocrine system, then we need to get the 

### 19 dose level correct. 

### 20 And if the dose level is too high and it 

### 21 elicits overt toxicity, then we'll have confounding affects and 

### 22 we won't be able to elaborate whether or not this is truly 

### 23 something that's interacting with the endocrine system or 

### 24 whether or not something else is going on. Because stress on 

### 25 the model system, be it the live animals or the cells in 

### 26 culture, can actually impact on the results.

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1 And moving forward, these are the suggestions 

### 2 that we would like to make. And these are probably more policy 

### 3 rather than scientist-oriented but they reflect and they are 

### 4 based on the previous presentations. 

### 5 CLA, CropLife America, strongly recommends 

### 6 that the EPA does not issue new Tier 1 screening orders for 

### 7 additional substances which is on the List 2, before the Agency 

### 8 has evaluated the lessons that have been learned from the 

### 9 initial List 1 substances. 

### 10 And because this is such an important program, 

### 11 because this is a definitive defining program, this program is 

### 12 being looked at by the eyes of the world. This is not going to 

### 13 be confined purely within the boarders of the United States, but 

### 14 Europe, Brazil, Japan, all these other countries will be looking 

### 15 to this program to see how it works and how it's structured, and 

### 16 they will also be evaluating the data. 

### 17 And so we feel it's very important that it's a 

### 18 very robust and very strongly science-based program. And this 

### 19 is why we would like to have more time and also to make sure 

### 20 that all 52 compounds are properly considered. Because then you 

### 21 can truly stand on this program and say this is it, this is the 

### 22 gold standard and we have evaluated it thoroughly. 

### 23 And again, CropLife America strongly 

### 24 recommends that the EPA deliberately evaluate the Tier 1 

### 25 screening results alongside the assays, the functionally 

### 26 equivalent data and the other scientific relevant information to

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1 identify the most effective and efficient path forward, and that 

### 2 the experiences from the contract research organizations and the 

### 3 test order recipients, as articulated in the previous 

### 4 presentation, have made it clear that there is a need to closely 

### 5 evaluate the test method procedures and endpoints with an eye 

### 6 towards improving the program. 

### 7 And so certain methods need to be upgraded and 

### 8 their corresponding test guidelines revised before EPA proceeds 

### 9 with issuing additional Tier 1 test orders for List 2 

### 10 substances. And so obviously, we support the comments that are 

### 11 made by the Endocrine Policy Forum. 

### 12 But we'd also like to direct you to the TERA 

### 13 workshop that was spoken about by Mike Dourson earlier which was 

### 14 an independent workshop, and it has a wide variety of 

### 15 stakeholders, not just industry. And some of the lessons 

### 16 learned and the messages came out of that were very clear and 

### 17 consistent across all the various representations that were 

### 18 there. 

### 19 My final point is around communication and, 

### 20 again, around the global implications of this program. 

### 21 As you move forward to evaluate the Tier 1, 

### 22 it's very clear that these are intended to screen and they are 

### 23 intended to look at the interaction with the endocrine system. 

### 24 The further studies and the ones that I've already articulated 

### 25 that are already done look at those level and look at 

### 26 information that can then be put forward into a risk-assessment.

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1 2 And we commend the EPA for very carefully 

### 3 articulating the purpose of the Tier 1 tests, and we encourage 

### 4 them to continue with this as the results are released because 

### 5 we have a lot of concern that this data will be misused to 

### 6 incorrectly conclude that substances with positive results in 

### 7 one or more Tier 1 assays are endocrine disrupters when in fact 

### 8 they have an endocrine activity. And an endocrine disrupter by 

### 9 definition is a compound that causes an adverse affect, and this 

### 10 is not what the Tier 1 screens are there to identify. 

### 11 And so we encourage EPA to explicitly state 

### 12 that the results of Tier 1 screening do not indicate an 

### 13 impending hazard or risk to humans or ecological species. Thank 

### 14 you very much for your time. 

### 15 **DR. DANIEL SCHLENK:**Thank you. Any questions 

### 16 from the Panel? Dr. Horseman? 

### 17 **DR. NELSON HORSEMAN:**If I might ask, so I've 

### 18 characterized the history of the whole notion of endocrine 

### 19 disrupter. It arises out of observations about effects that 

### 20 are, let's say quasi physiological, because they're happening in 

### 21 animals that aren't overtly suffering toxicologically 

### 22 life-threatening sorts of events, and generally with substances 

### 23 that had gone through at least some sort of testing as similar 

### 24 to what you're describing for these registered chemicals. So I 

### 25 think we're all having to deal with the issue of where do you 

### 26 set that window so that you're not looking at things that are

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1 just toxicologically active versus things that may have these 

### 2 endocrine effects. 

### 3 So if you could just comment on the fact that 

### 4 endocrine disrupters have been discovered amongst compounds that 

### 5 had already been screened for overt toxicity and how that plays 

### 6 into this, or are these tests that you're describing here modern 

### 7 enough that they would've picked up these compounds if they 

### 8 didn't know about them. 

### 9 **DR. CLARE THORP:**Well that's a big question 

### 10 and one in which I think I would rather prefer to have addressed 

### 11 by the Endocrine Policy Forum. 

### 12 But I think what you're talking about is the 

### 13 difference between something that interacts with an endocrine 

### 14 system and gives an effect but does not necessarily produce an 

### 15 adverse effect or is not at the potency or the dose level that 

### 16 would cause an adverse effect to be the lead toxic effect, 

### 17 because the risk assessment that takes place takes account of 

### 18 which is the most sensitive endpoint and which is the most 

### 19 sensitive species. 

### 20 So if you're going to get an issue with a 

### 21 different adverse effect that kicks in way before an endocrine 

### 22 affect, you're not going to regulate based on the endocrine 

### 23 affect, you're going to regulate based on the other one. But I 

### 24 would prefer Katie to answer that. 

### 25 **Ms. KATIE HOLMES:**Hello, I'm Katie Holmes. I 

### 26 work for BSF and I am the Policy Chair of the Endocrine Policy

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1 Forum. That's a really good question and I believe Clare did 

### 2 much better with the answer than I thought she was going to -- 

### 3 not in a bad way, but -- 

### 4 Our older testing -- and I'm not talking about 

### 5 testing done in the '50s, but '80s, '90s into 2000s, we were 

### 6 under a risk assessment policy so any of the data that were 

### 7 generated, we were looking for the most sensitive endpoints. So 

### 8 we did see affects possibly on endocrine-related mechanisms but 

### 9 those weren't necessarily identified as such, if it wasn't the 

### 10 most significant endpoint. So what we're looking at now is just 

### 11 sort of defining a specific mode of action rather than looking 

### 12 for the most sensitive endpoint for the driving of the 

### 13 assessment. 

### 14 When you do a risk-assessment, if you have an 

### 15 effect that is well above any potential exposure, then that's 

### 16 where it stopped. So that's why maybe the endocrine mode of 

### 17 action wasn't identified specifically in some of the compounds. 

### 18 **DR. DANIEL SCHLENK:**Thank you. Any other 

### 19 questions? Okay, thank you. Let's move on. Our next presenter 

### 20 will be Scott Slaughter from the Center for Regulatory 

### 21 Effectiveness. 

### 22 **DR. SCOTT SLAUGHTER:**My name is Scott 

### 23 Slaughter and I am commenting today on behalf of the Center for 

### 24 Regulatory Effectiveness and I'd like to make two points in my 

### 25 comments. 

### 26 First, do not use the amphibian metamorphosis

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1 assay in the EDSP. There are too many unexplained problems with 

### 2 the test. 

### 3 Second, I assume everyone knows that EPA 

### 4 intends to abandon the EDSP and use ComTox and high-throughput 

### 5 screening if and when the Agency can validate ComTox and 

### 6 high-throughput screening. We think that the use ComTox and 

### 7 HTS, if they could be properly validated, is a very good idea, 

### 8 and we recommend that the Agency do that sooner rather than 

### 9 later, and do that rather than issue another round of test 

### 10 orders for the EDSP that the Agency no longer even what's to 

### 11 use. 

### 12 The amphibian metamorphosis assay -- which 

### 13 will be known as AMA because I have a hard time pronouncing 

### 14 amphibian metamorphosis assay -- we have a history with it, we 

### 15 being CRE. In 2008, CRE filed a request for correction under 

### 16 the Information Quality Act regarding the AMA. This request for 

### 17 correction asked EPA to correct the Agency's publicly 

### 18 disseminated statement that the AMA had passed peer review and 

### 19 was properly validated. 

### 20 CRE's request for correction pointed out that 

### 21 the AMA had received a generally negative peer review report and 

### 22 asked EPA not to use the AMA in the EDSP Tier 1 test battery. 

### 23 Pages 2 to 4 of our written comments that you 

### 24 have, I hope in front of you, list some of the peer review 

### 25 comments back in 2008, 2007, 2009 on the AMA. 

### 26 Just a few of them were -- and I'm quoting,

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1 "With regard to whether the AMA is reproducible, one peer 

### 2 reviewer stated in the peer review report, 'This is a major flaw 

### 3 of the material provided.'" He also explained that 

### 4 inter-laboratory inconsistencies obviously just one table of the 

### 5 AMA validation study "Would convince any reviewer or a reputable 

### 6 scientific journal to recommend rejection" of the validation 

### 7 study. 

### 8 He further stated, "The conclusions regarding 

### 9 inter-laboratory validity are not warranted and it -- the AMA 

### 10 test protocol -- fails as a method for accomplishing the stated 

### 11 goal of the assay to be part of the endocrine disruptive 

### 12 screening program." 

### 13 There are more peer review criticisms of the 

### 14 AMA before it became a part of the EDSP, which are included on 

### 15 pages 2 to 4 of our written comments, and there are also links 

### 16 to our request for correction on the AMA and EPA's response, 

### 17 which was basically to make a few minor changes to the AMA test 

### 18 guidelines and go ahead and use it anyway. 

### 19 Now it looks like CRE and the peer reviewer 

### 20 may have been right. EPA now admits that there are serious 

### 21 problems with the AMA based on the EDSP Tier 1 test data that 

### 22 the Agency had reviewed. What's worse, I think, and probably 

### 23 more disturbing is that no one has an explanation of why these 

### 24 problems are occurring. 

### 25 These problems include the bent tails, which 

### 26 had been discussed extensively today, and also the more rapid

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1 development in the controls. Until and unless somebody can 

### 2 explain and hopefully solves these problems with the tests, 

### 3 we've asked this SAP to advise EPA not to use the AMA test data 

### 4 for any purpose. There are just simply too many unexplained 

### 5 problems in the assay resulting in the generation of flawed and 

### 6 questionable data at this point. 

### 7 We also asked this SAP to advise EPA not to 

### 8 use the AMA as part of the EDSP Tier 1 test battery until and 

### 9 unless these unexplained problems are explained and hopefully 

### 10 solved by someone. 

### 11 With regard to our second point, I must say 

### 12 that this all seems to maybe a waste of time and money and 

### 13 animal lives because EPA plans to abandon the current EDSP Tier 

### 14 1 test anyway. 

### 15 EPA told congress, and I'm quoting from 

### 16 several agency documents, "In fiscal year 2012, EPA will begin a 

### 17 multi-year transition from the EDSP to validate and more 

### 18 efficiently use computational toxicology methods and 

### 19 high-throughput assays that will allow the Agency to more 

### 20 quickly and cost-effectively assess potential chemical 

### 21 toxicity." 

### 22 We think that's an excellent thing for the 

### 23 Agency to do. We recommend that they do it sooner rather than 

### 24 later, and we recommend that they do that than spend their time 

### 25 and scarce resources and valuable resources on that process 

### 26 rather than sending out more Tier 1 test orders.

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1 Thank you for the opportunity to comment and 

### 2 I'll try to answer any questions you might have. 

### 3 **DR. DANIEL SCHLENK:**Thank you. Any questions 

### 4 from Panel? Dr. Green? 

### 5 **DR. SHERRIL GREEN:**I have a question for you 

### 6 concerning the AMA test. To my knowledge, there aren't many 

### 7 toxicological tests that are required to be performed on 

### 8 amphibians that are aquatic that go through metamorphic climax 

### 9 like these frogs. And as you are probably aware, there is a 

### 10 worldwide amphibian decline. 

### 11 So whether or not this test would be useful as 

### 12 a gatekeeper, clearly, you know, we have some discussion to 

### 13 think more about that -- but if not this test, could you 

### 14 recommend from your perspective what else the Panel might 

### 15 consider that would protect this species, in particular, in 

### 16 addition to the many others in case they are exposed to -- 

### 17 **DR. SCOTT SLAUGHTER:**Xenopus -- I can't 

### 18 pronounce that one either. You're talking about the one that 

### 19 was tested in the AMA, the one that begins with X? 

### 20 **DR. SHERRIL GREEN:**Xenopus laevis. 

### 21 **DR. SCOTT SLAUGHTER:**I believe that Syngenta 

### 22 Corporation under the supervision of EPA and the supervision of 

### 23 another SAP, in fact several other SAP, has extensively tested 

### 24 Xenopus using test protocols and looking primarily at -- you 

### 25 know. I'm not sure necessarily thyroid but certainly endocrine 

### 26 effect, and developed a series of test results and a series of

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1 test guidelines which prior SAP endorsed. So yeah, there are 

### 2 alternatives out there that have been developed that I'm aware 

### 3 of. 

### 4 **DR. SHERRIL GREEN:**Which company? 

### 5 **Mr. SCOTT SLAUGHTER:**I believe it's Syngenta. 

### 6 They're represented here and they can speak for themselves, but 

### 7 I think that they were cited in the White paper and I believe 

### 8 EPA commented on that particular SAP in that particular test. 

### 9 Once again, I am not sure, in fact, I'm not 

### 10 sure at all that it will address the thyroid issues, but yes, 

### 11 other amphibian tests have been developed. 

### 12 And we're talking about initial screenings to 

### 13 begin with. And I think the HTS and the ComTox, I think most 

### 14 people would agree that if they could be properly validated and 

### 15 standardized and prove to be of sufficient quality for 

### 16 regulatory purpose, then that is a preferable alternative to the 

### 17 current EDSP Tier 1 test. If people in the audience disagree 

### 18 with what I just said, I'm sure they will come and tell you 

### 19 that. 

### 20 But, you know, they use far less animal lives. 

### 21 They're far less expensive in general, I think, and they're 

### 22 easier and they hopefully would not have some of the problems 

### 23 that we're seeing in the AMA. And once again, the disturbing 

### 24 part of these problems is -- well not to mention that we told 

### 25 you so back in 2008, but no one really has an explanation as to 

### 26 why they're occurring.

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1 You have a bunch of crippled tadpoles swimming 

### 2 around and no one can explain why the test that crippled them 

### 3 crippled them, you know? And until those questions are answered 

### 4 satisfactorily, I would think that it would -- this test should 

### 5 not be used for regulatory purposes. Anything else? 

### 6 **DR. DANIEL SCHLENK:**Anybody else? Okay, 

### 7 thank you. Our next presenter will be Patricia Bishop 

### 8 representing PETA. 

### 9 **Ms. PATRICIA BISHOP:**Okay. Good afternoon 

### 10 everyone, I'm Patricia Bishop. I'm a research associate in the 

### 11 regulatory testing division of People for Ethical Treatment of 

### 12 Animals, otherwise known as PETA. 

### 13 This is kind of a summary of written comments 

### 14 that I already submitted, so you should have everything that I'm 

### 15 going to talk about here already. And also, these comments were 

### 16 submitted both on the behalf of PETA as well as the Humane 

### 17 Society of the U.S. and of Physicians Committee for Responsible 

### 18 Medicine. 

### 19 So just from an animals' protection 

### 20 perspective, these are some of our main concerns and I'll go 

### 21 through them a little bit more in detail. 

### 22 First of all, the large numbers of animals 

### 23 using the battery, we've been following this program for pretty 

### 24 much since its inception due to the potentially massive numbers 

### 25 of animals that could be used with all the chemicals that need 

### 26 to be tested.

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1 Also additional animals used in the 

### 2 range-finding studies that have come to our attention after 

### 3 reading the White Paper, animals killed in the culling process, 

### 4 the fact that the in vitro binding studies actually do use 

### 5 animals as they rely on the rat cytosol, the uterine and the 

### 6 prostate cytosol that are collected. 

### 7 I just want to touch again -- I think one of 

### 8 the other speakers has already mentioned this, but the 

### 9 replacement for the ERTA assay using the BG1 Luc which might 

### 10 have some advantages, elimination of the Hershberger and 

### 11 Uterotrophic and tying the results back to mechanistic 

### 12 interpretation. 

### 13 So again, the large numbers of animals; if we 

### 14 just look at the test protocols alone we can see that based on 

### 15 some estimates for the binding assays that we got from one of 

### 16 the labs, looks like it's about, in their case, 13 rats used in 

### 17 the ER binding to test one chemical. Again, just to test the 

### 18 chemical. AR binding they use of about 10 rats, uterotrophic 

### 19 18, Hershberger doing both the agonist and antagonist 48, 45 

### 20 each in the pubertals and the fish, 96, 320 tadpoles, so a total 

### 21 of 595 animals to test one chemical in just the protocols alone. 

### 22 So for the 52 List 1 chemicals that we are 

### 23 talking about here today, if you tested them all, in the entire 

### 24 battery you would use about 31,000 animals, that's 31,000. Some 

### 25 of the chemicals did receive waivers based on submittal of the 

### 26 OSRI, and that ended up saving about 3300 out of 31,000.

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1 So the range-finding studies to estimate the 

### 2 maximum tolerated dose; so it appears that -- and I'm not sure 

### 3 about this because I couldn't really tell from reading the 

### 4 document, but it appeared that potentially there were more than 

### 5 one range-finding study done for the same chemical due to maybe 

### 6 differences in the ages and gender differences of the various 

### 7 tests. 

### 8 For example, the pubertals used animals that 

### 9 are quite a bit younger. The Hershberger/Uterotrophic uses I 

### 10 think eight-to ten-week old animals and the other ones use maybe 

### 11 three-week old animals. 

### 12 So for the pubertal, the male pubertal, there 

### 13 was 12 out of 16 chemicals were evaluated in range-finding 

### 14 studies. A few of the others used the part 158 pesticide 

### 15 studies to determine the maximum tolerated dose. But again, for 

### 16 the example for the pubertal, 144 animals would have been tested 

### 17 upon before the actual assay was even performed. 

### 18 So you start multiplying that by many, many 

### 19 chemicals. You can see, beyond the test protocols you're using 

### 20 even more and more animals to get this maximum tolerated dose. 

### 21 The three-week animals in the pubertal versus 

### 22 the eight- to ten-week old adults in the ovariectomize 

### 23 uterotrophic. 

### 24 So we're thinking is there some other way to 

### 25 determine the maximum tolerated dose because, obviously, there 

### 26 was many cases of overt toxicity that you've all been hearing

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1 about. So however, these range-finding studies we're working, 

### 2 it doesn't seem like in all cases they were coming up with 

### 3 concentrations where animals were not still being subjected to 

### 4 overt toxicity. 

### 5 So we're recommending perhaps development of 

### 6 some kind of standardized method that hopefully does not have to 

### 7 subject more animals to testing that could draw fully upon the 

### 8 toxicity data from the part 158 studies and any reliable 

### 9 literature studies that might be available and try to use that 

### 10 to develop these higher doses rather than subjecting animals to 

### 11 more toxicity studies. 

### 12 So the issue of culled animals, I think Sue 

### 13 Marty in her presentation touched on this a little bit. But 

### 14 it's obvious that the culling of animals in many cases, or at 

### 15 least in some cases, may actually use more animals than the test 

### 16 itself is using. So again, using the pubertals as an example, 

### 17 45 animals are what's actually used in the test protocol. But 

### 18 to get there, you end up culling out about 200 animals and this 

### 19 just gives you a quick rundown on how that's done. 

### 20 You need apparently at least 15 litters which 

### 21 consist of about 13 or 14 pups. You usually get a few more dams 

### 22 just because you want to make sure you get 15 that meet your 

### 23 needs. Then these litters are culled after about three or four 

### 24 days, I believe, discarding about 130 animals. And then 

### 25 finally, to get down to your 45, you get rid of another 60 plus 

### 26 the dams.

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1 And interestingly, you can't use any of the 

### 2 excess animals usually to run another compound because the 

### 3 necropsies all have to be done the same day or two and this runs 

### 4 into personnel issues to be able to do all these necropsies. 

### 5 So the in vitro binding studies, as I 

### 6 mentioned, ER and AR binding also use animals because you need 

### 7 to collect the cytosol to run test, and again 13 rats for the ER 

### 8 -- and that was just to run the chemical. And apparently I was 

### 9 told that other rats were needed to optimize method and 

### 10 standardize things and kind of get it working. Same issue with 

### 11 the AR binding, that even though 10 were needed to run the 

### 12 chemical, we had to use additional animals to get the method up 

### 13 and running. 

### 14 Our suggestion here is to do away with the 

### 15 cytosol ER and AR binding methods. There's human recombinant ER 

### 16 binding assays that are available that don't use animals at all. 

### 17 18 I guess EPA has been participating in an 

### 19 OEC-sponsored validation study and a test guideline is expected 

### 20 in 2014. So we would urge the EPA to start looking toward 

### 21 switching over to something like that and getting rid of these 

### 22 animal so-call in vitro studies. 

### 23 The ERT assay, again, somebody did already 

### 24 mention the BG1 LUMI-CELL ERTA assay that was recently 

### 25 validated. It's an OECD test guideline now. It worked 

### 26 apparently just as well as the current STTA ERTA assay that's in

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1 the Tier 1 and it also has the advantage of detecting 

### 2 antagonists. 

### 3 What is interesting about this is, in the 

### 4 validation study, this assay showed a 92 percent concordance 

### 5 with the Uterotrophic. 12 out of the 13 reference chemicals 

### 6 matched up. 

### 7 So we would like to propose that with the data 

### 8 that are now available from the first round of List 1 chemicals 

### 9 that perhaps we could do more comparisons with this particular 

### 10 ERTA assay and hopefully strive for an elimination of the 

### 11 Uterotrophic if this assay could give us essentially the same 

### 12 information. 

### 13 The Hershberger, certainly from an animals' 

### 14 perspective, we would like to see some more animal tests be 

### 15 eliminated. We think this could happen based on if the issues 

### 16 with the fish and the male pubertal assay could be resolved. 

### 17 That was the proposal in the EPA's White Paper that perhaps this 

### 18 particular test is giving redundant results. And assuming the 

### 19 other two could have the issues worked out with them we would 

### 20 certainly support elimination of Hershberger once all the issues 

### 21 of overt toxicity and those types of things are resolved. 

### 22 And then just finally, again, we've been 

### 23 hearing this of tying the sort of apical affects that have no 

### 24 supporting binding TA or steroidogenesis responses; what do we 

### 25 do with these? 

### 26 For example, there was a chemical B, it had a

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1 positive Hershberger with an overt toxicity. It had a positive 

### 2 fish assay but there was potentially solvent interference and a 

### 3 positive male pubertal but it had negative in vitro responses. 

### 4 So the question is how do you interpret something like this? Is 

### 5 it just an artifice due to the experimental conditions? Is it a 

### 6 non-androgen mediated affect? Is it androgen-mediated but it's 

### 7 not covered under Tier 1 or is it a metabolic activation thing? 

### 8 9 So by trying to get away from the more apical 

### 10 effects in vivo and, you know, moving on to an adverse outcome 

### 11 pathway or mechanism of action mode, which I think is -- you 

### 12 know, again, we've heard moving from ComTox and high-throughput 

### 13 assays, I think this is really something the program needs to 

### 14 push toward and maybe get rid of some of these questionable 

### 15 results that we're seeing with the in vivo assays. 

### 16 This would also, I think, help to explain some 

### 17 of the odd patterns of complementarity and redundancy that we've 

### 18 seen and also the nonconcordance between what were supposed to 

### 19 be redundant assays. And that's it. 

### 20 And just to remind you all, you do have the 

### 21 Panel's charge question number 8 which is how to potentially 

### 22 minimize or reduce the use of animals in the Tier 1 assay, so 

### 23 hopefully I've given you a little bit of insight on maybe some 

### 24 responses to that. Thank you. 

### 25 **DR. DANIEL SCHLENK:**Thank you. Any 

### 26 questions? Okay. Let's move on to our next presenter. This

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1 will be Catherine Willett from the Humane Society. 

### 2 **DR. CATHERINE WILLETT:**Okay. Thank you very 

### 3 much for the opportunity to comment. I'm Catherine Willett. 

### 4 I'm currently at the Humane Society where I work on regulatory 

### 5 testing, risk-assessment and alternatives. And I also 

### 6 coordinate something called the Human Toxicology Project 

### 7 Consortium, which is a group of companies and other stakeholders 

### 8 who are interested in moving through a more predictive 

### 9 mechanism-based paradigm for toxicity testing. 

### 10 Well one of the benefits or hazards of going 

### 11 later in the commenting round is that a lot of what you have 

### 12 intended to talk about has already been spoken of. So some of 

### 13 this I can go through rather quickly. Let's just get straight 

### 14 to what I want to talk about. 

### 15 So let's talk about the animal use. Pat went 

### 16 through this in great detail so I don't need to do that. But 

### 17 roughly, there's about 600 animals per chemical and that, to 

### 18 remind you, does not count range-finding, repeats and anything 

### 19 else. That's just the bare minimum number of animals in each 

### 20 protocol. 

### 21 And the reason that's important is maybe 600 

### 22 animals doesn't sound like a lot to people, but the results of 

### 23 that test will lead to Tier 2 testing which can start to really 

### 24 impact the body count in a serious way. I don't know exactly 

### 25 what the numbers are yet because the protocols are not 

### 26 finalized, but this is a sort of gross approximation of how many

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1 animals will be used in the Tier 2 tests. 

### 2 And again, as Pat said, so far in the program, 

### 3 the 52 chemicals, about 30,000 animals have already been used. 

### 4 And when you consider that these are for evaluating pesticides 

### 5 in HPB chemicals, there's probably going to be very little 

### 6 actual information that's going to be used in a regulatory 

### 7 nature that comes out of this program. The body count starts to 

### 8 look like a tragedy in some respects. And I said this only to 

### 9 emphasize that I think it's critically important that we use 

### 10 this information to the maximum extent possible to help improve 

### 11 this program. 

### 12 And there is ample evidence that a better 

### 13 approach is warranted, one, some of them are listed on this 

### 14 slide. This program as we've been hearing is extremely 

### 15 resource, labor and animal intensive. It provides a lot of 

### 16 data, but only some of this data may have any regulatory 

### 17 applicability. 

### 18 The current structure of the program provides 

### 19 insufficient opportunity for chemically-relevant tailoring. We 

### 20 heard Clare talk about that extensively, and other people have 

### 21 suggested some other tiering approaches that might be more 

### 22 efficient. 

### 23 And critically, also, it does not link the 

### 24 mechanism to adverse outcome. You know, this idea really wasn't 

### 25 prevalent when the program was first conceived, but in 

### 26 intervening a long time a lot has been learned about this, and

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1 also, the value of moving toward this approach in terms of being 

### 2 able to create a predictive paradigm for toxicological 

### 3 assessment. 

### 4 And this current structure is not predictive. 

### 5 And in that way, we will never be able to progress in our 

### 6 ability to assess a chemical using less and less information, 

### 7 but we can if we have a different structure create something 

### 8 that will help us learn how to be predictive. 

### 9 Okay. So the first step toward doing this 

### 10 might be to use a more refined tiered system that we've heard 

### 11 suggested, and there are many different structures. I'm going 

### 12 to emphasis one particular structure. It would be nice if the 

### 13 initial tier took into account all existing information 

### 14 including physiochemical and exposure. 

### 15 A second tier would assess potential 

### 16 mechanisms of action. That's kind of where we are. I mean 

### 17 that's sort of what the Tier 1 currently intends to do but 

### 18 doesn't fully do. And then subsequent tiers would address the 

### 19 potential affects in more complex systems or on perhaps multiple 

### 20 modes of action. And then finally, the highest tiers would 

### 21 begin to address adverse outcomes and dose response from which 

### 22 you would be able to do a risk-assessment. 

### 23 And this tiered system would also apply a 

### 24 weight of evidence with clearly articulated criteria at each 

### 25 tier, so you can define positive and negative cut-offs for each 

### 26 tier level and then use the results to modify and design a

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1 strategy for further testing. And this way, the strategies 

### 2 designed to address information needs as well as the chemical 

### 3 activities. 

### 4 This has been articulated already by the OECD 

### 5 in the OECD conceptual framework, which is also been brought up 

### 6 previously today, and this was not conceived as a testing 

### 7 framework. However, it provides a logical approach to how one 

### 8 organizes information in a decision context. So depending on 

### 9 what level of decision you make, you need a certain level of 

### 10 information to make that decision. 

### 11 And this just puts into table form what I was 

### 12 talking about and it adds also the existing test guidelines into 

### 13 each one of these levels. And the good thing about using this 

### 14 kind of conceptual framework is that, as methods are developed 

### 15 and validated, you can add them to this conceptual framework. 

### 16 So it's sort of a way to phase in transition between where we 

### 17 are with these, like where we are now with this, and a future 

### 18 move away from the higher level testing. 

### 19 Okay. And then this is something that can be 

### 20 done relatively quickly, if not immediately; just a different 

### 21 way of organizing your testing procedure. 

### 22 We ran through how you might do this in a 

### 23 publication that we published in Toxci using Atrazine as an 

### 24 example, because at the time Atrazine had a full Tier 1 dataset. 

### 25 So we were able to sort of pretend that maybe you didn't have 

### 26 the information to go through this process and then make

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1 decisions based on the different levels of information and see 

### 2 what kind of testing you may have been able to eliminate using 

### 3 this approach. 

### 4 Okay, next. So this is where I think we're 

### 5 going. I think everybody agrees that this is where we're going 

### 6 to go more toward a pathway-based approach. And this helps with 

### 7 interpretation at every single level, would help with 

### 8 interpretation of the information you currently have to deal 

### 9 with now in the Tier 1. 

### 10 So adverse outcome pathways are basically 

### 11 conceptual construct that organizes the information in such a 

### 12 way that you would describe it from the direct molecular 

### 13 initiating event all the way to an adverse outcome in all of the 

### 14 intervening steps in between the different degrees of detail, 

### 15 depending on what you have. I'm just going to go quickly 

### 16 through some of the stuff that already exists. 

### 17 So EPA has been working on an ER immediate and 

### 18 reproductive impairment AOP for a number of years, which they 

### 19 have integrated into development of an integrated testing 

### 20 strategy, which I'll show here. This was mentioned earlier 

### 21 today. This is the ER SAR that someone mentioned earlier. They 

### 22 have a really very good SAR model for looking at chemicals that 

### 23 have weak to no binding. 

### 24 So this is the AOP and these are the kinds of 

### 25 tests right now that we have that can query the different levels 

### 26 of organization in the adverse outcome pathway. This is our

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1 current state of science. Of course the goal would be to, as 

### 2 you get more information and you can add more precision to these 

### 3 early steps and start to add linkages between these steps, you 

### 4 will progressively become more dependent on these early steps 

### 5 and less dependent on these later steps. 

### 6 But right now the way it goes is you do in 

### 7 vitro measurements or predictions of the binding. You can look 

### 8 at gene activation, vitellogenin, and you can start to look at, 

### 9 in liver slices, ex-vivo. You can look at changes in proteins 

### 10 and gonadal histopathology. And then you can also look in vivo 

### 11 in whole fish to see what's occurred and also the behavior and 

### 12 whether or not you get an effect on fecundity. 

### 13 In terms of ER and AR pathway efforts, there 

### 14 are a number in a variety of different organizations. So US EPA 

### 15 has worked on this to a large degree. As I mentioned, the ER 

### 16 AOP and high-throughput and other in vitro assays that they're 

### 17 looking that might satisfy the same pathway. 

### 18 The Hamner Institute has a project called Tier 

### 19 1 and Done, which is an estrogen receptor pathway their looking 

### 20 more globally at estrogen receptor. 

### 21 CAAT has a project called Pathways of 

### 22 Toxicity, similar thing. They're looking at bioinformatics and 

### 23 how that informs estrogen receptor pathways. The OECD 

### 24 Validation Management Group, Non-Animal is looking at the 

### 25 validations, so they're much more into the validation of assays 

### 26 themselves. And there's another group at OECD, an Advisory

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1 Group on Molecular Screening and Toxicogenomics which is also 

### 2 involved in AOP developments. 

### 3 Thyroid pathways are less well-developed and 

### 4 more complex. However, there are still some efforts that are 

### 5 designing these. Several things going on at the OECD. And 

### 6 also, the US EPA has a couple of case studies where they've been 

### 7 able to design AOPs based on chemicals to help in the 

### 8 risk-assessment for those chemicals. 

### 9 Okay. So I'm not going to go through this in 

### 10 detail but to get from where we are to creating this more 

### 11 predictive paradigm based on pathway understanding, there are a 

### 12 lot of things that are needed. But my point here is that all of 

### 13 these things are in development. This is moving forward. This 

### 14 is where we're going eventually and it would be nice if the EDSP 

### 15 could be structured in such a way that it could both feed into 

### 16 this process and allow the flexibility that it could be adapted 

### 17 to this process readily as it moves forward. 

### 18 So the current design needs to be improved. 

### 19 Let's change it using a more refined tiering system, combine 

### 20 with weight of evidence and then eventually allow progression to 

### 21 a pathway-based approach for all of this. 

### 22 **DR. DANIEL SCHLENK:**Okay. Quick questions 

### 23 from anyone? Okay, thank you. Our next presenter will be 

### 24 Colleen Toole representing CeeTox. 

### 25 **DR. COLLEEN TOOLE:**Good afternoon. My name 

### 26 is Colleen Toole. I'm a Director of Project Management at

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1 CeeTox. CeeTox is a small contract research organization 

### 2 located in Kalamazoo, Michigan. 

### 3 We've had lots of experience with these EDSP 

### 4 assays, specifically the five in vitro assays because we are an 

### 5 in vitro-only laboratory. We have also been doing endocrine 

### 6 screening and endocrine in vitro assays for 10 years since the 

### 7 company was first founded. We've run over 90 of these in vitro 

### 8 studies following the EDSP guidelines and we run approximately 

### 9 30 of these test materials that were on List 1 through our labs. 

### 10 So I want to touch on a couple of subjects 

### 11 that have sort of been tossed around in the group and bring some 

### 12 clarity to some of the things that were in the White Paper. One 

### 13 is the performance standards. So these are the performance 

### 14 standards for the ER binding assay. We're looking at a 

### 15 test-tube based assay, radioactive gold standard for in vitro 

### 16 binding assays. It's been around for decades. 

### 17 You can see estradiol on the left, and then on 

### 18 the right is the norethindrone in the weak positive. And you 

### 19 can see the shift in the IC50. 

### 20 When we talk about small changes in the white 

### 21 paper that went on with these performance standards, what you 

### 22 might see is the top plateau level at 112 versus 111. You might 

### 23 see the hill slope being minus 1.2 versus 1.1. Those are the 

### 24 types of small changes that they're talking about. If it was 

### 25 larger in magnitude, what we would have done is repeated the 

### 26 runs. So that just sort of outlines where I was going with the

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1 ER and those performance standards, and I'll get into the ERTA 

### 2 one soon. 

### 3 But comparison of the AR and the ER binding 

### 4 assays -- because this was brought up by Steve a little bit 

### 5 earlier -- there is differences in the guidelines because 

### 6 they're so specific, so they're both test-tube based assays. In 

### 7 the ER, you're going to use 100 micro liters of your cytosol 

### 8 prep. In your AR, you're going to use 300 micro-liters. And 

### 9 what happens is you're making up your assay into 300 

### 10 micro-liters and then you're removing a hundred micro-liters, 

### 11 putting that into a new test-tube, and you're discarding your 

### 12 200 micro-liters, so there's a lot of waste going on. 

### 13 And so what we'd like the guidelines to be is 

### 14 a little bit more free with the science, so that if we wanted to 

### 15 miniaturize this assay and put it into -- not test-tubes but 

### 16 maybe some 96 well-glassed tubes as something that's higher 

### 17 thru-put, we have the capability and the science behind it. We 

### 18 could still follow the performance criteria and allow us the 

### 19 freedom as a lab to do a little bit of that and upgrade the 

### 20 assays ourselves. 

### 21 Another thing that comes out of this is 

### 22 storage conditions for these assays. And this is, again, 

### 23 getting into the specificity of the guidelines. 

### 24 For the ER you can store the cytosol at minus 

### 25 80 for up to 90 days and then you have to re-characterize. But 

### 26 for AR, the guidelines say you can store and you have to discard

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1 after six months. Well that was a lot of work in characterizing 

### 2 the cytosol. You're running a protein determination, and then 

### 3 you're running three runs of your saturation binding and then 

### 4 you're going in to running competitive binding assays. 

### 5 So it's not trivial to prep this cytosol, and 

### 6 we certainly don't want to waste any of it because of the animal 

### 7 use too. And so these are just a couple of things to consider 

### 8 as we're looking at the guidelines. 

### 9 So one of the things that come out in the 

### 10 binding assays is these sharp decreases at these high 

### 11 concentrations, and I bring this up because the high 

### 12 concentrations have been mentioned a couple of times today, how 

### 13 relevant are they. And when you're looking at an in vitro 

### 14 system, you're going to start to get artifacts when you push the 

### 15 system out to these higher levels and concentrations of test 

### 16 materials. 

### 17 Susan Laws put out a paper a few years ago 

### 18 where you can look at these sharp decreases of these high 

### 19 contractions that fall like this in these non-monotonic curves 

### 20 and you can perform these Ki experiments. And I'm not going to 

### 21 go into the Ki experiments but it does go into talking about 

### 22 some of the issues that we face as a lab, even with the negative 

### 23 controls. 

### 24 And so basically we're going to determine 

### 25 these false positives by increasing concentrations of your 

### 26 tritiated E2 in the presence of your test material. We're going

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1 to construct Lineweaver-Burke plots and then we're going to 

### 2 replot these versus concentration of the test material. 

### 3 So I just wanted to show you what a true 

### 4 binding event looks like. You can see that all the trend line 

### 5 intersect at the same point. When you replot the data you get a 

### 6 nice linear slop. For a false positive what we see is this 

### 7 doesn't intersect at the same point and you get a nonlinear slop 

### 8 here. 

### 9 So when we start looking at things like the 

### 10 negative control that sometimes will fall out of solution 

### 11 because we have solubility issues here, it will look like a 

### 12 binder but we know it's not. There's going to be some variation 

### 13 in lab day to day. There's going to be some temperature 

### 14 changes, and we can see that we have solubility issues at this 

### 15 high concentration. And this is just a comparison of the curves 

### 16 for the atypical response with the negative control versus the 

### 17 false positive response. 

### 18 The next assay I want to talk about a little 

### 19 bit is the ERTA because we did have some challenges when we look 

### 20 especially at the 17 alpha-methyltestosterone. 

### 21 These are the performance criteria, so you 

### 22 have a strong positive, a weaker positive, your negative and 

### 23 then your weak positive. If you're looking at the logPC50, 

### 24 PC10, EC50, your hill slop, and these are the test range. 

### 25 So I just went back through the data and 

### 26 picked 10 runs at random and plotted it for E2. You see a nice

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1 dose-response curve. But what happens with 17 

### 2 alpha-methyltestosterone that makes it hard to reach these 

### 3 criteria is you basically have no response and a response, so 

### 4 it's not even a dose-response, it's all or nothing. And so you 

### 5 get small changes in that line and you're going to fall outside 

### 6 of these criteria. 

### 7 So what we do as an in vitro lab is we always 

### 8 keep track of all our historical data. We track this. And so 

### 9 what I would suggest is now we have lots of data from multiple 

### 10 labs and from multiple compounds. We're running these three 

### 11 times for each compound usually, and so we've got a lot of data 

### 12 that we could then go back and look at and build some more 

### 13 robust criteria around these controls, and I think that would 

### 14 really help the assays. The assays are not bad themselves. I 

### 15 think they just need more historical data and to firm up some of 

### 16 these points that we're looking at. 

### 17 So I know the BG1 assay has been talked about 

### 18 a couple of times now. Four our contract research lab we're 

### 19 looking at $10,000 to purchase that cell line and just get it up 

### 20 and running in the lab. So that differs a lot from the HeLa 

### 21 cell line which we can purchase for $500 and get up and going. 

### 22 When you're looking at in vitro assays you 

### 23 have to know your assay system. This system with the HeLa cell 

### 24 line is geared towards detecting false positive. And the reason 

### 25 I say that is because we have an introduced ER alpha as a 

### 26 plasma; so you've got high concentrations of ER alpha and the

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1 machinery of the cell just can't shut things down. The 

### 2 co-activators and co-repressors are just not there in levels 

### 3 that are going to really truly represent an in vivo response. 

### 4 And so that might be too wide, we see some 

### 5 discrepancies in the data. But I'll also remind you we're just 

### 6 looking at agonist and agonist activity here. So what's being 

### 7 picked up by an ER binding event has ER alpha and ER beta 

### 8 isolated from these cytosol preps. They're not always going to 

### 9 match. And if it's antagonism that's going on causing the 

### 10 binding event, we're not going to pick that up in this assay 

### 11 because we don't run antagonist. 

### 12 Stability of the cell line, what we're looking 

### 13 at here is this red line, a four-fold induction for the positive 

### 14 control. And so what I just wanted to show you is, is this is 

### 15 pretty stable over time. We can get induction up to 80 and 

### 16 normally we're probably around 20. 

### 17 But we're well over signal Denoise with this 

### 18 assay. It is a robust assay but there's limitations to it. And 

### 19 with every in vitro system, there's going to be limitation and 

### 20 in every in vivo, and we just have to understand this systems. 

### 21 Steroidogenesis can also be a challenge and 

### 22 this was discussed a little bit earlier today too. But in the 

### 23 guideline, 22R hydroxy cholesterol, which feeds in early in the 

### 24 pathway can be used according to the guideline at concentrations 

### 25 between 20 and 40 micromolar. 

### 26 You can see at 40 micromolar we've got a huge

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1 cytotoxicity issue. We've lost 60 percent in viability. Even 

### 2 at 20 micromolar, you can see that when we look at gene 

### 3 expression, you can see at 10 micromolar right before we're 

### 4 having viability issues, we see some increases in transcription. 

### 5 And so we'd backed off to 10 micromolar because this is good 

### 6 science and this is where we run our assays. 

### 7 So as far as laboratory proficiency goes, I 

### 8 want to give people a little bit of comfort in that, as a lab we 

### 9 do take great care and pride in what we're doing for every 

### 10 client that we have. These are GLP studies. We've been audited 

### 11 not only by the EPA but by our clients. 

### 12 And so our laboratory staff are proficient. 

### 13 They have to be to run these assays. They have to show they're 

### 14 proficient by running the proficiency compounds through the 

### 15 specific assays, and these are documented and put into their 

### 16 training records. There was no indication from the guidelines 

### 17 that we had to report those proficiency data in the reports that 

### 18 we were giving for our test chemicals. 

### 19 The DESTs were only available after our 

### 20 studies were completed. So at that point we were onto the next 

### 21 compounds, because there's five in vitro assays -- we're a small 

### 22 lab -- we didn't have time to go back at that time to finish up 

### 23 the DESTs. And we have now finished the DESTs for some of our 

### 24 clients who wanted to pay for that service. 

### 25 So there are some remaining challenges. One 

### 26 thing that would be helpful, especially if we're looking forward

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1 to List 2, is to have some sort of technical support available 

### 2 at the EPA for individual questions; and whether they can triage 

### 3 that to someone who can answer the question. It would just be 

### 4 helpful for us as a laboratory conducting these assays. 

### 5 Alternative vehicles are going to be a bigger 

### 6 issue on the second list. We had some compounds that were not 

### 7 soluble in any of the solvents listed in the guidelines, and 

### 8 those are the things that we would like to have more rapport 

### 9 with the EPA, more timely rapport to figure out how we should 

### 10 handle those, because it's not just running it into an alternate 

### 11 vehicle. And what we chose to do is, was now we have to run all 

### 12 the reference compounds in both DMSO and the alternative 

### 13 vehicle. What if we don't pass the performance criteria in the 

### 14 alternative vehicle? So these are things, as you know, as a lab 

### 15 doing laboratory work, we're always going to face. 

### 16 Outlier analysis is another thing that I 

### 17 wanted to briefly touch on because this could be why we fell 

### 18 outside of performance criteria by these small amounts. We're 

### 19 not removing any outliers. The validation study from the major 

### 20 lab that did the work for ERTA didn't have any edge effects. We 

### 21 do. We can see them, but we don't know really what freedom we 

### 22 have to remove outliers. We know they're there. We know it's 

### 23 edge effects, and we've reformatted our plates to address this, 

### 24 but we still see it, and how to acknowledge that and what we 

### 25 need to do that would be acceptable from the EPA. 

### 26 I touched on this before but revise the

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1 performance criteria. We now have more labs, more data. You 

### 2 know, can we build 95 percent confidence limits around the 

### 3 curves that we have, and then miniaturization of the assays 

### 4 because we feel pretty confident we could miniaturize some of 

### 5 these assays and get higher throughput and still get good data. 

### 6 And so with that, I'll take any questions. 

### 7 **DR. DANIEL SCHLENK:**Any questions from the 

### 8 Panel? Okay, Dr. Scott? 

### 9 **DR. GEOFFREY SCOTT:**Yes. I just wanted to 

### 10 follow-up on your comment about proficiency testing. You do 

### 11 maintain those records in your laboratory, I understood you. 

### 12 **DR. COLLEEN TOOLE:**Yes. 

### 13 **DR. GEOFFREY SCOTT:**But what you said is that 

### 14 was not always requested initially; is that correct? 

### 15 **DR. COLLEEN TOOLE:**It was not requested in 

### 16 any of the guidelines. However, when the DESTs came out 

### 17 afterwards they did ask for that information, but that was after 

### 18 we had finished a lot of studies for our laboratory. 

### 19 **DR. GEOFFREY SCOTT:**I thought that's what you 

### 20 said, I just wanted to make sure. Thanks. 

### 21 **DR. DANIEL SCHLENK:**Dr. McManaman? 

### 22 **DR. JAMES MCMANAMAN:**So can you briefly 

### 23 comment about the variability in the ERTA assays? It looks like 

### 24 you've got a maximum stimulation that ranges from 75 percent to 

### 25 over 200 percent. And so how would you assess a compound with 

### 26 that much variability in response?

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1 **DR. COLLEEN TOOLE:**It's all based upon 

### 2 positive control and relative to positive control. At that 

### 3 point, we have a robust signal. We're not downing the noise. 

### 4 In these transactivation assays, you'll always see some 

### 5 variability in that direction. It's not a perfect system. I 

### 6 don't make too much of it because it's just kind of some 

### 7 differences across the plate. 

### 8 **DR. JAMES MCMANAMAN:**But if you're looking 

### 9 for a 10 percent change to be a positive response and your 

### 10 maximum on that particular assay is 75 percent, that's going to 

### 11 be difficult to see. I think that's what the performance 

### 12 standards were for, for the ERTA assays, is looking at 10 

### 13 percent of the maximum response. 

### 14 **DR. COLLEEN TOOLE:**Oh, the maximum response, 

### 15 which is a hundred percent. So that is actually running one of 

### 16 the reference. So your positive control is just run at the one 

### 17 nanomolar, E2. And then you get your response curves from your 

### 18 dose response on the rest of the plate, so that's just the 

### 19 little bit of difference between the size of the plate from the 

### 20 positive control. 

### 21 **DR. JAMES MCMANAMAN:**Yeah, it looks like it 

### 22 could make a big difference in how much the signal is going to 

### 23 be, which could lead to some -- I could foresee that could lead 

### 24 to some problems with assessing reliability of an assay. 

### 25 **DR. COLLEEN TOOLE:**Well as long as your fold 

### 26 induction is high enough and you're not lost in the noise -- to

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1 Steve's point, what he was thinking is if you're just looking at 

### 2 a four-fold induction or a four-fold change, that's pretty low. 

### 3 So looking at 10 percent of that is pretty small. 

### 4 **DR. JAMES MCMANAMAN:**Right, so it could be 

### 5 problematic. 

### 6 **DR. COLLEEN TOOLE:**It could be. 

### 7 **DR. DANIEL SCHLENK:**Okay. Any other 

### 8 questions? Okay, thank you. 

### 9 **DR. COLLEEN TOOLE:**I'm sorry, I have one more 

### 10 comment. So as you all have the difficult job of next steps, 

### 11 what I would encourage you to evaluate is, looking at all the 

### 12 test chemicals -- 

### 13 **DR. DANIEL SCHLENK:**I'm sorry, you've kind of 

### 14 already exceeded your ten minutes. 

### 15 **DR. COLLEEN TOOLE:**All right, thank you. 

### 16 **DR. DANIEL SCHLENK:**Yeah, thanks. Our final 

### 17 presenter is Leah Zorrilla from Integrated Laboratory Systems. 

### 18 **DR. LEAH ZORRILLA:**I'd like to thank you for 

### 19 the opportunity to participate in this discussion. My name is 

### 20 Leah Zorrilla. 

### 21 I'm representing Integrated Laboratory 

### 22 Systems. I contract laboratory organization that participated 

### 23 in the conduct of the List 1, Tier 1 EDSP in vivo mammalian 

### 24 assays. 

### 25 So my objective is to really quickly discuss 

### 26 the in vivo conduct and performance from a CRO perspective.

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1 Our recent experience involved running 18 

### 2 chemicals in the Uterotrophic assays, 21 chemicals in the 

### 3 Hershberger bioassays, 11 chemicals in the female pubertal and 

### 4 10 chemicals in the male pubertal assays, and most of which of 

### 5 those were submitted to the Agency for evaluation. In addition, 

### 6 we have conducted over 30 range-finding studies to select a high 

### 7 dose according to the individual assay definitions for the 

### 8 maximum tolerated dose levels. 

### 9 For all test substances, more time and 

### 10 resources were required than originally anticipated to select 

### 11 and evaluate the appropriate test substance vehicles, along with 

### 12 performing dose formulation stability studies. This extended 

### 13 the timeframe of the studies by approximately 20 days prior to 

### 14 the initiation of the assay conduct. In addition, choosing the 

### 15 appropriate maximum tolerated dose for each of these assays took 

### 16 time and additional resources, which you've heard, which 

### 17 involved conducting range-finding studies that were not 

### 18 initially anticipated in the original timeline of the study 

### 19 conduct. And this really became difficult for the CROs because 

### 20 there weren't many of us that were able to conduct these assays, 

### 21 and the deadlines were pretty strict for submission of these 

### 22 reports. 

### 23 The range-finding study that I had last 

### 24 performed used age-matched animals and obtained daily body 

### 25 weights at clinical observations. And for the pubertal assays, 

### 26 we also evaluated liver and kidney weights to set those maximum

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1 tolerated dose levels. 

### 2 There were still studies in which the high 

### 3 dose level selected did exceed the maximum tolerated dose as 

### 4 ascribed and that has been well discussed here at this meeting. 

### 5 And as discussed in the white paper, though, it did make some of 

### 6 these findings difficult to interpret when there was a greater 

### 7 than 10 percent body weight loss. 

### 8 However, overall, the pubertal assays were 

### 9 conducted with ease. I did want to bring up several 

### 10 considerations that might be helpful in clarifying and refining 

### 11 the assays. 

### 12 As discussed, the performance criteria for the 

### 13 pubertal assays were overall met. However, now that several 

### 14 laboratories have historical controlled data from the 

### 15 performance of these assays, it would really be important to 

### 16 utilize the facility historical controlled data ranges, in 

### 17 addition to those available from the EPA guidelines. 

### 18 Another point, the liver is weighted in the 

### 19 pubertal assays, but it would be helpful to retain the liver in 

### 20 the event that the test chemical induces the enzyme activity 

### 21 which may help provide clarification and interpretation of 

### 22 selected endpoints. 

### 23 In terms of cost-savings, analysis of the 

### 24 clinical chemistry panel, which is a full clinical chemistry 

### 25 panel, adds costs to the conduct of these pubertal assays. And 

### 26 it's not clear that this has really been of value to study

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1 interpretation when range-finding studies have been performed. 

### 2 In our experience, these data did not provide any additional 

### 3 information to be observed, the body weight loss or clinical 

### 4 observations for the selection of the maximum tolerated dose 

### 5 and/or the definitive study interpretations. 

### 6 And finally, it would be useful for the 

### 7 pubertal guidelines -- if clarifications could be provided in 

### 8 regards to the appropriate covariate for the ANCOVA analyses, 

### 9 either the date of weaning, which is postnatal day 21 or the 

### 10 actual first day of dose substance administration which is PND 

### 11 22 for the females and 23 for the males. The guidelines, the 

### 12 DESTs and the SEPs have different days, so clarification would 

### 13 be great. 

### 14 In regard to the Uterotrophic assay, currently 

### 15 the maximum tolerated dose in the Uterotrophic assay guidelines 

### 16 is defined as a dose to ensure animal survival without 

### 17 significant toxicity or distress. And this is how maximum 

### 18 tolerated dose levels were chosen for this first test level, at 

### 19 least in our laboratory. And this is different from how it was 

### 20 defined in the white paper, as it suggests that greater than 10 

### 21 percent body weight loss should be taken into consideration when 

### 22 evaluating the study. 

### 23 Although the other three in vivo assays do use 

### 24 this criterion as a greater than 10 percent body weight loss in 

### 25 consideration in choosing the maximum tolerated dose, the 

### 26 Uterotrophic assay guidelines do not. And this really needs to

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1 be clarified moving forward as choosing the MTD based on a 10 

### 2 percent body weight loss or animal survival are different 

### 3 things. 

### 4 An additional consideration in the 

### 5 Uterotrophic assay is the oral gavage level of ethinyl 

### 6 estradiol, the positive control. The effective dose level is 

### 7 much higher in adult OVX animal than it is in the immature 

### 8 model. However, overall the performance criteria for this assay 

### 9 is easily met for both the immature and OVX models. 

### 10 The Hershberger bioassay was also easily 

### 11 conducted. However, there are two study considerations from our 

### 12 laboratory's experience. 

### 13 One would be to ensure the animals are 

### 14 sexually mature prior to castration and place and placed on to 

### 15 study so that all the tissues can be accurately evaluated. 

### 16 And the second is to weigh and retain the 

### 17 liver in these Hershberger assays to assess hepatic enzyme 

### 18 induction, particularly if the need arises for a follow-up study 

### 19 to interpret potential anti-androgenic effects. Overall, the 

### 20 performance criteria were easily met for these assays. 

### 21 So I'd like to summarize by saying that the 

### 22 four mammalian in vivo assays were easily conducted and 

### 23 performance criteria overall were met. 

### 24 The major challenges really involved meeting 

### 25 timelines for study conduct and submission that did not consider 

### 26 the time involved in selected the formulation, conducting

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1 formulation stability studies, which are required for GOP 

### 2 submission, and the dose level selections involve conducting 

### 3 these range-finger studies. And again, really, that all added 

### 4 time with the limited amount of time that we were given in order 

### 5 to submit these studies to the Agency. Thank you. 

### 6 **DR. DANIEL SCHLENK:**Thank you. Dr. Selvage, 

### 7 question? 

### 8 **DR. DANIEL SELVAGE:**I have a couple of 

### 9 questions particularly in running the pubertal assays. I didn't 

### 10 notice in the guidelines where it said that during necropsy, you 

### 11 were looking at the gut at all. I mean I'm a little concerned 

### 12 these (inaudible) are being introduced by gavage. So did you 

### 13 look at the stomach at all when you're doing necropsy? 

### 14 **DR. LEAH ZORRILLA:**For particular chemicals 

### 15 in which -- 

### 16 **DR. DANIEL SELVAGE:**Yes. 

### 17 **DR. LEAH ZORRILLA:**Yes, we did. 

### 18 **DR. DANIEL SELVAGE:**You have, okay. 

### 19 **DR. LEAH ZORRILLA:**But that was really test 

### 20 substance specific. So depending upon the information that our 

### 21 clients had prior to, you know, they would say "This might be a 

### 22 stomach irritant, potential irritant, so we would specifically 

### 23 have to follow just necropsy to look at the potential effects 

### 24 that were present. 

### 25 **DR. DANIEL SELVAGE:**Okay, but it's not 

### 26 standard?

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1 **DR. LEAH ZORRILLA:**It's not standard and it's 

### 2 not in every -- that's correct. 

### 3 **DR. DANIEL SELVAGE:**Another question in terms 

### 4 of the female pubertal assays, and this is kind of an opinion 

### 5 question, do you think something could be gained by, instead of 

### 6 taking the rats at day 42 or day 43, taking them for instance on 

### 7 their first estrous or synchronizing estrous cycles and taking 

### 8 them all the same day of the cycle? Do you think that might 

### 9 give you more stable results? 

### 10 **DR. LEAH ZORRILLA:**Right. I think it could 

### 11 add value to the study. It depended how long you wanted to 

### 12 continue the dose, which would also add cost to the study. But 

### 13 depending on the affects, in theory, you'd be able to -- you 

### 14 know, if you necropsied on the first day of diestrous, for 

### 15 example, it would make measuring hormone levels a lot easier I 

### 16 think. 

### 17 You would get rid of some of the variability, 

### 18 I think. And then you'd also have a longer timeframe to 

### 19 evaluate potential estrous cycle disruptions if there were. I 

### 20 actually published a paper on that a couple of years ago, but 

### 21 hormone levels, we didn't really see any significant differences 

### 22 when we did necropsy on the same day of the cycle. 

### 23 **DR. DANIEL SELVAGE:**Okay, thanks. 

### 24 **DR. DANIEL SCHLENK:**Dr. Green? 

### 25 **DR. SHERRIL GREEN:**Do you know what the 

### 26 original intent was for drawing blood to evaluate clinical

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1 chemistries, to look for what, liver and kidney enzymes? 

### 2 **DR. LEAH ZORRILLA:**Yes. So originally it was 

### 3 for BUN and creatine evaluations. And I think it makes sense if 

### 4 you're not doing a range-finder, and perhaps you don't see a 

### 5 body weight loss but there might be some toxicity. 

### 6 But then really, when you got into what the 

### 7 guidelines requested, it actually requested a full panel, and so 

### 8 it quickly added -- I mean a significant amount of money for 

### 9 what perhaps might not be adding a lot of scientific value. 

### 10 **DR. SHERRIL GREEN:**And there were no hormone 

### 11 analysis that were added to it or part of a -- because what a 

### 12 typical screen is can vary from lab to lab and what it includes, 

### 13 you know, T3, T4 or estradiol or things like that. 

### 14 **DR. LEAH ZORRILLA:**Well for the female 

### 15 pubertal, T4 and TSH are required, and any other hormone would 

### 16 be optional. Most of our clients decided not to do the optional 

### 17 hormone analyses. And in the males, it was T4, TSH and 

### 18 testosterone were required. 

### 19 **DR. SHERRIL GREEN:**Okay, thanks. 

### 20 **DR. DANIEL SCHLENK:**Any other questions? 

### 21 Okay, thank you. 

### 22 **DR. LEAH ZORRILLA:**Thanks very much. 

### 23 **DR. DANIEL SCHLENK:**Okay. This concludes 

### 24 our oral presentations of public comments. I do have an email 

### 25 that was received today that we'll read into the record real 

### 26 quick.

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1 "Dear Dr. Jenkins. I've just been scanning 

### 2 the proposed agenda for this week's SAP meeting, would 

### 3 like to contribute a remark relating to charge question 

### 4 8, how to minimize animal usage. In our view, a 

### 5 significant reduction in animal numbers and animal 

### 6 welfare could be achieved by allowing a different 

### 7 approach to the ER and AR binding assays allowing the 

### 8 use of recombinant cell lines in place of the current 

### 9 requirement for cytosol from rat uterus or rat 

### 10 prostate. 

### 11 The people we have working on these studies at 

### 12 Hunting Life Sciences at CRO are finding that for an AR 

### 13 assay, they are using the cytosol from about 60 male 

### 14 rats, all of which have to undergo surgical removal of 

### 15 testes 24 hours before necropsy to remove prostate. We 

### 16 consider this number of animals per in vitro test, 

### 17 ex-vivo in effect, highly undesirable when there are 

### 18 good cell line based assays available. 

### 19 I have seen no mention of this issue in EPA 

### 20 commentaries seen so far so I think it is worth drawing 

### 21 attention to it. 

### 22 Now that there is an extensive database of 

### 23 compounds tested with the current ER and AR ex-vivo 

### 24 designs, it would seen an opportune point at which to 

### 25 do a comparative validation with selected compounds in 

### 26 the cell line assays. Thanks for your attention and I

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1 hope the meeting goes well." 

### 2 And this was from Will Davies (phonetic) who's 

### 3 a consultant at LSR Associates in the U.K. 

### 4 Okay. With that, this concludes the public 

### 5 commenting period and let's take a 15-minute break. We'll be 

### 6 back here at twenty till. 

### 7 (Whereas a break was taken) 

### 8 **DR. DANIEL SCHLENK:**Okay. Mary, you want to 

### 9 go ahead and read into the record question 1? 

### 10 **DR. MARY MANIBUSAN:**Charge question 1: Based 

### 11 **on analyses of the data presented in Section III:**Please 

### 12 comment on the proficiency of the contributing laboratories to 

### 13 execute each assay in accordance with the test guidelines and 

### 14 achieve the performance criteria. 

### 15 **DR. DANIEL SCHLENK:**Thank you. The lead 

### 16 discussant for question 1 is Dr. Roby. 

### 17 DR. KATHERINE ROBY: Okay. Each test has an 

### 18 established guideline, and the guidelines define test 

### 19 parameters, include criteria for assessing testing proficiency 

### 20 and also include criteria defining acceptable limits for various 

### 21 positive and negative controls to be included in most tests. 

### 22 Thus, general proficiency can be monitored at several endpoints. 

### 23 Based on the information presented in Section 

### 24 III, and overall assessment at a high level would indicate that 

### 25 the testing laboratories demonstrated proficiency in carrying 

### 26 out the battery of tests. However, there were variations across

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1 laboratories in the ability to achieve proficiency criteria 

### 2 raising some concerns. 

### 3 At least some of the variation observed across 

### 4 testing laboratories is likely due to slight variation in 

### 5 methodology, interpretation or followthrough of testing guidance 

### 6 such as when solubility became an issue, laboratories not 

### 7 shifting to the recommended half dose log increment but instead 

### 8 shifting to a full dose and adding an additional dose at the 

### 9 lower end of the dosing series. 

### 10 Additional variation may also be introduced 

### 11 based on individual laboratory conditions such as the ability to 

### 12 maintain temperature control in the fish test. In addition, 

### 13 variation within a laboratory may occur based on the proficiency 

### 14 of individuals performing the test, and thus, individual 

### 15 performance criteria for each analysis should be documented and 

### 16 recorded over time. 

### 17 With the data that was provided in Section 

### 18 III, it was difficult at times to determine what laboratory 

### 19 tested what chemicals. In addition, within the description of 

### 20 the test given within the description of each individual test, 

### 21 at places there was discrepancy with regard to the number of 

### 22 testing labs that were included in that test and the numbers of 

### 23 chemicals tested; there are places within the text that do not 

### 24 agree that made some interpretation of the information 

### 25 difficult. 

### 26 Further, it appears that each chemical was

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1 tested by only one lab, possibly having more than one lab test 

### 2 the chemicals or even just a subset would provide an additional 

### 3 monitor for proficiency. 

### 4 With many of the in vitro assays, statements 

### 5 were often made within the white paper indicating that the 

### 6 testing laboratories did not provide complete proficiency data 

### 7 to the Agency. Incomplete datasets limit the ability of the 

### 8 Panel to carefully assess the proficiency in these instances. 

### 9 For example, with the steroidogenesis and the aromatase assays. 

### 10 If this is not clear within the guidance, it needs to be made 

### 11 clear. 

### 12 With the in vivo assays, it is not clear how 

### 13 many laboratories participated in each test, and thus, comments 

### 14 about multiple laboratories, proficiency cannot be made. 

### 15 Further, the apparent variation around established test criteria 

### 16 together with the limited data provided within the White Paper 

### 17 make interpretation of the outcomes difficult, and then 

### 18 interpretation of laboratory proficiencies sometimes difficult. 

### 19 In regard to the aquatic tests, data again is 

### 20 not presented by lab, and thus, variation across laboratories or 

### 21 within laboratories cannot be judged. As with the other Tier 1 

### 22 tests, the aquatic test had variation around performance 

### 23 criteria. Similar to the other tests, a clear definition of the 

### 24 acceptable limits are needed for these tests. Although it is 

### 25 understood, especially with the aquatic tests and across tests, 

### 26 the acceptable deviation may differ.

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1 To the point of variation around testing 

### 2 criteria; in many cases, the established criteria were not met, 

### 3 but they were not met by what would be considered a slight 

### 4 reminder variation. If analyses indicated, as stated in the 

### 5 White Paper, slight deviation from the established criteria did 

### 6 not affect interpretation of the study outcomes, the guidance 

### 7 performance criteria should be revisited. 

### 8 It's further suggested that the Agency 

### 9 establish a quantitative definition for the variation around 

### 10 specific performance criteria, thus avoiding the more 

### 11 non-descript slight and minor terminology. 

### 12 An example might be defining minor and major 

### 13 nonconformities and then define quantitatively what these two 

### 14 ranges of deviation from the norm include. This may be based on 

### 15 a percentile deviation such as a minor being less than 10 

### 16 percent and a major being greater than 10 percent. It may also 

### 17 be a statistical measure such as plus or minus one or two 

### 18 standard deviations. 

### 19 Some specific criteria should be established 

### 20 to aid in the identification of unreliable test runs. In 

### 21 addition, more specifically in regard to the CVs reported in the 

### 22 in vivo studies, criteria defining acceptable performance CVs 

### 23 may need to be revisited in order to account for the variations 

### 24 existing in controlled populations. 

### 25 In addition to further defining 

### 26 nonconformities, it's considered that a decision tree approach

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1 would allow for a clear assessment of when test criteria have or 

### 2 have not been met and when a given test can be accepted or 

### 3 rejected or should be rerun. 

### 4 For example, when negatives controls included 

### 5 in a test run are read as positive, as indicated in at least one 

### 6 instance in the data summary that was provided, the run should 

### 7 likely be rejected. In addition, guidance on how to consider 

### 8 data in the presence of cytotoxicity and toxicity should be 

### 9 included. 

### 10 Although the majority of the deviation from 

### 11 proficiency criteria was not considered to impact the 

### 12 interpretation of data, there were some deviations that may be 

### 13 cause for concern. In regard to the fish short-term 

### 14 reproductive bioassays, there are major deficiencies in dosing 

### 15 conformance. If this is due to analytical limitations, 

### 16 absorption or biodegradation needs to be determined. 

### 17 There is some concern that if laboratories 

### 18 alter the rate of volume replacement, the total volume of 

### 19 chemical delivered per aquaria per day will be different. This 

### 20 would introduce a source of variation and effect. The 

### 21 parameters of the protocol should assure that the initial target 

### 22 nominal dose is being delivered. An additional measure in an 

### 23 aquaria not containing fish may be recommended, although it is 

### 24 understood this would add additional cost. 

### 25 In questions earlier with the EPA, it was 

### 26 mentioned that there are routines stock concentration

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1 measurement requirements, as well as daily flow determinations. 

### 2 3 This combination of measurement should be 

### 4 reported. It can also then be used to predict estimated nominal 

### 5 concentration being delivered throughout each bioassay, and this 

### 6 would at least provide an accurate estimate of the delivered 

### 7 nominal dose and without having to directly measure it. Also, 

### 8 it's important to use only one stock for dosing purposes. 

### 9 The second concern with the fish assay relates 

### 10 to the cumulative affects of several non-conformities, including 

### 11 low dissolved oxygen levels, elevated exposure temperatures, and 

### 12 elevated levels of chlorine. 

### 13 Fish will respond to these changes with 

### 14 increased ventilation, which may damage fish gill and may also 

### 15 result in increased up-take of the chemical being tested. These 

### 16 three non-conformities for performance criteria are easily 

### 17 corrected and participating labs should be taking steps and 

### 18 should have taken steps to assure compliance within these 

### 19 criteria. In addition, there are a couple of concerns raised 

### 20 with the amphibian metamorphosis assay. Again, we've discussed 

### 21 these today. 

### 22 First, related to the tail curvatures, the 

### 23 high rates of this observed in controls and exposure groups does 

### 24 raise concern as to the health of the animals being used in the 

### 25 bioassay. There are obvious issues with the abnormality being 

### 26 tied to diet as well as the source of the test organisms. EPA

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1 should examine the underlying health of the frogs post 

### 2 metamorphosis since the tail is resorbed during the 

### 3 developmental process. 

### 4 If the abnormality does not affect the growth, 

### 5 health and survival later in life, this issue may not be as 

### 6 significant as it would be if the animals are affected later in 

### 7 life. Also, maintaining accurate records of the source of the 

### 8 frogs is a critical performance criteria to maintaining good 

### 9 recordkeeping. 

### 10 The second issue is related to the potential 

### 11 affects of carriers used in relation to chemical solubility. 

### 12 The performance criteria presented clearly demonstrate that it 

### 13 is possible to discern the affects of carriers used within the 

### 14 assay. The question, however, is how to use the data or how not 

### 15 to use the data when a carrier effect is observed, and the EPA 

### 16 should formally address this within the performance criteria. I 

### 17 think that's it for me for now. 

### 18 **DR. DANIEL SCHLENK:**Thank you. Our first 

### 19 associate discussant is Dr. Ehrich. 

### 20 **DR. MARION EHRICH:**I think everything I had 

### 21 is already in what she said. I have no further comment. 

### 22 **DR. DANIEL SCHLENK:**Thank you. Dr. 

### 23 Maclatchy? 

### 24 **DR. DEBORAH MACLATCHY:**I have no additional 

### 25 comments. 

### 26 **DR. DANIEL SCHLENK:**And Dr. Scott?

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1 **DR. GEOFFREY SCOTT:**All of my comments were 

### 2 well captured by the lead discussant. 

### 3 **DR. DANIEL SCHLENK:**Thanks. Other Panel 

### 4 members for question 1? Dr. Green? 

### 5 **DR. SHERRIL GREEN:**I would like to second the 

### 6 recommendations of the working group about the achievement of 

### 7 appropriate doses in the aquatic environment. The same would 

### 8 hold true for Xenopus laevis tadpoles in their tanks. Thanks. 

### 9 **DR. DANIEL SCHLENK:**Any other comments for 

### 10 question 1? Okay. Let me go to the Agency. Any questions or 

### 11 clarifications regarding the comments of the Panel? 

### 12 **DR. CATHERINE AUBEE:**This is Catherine Aubee. 

### 13 Thank you for your comments and advice. I'd just like to ask 

### 14 Dr. Roby to clarify your last recommendation regarding the 

### 15 interpretation of effects when there is apparent solvent 

### 16 interference in this study. I believe that's what you were 

### 17 discussing. 

### 18 Could you just clarify what exactly you were 

### 19 recommending? Are you recommending that additional criteria be 

### 20 added to the guideline or are you just recommending that some 

### 21 sort of formal analytical framework be set forth? I would 

### 22 appreciate more information on that. Thank you. 

### 23 **DR. DANIEL SCHLENK:**Dr. Scott? 

### 24 **DR. GEOFFREY SCOTT:**If I could, I'll answer 

### 25 that but I'm not sure if I could. I think what we were asking 

### 26 is if it's in the protocol already, how to address that, that's

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1 fine. I could not discern from the document if that information 

### 2 is in the protocol in a prescriptive way or in a general 

### 3 guidance way. 

### 4 If it's in a general guidance way, then I 

### 5 think you may want to make it a little more prescriptive as to 

### 6 how you address that and that was really the intent there. It's 

### 7 not that you guys are ignoring it; we know you're looking for 

### 8 it, you reported it. 

### 9 So I think what we're really asking is if you 

### 10 haven't given more specific criteria, you may want to think 

### 11 about that just to help. Because I think we heard from several 

### 12 of the labs today about the issues on some of this. 

### 13 **DR. CATHERINE AUBEE:**Thank you. 

### 14 **DR. DANIEL SCHLENK:**Thanks. Dr. Patino, you 

### 15 have something to add? 

### 16 **DR. REYNALDO PATINO:**Yes, follow-up. 

### 17 **DR. DANIEL SCHLENK:**Okay. 

### 18 **DR. REYNALDO PATINO:**We had a little bit of 

### 19 discussion on this yesterday and I expressed an opinion that I 

### 20 wanted to reiterate today, that if there is a solvent effect, I 

### 21 think the proper comparison of effects of the compounds is 

### 22 against the solvent control, not the native control. I just 

### 23 want to repeat that comment. Thank you. 

### 24 **DR. DANIEL SCHLENK:**All right. Let me go 

### 25 around again one more time. Anybody else have any other 

### 26 comments for question 1? Okay. Ready to move on to question 2?

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1 **DR. MARY MANIBUSAN:**Charge question number 2. 

### 2 The performance criteria for each in vitro assay are clearly 

### 3 stated in the test guidelines for the ER binding, AR binding, ER 

### 4 alpha Transcriptional Activation, H295R steroidogenesis and 

### 5 aromatase human recombinant assays. 

### 6 Although contributing laboratories did not 

### 7 always demonstrate that results were within the specified 

### 8 boundaries of the performance criteria, the majority of the 

### 9 deviations were still close to the performance criteria. In 

### 10 this regard, the EPA concluded that the data was still adequate 

### 11 for use. 

### 12 Please comment on the EPA's conclusion. 

### 13 Please comment on when a deviation from the recommended 

### 14 performance criteria would render the study unreliable. 

### 15 **DR. DANIEL SCHLENK:**Lead discussant for 

### 16 question 2 is Dr. Schultz. 

### 17 **DR. TERRY SCHULTZ:**Thank you. Clearly the 

### 18 answer to question 2 has to be linked to the comments that came 

### 19 out of question 1, so we asked the Agency to look at the answers 

### 20 as being provided to question 2 in the context of what has been 

### 21 provided already in question 1. 

### 22 It was felt by the Panel that question 2 

### 23 related to the first purpose of this review of Tier 1 assays, 

### 24 specifically the evaluation of how well each Tier 1 assay test 

### 25 guideline can be consistently executed based on defined 

### 26 performance criteria and to highlight any issues associated with

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1 the interpretation of responses within each assay. 

### 2 So what the Panel has done is addressed each 

### 3 of the five assays separately, and then we have some additional 

### 4 comments which are kind encompassing all of those. The Panel 

### 5 has added some boilerplate to define the understanding of that 

### 6 and I will briefly refer to that and then remind you to read 

### 7 that in more detail. This is simply to note that the Panel 

### 8 understands how the question was being asked. 

### 9 So the Panel understands that the endocrine 

### 10 disruptive screening program White paper on Tier 1 screening 

### 11 assay and battery performance -- hereafter I would approach that 

### 12 just as the White Paper -- consists of a battery of 11 assays, 

### 13 however, only five of these assays are defined as in vitro 

### 14 assays, and we list those five. 

### 15 The Panel understands that the Tier 1 

### 16 screening by plan consist of a battery of less complex in vitro 

### 17 and in vivo assays designed to effectively and efficiently 

### 18 detect the potential of a chemical to interact with any of the 

### 19 three hormonal pathways noted, that is estrogen, androgen and 

### 20 thyroid. And that based (inaudible) analysis will determine 

### 21 what chemicals will be candidates for the Tier 2 testing. 

### 22 In contrast to the Tier 2 testing, which 

### 23 involves more complex in vivo tests, the Tier 1 basically is the 

### 24 screening that lies between a T0 or the prescreen or a 

### 25 prioritization step, and the more definitive Tier 2 that's 

### 26 associated with the risk-assessment, so that there are some

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1 trade-offs associated with that. 

### 2 Specifically, that the Tier 1 assays by design 

### 3 are shorter-term, lower cost -- that's a relatively term 

### 4 obviously -- designed to minimize false negatives, basically 

### 5 report the potential to interact in a yes or no format and use 

### 6 limited dose regimes, especially at the higher end. With that 

### 7 statement, basically the Panel is trying to couch our responses 

### 8 in the way that we think that the question was being presented. 

### 9 Each one of the Tier 1 assays which formed the 

### 10 basis for this scientific Panel was previously validated with an 

### 11 onset of referenced compounds and I believe that was reported in 

### 12 2009, and that each one of these has gone through a methods 

### 13 development, a pre-validation and some sort of inter-laboratory 

### 14 validation. 

### 15 So that all of this was basically put together 

### 16 and culminated in the panel of 2008, which basically reviewed 

### 17 this and came up with the proposal of using these 11 assays, and 

### 18 then to identify this in this two-step process looking at some 

### 19 chemicals that have known mechanisms of affecting endocrine 

### 20 disruption. And then the second step is screening a series of 

### 21 chemicals that fit under the legislative mandate from FIFRA 

### 22 where the effect on endocrine pathways is unknown. So the Panel 

### 23 clearly understands how that came about. 

### 24 The Panel also understands that in evaluating 

### 25 the performance of the assays within this SAP that it is 

### 26 difficult to separate that from the weight of evidence, since in

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1 the document that laid this out from 1998 it talked about the 

### 2 different steps. However, we realized that the weight of 

### 3 evidence is not part of this SAP. 

### 4 With all of that said, I'm sure that is 

### 5 agreeable for all the questions. We just want to get that on 

### 6 the record. 

### 7 The Panel would like to congratulate the U.S. 

### 8 Environmental Protection Agency on providing the summary the 

### 9 results for the 21 compounds brought forth as part of this SAP. 

### 10 11 The Panel also thanks the public commenters 

### 12 for their timely input because it clearly, in many cases, 

### 13 address specific issues associated with the 11 assays, and these 

### 14 were being provided by people that had on-hand experience with 

### 15 these assays, and that's a great help. 

### 16 The Panel realizes that the 21 test compounds 

### 17 represents the 50 active and 2 inactive or inert ingredients 

### 18 that's been screened as part of the Tier 1 battery as an 

### 19 intermediate step towards the recommendation of 2008 that 

### 20 basically somewhere between 50 and 100 chemicals be screened in 

### 21 that sort of situation. And that basically this is following 

### 22 the recommendation of the 2008 SAP. 

### 23 While we understand what the Agency said, that 

### 24 the 52 chemicals were selected from the Tier 1 test to be 

### 25 representative in regards to the physical chemical properties in 

### 26 modes of toxic action from the specific pesticides, we're

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1 unclear why these 21 were selected out of the 52. Were they the 

### 2 first ones that information came in on, and those types of 

### 3 situations? But we believe that, as the Agency said, it is 

### 4 likely that the 21 chemicals reflect the spectrum of issues that 

### 5 you would have with all 52. 

### 6 The Panel realizes that while the assays in 

### 7 total were designed to identify whether a chemical has potential 

### 8 to interact with an estrogen, androgen or thyroid hormone 

### 9 system, the in vitro assays, because of the nature of their 

### 10 endpoints, focus only on the estrogen and androgen systems. 

### 11 Furthermore, the Panel understands that not all in vitro data 

### 12 and in vivo data are of equal value. And the in vitro data may 

### 13 or may not be informative of results from the in vivo assays. 

### 14 Specifically, the Tier 1 in vitro assays are 

### 15 intended to provide some mechanistic data for a single known 

### 16 pathway for endocrine disruption while the in vivo Tier 1 assays 

### 17 are more amenable to detecting multiple mechanisms of action. 

### 18 So whether a particular in vitro assay had a negative or a 

### 19 positive or non-response compared to another assay was less 

### 20 critical at this point. 

### 21 As noted in Table 1 of the white paper, the in 

### 22 vitro assays in the current Tier 1 test batteries addressed only 

### 23 the ANE systems, but they quantify key endpoints that are most 

### 24 likely to be mode of actions that are associated with the 

### 25 receptor binding and steroidogenesis, so it's much more narrowly 

### 26 defined.

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1 I'm going to skip reading the discussion that 

### 2 is in here about the specific assays that basically is in the 

### 3 White Paper, and I'm not sure I can say all the letters and 

### 4 number combinations correctly and it'll just cause you 

### 5 confusion. 

### 6 But basically we're talking about the estrogen 

### 7 system, binding and the gene expression, and we're talking about 

### 8 the androgen system, aromatase and steroidogenesis. All of that 

### 9 is rather obvious. 

### 10 Okay. I was just scrolling through all this 

### 11 stuff to make sure that I kind of captured all that. Obviously 

### 12 there are several paragraphs in there, but they're just pulled 

### 13 out from the documents. 

### 14 ER binding. As noted in the White Paper, 19 

### 15 neurethrone (phonetic) -- did I say it correctly? Somebody over 

### 16 here should nod. They know what I'm talking about -- okay -- 

### 17 which is basically replacement for the negative weak positive 

### 18 control ligand. And that was used in the assay. 

### 19 Since the guideline doesn't contain any 

### 20 performance criteria for this particular weak positive, the 

### 21 Panel recommends the Agency establish performance criteria for 

### 22 this chemical since this chemical seems to be used universally 

### 23 in place of the initially recommended positive weak control. 

### 24 The Panel agrees that ER binding assay 

### 25 performance was, by and large, consistent across all 18 

### 26 chemicals tested and that general issues were consistent with

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1 individual runs. 

### 2 In addition, while a number of the evaluated 

### 3 assays did not meet all the performance criteria, overall, the 

### 4 laboratory performance of the ER binding assays were acceptable. 

### 5 Again, it was noted the use of the word slight would better be 

### 6 served if performance guideline could be readdressed to 

### 7 particular values in there. And as was mentioned earlier, the 

### 8 idea of 10 percent or less being acceptable and greater than 10 

### 9 percent being unacceptable was listed as an example. 

### 10 One Panel member felt that the most 

### 11 problematic aspect of the ER binding assay is use of a highly 

### 12 complicated and labor-intensive protocol for the extraction of 

### 13 the uterine cytosol or different laboratories and had varying 

### 14 success, and recommended that the agency look into standardizing 

### 15 this parameter for the assay. And this kind of leads into the 

### 16 email that was mentioned that might look into replacing this 

### 17 with a recombinant assay. 

### 18 The Panel also feels the results and 

### 19 conclusions of this assay are supported in the weight of 

### 20 evidence by the Agency's work on their prioritization, in 

### 21 particular, their in silico methods which involved work on the 

### 22 rainbow trout ER binding assay, and that the work here on the 

### 23 cytosolic assay should not be looked at just in isolation, but 

### 24 should be looked at in regards to all the information that's 

### 25 available on this. 

### 26 Again, and in particular, look at the report

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1 from the January 2009 SAP on the prioritization. 

### 2 ER alpha transcriptional assay, obviously this 

### 3 assay as noted in the White Paper basically looks at the 

### 4 activation of transcription estrogen dependent reporter gene 

### 5 protocol. It's obviously valid for detection of estrogen 

### 6 agonist. 

### 7 As noted in the White Paper, the response 

### 8 curve parameters for the weak estrogen/androgen were met in only 

### 9 five of the 19 chemicals. Since the outer range of values were 

### 10 very close to the test guideline ranges for the majority of 

### 11 these outliers, the Panel recommends the Agency look into 

### 12 revising the guideline ranges for these control concomitant with 

### 13 reviewing whether the issue are within laboratory performance, 

### 14 and that's minimizing the need to revise the guideline and focus 

### 15 more on the laboratory compliance within the guidelines. 

### 16 The Panel generally agrees that the majority 

### 17 of the deviation for the transcriptional activation assays can 

### 18 be considered minor. However, some Panel members raise the 

### 19 issue concerning that cytotoxicity observed with a couple of the 

### 20 laboratory experiments. 

### 21 I think this is, again, a reflection of a 

### 22 general comment that we see that's putting the Agency between a 

### 23 rock and a hard place because one time we're asking them to do a 

### 24 full range of solubilities to the testing and at the same time 

### 25 we ask them to back off when they get into the cytotoxicity. 

### 26 And it's hard to do one without the other, but perhaps the

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1 weight of evidence issue will give us some guidance or clearance 

### 2 on this. 

### 3 Moving on to AR binding; the AR binding assay 

### 4 as noted by the Agency is basically using radioactive labeling 

### 5 and is considered to not be able to distinguish between agonist 

### 6 and antagonist. 

### 7 The Panel agrees with the Agency with the 

### 8 findings that overall the laboratory performance and the AR 

### 9 binding assays were generally acceptable, and that the data for 

### 10 the 17 chemicals that were tested were reliable. The Panel 

### 11 agrees the results demonstrate that AR binding assay as 

### 12 performed can distinguish between chemicals that bind to the 

### 13 androgen receptor in vitro. That was about all. 

### 14 Steroidogenesis; again, steroidogenesis 

### 15 pathway, looking at production of testosterone and estradiol and 

### 16 that sort of aspect, it measures changes in these two hormones. 

### 17 The Panel agrees with the agency that the laboratory's 

### 18 performance of the assays was in demand consistent across all 18 

### 19 test compounds and the performance criteria were by and large 

### 20 generally met for all compounds. 

### 21 In the majority of cases where the performance 

### 22 criteria were not met, the values only slightly exceeded the 

### 23 expected values and slight deviations did not impact the 

### 24 interpretation of the reliability. Again, perhaps the best way 

### 25 of addressing this would be to revise the performance criteria 

### 26 and add a quantitative component to that and get away from

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1 slight changes and things like that. 

### 2 The Agency reported in the White Paper that 

### 3 proficiency testing for both ER binding and the steroidogenesis 

### 4 in vitro assays were often omitted from the laboratory reports. 

### 5 If the Agency feels that these are important, the Panel would 

### 6 suggest that these proficiency tests be routinely run and 

### 7 reported because it's going to add to the competence in the 

### 8 result that you had. 

### 9 And the Panel agrees that the overall results 

### 10 demonstrate that steroidogenesis as performed can distinguish 

### 11 the (inaudible) and alter and do not alter testosterone and 

### 12 estrogen levels in vitro. 

### 13 Moving on to aromatase, the Panel concurs with 

### 14 the Agency that, overall, the aromatase assay as performed by 

### 15 the testing laboratories was able to distinguish between 

### 16 inhibitors and non-inhibitors. And the performance for the 

### 17 aromatase assay was mainly consistent across all 18 compounds 

### 18 that were reported. 

### 19 Moreover, the Panel agrees with the Agency 

### 20 that the performance criteria for the assays were generally met 

### 21 in each study and the deviation noted were minor. Again, if we 

### 22 can quantify what is met by minor, it's going to be a lot easier 

### 23 for acceptance of these results. 

### 24 As we move on to other comments, as noted in 

### 25 the White Paper, the laboratory performance of each of the Tier 

### 26 1 assays were evaluated across 15 to 21 compounds. Although

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1 there were situations where performance criteria were not met, 

### 2 the Agency considered these deviations did not matter and did 

### 3 not impact the interpretation or validity of the data. 

### 4 In summary, the Panel is in basic agreement 

### 5 with the Agency's findings that overall laboratory performance 

### 6 for each of the Tier 1 in vitro assays met expectations and the 

### 7 submitted Tier 1 assay data provided the expected useful 

### 8 information to evaluate potential interactions for test 

### 9 chemicals with the A and the E signaling pathway. 

### 10 The Panel notes that while the number of test 

### 11 chemicals, 21 or less, is small, they Agency should look to 

### 12 adjust the performance criteria for the individual assays to 

### 13 better reflect the experience gained since 2008. In other 

### 14 words, as the sciences move forward there's no reason for the 

### 15 Agency to keep themselves handcuffed by what was considered the 

### 16 state-of-the-art of 2008 when we know it's a lot better than 

### 17 that in 2013. 

### 18 Specially, the Panel recommends more guidance 

### 19 on how much deviation from the norm is appropriate for an 

### 20 individual run to be acceptable, and it should be included in 

### 21 the Tier 1 guidance. This type of guidance should alleviate 

### 22 conclusions of cases where there are questions about assay 

### 23 parameters. 

### 24 The Panel further notes that this guidance 

### 25 could be to take the form of a decision tree that would assist 

### 26 laboratories in deciding when to redo an individual run which

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1 has not met the criteria. 

### 2 Additionally, the Panel recommends the Agency 

### 3 rephrase from using such terms as slight and rather report 

### 4 percent differences in the performance criteria. 

### 5 In general, the Panel feels that small 

### 6 deviations less than 10 percent are likely to be insignificant. 

### 7 Generally, the Panel feels that deviations from the recommended 

### 8 performance that would render the study unreliable are likely to 

### 9 vary between the specific assays, but as a rule of thumb, 

### 10 greater than 10 percent and maybe up to 20 percent on the assay 

### 11 that the Panel seems to be comfortable with. 

### 12 The Panel understands that the protocols used 

### 13 in the Tier 1 assay represents the state of the science in 2008 

### 14 when the initial Tier 1 testing order was issued. The Panel 

### 15 agrees with the Agency of not altering the protocol until the 

### 16 first round of testing was complete. 

### 17 While this SAP is based on data for only 21 of 

### 18 the 52 chemicals being tested in the order, data for the 

### 19 remaining 31 chemicals will soon because available. The Panel 

### 20 recommends that the Agency make these data available as soon as 

### 21 possible as they will impact any weight of evidence 

### 22 deliberation. 

### 23 With that said the Panel feels there is 

### 24 sufficient evidence with selected assays to suggest the 

### 25 reevaluation of the test protocols may be in order. And I guess 

### 26 the poster child for that would be the AMA, based on the

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1 discussions that we've had. 

### 2 Based on both report in the January 2013 FIFRA 

### 3 SAP on a prioritization scheme and public comment, some Panel 

### 4 members question the redundancy of the ER binding and ER alpha 

### 5 transcriptional assay used in the Tier 1 in the context of the 

### 6 rainbow trout, ER binding and rainbow trout liver/gene 

### 7 activation assays that were used by the program in the 

### 8 development of the computational toxicological tools that are 

### 9 used in the prioritization of prescreening. 

### 10 Specifically, it's recommended in light of the 

### 11 fact that "The hormone binding domain of the ER is highly 

### 12 conserved across species." And I believe that comes out of the 

### 13 document. 

### 14 The agency may want to look at what value the 

### 15 Tier 1 in vitro assay has in relation to the ER pathway and 

### 16 adding the information that is already available from the 

### 17 screening exercise. 

### 18 This recommendation for the Agency to look at 

### 19 ER binding and ER transcriptional activation assays in total to 

### 20 reinforce its interest in using high-throughput screening data 

### 21 to expand the structural domain of prescreening activities. 

### 22 This is consistent with some of the recommendations that were 

### 23 made in the open comment session is that we can use all relative 

### 24 information to make these decisions for what we end up doing in 

### 25 Tier 1 testing and moving on to Tier 2. 

### 26 And that, while very long, summarizes all of

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1 the comments that were put together. And I may add that the 

### 2 reason that these were a little bit long is that we had six 

### 3 members responding on this particular question, so there's a lot 

### 4 of comments to integrate. Thank you. 

### 5 **DR. DANIEL SCHLENK:**Thank you, Dr. Schultz. 

### 6 Dr. Ehrich, anything to add? 

### 7 **DR. MARION EHRICH:**Well I question the value 

### 8 of aromatase assay since the steroidogenesis assay covers some 

### 9 of the same things, but that would be a minor, since the comment 

### 10 was made that you're going to look at all of them any way. I 

### 11 think that's included in what Dr. Schultz said. 

### 12 **DR. DANIEL SCHLENK:**Okay. Dr. MacLatchy? 

### 13 **DR. DEBORAH MACLATCHY:**I just have one 

### 14 additional comment that I just want to make sure is highlighted, 

### 15 in that the Panel does agree that the results demonstrate that 

### 16 the AR binding assays performed can distinguish between chemical 

### 17 that bind or do not bind the AR in vitro. 

### 18 And I just want to note that I think this 

### 19 conclusion is supported by the result that the control compounds 

### 20 tested appropriately in the assay even if the assay preparations 

### 21 do not fully meet all the assay criteria; and that that really 

### 22 demonstrates the robustness of the assay. That's noted in the 

### 23 White Paper and I think it's a point that shouldn't be lost as 

### 24 we move forward. And I think it gets to this issue around the 

### 25 guidelines and the preparations, and then the value of the 

### 26 controlled substance and what information that adds to on the

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1 efficacy of the assay and its interpretation. So I just didn't 

### 2 want that to be lost. 

### 3 **DR. DANIEL SCHLENK:**Dr. Roby? 

### 4 DR. KATHERINE ROBY: I have nothing more to 

### 5 add. Thank you. 

### 6 **DR. DANIEL SCHLENK:**Dr. Selvage? 

### 7 **DR. DANIEL SELVAGE:**I have nothing to add 

### 8 either. 

### 9 **DR. DANIEL SCHLENK:**Dr. Tillitt? 

### 10 **DR. DONALD TILLITT:**I have nothing more to 

### 11 add. Thank you. 

### 12 **DR. DANIEL SCHLENK:**Other Panel members want 

### 13 to weigh in? Dr. Scott? 

### 14 **DR. GEOFFREY SCOTT:**Just one comment. Today 

### 15 we had a presentation in the public comment section regarding 

### 16 cytosol fractions and we heard a lot of testimony about the cost 

### 17 of the assays and the number of animals involved. 

### 18 We're using performance criteria assuring that 

### 19 that fraction is active and useful. Let's use those performance 

### 20 criteria and I would recommend consideration of a storage study 

### 21 of some sort to look at extending the time requirements that are 

### 22 currently in place since we do have performance criteria to 

### 23 assure that the fractions are active. That will reduce costs 

### 24 and also reduce the number of animals. 

### 25 **DR. DANIEL SCHLENK:**Thanks. Dr. Ehrich? 

### 26 **DR. MARION EHRICH:**One of our questions was

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1 to say should any of the data be discarded based on you 

### 2 performance criteria. But you had good positive control in all 

### 3 these assays, and therefore underneath Dr. Schultz's, they were 

### 4 saying that the date is okay to include. 

### 5 **DR. DANIEL SCHLENK:**Okay. Any other input? 

### 6 Dr. Delclos? 

### 7 **DR. K. BARRY DELCLOS:**Just one minor comment 

### 8 based on the public comments and the comments about the cytosol 

### 9 assays. I'm pretty sure the 2008 SAP also recommending using 

### 10 recombinant, at least the androgen receptors, so that's been 

### 11 pretty well support that that should be looked into. 

### 12 **DR. DANIEL SCHLENK:**Okay. Any other input? 

### 13 Okay. Let me go to the Agency. Any questions or clarification? 

### 14 **DR. GREGORY AKERMAN:**This is Greg Akerman. 

### 15 When you're referring to the performance criteria for the 

### 16 transcriptional activation assay and potentially revisiting or 

### 17 looking at the performance criteria, were you referring to all 

### 18 of the referenced chemicals or just one specific reference 

### 19 chemical, the very weak positive control? The transcriptional 

### 20 activation, ERTA, that's probably about three pages back. 

### 21 **DR. TERRY SCHULTZ:**The White Paper mentions 

### 22 cytotoxity observed with two particular chemicals done with 

### 23 laboratory one, whether that's of any help to you, and how that 

### 24 toxicity might impact the interpretation of the assay. So the 

### 25 performance criteria that were talked about in that situation; 

### 26 that wasn't it?

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1 **DR. GREGORY AKERMAN:**No. I thought you 

### 2 reference -- you know, it was like the four reference group the 

### 3 study used. I may have misunderstood you. 

### 4 **DR. DEBORAH MACLATCHY:**Yeah, it was with 

### 5 corticosterone and methyltestosterone in those labs, and the 

### 6 issues with the cytotoxicity with some of those labs. 

### 7 **DR. GREGORY AKERMAN:**So it was just the issue 

### 8 with cytotoxicity and not the actual performance criteria 

### 9 values? 

### 10 **DR. TERRY SCHULTZ:**No. But the issue of 

### 11 cytotoxicity is part of the performance evaluation, because 

### 12 where you're being asked to raise the concentration of the test 

### 13 and you're bumping in to the cytotoxity. Now how the Agency is 

### 14 going to address that, I don't know, but it's going to have to 

### 15 obviously be addressed in those cases where we're getting into 

### 16 those issues. Whether you just drop out the highest 

### 17 concentration because it overlaps with cytotoxicity, I don't 

### 18 know if the performance criteria states those things. 

### 19 **DR. GREGORY AKERMAN:**Yes. I mean if there's 

### 20 20 percent cytotoxicity, unusually they don't consider that 

### 21 concentration because that concentration is considered 

### 22 cytotoxic. 

### 23 **DR. TERRY SCHULTZ:**Right. 

### 24 **DR. GREGORY AKERMAN:**I guess I thought you 

### 25 were talking about the performance criteria for those specific 

### 26 reference chemicals because some of them had trouble meeting

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1 performance criteria, especially for the very weak positive 

### 2 controls. I thought you were referring to revisiting that. 

### 3 Maybe I just misunderstood. Were you just talking about 

### 4 cytotoxicity only? 

### 5 **DR. TERRY SCHULTZ:**No. I'm talking about 

### 6 relation to cytotoxicity. 

### 7 **DR. GREGORY AKERMAN:**Okay. Okay. Thanks you. 

### 8 **DR. DANIEL SCHLENK:**Okay. Mary? 

### 9 **DR. MARY MANIBUSAN:**I have a couple of 

### 10 clarification questions. Dr. Schultz, you did mention the 

### 11 difficulties, especially within a screening approach to follow 

### 12 the recommendations of running an in vitro assay up to limits of 

### 13 solubility then bumping against cytotoxity. 

### 14 If there are any explicit recommendations to 

### 15 the Agency from you or the entire Panel, we would certainly 

### 16 welcome that today. 

### 17 And another clarification on the mention of 

### 18 the prescreening, the rainbow trout, ER binding and gene 

### 19 activation assays that were used to develop the (Q)SAR expert 

### 20 system, and then mentioning the high-throughput data. 

### 21 Is your recommendation to look for that 

### 22 information if that data is available and not be redundant in 

### 23 requesting the Tier 1 ER binding and gene trans-activation 

### 24 study? 

### 25 **DR. TERRY SCHULTZ:**As one I would make that 

### 26 recommendation, but until I contact the rest of my cohorts on

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1 that, it wouldn't go so forward on that. 

### 2 I think that, in light of all the information 

### 3 that you're getting from other sources on ER binding, that there 

### 4 is a great redundancy in those two Tier 1 tests than there is in 

### 5 any of the other test. And I would go so far as to say I 

### 6 wouldn't have expected that those things should not have been 

### 7 positive in your Tier 1 test because those things should've been 

### 8 selected out a long time ago. No one would be developing a 

### 9 pesticide that had an overt ER binding activity. 

### 10 So in this case, some of those things where 

### 11 you get negatives are a good thing. It's what you would expect. 

### 12 And I don't know if you want to look at that as a confirmation 

### 13 or what. It gets to be an expensive type of conformation. 

### 14 **DR. DANIEL SCHLENK:**Do we went to go around 

### 15 to our associates to see if they concur on that one? Would that 

### 16 help? Mary? 

### 17 **DR. MARY MANIBUSAN:**I think that would be 

### 18 very helpful as the Agency is looking towards building 

### 19 efficiency in the Tier 1 assays. And if there's opportunity to 

### 20 do so, with respect to what we've learned from the January SAP 

### 21 to build into this conversation, I think will be helpful to have 

### 22 that on record. 

### 23 **DR. DANIEL SCHLENK:**Okay. Dr. Ehrich? 

### 24 **DR. MARION EHRICH:**Well if it was the same 

### 25 compound that was tested in the fish assay that we talked about 

### 26 in January and the one that you're proposing to run through Tier

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1 1, why not just use that data? 

### 2 **DR. DANIEL SCHLENK:**So that would be a yes. 

### 3 Okay. Dr. MacLatchy? 

### 4 **DR. DEBORAH MACLATCHY:**I would agree. If 

### 5 you've got data on ER binding and in some system that you've got 

### 6 data that's solid, then you don't need to do it in multiple 

### 7 systems. 

### 8 **DR. DANIEL SCHLENK:**Dr. Roby? 

### 9 DR. KATHERINE ROBY: I agree. 

### 10 **DR. DANIEL SCHLENK:**Dr. Selvage? 

### 11 **DR. DANIEL SELVAGE:**Yes. 

### 12 **DR. DANIEL SCHLENK:**Dr. Tillitt? 

### 13 **DR. DONALD TILLITT:**Yes, I agree. I think 

### 14 that, as you move forward, you'd want to go to these more in 

### 15 silico approaches to do the screening. 

### 16 **DR. DANIEL SCHLENK:**Is there anyone who does 

### 17 not agree with that on the Panel? Okay. I think you got your 

### 18 answer there. 

### 19 **DR. MARY MANIBUSAN:**Thank you very much. 

### 20 **DR. DANIEL SCHLENK:**Okay. Let's go ahead and 

### 21 move on to question 3. 

### 22 **DR. MARY MANIBUSAN:**Charge question 3, unlike 

### 23 the Hershberger and Uterotrophic assays, a positive control is 

### 24 not required in the male and female pubertal assays. For these 

### 25 in vivo assays in rats, coefficient of variation limits are 

### 26 specified in the test guidelines for most endpoints.

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1 Submissions from different laboratories sometimes fell short of 

### 2 meeting all the test guideline-recommended CV limits for the 

### 3 endpoints evaluated. However, in most cases these shortcomings 

### 4 were considered of minor importance to the overall results, and 

### 5 EPA concluded that the data are still adequate for endocrine 

### 6 screening. 

### 7 Please comment on the EPA's conclusion. 

### 8 Please comment on when a deviation from the recommended CV 

### 9 limits would render the study unreliable. 

### 10 **DR. DANIEL SCHLENK:**Lead discussant for 

### 11 question 3 is Dr. Selvage. 

### 12 **DR. DANIEL SELVAGE:**Okay. I've revised a 

### 13 little bit of what I was going to say, so I'll have to try and 

### 14 read my own writing here, so forgive me if it's a little rough. 

### 15 16 First, just for the record, so we can put it 

### 17 in the minutes, I wanted to just briefly describe the assay. So 

### 18 the male and female pubertal assays, these rats are undergoing 

### 19 puberty, which is a period that is highly depended on normal 

### 20 endocrine function to transition to early adulthood 

### 21 successfully, and so this is used to test the disrupting affects 

### 22 of various compounds that are found in the environment. The 

### 23 tests used are very relevant administrative paradigm, gavage, so 

### 24 metabolism, et cetera, is going to happen, which is good. 

### 25 It is noted, and it was noted earlier today 

### 26 that these tests do use a significant number of animals to

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1 ensure proper study population control and that sort of thing, 

### 2 and if that could be limited in some way, shape or form, that 

### 3 would be a good thing, obviously. 

### 4 Okay. Just to get into the variations and 

### 5 their acceptability, I think overall, the CVs were frequently 

### 6 exceeded in the assays -- well assays for 16 out of 17 

### 7 chemicals, but most of these were considered minor. And the EPA 

### 8 concluded that that did not really overall affect 

### 9 interpretation. And I think the Panel in general agrees with 

### 10 that evaluation. I do, however, want to get into specifics for 

### 11 a little bit on each of the assays. 

### 12 You invited an HPA axis guy to the table, so I 

### 13 do have a little bit of concern about the CVs and what's 

### 14 acceptable, and also about developing CVs with randomly cycling 

### 15 female rats, in particular. 

### 16 We'll go to the female rat assays. So these 

### 17 have relatively high acceptable CVs for a number of parameters 

### 18 that are presumably due to cyclicity affects that aren't 

### 19 accounted for in the assays. 

### 20 It is noted that the cyclicity data were not 

### 21 included in the White Paper and so we can't really see if that 

### 22 was taken into account with the data. It would be useful to 

### 23 know the day of estrous at the time of sacrifice for these 

### 24 animals if they are indeed cycling for the treated ones. 

### 25 I think the overall thought is that even the 

### 26 minor deviations were acceptable. However, I'm going to be a

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1 little bit cautionary in some of my comments here regarding 

### 2 proceeding, in part because I'm an HPA axis guy and I think you 

### 3 might have a stress parameter going on with some of these 

### 4 studies that you could influence them. 

### 5 So briefly, female reproductive parameters 

### 6 that are being measured here, they're highly subjected 

### 7 stress-related effects, and that could also affect the 

### 8 variability of the data that you see. Thus, high CVs that are 

### 9 found in treatment groups should be really interpreted carefully 

### 10 when they're considered acceptable because you already have the 

### 11 parameter of relatively large acceptable variation because 

### 12 you're not cycling the rats or checking them on the same day of 

### 13 the cycle for your measurements; so that could be an issue that 

### 14 you'd want to take into account. 

### 15 But also just to suggest to the Agency that in 

### 16 doing these studies, either during the range-of-finding studies 

### 17 or the studies themselves, you look at some HPA activation 

### 18 parameters that might influence your results down the road. 

### 19 For instance, if you're getting low chronic 

### 20 stress activity that you're not picking up at the overt 

### 21 observation, behavioral observation et cetera, that could be an 

### 22 issue. And these could contribute to the higher CVs that you're 

### 23 seeing. And one worry is that you get a higher CV, get higher 

### 24 variability, and you might be getting a negative that you're not 

### 25 picking up with those, so that is a concern. 

### 26 Also, I would say there is a bit of a way

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1 around estrous cycling problem and I would just like to suggest 

### 2 that you could account for some of the variation that you're 

### 3 seeing, for instance, in ovarian weight by either synchronizing 

### 4 the estrous cycles of the rats prior to sacrificing them, so 

### 5 that they're on the same day of the cycle. And then your values 

### 6 would be much more likely to be at a lower variation, and then 

### 7 that would be easier to tell if the chemicals that you're 

### 8 testing are having an effect. 

### 9 Also, if you cycled the rats using a GnRH 

### 10 agonist that would also, with these rats, test the integrity of 

### 11 the pituitary in terms of responding to GnRH and then the 

### 12 ovaries in terms of responding to LH and FSH. So I think you 

### 13 might gain something from doing that, in addition to being able 

### 14 to sacrifice the rats on the same day of the cycle and at the 

### 15 same age. 

### 16 Or, alternatively you could use "reproductive 

### 17 age" as your target for sacrificing the rats, i.e., as a woman 

### 18 said earlier today, the first day of diestrous and use that as 

### 19 the parameter rather than just absolute age. So you might be 

### 20 able to tighten up your numbers that way. 

### 21 Okay. That's really about all I'm going to 

### 22 say for the female part of the assay. I think, once again, 

### 23 mostly what was said was acceptable, and the right 

### 24 interpretations, I think, were being made. 

### 25 For the males, there were also a fair number 

### 26 of CVs that were out of range, but interpreted as acceptable and

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1 I think the Panel overall agreed with that. 

### 2 Once again, just as an HPA axis guy, though, I 

### 3 would really caution about stress and stimulating stress in 

### 4 these animals which the chemicals that you're testing and having 

### 5 that go undetected. Because in particular, one of your measures 

### 6 is of course testosterone which bounces all over the place in 

### 7 rats, that could be down to about a zero in a perfect fine rat. 

### 8 And if you're only taking one measure, you know, you might see 

### 9 that. 

### 10 What I would personally do if I wanted to 

### 11 measure a testosterone, which obviously is going to drive your 

### 12 other measures to a very large extent, would be to -- you could 

### 13 give the rats, for instance, and HCG challenge where you give 

### 14 them human chorionic gonadotropin and stimulate testosterone 

### 15 release. 

### 16 And so you can get a much more uniformed view 

### 17 of what's happening with testosterone production and also the 

### 18 responsiveness of the testes to LH. If you did that, I don't 

### 19 think you would affect your other parameters, your weights of 

### 20 various testes, et cetera, by doing that, and you would get a 

### 21 better idea of what's happening with testosterone itself. 

### 22 Other than that, in terms of the overall Panel 

### 23 in general, I think, once again there was concern about the high 

### 24 doses that were being used in the interpretation from those high 

### 25 doses and what that might do the CV criteria and it's caution 

### 26 that a weight of evidence approach needs to be utilized to

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1 determine if the compound moves forward. You can't really take 

### 2 either one of these assays on their own because they both have a 

### 3 big likelihood of giving you a false positive. 

### 4 Also it's noted that, to what extent you can 

### 5 -- supplier of rats. I mean I realize these rats are raised in 

### 6 house by each of the labs that make them. Sorry, I can't ask 

### 7 you questions, can I? 

### 8 The rats are raised inhouse so, you know, 

### 9 there's all kinds of parameters that should go into the 

### 10 variations between labs, and I'm sure you guys are aware that 

### 11 you need to look out for that and communication between labs 

### 12 would be good so that they could account for that. And what 

### 13 else do we have? I believe that's about it. Hopefully my 

### 14 colleagues on the Panel can add more to what I missed. 

### 15 **DR. DANIEL SCHLENK:**Okay. Dr. Delclos? 

### 16 **DR. K. BARRY DELCLOS:**I don't have much to 

### 17 add. I'll just say that, from the discussions yesterday, it 

### 18 seems that, between the EPA, it was indicated that the test 

### 19 guideline recommended CV limits were recommendations rather than 

### 20 set assay. I think that needs to be clarified and perhaps 

### 21 revise the guideline, or whatever. 

### 22 And as Dan just mentioned, while 

### 23 Sprague-Dawley rats were used for most of the pubertal assays 

### 24 reported, it wasn't clear that the rats were from the same 

### 25 supplier and factors such as substrain or diet can certainly 

### 26 attribute to variability and the means and variances among labs

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1 and from the guidance that was given in the document. 

### 2 In any case, as stated, the deviations or 

### 3 recommended CV limits does not occur in all endpoints for a 

### 4 given chemical in the most part. On the assays for chemicals 

### 5 that did have CV limits that deviated from recommendations are 

### 6 listed as showing positive effects, and the concern with the 

### 7 large CVs of course is that the extent of variability weakens 

### 8 statistical power and make the assay unreliable for the 

### 9 detection of biologically significant effects. 

### 10 So I thought in the event that other assays, 

### 11 uterotrophic or the Hershberger that are complimentary or 

### 12 redundant for these endpoints, that are removed from the 

### 13 battery, perhaps the inclusion of positive controls should be 

### 14 considered, or at least periodic laboratory proficiency assay 

### 15 with positive control. That's all. 

### 16 **DR. DANIEL SCHLENK:**Thank you. Dr. Horseman? 

### 17 **DR. NELSON HORSEMAN:**Have captured most of 

### 18 our consideration, I think. However, I did want to add a couple 

### 19 of thoughts. 

### 20 This is related to a performance criteria for 

### 21 a coefficient of variation, which is really a statistical issue. 

### 22 And the charge questions ask whether the fact that these 

### 23 sometimes fell short of meeting the guidelines was meaningful. 

### 24 And I'm sure a lot of this is going to come up later in the 

### 25 weight of evidence discussion. 

### 26 But I think, overall, the challenge with

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1 accepting evidence from experience that fall short of meeting 

### 2 this performance criterion, it relates to the weight one assigns 

### 3 to committing either type 1 or type 2 errors in this stage in 

### 4 this Tier 1 assessment. So higher coefficient of variation will 

### 5 tend to increase the likelihood of committing a type 2 error, in 

### 6 other words, failing to detect a true difference or having a 

### 7 false negative. 

### 8 And if there's a bias at Tier 1, one might 

### 9 suggest that you would be more willing to accept a type 1 error 

### 10 than a type 2 error. So that would likely increase the number 

### 11 of compounds moving forward, but it would minimize the chances 

### 12 of failing to move a compound forward that was impacted from 

### 13 endocrine disrupters. So there is a balance to be met here, and 

### 14 the tighter you get these coefficients of variation, the easier 

### 15 it's going to be to meet that balance. That's the point. 

### 16 The problem is that there's a potential for 

### 17 piling type 2 errors on top of one another in this situation so 

### 18 that the battery admits multiple assays that don't meet that 

### 19 coefficient variation performance criterion, you're adding type 

### 20 2 errors on top of one another, so it's a challenge. 

### 21 So the other side of this is a consideration 

### 22 related to whether the criterion CVs are set at the correct 

### 23 level or not, or whether they need to be revisited so they can 

### 24 be met without compromising utility of this Tier 1 screening. 

### 25 And this question is relevant not just to the two types of 

### 26 assays in this charge question, but across the spectrum of this.

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1 2 Ultimately the acceptable level of variance in 

### 3 the screening assay needs to be sufficiently broad to capture 

### 4 the range of biological variability, but not so large as to 

### 5 allow for careless execution to assays. 

### 6 And it'd be very helpful if the Agency could 

### 7 provide an explicit rationale for how these criteria for assay 

### 8 variances are actually set and documentation regarding how well 

### 9 the variance criteria captures biologically meaningful spectrum 

### 10 while maintaining the high standards that are necessary for the 

### 11 testing. 

### 12 **DR. DANIEL SCHLENK:**Okay. Dr. Roby? 

### 13 DR. KATHERINE ROBY: Just to continue on to 

### 14 the thought of the CVs, and that I think some of the comments 

### 15 with regards to source of animals and large variation across 

### 16 source. If some consideration could be given to assessment of 

### 17 inhouse CVs or historically inhouse CVs, this might reduce the 

### 18 deviation from what you consider to be the criteria parameters. 

### 19 20 So within a given lab, their own CVs may 

### 21 differ from what you consider would reduce, if you were allowing 

### 22 the individual CROs or testing labs to give consideration to 

### 23 their inhouse historical CVs for these test parameters. 

### 24 **DR. DANIEL SCHLENK:**Okay. Other panel 

### 25 members, question 3? No? Okay. Let me go back to the Agency. 

### 26 Any questions or clarification?

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1 **DR. JOHN LICCIONE:**John Liccione from EPA. I 

### 2 want to thank Dr. Selvage for his comments. We appreciate the 

### 3 complexity of the HPG axis. I just wanted to make sure I 

### 4 understood one of your comments on the female pubertal and the 

### 5 male pubertal. 

### 6 I understand the suggestion about the 

### 7 synchronizing the cyclicity for necropsy. So are you suggesting 

### 8 also the possibility for amending the assay to include GnRH 

### 9 agonist? Is that something to explore for us? 

### 10 And just to finish up, because it's related to 

### 11 the other suggestion on gonadal is that the HCG challenge to 

### 12 stimulate testosterone would lead to then also to look at the 

### 13 responsiveness of the testes to LH. So these are things you are 

### 14 recommending as things that we should look at to incorporate in 

### 15 the assay, because we do know the testosterone is a variable 

### 16 thing that could be influenced by stress. 

### 17 **DR. DANIEL SCHLENK:**Dr. Selvage? 

### 18 **DR. DANIEL SELVAGE:**Yes. If I were running 

### 19 these studies, I would look at both of those as ways to try and 

### 20 stabilize the data that you're seeing, and also as something of 

### 21 the test of the integrity of the HPG axis of the females or the 

### 22 males; because, as it is now, it doesn't seem like you have a 

### 23 really good feel for that. The females are young and they're 

### 24 not going to be cycling very regularly anyway. 

### 25 So yeah, I would look at that if I were you. 

### 26 I think it could help. It would need some analysis though.

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1 **DR. JOHN LICCIONE:**Thank you. 

### 2 **DR. DANIEL SCHLENK:**Okay. So we have one 

### 3 more question. How about a 10-minute break before we move on 

### 4 just to take a breath and regroup a little bit for our last 

### 5 question in? So let's be back at four o'clock. 

### 6 (Whereas a break was taken) 

### 7 **DR. DANIEL SCHLENK:**Let's go ahead and read 

### 8 question 4 into the record. 

### 9 **DR. MARY MANIBUSAN:**Charge question number 4, 

### 10 The test guidelines for the six in vivo assays, Hershberger, 

### 11 Uterotrophic, the male pubertal, female pubertal, the fish 

### 12 short-term reproductive assay and the amphibian metamorphosis 

### 13 assay offer some guidance on setting the dose/concentration 

### 14 range when testing for specific effects on the E, A or T 

### 15 signaling pathways. 

### 16 In some of the in vivo assays, overt toxicity 

### 17 was noted based on the effects on growth, other sub-lethal 

### 18 effects and even mortality at the highest dose/concentration 

### 19 tested. Positive Tier 1 findings indicating the potential for 

### 20 endocrine activity can be difficult to interpret in the presence 

### 21 of overt toxicity. 

### 22 Please comment on the nature and severity of 

### 23 overt toxicity that would render study results unreliable for 

### 24 the purpose of Tier 1 screening of a chemical for endocrine 

### 25 activity. 

### 26 Please comment on what, if any, additional

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1 guidance is needed to minimize overt toxicity while ensuring 

### 2 adequate dose selection for these screening assays. 

### 3 Specifically comment on the validity of 

### 4 including treatment concentrations with apparent body weight 

### 5 effects in the analysis for potential endocrine interaction for 

### 6 the fish short-term reproductive assay. 

### 7 **DR. DANIEL SCHLENK:**The lead discussant for 

### 8 question 4 is Dr. Selvage. 

### 9 **DR. DANIEL SELVAGE:**Okay, you've got me 

### 10 again, but I'm going to be fairly short with this one. What 

### 11 I've decided to do because I'm an in vivo person doing mostly in 

### 12 vivo research, I'll do a quick summary and then we've all pretty 

### 13 much focused on our own areas, so what I'm going to do is turn 

### 14 it over then to the other discussants and let everybody say 

### 15 their piece, so I'll be pretty quick here. 

### 16 I think everybody on the Panel understands 

### 17 that the use of high doses when investigating potential 

### 18 environmental endocrine disrupting affects of different 

### 19 compounds is of great importance and we need to test the range 

### 20 of doses, including high doses. 

### 21 However, overt toxicity reported during 

### 22 testing can really complicate the interpretation of data in all 

### 23 the in vivo assays that are employed by the EPA for testing 

### 24 compounds for the endocrine disrupting affects. And while the 

### 25 use of high doses are important, it's also important for the EPA 

### 26 to take into account the high potential for confounding affects

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1 elicited by test substances, their dose at levels resulting in 

### 2 toxicity, you know, for instance, in the rat, the overt toxicity 

### 3 (inaudible), et cetera. 

### 4 Okay. So this is obviously true for all the 

### 5 in vivo assays performed in the endocrine disruptor program that 

### 6 are being discussed at this meeting. So as when activation of 

### 7 stress parameters are involved, it affects all endocrine 

### 8 systems. And once again, they brought an HPA axis guy to the 

### 9 table, and when you activate the stress axis that overrides 

### 10 pretty much everything and a lot of the parameters you're 

### 11 looking at. And I know you're aware of that but I want to put 

### 12 it out there. 

### 13 So in my previous statement I talked a little 

### 14 bit about potentially confounding stress axis related factors 

### 15 that can be involved in mammalian studies and the pubertal 

### 16 assays, in particular. This can also play an effect in the 

### 17 Hershberger and uterotrophic assays; in other words, it you get 

### 18 stress levels that are too high, whether you detect them or not, 

### 19 you're going to have problems measuring your endocrine endpoints 

### 20 for your reproductive parameters, and also thyroid. 

### 21 I think, because of these stress issues and 

### 22 other issues that are going to be noted by the other 

### 23 discussants, it's highly suggested that every effort we made to 

### 24 eliminate the use of doses that are going to cause overt 

### 25 toxicity during initial testing just because of what this does 

### 26 to all the parameters that you're looking at. So I think the

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1 general feeling of the Panel is that you really don't want to go 

### 2 there where you're seeing really big overt toxicity when you're 

### 3 doing a screening. 

### 4 So just a couple other points that I picked up 

### 5 from the Panel, then I'll turn it over to the rest of the 

### 6 discussants. 

### 7 One thing that I felt was really important 

### 8 that was brought up was that toxic compounds are going to 

### 9 disrupt basic cellular machinery that are necessary for a normal 

### 10 endocrine function. So this really needs to be taken into 

### 11 account, and that was in part from a comment by Dr. Leonard. 

### 12 And also, when you have a dose that's 

### 13 affecting body weight in any of these systems, I think you're 

### 14 going to have a problem in terms of the endpoints you're 

### 15 measuring. 

### 16 And then finally -- well I was going to say 

### 17 something about the fish, but I think I'll let that go to the 

### 18 other discussants and I'll end right there. 

### 19 **DR. DANIEL SCHLENK:**Dr. Delclos? 

### 20 **DR. K. BARRY DELCLOS:**Well I think, as 

### 21 already been said, in most cases the overt toxicity is going to 

### 22 make it impossible to clearly link observed effects to an 

### 23 endocrine-related mechanism. It might not always be the case. 

### 24 I could imagine, for example, a body weight being significantly 

### 25 reduced and still seeing an accelerated vaginal opening. That 

### 26 wouldn't interfere with the interpretation of that affect.

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1 But it is going to be problematic, 

### 2 particularly if it's a critical study. Affects are observed 

### 3 only in the dose group of overt toxicity. If only one screening 

### 4 assay shows a positive affect it would be difficult to weigh 

### 5 that in to deciding whether compounds should go on to Tier 2 

### 6 without a repeat experiment of a lower dose. 

### 7 It's been indicated in the White Paper and in 

### 8 the discussions yesterday that sometimes two or three doses are 

### 9 used. If it continues to be necessary to use a dose at or near 

### 10 the MTD, to set the high dose, perhaps it should be required 

### 11 that at least three doses be used in the screening assay to 

### 12 increase the chance if there's a dose without overt toxicity or 

### 13 even add an additional dose. 

### 14 Personally I would like to see, where 

### 15 possible, moving away from the MTD and using either an 

### 16 exposure-based, larger exposure type setting for the high dose 

### 17 or previous information, even if it's not exactly the same 

### 18 exposure regimen. And I think I'm not going to comment on the 

### 19 fish assay because I don't know much about it. 

### 20 **DR. DANIEL SCHLENK:**Dr. Leonard? 

### 21 **DR. JACK LEONARD:**I'll just amplify a little 

### 22 bit on what Dan Selvage said about the basic machinery. Having 

### 23 worked with rats for 30 plus years, I know that toxic compounds 

### 24 will impact almost all the basic machinery in the cell, energy 

### 25 utilization, (inaudible) precursors, genome stability, and 

### 26 transcription.

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1 And as such, if the impact of toxicity on the 

### 2 organism of well-being is so large that you see weight loss or 

### 3 overt symptoms, it's going to be very difficult then to sort out 

### 4 whether we're talking about toxicity or endocrine affects. So 

### 5 from that perspective, I think that an overt toxicity finding 

### 6 will probably be a disqualifying attribute for that specific 

### 7 data subset and a smaller dose is necessary, so your 

### 8 range-finding becomes very important for looking at these 

### 9 compounds that have an opportunity to be toxic. 

### 10 A good example of what I mean by that is 

### 11 energy disruptors which will impact the mitochondria, will alter 

### 12 energy dynamics led to really well characterize changes in 

### 13 signal transduction pathways. Commonly, the Akt pathway which 

### 14 is really one of the key elements looking at carbon utilization 

### 15 in the cell, hypotrophy gets kicked in and all of those events 

### 16 will impact negatively on any endocrine downstream effects that 

### 17 take place. 

### 18 So that, at all costs, I think it's important 

### 19 when overt toxicity is looked at to look at that as a toxic 

### 20 event and not an endocrine. 

### 21 Finally, with respect to body weight change, I 

### 22 can refer most likely to rodents. Whenever you see a dramatic 

### 23 loss of 10 percent or so, usually that's due to loss of glycogen 

### 24 from the liver, as well as other sources. The liver is a source 

### 25 of all the serum binding proteins that are necessary for your 

### 26 EAT targets that you're using downstream. And in fact, when you

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1 make an animal sick, the liver of rats in particular make a 

### 2 thyroid binding globulin that binds thyroxin much more avidly 

### 3 than the serum does albumin. 

### 4 And in that case, you're going to get an 

### 5 increased longevity of thyroid hormone thyroxin and (inaudible) 

### 6 in the serum and the circulation, so it's going to look like 

### 7 you're going to have more thyroid hormone than you really do 

### 8 have; and the animal is not really hyperthyroid, it's just 

### 9 you've got them sick. 

### 10 And so those are some events that I can 

### 11 foresee might confound the issue. 

### 12 **DR. DANIEL SCHLENK:**Thank you. Dr. Patino? 

### 13 **DR. REYNALDO PATINO:**I'll make comments 

### 14 specific to the metamorphosis assay, which is what I was asked 

### 15 to do. 

### 16 So the metamorphosis amphibians is a 

### 17 particularly well characterized process that is known. They're 

### 18 well established; a primary regulator of that is thyroid 

### 19 hormones, so that's pretty well established. It is also known 

### 20 that the growth and development during pre-metamorphosis and 

### 21 during pro-metamorphosis in xenopus are concurrent but not 

### 22 necessarily associated events. 

### 23 For example, tadpole development can be 

### 24 inhibited by a number of well-characterized (inaudible) without 

### 25 considerable or significant effects on growth. On the other 

### 26 hand, growth can be accelerated by compounds such as triclosan.

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1 And by the way, I'm providing some references here; I'm sure you 

### 2 have them, but just to backup my statements, so they'll be 

### 3 references associated with this statement or this comment. 

### 4 So growth can be accelerated by compounds such 

### 5 as triclosan without detectable affects. And there was another 

### 6 perhaps more complicated or interesting example where tadpole 

### 7 growth and certain aspects of development, such as hind limb 

### 8 length, can be inhibited by environmentally relevant 

### 9 concentrations of cadmium, but without notable effects on 

### 10 thyroid and histopathology, particularly thyroid epithelial cell 

### 11 height. And for full disclosure that's a study that my lab 

### 12 conducted. 

### 13 So although it is important to measure and 

### 14 differentiate effects on growth from effects on development in 

### 15 this assay, given the wide variety of responses that have been 

### 16 already characterized in terms of growth and development to a 

### 17 wide variety of chemicals, in my opinion, there is not a priori 

### 18 reason to conclude that overt toxicity as defined by effects on 

### 19 growth will render the assay results unreliable for the purposes 

### 20 of Tier 1. 

### 21 So it is a concern, effects on growth, but in 

### 22 my opinion they don't necessarily invalidate the results for the 

### 23 purpose of Tier 1. 

### 24 A relevant issue in regards to growth and 

### 25 growth measurements in the metamorphosis assay is the method 

### 26 used for normalizing hind limb length in tadpoles of different

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1 size, and this has come up a couple of times yesterday in the 

### 2 questions and during the questions today and several of the 

### 3 public comments. 

### 4 In the White Paper, the Agency recommends 

### 5 normalizing hind limb length based on snout-vent length mainly 

### 6 by dividing one over the other. This method, however, assumes 

### 7 that the relationship between the two variables is isometric. 

### 8 And generally speaking, an isometric scaling relationship is one 

### 9 where, on a double-log plot of the data, you have a slope of 

### 10 one. However, if the slope is different than one, either higher 

### 11 or lower, the method which would render that relationship 

### 12 allometric -- not isometric -- the method of correction could 

### 13 lead to errors. That would depend on the deviation from one of 

### 14 the slops and also the size range of tadpoles that you're 

### 15 considering in population. 

### 16 And it is well-known that the size of most 

### 17 body parts across organisms, that relationship is not isometric. 

### 18 I mean with some exceptions, it is allometric. So a suggestion 

### 19 would be that the Agency should confirm what type of 

### 20 relationship there is between those two variables. And it 

### 21 indeed is allometric, then there's actually and alternative 

### 22 method. I'm not a biometrician, so this is a suggestion from 

### 23 just a practicing biologist. 

### 24 But there is a alternative method that could 

### 25 be used to correct for tadpole size that would get around the 

### 26 problem, and that would be to use an appropriate regression

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1 analysis for hind limb length against snout-vent length to 

### 2 calculate residuals, and then to use the residuals as opposed to 

### 3 the raw data for ratios in this statistical analysis or 

### 4 comparison. 

### 5 And that has been done by others. We've done 

### 6 it in our lab. Again, I'm not a biometrician. You need to 

### 7 consult with somebody who knows, but that's a method that has 

### 8 been used and again it's published in the literature. 

### 9 So those are my suggestions on the topic of 

### 10 growth or affects of growth in metamorphosis and impacts on the 

### 11 utility of the assay. Thank you. 

### 12 **DR. DANIEL SCHLENK:**Dr. Scott? 

### 13 **DR. GEOFFREY SCOTT:**The overt toxicity may 

### 14 affect the ability to discriminate endocrine affects in these 

### 15 battery of assays. If there is actual mortality or lethality 

### 16 itself, one of the things you're doing is you possibly are 

### 17 removing some of your most sensitive organisms out of the 

### 18 population, and they may be sensitive to a broad spectrum of 

### 19 things including affects for endocrine disruption. So that is a 

### 20 very significant thing to consider when we see actual lethality. 

### 21 I think when overt toxicity is observed within 

### 22 the guidelines going back and running the lower doses, 

### 23 particularly at the half logs and things, that's a good way to 

### 24 go back and discern that affect. And if you do not see those 

### 25 affects in the absence of overt affects, then you either 

### 26 conclude that the affect at the higher dose was a combination of

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1 overt stress and endocrine disruption or, if you do not see any 

### 2 endocrine disruption at all in those lower doses, you may decide 

### 3 it was only that overt stress. So I think the guidelines allow 

### 4 you to make those determinations. 

### 5 Now we were asked to comment on what would 

### 6 render results unreliable, and I have a couple of suggestions, 

### 7 at least that I would make to consider. Again, if there's 

### 8 actual mortality/lethality in the assay, that's one. What is 

### 9 the severity of the affect? Is it a 5 percent, 10 percent, 20 

### 10 percent, 50 percent change in growth of whatever behavior or 

### 11 whatever it may be? I think the severity is very important. 

### 12 Also the size of severity magnitude is the frequency that that 

### 13 occurs across the number of animals in the test. 

### 14 For example, if it occurs in more than 50 

### 15 percent of your animals -- I happen to believe 50 percent is 

### 16 used in a lot of assay because it has a population connotation. 

### 17 And so I would say that that is something to really look at is 

### 18 the proportion in the population of your bioassay at that 

### 19 treatment effect is a very significant thing. 

### 20 So if you consider those three things, I think 

### 21 it really gives you a way to look at when you may or may not 

### 22 disqualify an assay. 

### 23 Another thing we were asked to do is please 

### 24 comment on any additional guidance as needed to minimize overt 

### 25 toxicity. And I think the real guidance here is on the 

### 26 selection of the maximum tolerated dose. We've heard a lot

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1 about range-finding assays, how we set those, and particularly 

### 2 in the fish where we're setting that in the third of the LC50. 

### 3 We also have had a lot of information about 

### 4 high coefficients of variation on exposure concentrations. Well 

### 5 if you're testing on that threshold between effects and 

### 6 non-effects and then you have an alteration in dose that can be 

### 7 a deadly combination. 

### 8 And so one of the ways that we might want to 

### 9 look at this -- and I'm just thinking out loud with you here and 

### 10 giving you some suggestions. One thing that you could possibly 

### 11 do to reduce the occurrence would be to better define the 

### 12 maximum tolerated dose from the range-finders, and that may be 

### 13 to use the correction that you're using when you have overt 

### 14 toxicity in better defining using half-log doses in the 

### 15 range-finder to help you better define with greater precision 

### 16 the maximum tolerated dose. 

### 17 So if it can help you on one hand when you 

### 18 have a problem, it may also help you in preventing the problems 

### 19 we're trying to suggest to you. 

### 20 The other thing to do would be to evaluate the 

### 21 95 percent confidence limit from the range-finder and look at 

### 22 that dose. And if you were to actually run a couple of 

### 23 compounds and do half-log doses, if that more precise 

### 24 range-finder number is closer to that lower 95 percent 

### 25 confidence limit, that could be a potential way around dealing 

### 26 with the confounding problems that you have with maintaining the

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1 dose. 

### 2 So those are just sort of suggestions of how 

### 3 you can look at maybe solving some of this problem because I 

### 4 think as we were presented information today from the public 

### 5 comment, we asked some questions about no X and low X and really 

### 6 where do those really fit? 

### 7 We want to be at a level to induce and 

### 8 endocrine effect if it's going to be induced. At the same time 

### 9 we want to maintain that fine line between not causing other 

### 10 related toxicity that's nonendocrine related. That's a fine 

### 11 line to walk, so I'm only saying the better we can define those, 

### 12 the better we will probably prevent some of this. 

### 13 We were also asked to comment specifically on 

### 14 concluding body weight looking at fish assay, and I'm going to 

### 15 just make a few comments on that. 

### 16 Body weight determinations are easy to measure 

### 17 and may be directly related to altered behavior and reduced 

### 18 feeding rates that may be contributing to overt toxicity. So it 

### 19 may be an integrated measure of a lot of things that may be 

### 20 important. Body weight effects are directly measurable and are 

### 21 not subjected as opposed to behavioral observations which may be 

### 22 a little more subjective. So I really think that is an 

### 23 important point to make. 

### 24 The percentage of feeding response is also 

### 25 very important in addition to body weight because it is 

### 26 quantitative. So I really think if you're going to use body

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1 weight, use a more quantitative endpoint, and the fish assay is 

### 2 going to help you. 

### 3 Just to read out of the report in the fish 

### 4 assay, only one compound out of 21 compounds tested resulted in 

### 5 significantly reduced lengths and weight determinations. And 

### 6 there were some other results, 5 out of 21 compounds as 

### 7 significant differences in male or female body weight at doses 

### 8 where other sub-lethal effects occurs. 

### 9 So it seems like there was some concordance 

### 10 where we wanted to see with this. We do recognize the changes 

### 11 in the body weight of females were problematic and could lead to 

### 12 some exclusion of some data at higher doses at some points in 

### 13 time. 

### 14 I think, despite this complication, I think 

### 15 tracking the body weight is an important endpoint. 

### 16 At the Duluth meeting after the Raleigh 

### 17 conference, we said there were three things you need to pay 

### 18 attention to; growth, development, reproduction. And I still 

### 19 think growth is one of those that we need to be paying attention 

### 20 to, and body weight is a certain measure of that. Thank you. 

### 21 **DR. DANIEL SCHLENK:**Dr. Tillitt. 

### 22 **DR. DONALD TILLITT:**I think many of my 

### 23 comments have been gone over, but I'll try not to be redundant. 

### 24 So I'm going to start out with the fact that 

### 25 the Panel agrees, or at least this Panel member agrees with the 

### 26 Agency that the FSTRA test protocol is very robust in the sense

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1 of having study metrics at multiple endpoints of biological 

### 2 organizations. You know, this really enhances the 

### 3 complementarity of the assay in the system. It's a real 

### 4 strength of the test and it really is, I think, an integral part 

### 5 of the EDSP Tier 1 Panel. 

### 6 I think it's already been discussed, the idea 

### 7 of getting at this idea of overt toxicity, that the starting 

### 8 initial dose at one-third 96 hours LC50 of a compound and 

### 9 expecting not to see overt toxicity in a 21-day assay is 

### 10 unreasonable, and that that dosing scheme needs to be addressed. 

### 11 12 Now with regard to the signs of overt 

### 13 toxicity, absolutely there's going to be potential confounding 

### 14 factors with interpretation of endocrine related results. 

### 15 The Agency plans to evaluate results of the 

### 16 assay and a systematic case-by-case stance will be required to 

### 17 properly tease out which endocrine related affects are clear and 

### 18 which ones are going to be confounded by overt toxicity. 

### 19 Overt toxicity including reduced survival and 

### 20 body weight loss can confound the results of the study at 

### 21 exposure concentrations where you see this expressed toxicity. 

### 22 It may not be possible in some cases, for example, the GSI to 

### 23 discern these effects. However, there are certain cases where 

### 24 even through the signs of overt toxicity, for example, ovotestes 

### 25 or secondary sex characteristics where you can have clear 

### 26 endocrine related effects through this overt toxicity.

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1 But again, I think the idea here is that 

### 2 you're going to have to go case-by-case looking for consistency 

### 3 of endpoints with this assay to determine whether you can or 

### 4 cannot determine whether there's an endocrine related effect, 

### 5 and that's going to be addressed, I think, in this weight of 

### 6 evidence Panel. 

### 7 You know, solutions to how you address this 

### 8 overt toxicity, I think, reducing the starting point. And a 

### 9 selection has to be made and there have been some good 

### 10 selections, ideas brought up already. You know, you could go to 

### 11 the half-log idea that is used with binding assays, but I think 

### 12 maybe a starting point that is determined in the range-finding 

### 13 test at the beginning could give you a better starting point. 

### 14 That's it. 

### 15 **DR. DANIEL SCHLENK:**Thanks. Other Panel 

### 16 members? Yes, Dr. Schultz? 

### 17 **DR. TERRY SCHULTZ:**I want to point out the 

### 18 nature and severity of overt toxicity and what the definition is 

### 19 there, and then down to the specific issue of apparent body 

### 20 weight effects. 

### 21 When you're dealing with fish at the 

### 22 individual chronic endpoint, the most sensitive chronic endpoint 

### 23 it's typically growth. And obviously body weight is going to be 

### 24 a parameter that fits into that mix. So you can have chemicals 

### 25 that cause chronic effects via endocrine pathway modes of 

### 26 action, but you will also have chemicals that have chronic

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1 effects by other mechanisms. 

### 2 And so to say that the body weight is a sign 

### 3 of overt toxicity and to disregard that with the endocrine 

### 4 function, in some cases it may be valid. In other cases, it 

### 5 won't be valid. So I just caution you in that type of situation 

### 6 because that is a place where you have individual chronic 

### 7 toxicity and aquatic species bumping into the endocrine thing, 

### 8 so there are the changes that they're going to be chemical and 

### 9 concentration schemes where the interpretation is going to be 

### 10 very pleasant. I don't know if that helps you. 

### 11 **DR. DANIEL SCHLENK:**Dr. Horseman. 

### 12 **DR. NELSON HORSEMAN:**My comments may differ 

### 13 in degree but not in kind from what the other Panelists 

### 14 remarked. And probably because I'm old enough to remember when 

### 15 there was no such thing as an endocrine disruptor, right? 

### 16 So the theory of an endocrine disruptor is 

### 17 sort of a relatively new kid on the block, and it seems to me 

### 18 that it's based on a very explicit notion that it is only useful 

### 19 in absence of overt toxicity, because endocrine disruption was 

### 20 discovered as a phenomenon related to exposures to things that 

### 21 were not overt toxic, right? The feminization of fish; the fish 

### 22 weren't dying. 

### 23 So if that's the theory behind endocrine 

### 24 disruption, I think that this issue is really critical and 

### 25 getting it right, whatever that is -- and I understand EPA's 

### 26 interest in making sure the doses are always high enough, but if

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1 chemicals have been defined as toxic and therefore you're in a 

### 2 concentration range at which they're already regulated, it seems 

### 3 to me the information about endocrine disruption isn't very 

### 4 useful there because it's not going to contribute anything new 

### 5 to regulation at those concentrations. 

### 6 So getting data on endocrine disruption at 

### 7 lower concentrations would seem in the long run to be more 

### 8 useful. 

### 9 **DR. DANIEL SCHLENK:**Okay. Dr. Patino. 

### 10 **DR. REYNALDO PATINO:**Just a follow-up to what 

### 11 Dr. Tillitt said or was said earlier about, I think it was the 

### 12 effects on growth in the fish assay, especially in the females, 

### 13 could be an indirect effect on spawning, for example. If the 

### 14 females are not spawning, they may still be producing eggs. So 

### 15 I mean that probably can be captured in the GSI information, but 

### 16 it needs to be considered. It would need to be considered in 

### 17 order to really say it was an effect on growth. If you're using 

### 18 mass as an indicator of growth and compensate for that, based on 

### 19 the GSIs, or the gonad weight. I think that's all I want to 

### 20 say. Thank you. 

### 21 **DR. DANIEL SCHLENK:**Other Panel members? Dr. 

### 22 Scott? 

### 23 **DR. GEOFFREY SCOTT:**If I could just correct 

### 24 one thing I said earlier. When I was mentioning the use of the 

### 25 lower 95 percent confidence limit, I meant that within the 

### 26 connotation of one-third of the lower 95 percent confidence.

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1 **DR. DANIEL SCHLENK:**Okay. Any other 

### 2 comments? Okay, let me go to the Agency, any questions or 

### 3 clarification regarding the comments? 

### 4 **DR. THOMAS STEEGER:**This is Thomas Steeger. 

### 5 I think the discussion about how to define maximum tolerated 

### 6 dose has been really helpful. I believe that the one-third of 

### 7 the LC50 has been proven to be problematic. 

### 8 Using the guidance that was bulleted out that 

### 9 maybe one way to approach this would be to say that you want to 

### 10 have a dose that doesn't result in mortality with severity of an 

### 11 effect, say, on growth would be less than 10 percent and that 

### 12 the frequency of effects across animals would be defined. 

### 13 Does the Panel agree that that's a reasonable 

### 14 standard to apply in terms of setting a maximum tolerated dose? 

### 15 We would like to give some kind of guidance towards eliminating 

### 16 or reducing this problem. As a follow-up to that, when you do 

### 17 have an effect, and overt effect, am I correct in understanding 

### 18 that the Panel is telling us that, yes, it would be considered 

### 19 in the context of what's going on at lower doses, but it's 

### 20 reasonable to consider dropping that dose and evaluating a 

### 21 potential for an effect. 

### 22 **DR. DANIEL SCHLENK:**Dr. Selvage, do you want 

### 23 to lead off on that? 

### 24 DR. DANIEL SELVAGE: No. 

### 25 **DR. DANIEL SCHLENK:**Do you want to pass that 

### 26 to one of your associate discussants then? Dr. Delclos, do you

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1 have a comment? No? Dr. Leonard? 

### 2 **DR. JACK LEONARD:**I think that when you have 

### 3 two events taking place, you have overt toxicity and a presumed 

### 4 endocrine disruptor event, that if the overt toxicity may 

### 5 overweigh or overshadow so that dataset, that particular set of 

### 6 points might be removed from the evaluation so that you were 

### 7 then potentially looking at lower concentration. That's what 

### 8 Dr. Scott was talking about, one-third of the LC50 to start with 

### 9 so that you could sort out the difference between the stress 

### 10 events and the endocrine disruptors events. 

### 11 I think putting those two together makes it 

### 12 difficult to decide which is the cause and which is the effect. 

### 13 So I think yes, if you dropped your dosage, that would be much 

### 14 better. So I would encourage you to remove that high 

### 15 concentration datapoint from your analysis and then reevaluate. 

### 16 **DR. THOMAS STEEGER:**Could you give us a 

### 17 specific example of that? 

### 18 **DR. JACK LEONARD:**No. Since I haven't looked 

### 19 at all of your data, no. But I can tell you an example from my 

### 20 laboratory which we've looked at the thyroid action for 30 plus 

### 21 years, and perchlorate is a very big endocrine disruptor there. 

### 22 You can give quite a bit of perchlorate to the animal without 

### 23 having an overt effects, yet it will really (inaudible) the 

### 24 thyroid gland very quickly. 

### 25 But when you do raise it to a level that you 

### 26 begin to see toxic events in the liver, then the stress events

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1 eliminate what you're sorting. So at that point, we would chose 

### 2 to evaluate perchlorate levels at less than when we see body 

### 3 weight loss. And that's just how operationally I would suggest 

### 4 that would be something to look at. 

### 5 **DR. DANIEL SCHLENK:**Okay, thanks. Dr. 

### 6 Patino? 

### 7 **DR. REYNALDO PATINO:**I don't have any 

### 8 comment. 

### 9 **DR. DANIEL SCHLENK:**Okay. Well I think he's 

### 10 asking if -- I could be wrong here -- but you're asking if 10 

### 11 percent is a good number, is that correct? 

### 12 **DR. REYNALDO PATINO:**Oh, I'm sorry. Okay. 

### 13 Yeah. Like I said for the metamorphosis assay, I don't think 

### 14 that an impact on growth is necessarily a sign of a problem with 

### 15 the assay, so I don't know that a 10 percent -- that may be a 

### 16 little too restrictive. So with that caveat, I can't comment on 

### 17 the other -- you know, I'm not familiar with mammalian systems 

### 18 or others. 

### 19 With the fish assay, you know, they're 10 

### 20 percent might be reasonable just because these fish are adults. 

### 21 I mean fish have an indeterminate growth which means that they 

### 22 grow throughout life more or less until they die, but there is 

### 23 not a whole lot in the growth rate that diminishes. 

### 24 So at that time, in a three week period there 

### 25 is probably not a whole lot of difference in size, I would 

### 26 suspect. And there may be some but not a whole lot. And the

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1 effects on body mass may be a different thing. They could 

### 2 starve, you know, lose appetite and starve and be emaciated, 

### 3 that sort of thing, but given a toxic effect. But I suspect 

### 4 that would occur probably at very high doses of chemicals. 

### 5 So 10 percent might be a reasonable estimate 

### 6 for a fish but I think for frogs, it may be a little too 

### 7 restrictive. And in the context of growth and development being 

### 8 regulated by different -- they're joined at the hip a little 

### 9 bit, but they're really regulated primarily by different 

### 10 systems. 

### 11 **DR. DANIEL SCHLENK:**Okay, great. Let me just 

### 12 remind Dr. Selvage, I hope you're getting all this down. Okay. 

### 13 Dr. Scott? 

### 14 **DR. GEOFFREY SCOTT:**Tom, let me just try to 

### 15 address your question. 

### 16 I think I heard you considering a percent drop 

### 17 in body weight in the extent of incidence. And those were the 

### 18 two things I said I really felt were important. One suggestion 

### 19 I would have would be to go back and look at the compounds 

### 20 you've tested and see if you can generate that. I mean I don't 

### 21 want to put too much onus on y'all but I think it would be nice 

### 22 to know what that number is, because this might come up in the 

### 23 weight of evidence discussion subsequent to this. 

### 24 And I would think if you could at least 

### 25 provide that, I might have a better indication of whether 10 is 

### 26 the right number or not. I will say that in differentiating

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1 between dose response effects in fish, a 10 percent difference 

### 2 usually may not -- given the variance in the population -- may 

### 3 not be significant. And so it would really depend -- and I 

### 4 don't work -- I work with marine fish. You know, they do the 

### 5 exact opposite with salts and things. I can only say that the 

### 6 work I've done, that would be pushing your ability to discern 

### 7 differences, so it may need to be higher than that, I'm not 

### 8 sure. 

### 9 **DR. DANIEL SCHLENK:**Dr. Tillitt? 

### 10 **DR. DONALD TILLITT:**Yes. And my comments are 

### 11 related to the fish assay. I would absolutely tend to agree, 

### 12 I'm saying agree with Dr. Scott's comments. Ten percent may be 

### 13 very difficult. You're talking about fish that are one gram, 

### 14 two grams, and so that amount of difference can easily be seen 

### 15 and so it may be greater than 10 percent as a number. But I 

### 16 think, analyzing the data that you have and looking at variation 

### 17 of that data would be maybe an appropriate step. 

### 18 **DR. DANIEL SCHLENK:**Okay. Dr. Green? 

### 19 **DR. SHERRIL GREEN:**So in terms of growth of 

### 20 Xenopus through the metamorphic period, I don't believe 

### 21 typically you're measuring snout to vent length in the tadpoles, 

### 22 but it may depend on the lab. And clarification on what they're 

### 23 measuring in terms of assessing growth rate will be important 

### 24 because some labs report the time it takes to progress from one 

### 25 stage to the next, and that's a growth rate over the number of 

### 26 days, which you could compare to controls.

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1 But turning tadpoles upside down and looking 

### 2 for the vent, that may be another way to do it that some people 

### 3 report, but typically you're not measuring from the snout to the 

### 4 tip of the tail. And using that as a growth rate, or it could 

### 5 be some combination in -- it wasn't clear to me in the 

### 6 guidelines how exactly as we progress through stages, but that 

### 7 would be something worth clarifying in that how are the labs 

### 8 reporting growth rates. Is it how long it takes to get through 

### 9 stage 4 through 57 or are they measuring snout to vent or snout 

### 10 to tail, or is it body weight? Are they weighing the tadpoles 

### 11 and looking at mass increase? 

### 12 **DR. DANIEL SCHLENK:**Okay. Yes, Dr. Roby? 

### 13 DR. KATHERINE ROBY: I just want to double 

### 14 check, you were also talking about the rodent assays here. I 

### 15 think 10 percent is probably too restrictive, especially with 

### 16 gavaging, certainly with the onset of dosing. There will 

### 17 probably be at least a transient loss of easily 10 percent 

### 18 weight. So maybe again looking at -- not to give you more work 

### 19 -- but looking at some of the data you already have might be 

### 20 insightful as to setting that criteria. I would guess 10 

### 21 percent is too restrictive. 

### 22 **DR. DANIEL SCHLENK:**Dr. Green? 

### 23 **DR. SHERRIL GREEN:**And one more comment. I 

### 24 apologize for talking about the xenopus on the rodent question. 

### 25 But with regards to the 10 percent, in terms of animal's welfare 

### 26 and compliance, typically the IACUCs will have a body loss that

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1 they will approve of on a toxicity study. So some animal use 

### 2 and care committees may recommend that it is no more than 10 

### 3 percent. 

### 4 So if that is an estimate, we're within the 

### 5 ballpark of what would be expected over a period of time when 

### 6 exposed to a potential toxic substance, so no more than 10 

### 7 percent. And I agree, I think 10 percent growth rate might be a 

### 8 little fast for some specific strains. 

### 9 **DR. DANIEL SCHLENK:**Okay. Dr. Leonard. 

### 10 **DR. JACK LEONARD:**At our institution, the 

### 11 IACUC has established an acute loss 10 percent body weight as an 

### 12 overt toxic event. And in our studies we are obliged to 

### 13 terminate the animal at that point, so that colors how I view 

### 14 the weight gain or weight loss. 

### 15 It'll be really interesting to go back and 

### 16 evaluate whether or not that loss was acute. Did it occur over 

### 17 a very short time or was it gradual over the time period? That 

### 18 would clearly change your view point what the event was taking 

### 19 place specifically in the rodent. 

### 20 **DR. DANIEL SCHLENK:**Dr. Patino? 

### 21 **DR. REYNALDO PATINO:**Yes. I think we 

### 22 probably need to differentiate between animals that are not 

### 23 growing in the different assays and animals that do. And I 

### 24 don't know if that question there refers to weight loss or 

### 25 difference between the treatments and the controls, which is a 

### 26 different measure. So just clarifying that, we need to be clear

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1 on that. And that may be assay specific. 

### 2 **DR. DANIEL SCHLENK:**Okay. Dr. Scott? 

### 3 **DR. GEOFFREY SCOTT:**Yeah, one additional 

### 4 comment. If indeed you have length measurements on the fish, it 

### 5 would be good to use it conditioning -- in my opinion, and Don 

### 6 you may comment on this. In my experience, normalizing its 

### 7 length with weight changes is a condition -- actually we used 

### 8 length and then you divide it by weight -- is a good metric. 

### 9 And if you're collecting both of those, it allows you to 

### 10 normalize for differences in size. So if you have those -- I 

### 11 wouldn't suggest adding those as additional measurements -- but 

### 12 if you have them and can do that, it may be helpful. 

### 13 **DR. DANIEL SCHLENK:**Okay. Anyone else want 

### 14 to weigh in on this one? No pun intended. Don? 

### 15 **DR. DONALD TILLITT:**I don't believe they take 

### 16 the length. They do take lengths, so yeah. Okay, so that would 

### 17 be useful, absolutely. 

### 18 **DR. DANIEL SCHLENK:**Okay. Let me go back to 

### 19 Dr. Steeger, did you get the information that you needed from 

### 20 that? 

### 21 **DR. THOMAS STEEGER:**Yes, I did. Thank you. 

### 22 **DR. DANIEL SCHLENK:**Okay. All right. We are 

### 23 done for the day, I think, so let's adjourn. We will reconvene 

### 24 tomorrow morning at nine o'clock. 

### 25 (Whereas the meeting was adjourned for the 

### 26 day)

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1 **DR. DANIEL SCHLENK:**Good morning, everyone. 

### 2 We've got a long day ahead us. So just to sort of streamline 

### 3 things a bit, we'll skip introductions this morning and kind of 

### 4 get straight into the questions. 

### 5 A couple of things before we get started, a 

### 6 couple of Panel members approached me and said that they had 

### 7 talked with some folks during the break yesterday and I wanted 

### 8 to put that on the record. I think Dr. Leonard had -- 

### 9 **DR. JACK LEONARD:**Yesterday afternoon Dr. 

### 10 Manibusan and I had brief conversation that amplified slightly 

### 11 on the percentage loss of weight during some of the in vivo 

### 12 screening tests. And I made a clarification there that I want 

### 13 to make sure it's on record that it was my opinion that an acute 

### 14 loss of 10 percent weight should be viewed very differently than 

### 15 a gradual loss of weight of 10 percent over the entire period, 

### 16 which I considered less of a biological problem. Thank you. 

### 17 **DR. DANIEL SCHLENK:**Great. Thanks. 

### 18 Anybody else have any comments on that? Dr. Green. 

### 19 **DR. SHERRIL GREEN:**Yesterday, I spoke briefly 

### 20 with a representative from Syngenic -- Syngenta, right? -- 

### 21 about an alternative assay to the AMA, and they clarified for me 

### 22 that they did not have a different or more detailed approach to 

### 23 the current AMA assay that's being used in Tier 1 testing. 

### 24 That's it. 

### 25 **DR. DANIEL SCHLENK:**Thanks. Dr. Selvage. 

### 26 **DR. DANIEL SELVAGE:**Sorry. Since we're doing

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1 this, I thought I would say I spoke to a gentleman from EPA 

### 2 regarding the corticosteroid test in the rat species. So that 

### 3 having be my comments. 

### 4 **DR. DANIEL SCHLENK:**Great. Thanks. 

### 5 Anyone else? Any other comments. 

### 6 Fred has informed me that there are some 

### 7 questions of clarification from the Agency for Question 4; is 

### 8 that right? Do you want to put that on the record? 

### 9 **MS. CATHERINE AUBEE:**Thank you. Good 

### 10 morning, everyone. I just had a follow-up clarification 

### 11 question for Dr. Patino regarding his discussion yesterday of 

### 12 hind limb length normalization. You had made some 

### 13 recommendations for testing for an isometric relationship prior 

### 14 to doing any sort of normalization. 

### 15 I wanted to know, 1) if you're recommending 

### 16 that we only do that in controls or across treatments. I want 

### 17 to make sure I understand the recommendation. 

### 18 2) How much of a deviation from that one/one 

### 19 isometric relationship would be too much that you would not 

### 20 recommend doing the normalization? 

### 21 Additionally, I also wanted to request, if 

### 22 possible, you said that you had some references to give to us. 

### 23 If it would be possible for you to read those into the record, 

### 24 that would be very helpful since we won't actually get your 

### 25 report for some time. Thank you. 

### 26 **DR. REYNALDO PATINO:**Yes. That is something

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1 that I think needs to be, as far as the deviation from the slope 

### 2 of one needs to be empirically determined. Also, that depends. 

### 3 It may not be a problem. Let me say that first. It depends on 

### 4 the deviation from the slope of one and also on the range of 

### 5 sizes that they're using because if you have a very short range 

### 6 of sizes, the impact of a deviation of a slope of one may be 

### 7 very limited. 

### 8 So I think it's something that needs t to be 

### 9 empirically determined. I come mostly from the fish literature, 

### 10 from the fish field, biology field, and it's not commonly 

### 11 considered, but morphometricians do care about these things and 

### 12 I've learned in the last few years about that. And the errors 

### 13 -- if you don't consider those things, if you don't show that 

### 14 there is not a problem -- then you may be introducing errors 

### 15 into your data that actually does not exist before you do the 

### 16 transformation. 

### 17 So I have some references. I'm allowed to do 

### 18 it a little bit later because I'm going to have to find them. I 

### 19 can read those into the record. Essentially, a colleague, 

### 20 morphometrician, who taught me about this is, you know, some of 

### 21 this papers is what I'm citing into the record. 

### 22 **MS. CATHERINE AUBEE:**Thank you. 

### 23 **DR. DANIEL SCHLENK:**Dr. Steeger. 

### 24 **DR. THOMAS STEEGER:**I spoke briefly to Dr. 

### 25 Selvage this morning and want to just make clear that what we're 

### 26 looking for, in terms of the maximum tolerated dose, as I

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1 indicated yesterday, we appreciated the panel's comments on how 

### 2 better to define that. 

### 3 To the extent that you can put boundaries 

### 4 around that would be very helpful so that when we make 

### 5 recommendations to the regulated community and how to go about 

### 6 testing for maximum tolerated dose, we can be more specific. 

### 7 Thank you. 

### 8 **DR. DANIEL SCHLENK:**All right. Let's go 

### 9 ahead and move forward with Question 5. 

### 10 **DR. MARY MANIBUSAN:**Charge Question Number 5: 

### 11 Spinal curvature usually manifesting as bent tail and Xenopus 

### 12 laevis tadpoles was reported in 15 of 18 amphibian metamorphosis 

### 13 assay studies reviewed thus far. The anomaly appears to be the 

### 14 first observed several days after study initiation and 

### 15 prevalence increases with time. 

### 16 Overall, the prevalence of spinal curvature in 

### 17 these studies range from a "few per replica" to 92 percent of a 

### 18 given treatment group by test termination. Experimental work by 

### 19 the EPA Office of Research and Development suggests that certain 

### 20 feeding regimes are associated with spinal curvature in their 

### 21 Xenopus test populations; however, spinal curvature remains 

### 22 prevalent in the range of 16 to 92 percent in the five industry 

### 23 AMA studies in which feed was reduced by 50 percent, compared to 

### 24 the guideline recommendations. 

### 25 Overall, the incidence of spinal curvature 

### 26 appears to be highly variable. From a qualitative review of the

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1 data, there appears to be no consistent differences in the 

### 2 incidence or variability of spinal curvature when studies using 

### 3 guideline versus reduced feeding regimes are compared. 

### 4 Please comment on whether the presence or 

### 5 prevalence of spinal curvature in test species, including 

### 6 controls, compromises the utility or validity of an AMA 

### 7 submission. If so, when does the prevalence of spinal curvature 

### 8 render the study unreliable? 

### 9 What technical guidance may be useful for 

### 10 laboratories in reducing the occurrence of spinal curvature and 

### 11 determining if or at what point within the study a study may be 

### 12 compromised because of this phenomenon? 

### 13 **DR. DANIEL SCHLENK:**Thank you. Our lead 

### 14 discussant for that is Dr. Schultz. 

### 15 **DR. TERRY SCHULTZ:**Thank you. The Panel 

### 16 realizes that there is a very high instance, 83 percent of the 

### 17 studies that show this issue of bent tail or spinal curvature, 

### 18 and they do feel that this has potential of impacting the assay. 

### 19 20 I will not read into the record, but within 

### 21 the report we will refer to basically Table 32 and the 

### 22 information presented there. And from that came out, the Panel 

### 23 agrees that the difference in iodine in the food and water, does 

### 24 not seem to explain the syndrome, per se. And that the 

### 25 examination of the data presented in the White Papers suggested 

### 26 response seems to be somewhat laboratory dependent.

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1 With that said, the Panel understands that the 

### 2 AMA is a screening assay in the Tier 1 battery, intended to 

### 3 empirically identify substances which may interfere with normal 

### 4 function of the HPT axis. The AMA is a general vertebrate model 

### 5 to the extent it is based on conservative structures and 

### 6 functions of the HPT axis. As such, it's an important as an ex 

### 7 vivo model or thyroid degenerative processes which respond via 

### 8 the HPT axis. 

### 9 The Panel further understands the AMA is 

### 10 related to altered hypothalamic pituitary function antithyroid 

### 11 activity and thermometric activity. In the case of the altered 

### 12 hypothalamic pituitary function, the AMA is complementary to the 

### 13 rat pubertal assays and the fish reproductive screening. In the 

### 14 case of altered antithyroid activity, the AMA is complimentary 

### 15 to the rat pubertal assay. 

### 16 In the case of the thermometric activity, AMA 

### 17 has no complimentary assays within the Tier 1 fabric. So there 

### 18 is an overlap with other assays, other in vitro assays in the 

### 19 Tier 1 battery, but there are some mechanistic or mode of 

### 20 actions that are covered only by the AMA. 

### 21 The general experimental design, of course, 

### 22 comes from the work of Fort, et al. (2007). Briefly, the 

### 23 Nieuwkoop and Faber Stage 51, Xenopus tadpoles are exposed to 

### 24 treatment groups and minimum of three concentrations with a 

### 25 negative control. The assay runs 28 days, there are replicates 

### 26 per tank.

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1 So basically, the density of 20 tadpoles per 

### 2 tank with the replicates gives you 80 larvae that you're going 

### 3 to be looking at. The observed endpoints include the hind limb 

### 4 length, the snout to vent length, the Nieuwkoop and Faber 

### 5 developmental stage at the end of 21 days, body weight, thyroid 

### 6 histology or histopathology and daily observance mortality and 

### 7 general clinical signs. 

### 8 The Panel feels that without an evaluation of 

### 9 the weight of evidence, the AMA must today be considered 

### 10 important as a Tier 1 screen. But the prevalence and 

### 11 variability and the percent of clutches expressing this general 

### 12 tail malformation or bent tail, does affect competence in the 

### 13 utility of the assay. 

### 14 The Panel further understands that the 

### 15 protocol represented the state of the science in 2008 when the 

### 16 initial Tier 1 testing order was issued. The Panel agrees with 

### 17 the Agency in not altering that protocol until the first round 

### 18 of testing was completed. 

### 19 While this SAP is based on only 21 of the 52 

### 20 chemicals being tested in this initial order, the Panel feels 

### 21 there is sufficient evidence with the AMA that the protocol be 

### 22 reevaluated as soon as possible. 

### 23 However, the Panel also recommends not 

### 24 changing the experimental design of the actual study outcomes 

### 25 until all 52 chemicals are evaluated. The reason for the 

### 26 discrepancy here, the Panel wasn't sure whether all 52 chemicals

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1 have been tested, and only 21 have been evaluated by the Agency 

### 2 or there are others that are in the process. 

### 3 We're not asking the Agency to change the 

### 4 horse in the middle of the stream, but as soon as you get to 

### 5 shallow water on the other side, start looking at another way to 

### 6 get across the river. 

### 7 Since they stopped the vent length as a key 

### 8 measurement in the AMA, the Panel feels the bent tail issue has 

### 9 the potential to impact both performance criteria for acceptance 

### 10 of NGO test results and scientific competence in the utility and 

### 11 the validity of the AMA. Therefore, the Panel recommends the 

### 12 Agency determine the cause and possible mode of action for the 

### 13 bent tail syndrome so guidance can be given to the laboratories 

### 14 in how to eliminate it or at least reduce its morbidity response 

### 15 to less than 10 percent of the population. 

### 16 The Panel understands that the assay typically 

### 17 begins with Stage 21 larvae that do not show tail curvature, 

### 18 however, curvature is typically not observed until the larvae 

### 19 are further along in the pro metamorphose stage. That is, 

### 20 window of susceptibility for the AMA is basically Stage 51 

### 21 through 55. 

### 22 Thus, laboratories cannot simply select the 

### 23 effect out of the test; however, the Panel feels there may be 

### 24 signs in the Stage 51 embryos they can use to assist the 

### 25 laboratory in doing a better job of selecting against this 

### 26 response.

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1 The Panel further recommends monitoring a 

### 2 portion of each clutch not used in a given study. It would be 

### 3 prudentially useful to possibly report these results. The Panel 

### 4 understands that summation of this competence is in the guidance 

### 5 document, but maybe it needs to be elaborated. 

### 6 In other words, once you set up the tests, you 

### 7 had some things in the clutch monitoring you may identify early 

### 8 on of whether you want to proceed to the full 21 days on that 

### 9 test or make some decision at day 7 to discontinue that test 

### 10 because clutch performance was so poor that it wouldn't be 

### 11 useful. 

### 12 The Panel further recommends that with the 

### 13 acceptance of the AMA test results, overall, the clutch should 

### 14 not have a rate of mortality or morbidity combined to be greater 

### 15 than 20 percent. 

### 16 Evidence presented in the White Paper and the 

### 17 oral presentations at SAP does not allow for definitive 

### 18 understanding of the bent tail syndrome. With that said, the 

### 19 Panel acknowledges the presentation of Dr. Olmstead during the 

### 20 public comment period was of real value. 

### 21 The Panel believes that pathological 

### 22 examinations of the tadpoles may reveal information that would 

### 23 be more helpful. And in that regard we're referring not only to 

### 24 gross anatomical pathology, but actually being some 

### 25 histopathology of the lesion area. And the additional comments 

### 26 will explain why we're interested in that.

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1 The Panel benefits from having members that 

### 2 have expert, extensive experience with Xenopus husbandry. These 

### 3 Panel members feel that the bent tail syndrome is mainly a 

### 4 husbandry issue. The Panel believes that the potential causes 

### 5 of this response are 1) genetic, 2) nutrient deficiencies, and 

### 6 3) ionic imbalance. 

### 7 The Panel further thinks that it is highly 

### 8 probable that all three factors may come into play in some sort 

### 9 of multifactorial causation scheme. In other words, it's very 

### 10 difficult to tease out the genetics from the nutrition from the 

### 11 (inaudible) because of the complexity of running those types of 

### 12 assays, but there is both trend and anecdotal information that 

### 13 says all three of those are particularly important. 

### 14 In particular, as anecdotal information 

### 15 leading to such syndrome is related to ionic balance which is 

### 16 expected by both the composition of the water and the 

### 17 nutritional deficiency. 

### 18 One panel member has extensive experience in 

### 19 performing the frog embryo teratogenesis assay Xenopus, better 

### 20 known as FETAX. Where this assay is different in protocol from 

### 21 the AAM, the test species is the same. Work by this panel 

### 22 member found that softer water, water with less calcium and 

### 23 potassium caused a greater incidence in the lateral tail 

### 24 flexures, and other general toxicity -- I'm putting "toxicity" 

### 25 in quotes in here -- effects such as edema and abnormal gut 

### 26 coiling.

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1 We found edema, abnormal gut coiling and 

### 2 lateral tail flexures to be general responses we see in a wide 

### 3 variety of exposures to different toxicants. They are not a 

### 4 specific teratogenic response that can be linked to a particular 

### 5 mode of action. 

### 6 Moreover, experience by selected Panel members 

### 7 has shown that by keeping track of clutch performance, 

### 8 specifically the number and the quality of the embryos from each 

### 9 spawn, for high quality breeding pairs can assist in reducing 

### 10 these general effects. 

### 11 Moreover, breeding these selected pairs on a 

### 12 regular schedule, regardless of the need for the embryo, does 

### 13 improve clutch quality. So if people are people are purchasing 

### 14 pairs, bringing them in, using them one or two times for this or 

### 15 that, you get less overall clutch quality than if you bring in 

### 16 matched pairs and keep track of them on the frontend and then 

### 17 read them regularly so you're not getting older eggs in the 

### 18 system. All improves clutch quality and all those things reduce 

### 19 the general toxic observation. 

### 20 The panel also notes that in fish, nutrient 

### 21 deficiencies such as micronutrients, can lead to development 

### 22 curvature of the spine. Also, excess of certain nutrients, for 

### 23 example, selenium and copper, can also cause this lesion. 

### 24 Lastly, dissolved oxygen also leads to spinal 

### 25 curvature in fish embryos. So there is evidence for a number of 

### 26 aquatic species that the husbandry issue is very, very important

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1 in identifying this particular lesion. 

### 2 In relation to other comments, while not 

### 3 directly asked in Question 5, the Panel strongly feels that the 

### 4 issue of the bent tail in Question 5 and thyroid histology in 

### 5 Question 6 and their interaction with other endpoints associated 

### 6 with the AMA, leads to a recommendation that the Agency look at 

### 7 the change in the experimental design of the AMA study. 

### 8 It is further recommended that the Agency 

### 9 specifically look into the development of stage-dependent, 

### 10 rather than time-dependent protocol. And I think this will be 

### 11 addressed more specifically in Question Number 6, so I will 

### 12 leave that right there and it will appear up again. 

### 13 Since neither the White Paper, nor the 

### 14 Agency's oral presentation have tried the histopathology of the 

### 15 bent tail lesion or presents photomicrographs of the conditions 

### 16 - this is looking at it with either a live microscope or the 

### 17 electron microscope -- the Panel found it difficult to provide 

### 18 meaningful guidance. 

### 19 The Panel members with experience with the 

### 20 Xenopus testing realize that the issue where tail mouth 

### 21 (inaudible) lateral tail afflictions are typically considered 

### 22 non-specific effect and associated with a muscular phenomenon 

### 23 where malformation, such as the tail, a dorsal ventral kinking 

### 24 tend to be more related to connective tissue or skeletal 

### 25 phenomenon. And those are two very different mechanisms which 

### 26 show different histopathologies, but show very similar growth

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1 pathologies. 

### 2 I will then like to read into the record that 

### 3 we're noting the study of Schultz, et al. (1985), examining 

### 4 osteolathyrogenic effects and the history of that and the study 

### 5 of Hitch and Dale (2005), which talks about the lysyl oxidase 

### 6 family and expression of frog which reports the importance of 

### 7 lysyl oxidase and the copper co-factor in the formation of 

### 8 elastin and collagen fibers. 

### 9 Lastly, the Panel understands there is no 

### 10 biochemical data to date that demonstrates there is alteration 

### 11 in elastin and collagen formation. The Panel understands that 

### 12 functionally, the tail bending that is reported does not 

### 13 actually affect the reabsorption of the tail, but the Panel is 

### 14 not convinced that the site of a lesion could not affect the 

### 15 measurement of the snout to vent length. 

### 16 And since that is a key measurement that's 

### 17 used in the total package compared to other components, it can't 

### 18 be eliminated as that the tail bending does not have an impact 

### 19 on the interpretation of the results of the AMA. And those are 

### 20 the general comments, but there will be some other comments made 

### 21 by some of the Panel members. Thank you. 

### 22 **DR. DANIEL SCHLENK:**Thank you, Dr. 

### 23 Schultz. Our next discussant is Dr. Green. 

### 24 **DR. SHERRIL GREEN:**I'd like to elaborate, 

### 25 particularly on some elements of information that we would need 

### 26 in addition to assess the bent tail, as reported by some of the

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1 contract laboratories, and focus a little bit more on some of 

### 2 the husbandry and the recommendations that I can make from 

### 3 personal experience and also from what is published on Xenopus 

### 4 and other species about the occurrence of bent tail. 

### 5 Some of the additional information that would 

### 6 be required, that would be helpful would be for the panel to 

### 7 understand, in addition to photographs and histopathology of 

### 8 these affected specimens, the source of the stock used in this 

### 9 study -- and I'll explain why that's important in a moment -- 

### 10 and details regarding the husbandry conditions, especially water 

### 11 quality; especially water quality -- I need to reiterate that -- 

### 12 under which these conditions occurred. 

### 13 For someone like myself who is maintaining 

### 14 large populations of adult Xenopus in laboratory conditions and 

### 15 dealing with researchers who reared tadpoles, not on the 

### 16 production scale that would be required in these situations for 

### 17 laboratory testing. There are very few labs in academics that 

### 18 do raise the large number of tadpoles. I can probably think of 

### 19 three, maybe across the nation, and one or two vendors that do 

### 20 it. 

### 21 The reason I bring this up is because the 

### 22 raising of tadpoles is very much an art, as most of you who've 

### 23 done it know, and part of it is science, but it really takes 

### 24 experience. You learn a lot by trial and error. Somebody who 

### 25 is new to the business of Xenopus, and particularly raising 

### 26 Xenopus tadpoles beyond 96 hours, would be faced with some of

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1 these husbandry issues no matter what. So I wouldn't say that 

### 2 they are entirely novel or unexpected. 

### 3 Moving forward, though, I think it's important 

### 4 to point out that as a laboratory animal veterinarian in charge 

### 5 of several thousand Xenopus laevis in a laboratory environment, 

### 6 when we come across the occasional tadpole with a bent tail we 

### 7 attribute it to infrequent recessive mutations that are 

### 8 well-documented in this species. 

### 9 In fact, the mutation load in Xenopus laevis 

### 10 is 1.875, and that's slightly lower than what is actually seen 

### 11 in humans. So it does occur in ideal conditions where husbandry 

### 12 is carefully assessed every day and monitored continuously. So 

### 13 they're not unusual, but they are considered rare. 

### 14 And usually, we can trace back those rare bent 

### 15 tail tadpoles to matings with wild-caught stock. And this has 

### 16 been published in a 2005 paper which I will supply to the 

### 17 agency. Some of this information comes directly from this 

### 18 paper. 

### 19 Again, as a laboratory animal veterinarian, if 

### 20 I went into a room and somebody reported to me an entire tank of 

### 21 tadpoles suddenly developed bent tail -- and this can happen, 

### 22 literally, overnight. There are good hobbyist videos online 

### 23 where you can see they've set up a webcam and they're watching 

### 24 their tadpoles grow and then they'll report, "Oh my gosh, I came 

### 25 in today and the entire tank of tadpoles has curved tails." 

### 26 The first thing I would think of, as a

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1 laboratory animal veterinarian, diagnostically, is this is a 

### 2 water quality problem. The sporadic morbidities and mortalities 

### 3 in large colonies of Xenopus and tadpoles may be other causes, 

### 4 infectious diseases, and you can usually look at your morbidity 

### 5 reports and see that it happens over time, over weeks. But when 

### 6 you have large numbers of animals affected, it's water quality 

### 7 that we look to first. 

### 8 Again, Noramly, et al. published that these 

### 9 bent tail occurrences under these husbandry conditions are 

### 10 considered epigenetic effects, usually due to less than ideal 

### 11 husbandry conditions. 

### 12 So what are those conditions? There are a 

### 13 couple of them that had been reported in Xenopus. And I would 

### 14 refer the Agency and the labs that they work with to that 

### 15 Noramly paper published in 2005. It's from the Grainger Lab at 

### 16 the University of Virginia, and they have vast experience and 

### 17 they also refer to the Harland Lab at UC Berkeley. 

### 18 The tadpole rearing conditions must be very 

### 19 fine-tuned, as they report, especially during the first seven 

### 20 days. They see bent tail then and then beyond and they feel 

### 21 that it's largely due to water quality changes and issues in the 

### 22 first seven days. 

### 23 But other things affect the husbandry and they 

### 24 go through in detail what they recommend for stock and density. 

### 25 One of the important points that they have 

### 26 raised, particularly with tadpole rearing, and this is for

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1 Xenopus tropicalis, but I can infer by reading that paper that 

### 2 probably for Xenopus laevis as well; pH seems to be very 

### 3 important. And they have a very narrow pH range, as published 

### 4 in that paper. On their website URL that they refer readers to 

### 5 about dealing with bent tail. 

### 6 Let me move through the document a little bit 

### 7 more. Recommendations here, as Dr. Schultz pointed out and the 

### 8 rest of the group that studied this on the panel, it will be 

### 9 important to keep track of clutch performance. So you need to 

### 10 know where your mating pairs came from. And some vendors, 

### 11 wild-catch their Xenopus laevis and one big vendor has only 

### 12 laboratory-reared and conditioned frogs. 

### 13 I might make the recommendation that that 

### 14 should be considered that wild-caught frogs not be used in the 

### 15 matings and that would eliminate some of the problem, not all of 

### 16 the problem. 

### 17 Usually, even if they are wild caught by two 

### 18 or three generations, you can pick out the frogs that don't seem 

### 19 to be showing, if it is a genetically related recessive mutation 

### 20 and get rid of the problem significantly by doing that. 

### 21 Another recommendation would be to closely 

### 22 review the water quality conditions under which those bent tail 

### 23 tadpoles were kept. And daily recording observations, 

### 24 minimally, of pH, water temperature, ammonia content, water 

### 25 hardness, DO. Those are pretty standard water quality tests, 

### 26 with the YSI monitors that sit in the tanks that continuously

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1 read out which most systems use, or even by hand, would be 

### 2 really important. 

### 3 Temperature, pH, some nutritional deficiencies 

### 4 such as calcium and Vitamin C are reported to cause bent tail in 

### 5 other amphibian species that are laboratory-reared. The 

### 6 American bullfrog and the leopard frog, you know, be aware that 

### 7 those are not fully aquatic species. They differ in eco-habitat 

### 8 and in some of their nutritional requirements. But they're 

### 9 close enough as an example of what can happen in a laboratory 

### 10 environment without careful attention to some of these other 

### 11 conditions. 

### 12 Temperature and pH have been associated with a 

### 13 condition in Xenopus tropicalis, as well as water softness, 

### 14 which in some institutions, the water filters out certain ions: 

### 15 calcium, magnesium, potassium, and can make the water softer. 

### 16 And that has been experienced by a panel member with Xenopus 

### 17 laevis. 

### 18 I studied carefully the overfeeding and there 

### 19 is a different perspective, from a laboratory animal 

### 20 veterinarian's review, that it isn't overfeeding and excessive 

### 21 growth as a result of it that may be contributing to bent tail, 

### 22 but in my opinion, overfeeding can really pollute the water. 

### 23 You have ammonia and pH changes that comes with the degradation 

### 24 of the uneaten food and also the amount of feces and body wastes 

### 25 that growing frogs will produce. So that all comes back to 

### 26 water chemistry and scrupulous husbandry practices.

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1 Uneaten food and excess food, again, will 

### 2 cause those changes and could potentially cause the bent tail 

### 3 seen in many of these labs. So when they backed off by 50 

### 4 percent, you could eliminate some of it, but not all of it 

### 5 because you would still need to pay attention to what's 

### 6 happening in the water. I would suggest that again, water 

### 7 quality is a big issue here. 

### 8 There are several papers that have been 

### 9 published, maybe five years ago at this point, that show certain 

### 10 herbicides and pesticides are known to cause bent tail in 

### 11 Xenopus laevis tadpoles. So it makes sense that under these 

### 12 testing conditions that we might screen both the water and the 

### 13 food for herbicides and pesticides. Maybe not an entire 

### 14 toxicological screen because that would be an added expense, but 

### 15 certainly for the ones that we know cause bent tail. And that 

### 16 too, I believe, has been recommended by Noramly in Robert 

### 17 Grainger's paper. 

### 18 All in all, I think those recommendations 

### 19 would take the Agency and the contract labs fairly far in 

### 20 getting a handle on what's happening with bent tail in some of 

### 21 these animals. 

### 22 As the Panel stated, morbidity and mortality 

### 23 less than 20 percent is a typical acceptance rate, and bent tail 

### 24 less than 10 percent is probably a reasonable target to try and 

### 25 meet, I'd say. 

### 26 Again, even some of the research labs have

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1 recognized that some of these animals will go on with a bent 

### 2 tail and go through metamorphosis and do quite well. In fact, 

### 3 the webcam hobbyist had a tank of 15 tadpoles that literally, 

### 4 overnight, developed bent tail. And if you look closely, you 

### 5 also made a comment, "I noticed some brown fuzzy stuff growing 

### 6 on one of them." 

### 7 Well, that's probably a water mold that's an 

### 8 opportunistic pathogen, saprolegnia or some related. And that 

### 9 doesn't usually appear unless there is a water quality problem. 

### 10 At any rate, with the exception of that tadpole, the other 15 

### 11 progressed through metamorphosis quite fine and normally. 

### 12 And again, if it is related to some of these 

### 13 mutations where the kink affects the spine, then it will, as Dr. 

### 14 Schultz pointed out, affect your ability to get an accurate read 

### 15 on snout to vent length because the back is crooked. But if 

### 16 it's beyond that and it's just the tail, many of those animals 

### 17 will go on to develop just fine. 

### 18 Whether or not they should be included in a 

### 19 study, ideally, I wouldn't include them and that's why it would 

### 20 be important to get the effect rate, the prevalence of the 

### 21 condition to 10 percent or below, and then you would still have 

### 22 plenty of animals that are not affected and are developing 

### 23 normally. 

### 24 So I think that's all I have. 

### 25 **DR. DANIEL SCHLENK:**Thank you, Dr. Green. 

### 26 Dr. Scott.

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1 **DR. GEOFFREY SCOTT:**Just a couple of comments 

### 2 to add to what Terry and Dr. Green had said. One of the issues 

### 3 that I think is really important, we talked about, in this 

### 4 assay, alterations in feeding, both overfeeding and reducing 

### 5 feeding, and I want to talk, first of all, about the quality of 

### 6 the food. 

### 7 One of the things that I think is very 

### 8 important in looking at that is to know the active ingredients 

### 9 in the food. And I only say that from some experience dealing 

### 10 with some fish assays. 

### 11 We were dealing with fish bioassays, looking 

### 12 at multi-drug resistant transporter proteins expression and PGP, 

### 13 which will prevent uptake of many things, chemicals as well as 

### 14 other nutrients. And what we found was in some work we did, 

### 15 taking fish from a reference area that had very little PGP 

### 16 expression, after a week in the lab of feeding with tetramin 

### 17 fish flakes, they suddenly had levels of that expression 

### 18 comparable to what we found at EPA superfund sites in the same 

### 19 species, which before, they had none. 

### 20 We went back and looked; the only thing that 

### 21 was different was the food and we found in the food ethoxyquin 

### 22 which is a stabilizer that basically revs up the PGP and NDR. 

### 23 Now, the reason that's important, it prevents uptake of certain 

### 24 things. So if you are reducing the uptake of essential 

### 25 nutrients by that type of response that could -- and you're 

### 26 overfeeding and giving more of those types of ingredients, then

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1 you're really compounding a situation. 

### 2 So one of the things that I would highly 

### 3 recommend would be to look at the quality of the food and look 

### 4 at those types of agents to see if any of those types of 

### 5 compounds could play a role in some of this. It may not, but I 

### 6 know that really surprised us that we could see those types of 

### 7 gene expression get so increased dramatically. 

### 8 The second point I would make about this, this 

### 9 is not the only species we have seen problems with confounding 

### 10 variables in testing. Those of us that have done molluscan 

### 11 testing for FIFRA, we know, all of a sudden in the '80s, oysters 

### 12 suddenly got a lot of oyster disease. 

### 13 We were getting annual mortality rates of 50 

### 14 percent and we had to adjust our testing schedule to test around 

### 15 that, but we did, as Dr. Green, I think suggested, limits of 10 

### 16 percent. We did have to set limits control mortality and 

### 17 bioassays, and you had to perform in order to do that. We've 

### 18 had similar problems in shrimp assays with disease. 

### 19 So we have had those problems in other 

### 20 species. One of the things, like I'll say in the oysters, we 

### 21 did move towards a disease-free oyster. And I think Terry made 

### 22 that in his comments, trying to get to those select strains so 

### 23 we can minimize this is another approach to control of this 

### 24 issue. 

### 25 I'd like to talk a little bit about how we can 

### 26 improve QA/QC on the husbandry side. Dr. Green hit on numerous

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1 points and Terry did too, I just want to add, I think really 

### 2 getting that information on clutch performance, the water 

### 3 quality data Dr. Green talked about, and again, I would make 

### 4 sure we have very good records on the food, again, the quality 

### 5 of the food. 

### 6 And in that screening, as you mentioned, for 

### 7 contaminates, I would add to that screening for biology toxins, 

### 8 which could play a role in this. Possibly. Just to be sure 

### 9 that that food is of good quality. And those should be 

### 10 recordkeeping as part of this, in my opinion. 

### 11 The manufacturers are probably doing this, 

### 12 asking for those records and keeping that information, it would 

### 13 seem like would not be inordinate to ask and it would help us 

### 14 move this along without increasing additional cost in testing. 

### 15 Finally, one other suggestion. If we can 

### 16 minimize this problem, but we still have some rate of bent back 

### 17 that we're not happy with, one other option would be to look for 

### 18 a positive control that would induce bent back that we know that 

### 19 would do this. And I certainly can think of compounds for fish 

### 20 that can do this and then see whether or not if you have -- what 

### 21 you can do is take your data and use a large enough end in 

### 22 developing some kind of positive control assay that referencing 

### 23 this. 

### 24 If you do have ones that develop the syndrome, 

### 25 you can compare the ability to discern an effect with or without 

### 26 that syndrome. But I think using a positive control that would

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1 induce it will tell you whether or not you can discriminate and 

### 2 whether with that confoundingness (sic) of that, you can still 

### 3 discern an effect. At least that would allow you to standardize 

### 4 and try to control for that. And I think that's all my 

### 5 comments. 

### 6 **DR. DANIEL SCHLENK:**Thank you, Dr. Scott. 

### 7 Dr. Tillit. 

### 8 **DR. DONALD TILLIT:**I have no further comment. 

### 9 **DR. DANIEL SCHLENK:**Other Panel members? 

### 10 Dr. Green. 

### 11 **DR. SHERRIL GREEN:**Following up on Dr. 

### 12 Scott's comment about quality control of a clutch, I do agree, 

### 13 too, that it takes some nominal technical expertise, but when 

### 14 the female frogs lay eggs, the best way to get good control from 

### 15 the get-go is have somebody go in there and put the Petri dish 

### 16 under a dissecting scope and pick out all the ones that are 

### 17 crinkled or misshapen, or have bacteria, or debris stuck to the 

### 18 surface. And you can call a number of them that normally 

### 19 wouldn't go on. 

### 20 After fertilization, you can go back and look 

### 21 again, and with a pipette, suck out all the ones that look 

### 22 deformed. That way you'll get a better quality hatch and 

### 23 probably, at least the Grainger Lab has suggested that you can 

### 24 eliminate some of the bent tail from the very beginning. 

### 25 **DR. DANIEL SCHLENK:**Any other comments? 

### 26 Okay. At this point, we'll go to the Agency. Any questions or

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1 clarification regarding Question 5? 

### 2 **MS. CATHERINE AUBEE:**I have a clarifying 

### 3 question for Dr. Scott. One of your recommendations was to add 

### 4 the screening for biotoxins to the screening of the diet. Are 

### 5 there specific biological toxins that you had in mind? 

### 6 **DR. GEOFFREY SCOTT:**I can get back with some 

### 7 suggestions on that. Generally speaking, you can extract by 

### 8 different solvent techniques and get classes. And I think I can 

### 9 come back some more detailed information on that later. It may 

### 10 not be today, but I can get you that information. 

### 11 **DR. DANIEL SCHLENK:**Okay. Ready to move 

### 12 forward. 

### 13 **DR. MARY MANIBUSAN:**Yes. No additional 

### 14 comments. Thanks. 

### 15 **DR. DANIEL SCHLENK:**Okay. 

### 16 **DR. MARY MANIBUSAN:**Charge Question Number 

### 17 6, (a) and (b): With the exception of thyroid gross pathology 

### 18 findings, thyroid gland atrophy and hypertrophy in the AMA, 

### 19 severity grades are generally assigned based on comparison to 

### 20 normal Xenopus laevis thyroid findings depicted in the guidance 

### 21 or based on the professional opinion of the pathologist 

### 22 conducting the assessment; they are not assigned in comparison 

### 23 to concurrent control findings from a given study. 

### 24 In one study, the pathologist's report 

### 25 identified a lower incidence in severity of follicular cell 

### 26 hypertrophy when compared to the incidence and severity of this

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1 trait in control specimens. Similar trends have been observed 

### 2 in other studies. 

### 3 In this case, the pathologist concluded that 

### 4 the finding was potentially consistent with treatment-related 

### 5 delay of metamorphosis because thyroid follicular cells normally 

### 6 increase in height during tadpole development. 

### 7 Please comment on the validity of this 

### 8 conclusion: What guidance may be given to better distinguish 

### 9 between histological changes in the thyroid associated with 

### 10 normal progression of metamorphosis and treatment-related 

### 11 effects? 

### 12 Are there certain lesions or diagnoses which 

### 13 may, by their absence or lessened severity as compared to 

### 14 controls, be indicative of treatment-related HPT effects such as 

### 15 delayed metamorphosis? 

### 16 **DR. DANIEL SCHLENK:**Our lead discussant 

### 17 is Dr. Patino. Let's handle (a) first if that's okay. 

### 18 **DR. REYNALDO PATINO:**Thank you. The 

### 19 discussants for this question met, or we emailed and exchanged 

### 20 information and I'm going to try to represent the consensus 

### 21 opinion of the group. I'm sure they will chime in later if I 

### 22 missed or stated something incorrectly. 

### 23 Before addressing these specific questions, I 

### 24 would like to provide some general information to provide 

### 25 context and also to maybe describe some of the issues that have 

### 26 been raised in regards to the question.

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1 The AMA test guidelines states that the assay 

### 2 should begin with NF Stage 51 tadpoles and that the median NF in 

### 3 controls at test termination, which is 21 days, should be 

### 4 greater than 57, with the difference between the 10th and 90th 

### 5 percentile, being no more than four stages. What this means is 

### 6 that you can get the control stage at Day 21 to be less than 

### 7 Stage 60. 

### 8 As has been noted a number of times already 

### 9 during this meeting, the activity of the HPT axis normally 

### 10 increases during amphibian metamorphosis and this phenomenon 

### 11 makes an assessment of treatment effects on thyroid 

### 12 histopathology relatively difficult because the control 

### 13 condition is literally a moving target. 

### 14 To compensate for this complication, or 

### 15 potential complication, the AMA test guidelines recommends using 

### 16 stage match individuals for histological analysis beyond 

### 17 exposures. The guidelines states five tadpoles for treatment 

### 18 per replicate for up to a total of 20 should be matched to the 

### 19 median stage of full control tadpoles whenever possible. 

### 20 Comments provided yesterday by the Endocrine 

### 21 Policy Forum indicated that there is variability in the median 

### 22 stage of developmental control tadpoles reported by the various 

### 23 studies, and also included concern about the possibility of 

### 24 inconsistency among the studies with respect to developmental 

### 25 stage at which treatment effects are being compared. 

### 26 Another point worth noting is that the test

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1 guideline calls for the use of a standard scheme to grade 

### 2 thyroid lesion severity, but it's based on normal changes that 

### 3 occur during metamorphosis in Xenopus. 

### 4 The lack of quantitative endpoint of thyroid 

### 5 activity was also a concern expressed in some of the written 

### 6 statements provided by some of the public commenters. We will 

### 7 explain in a few moments, this concern is also shared by the 

### 8 panel. 

### 9 Specifically going through Question A, in the 

### 10 opinion of this panel, the answer to Question 6a is "Yes." The 

### 11 pathology's conclusion that a lower incidence and severity of 

### 12 follicular cell hypertrophy is consistent with the treatment 

### 13 recommendation of metamorphosis is valid, in our opinion. 

### 14 And in this context, the finding is also 

### 15 consistent with the potential disruption of the HPT axis; 

### 16 however, this particular case also presents what may be an 

### 17 atypical disruption of the HPT axis in that it is not based on 

### 18 classical anti-thyroid mechanisms such as those observed after 

### 19 exposure to thyroid synthesis inhibitors, but on a failure, 

### 20 perhaps, of the axis to activate as it should, or normally. 

### 21 A similar finding, as a reference, a similar 

### 22 finding by the laboratory of a Panel member has been reported 

### 23 for Xenopus tadpoles exposed to very high concentration of 

### 24 (inaudible), and I will provide that reference in the comments. 

### 25 Possible interpretations of these findings 

### 26 include a reduced ability of thyrocytes to respond to TSH

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1 stimulation. That would be a thyroid lesion, and/or a reduced 

### 2 ability of the pituitary to produce TSH. That would probably 

### 3 represent the hypothalamic or pituitary lesion. 

### 4 And a better understanding of the pathology's 

### 5 finding would require not only consideration of the actual 

### 6 effects on development, which are also available. That would be 

### 7 hind limb length in that stage, but also clarification of 

### 8 mechanisms responsible for the apparent failure of the HPT axis 

### 9 to activate properly, if in fact that is what happened. 

### 10 These questions may be appropriately 

### 11 considered more in-depth later, maybe when the Agency considers 

### 12 mechanisms. Maybe even Tier 2 axis. 

### 13 As far as Question 6a, that's what I have. 

### 14 **DR. DANIEL SCHLENK:**Dr. Green. 

### 15 **DR. SHERRIL GREEN:**I have no further 

### 16 comments. 

### 17 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 18 **DR. JACK LEONARD:**I would just like to 

### 19 amplify one confounding issue that I think highlights what some 

### 20 of the trouble here is. For context, the height of the 

### 21 follicular cell epithelial is really a direct read out of tropic 

### 22 hormone. So it is in the absence of TSH. The height is small 

### 23 or thin, and in the presence of PSH, it's tall. 

### 24 What pathologists here found was a flattened 

### 25 epithelial, which suggests the silencing of the HPT axis 

### 26 directly. A thyromimetic would do that. And a thyromimetic, in

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1 this instance, would silence the axis, but still allow a 

### 2 metamorphosis to proceed. 

### 3 So in the absence of corroborating data, 

### 4 pointing to the metamorphic state of the animal, this, in 

### 5 isolation, just gives you insight into the HPT axis itself. So 

### 6 it's consistent but not diagnostic. Thank you. 

### 7 **DR. DANIEL SCHLENK:**And Dr. Schultz. 

### 8 **DR. TERRY SHULTZ:**Yes. I would just like to 

### 9 emphasize that change in the protocol to include software for 

### 10 morphometric analysis would clearly eliminate some of the 

### 11 subjectivity in looking at this follicular cell height, since 

### 12 this is obviously a critical issue coming out of the assay. And 

### 13 this eliminates them having to have a high level of experience 

### 14 by the operator to do that. 

### 15 Many of these have gotten to the point where 

### 16 they are fairly sophisticated, inexpensive and it kind of does 

### 17 all the things for you, dump it in and do the analysis, so 

### 18 really streamline the whole operation. 

### 19 **DR. DANIEL SCHLENK:**Okay. Other Panel 

### 20 members for 6a? Dr. Leonard. 

### 21 **DR. JACK LEONARD:**Just one additional 

### 22 amplification of Dr. Schultz's comments. When you look at the 

### 23 histological preparation of a thyroid in cross-section, the 

### 24 gland itself is functionally heterogeneous. So there are 

### 25 portions of the gland that are quite active. Portions of the 

### 26 gland that are actually silent. And it's evident; if you look

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1 at a lot of published images, you'll see large follicles, you'll 

### 2 see small follicles. Partly it's because the follicles aren't 

### 3 circled. But that confounds the issue of using -- it requires 

### 4 the pathologist to make a very concerted effort to screen the 

### 5 whole slide. And I think they are usually pretty good about 

### 6 that. 

### 7 But at times when things are oppressed, those 

### 8 screens can be more superfluous than statistical. So a 

### 9 morphometric measurement is recommended. 

### 10 **DR. DANIEL SCHLENK:**Sounds like a perfect 

### 11 segue into (b), actually. Do you want to go ahead, Dr. Patino, 

### 12 for 6b? 

### 13 **DR. REYNALDO PATINO:**As we just mentioned, 

### 14 the change in histological appearance of the thyroid during 

### 15 metamorphosis complicates analysis and interpretation of 

### 16 crippling effects. 

### 17 Unfortunately, this panel is unaware of any 

### 18 other standard histological feature of the thyroid that does not 

### 19 normally change during metamorphosis, but there may be one 

### 20 histochemical feature, specifically in the Xenopus thyroid, but 

### 21 maybe independent of development. 

### 22 A recent study involving collaboration with a 

### 23 panel member reported that T4 immunoreactivity is concentrated 

### 24 in the thyroid follicle, at the periphery of the colloid and 

### 25 that the intensity of this ring remains fairly constant during 

### 26 metamorphosis. It's fairly independent of development.

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1 Moreover, the changes in the intensity of this 

### 2 ring, following exposure to (inaudible) in this particular case 

### 3 was recurring, had better sensitivity to detect anti-thyroid 

### 4 activity than changes in standard histological features such as 

### 5 thyrocyte height. 

### 6 This marker, obviously, it's only one study, 

### 7 has not been validated for standard testing and is valued for 

### 8 situations such as the one percent in Question 6, which is the 

### 9 figure of activating -- an apparent failure to activate the HPT 

### 10 axis. The applicability of this marker is uncertain. 

### 11 Nonetheless, the availability of a development 

### 12 independent marker of thyroid activity will certainly avoid 

### 13 most, if not all of the issues of concern to Question 6. 

### 14 In addition, there are other assays that could 

### 15 also be useful, such as an iodide optic assay that may provide 

### 16 quantitative information. It may be worth, to the Agency, to at 

### 17 least consider the possibility of modifications to this 

### 18 guideline in the future to incorporate some of these assays, or 

### 19 perhaps properly validate it to use them as replacement for some 

### 20 of the current influence that have some of these questions. 

### 21 There is another comment that has already been 

### 22 partially mentioned in the preceding moments that is relevant to 

### 23 Question 6 that this panel would like to offer and that is that 

### 24 although categorical rank variables typically do not yield the 

### 25 same statistical part of continuous variables, the current 

### 26 amphibian metamorphosis test guidelines relies on the

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1 semi-quantitative grading scheme to assess thyroid effects. 

### 2 Also, an additional limitation of grading 

### 3 schemes is that they are subject to conditions biases and 

### 4 therefore may yield inconsistent results between studies as well 

### 5 as between laboratories. The Panel therefore advises the Agency 

### 6 to consider the use of quantitative histological data to further 

### 7 standardize the test. 

### 8 As has already been mentioned, there are a 

### 9 number of computer-assisted image analysis procedures that could 

### 10 be adopted for quantitative measurements. Many of these assays 

### 11 are actually publically available for free. To some degree -- I 

### 12 don't make the procedure -- and therefore avoid the bias that 

### 13 has been of some concern to some people. Thank you. 

### 14 **DR. DANIEL SCHLENK:**Dr. Green. 

### 15 **DR. SHERRIL GREEN:**I have no further 

### 16 comments. 

### 17 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 18 **DR. JACK LEONARD:**No further comments. 

### 19 **DR. DANIEL SCHLENK:**And Dr. Schultz. 

### 20 **DR. TERRY SCHULTZ:**I apologize for adding my 

### 21 comment. It was meant for (b) and (a). 

### 22 **DR. DANIEL SCHLENK:**Kind of jumped the gun a 

### 23 little bit there. Any other panel input on Question 6? 

### 24 Okay. At this point let me go to the Agency. 

### 25 Any questions or clarification for (a) or (b) for Question 6? 

### 26 **MS. CATHERINE AUBEE:**So I have two questions.

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1 One is specifically for Dr. Patino and the other one is for 

### 2 whoever might care to answer. First, could you just repeat a 

### 3 little bit more slowly for me what you are recommending with 

### 4 respect to potentially looking at immunoreactivity in the 

### 5 thyroid? 

### 6 I didn't quite catch what you were saying 

### 7 something about T4 and the follicular cells. If you could just 

### 8 repeat what you said for me, maybe a little bit more slowly. 

### 9 **DR. REYNALDO PATINO:**About the procedure or 

### 10 about the recommendations? 

### 11 **MS. CATHERINE AUBEE:**About both. 

### 12 **DR. REYNALDO PATINO:**Okay. Maybe because I 

### 13 was reading that it was not very clear. So the procedure 

### 14 involved doing immunocytochemistry for thyroid hormone 

### 15 thyroxine. It has been shown in Xenopus, in the study that I 

### 16 collaborated with, and also in fish that the immunoreactivity 

### 17 concentrates at the periphery at the colloid so it kind of looks 

### 18 like a ring. Both in fish and in Xenopus, this ring has been 

### 19 shown to be a very sensitive marker of exposure to anti-thyroid 

### 20 compound, such as for chloride. 

### 21 In xenopus, the study that I'm referring to, 

### 22 also shows that the ring, that intensity of the ring, which we 

### 23 don't understand the mechanisms fully, but seems to be fairly 

### 24 constant during development. It doesn't increase or decrease 

### 25 like the other histological features do; they kind of increase 

### 26 in severity. This one seems to remain constant and potentially

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1 could avoid the complicated issues of a moving target. So the 

### 2 suggestion was that you could consider that. That has not been 

### 3 validated. 

### 4 I'm not saying you must do it, but potentially 

### 5 could be a better marker for disruption of thyroid activity, 

### 6 particularly of the anti-thyroid, those that you know are based 

### 7 on anti-thyroid mechanisms. It could potentially be a better 

### 8 marker of that than the current histological markers. 

### 9 **MS. CATHERINE AUBEE:**Okay. Thank you. And 

### 10 my other question was because we also look at thyroid histology 

### 11 in the male and female pubertal rats and we don't really talk 

### 12 about it because it's something that is more of a routine 

### 13 measure, whereas with this, it's newer to us. We're getting 

### 14 familiar with the data, as we mentioned, as the thyroid 

### 15 undergoing metamorphosis. 

### 16 So your recommendations could be especially 

### 17 useful there, especially in terms of the quantitative 

### 18 morphometric evaluation. But my question then is whether you 

### 19 think that approach would also be useful for the evaluation of 

### 20 the mammalian thyroid in those assays? 

### 21 **DR. DANIEL SCHLENK:**Dr. Leonard? 

### 22 **DR. JACK LEONARD:**Yes. 

### 23 **DR. DANIEL SCHLENK:**Dr. Shultz. 

### 24 **DR. TERRY SCHULTZ:**Absolutely. 

### 25 **DR. DANIEL SCHLENK:**Any other synonyms 

### 26 out there? Dr. Green.

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1 **DR. SHERRIL GREEN:**I totally agree, yes. 

### 2 **DR. DANIEL SCHLENK:**Okay. With that, 

### 3 let's go ahead and take a break before we do Question 7. That's 

### 4 a fairly extensive question. Oh, Dr. Scott. 

### 5 **DR. GEOFFREY SCOTT:**If I could just respond 

### 6 back on that toxin question on Number 6, please. 

### 7 **DR. DANIEL SCHLENK:**Okay. Sure, if you 

### 8 want to at this point. 

### 9 **DR. GEOFFREY SCOTT:**At least on the algal 

### 10 toxins. The question had been what biological toxins might you 

### 11 want to consider to look at. I think I would like at the 

### 12 microcystin toxin and the antitoxin, which are freshwater toxins 

### 13 that might be worth looking at. But I will get you some more 

### 14 detailed information. Those would at least be two that I would 

### 15 get back on the microbial toxins. 

### 16 **DR. DANIEL SCHLENK:**Okay. Let's go ahead 

### 17 and break and be back at 15 after. 

### 18 (Brief recess.) 

### 19 **DR. DANIEL SCHLENK:**We're going to move 

### 20 on to Charge Question 7. Dr. Patino wants to provide the 

### 21 references for the record that Catherine asked for here. Do you 

### 22 want to go ahead and do that? 

### 23 **DR. REYNALDO PATINO:**I'll just read the 

### 24 author names and year and maybe the journal or book so that you 

### 25 can find it. More or less conceptual statements about the 

### 26 procedure can be found in -- first author is Strauss, R.E., a

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1 chapter in a book called "Methods for Fish Biology." American 

### 2 Fishery Society, also same author; this is a journal review 

### 3 paper, Strauss, R.E., Systematic Zoology (1982), Volume 31. 

### 4 That's more conceptual. 

### 5 There is a paper that I found from another 

### 6 colleague of mine too that has more specific procedures because 

### 7 it was applied on the original research. The first author is 

### 8 Hoverman, H-O-V-E-R-M-A-N, et al. Oecologia (2005), Volume 144. 

### 9 Thank you. 

### 10 **DR. DANIEL SCHLENK:**All right. Thanks. 

### 11 Okay, let's just read in 7a if we can. 

### 12 **DR. MARY MANIBUSAN:**Charge Question 7: In 

### 13 2008, the SAP acknowledged that the in vivo assays included in 

### 14 the Tier 1 battery provide both redundancy and complementarity 

### 15 for evaluating interactions with the E-, A-, or T- signaling 

### 16 pathways. 

### 17 The Panel also noted that all of the Tier 1 

### 18 assays and the broad range of endpoints appeared to be necessary 

### 19 to "discriminate positive and negative results." 

### 20 7a: Please comment on the battery 

### 21 performance with respect to the anticipated complementary nature 

### 22 of the more complex, multi-parameter in vivo assays in the 

### 23 context of the observed responses with the case studies. Please 

### 24 comment separately on the E-, A-, and T-related assays and 

### 25 endpoints. 

### 26 **DR. DANIEL SCHLENK:**Okay. Thank you.

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1 Our lead discussant is Dr. Delclos. 

### 2 **DR. BARRY DELCLOS:**Okay. Thanks. I hope not 

### 3 to sound like a broken record going through here, but most of 

### 4 the discussants listed on the question had a feeling that there 

### 5 was a limited amount that we could do with this question, given 

### 6 the level of information provided. 

### 7 So it would've been useful to have at least 

### 8 directionality, that sort of thing, in terms of instruction, but 

### 9 given that, we did come up with answer. First, I'll through the 

### 10 estrogen. 

### 11 The current Tier 1 battery contains three in 

### 12 vivo assays that are designed to detect estrogenic activity. 

### 13 The uterotrophic assay includes only one required endpoint, 

### 14 uterine weight, but is a well-established and reliable assay for 

### 15 the detection of estrogenic activity. 

### 16 The female pubertal assay includes vaginal 

### 17 opening, time to first estrus, reproductive organ weights and 

### 18 histology as complementary endpoints. The guideline also 

### 19 includes monitoring the estrous cycle, but this was not 

### 20 discussed and the contribution of this endpoint to the female 

### 21 pubertal assay is not clear. 

### 22 The FSTRA assay has five complementary 

### 23 endpoints. An optional endpoint of plasma concentrations of sex 

### 24 steroids was stated by EPA to be useful and potentially should 

### 25 be added as a requirement for the assay. Overt toxicity or 

### 26 other issues cloud the interpretation of the effects observed in

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1 the majority of the FSTRA assays, nine of the 17 chemicals that 

### 2 showed effects in those assays, and the female pubertal assay, 

### 3 four of the seven chemicals showing effects. 

### 4 Overall, the results presented suggest that 

### 5 the complementary performance of the multiparameter E battery 

### 6 tests was relatively poor, although there were test chemicals 

### 7 that affected 2 endpoints, both the FSTRA and the female 

### 8 pubertal assay. 

### 9 Since the present exercise does not provide 

### 10 the panel members with the magnitude or the direction of the 

### 11 effects, it is difficult to say whether or not these two effects 

### 12 will be determined to strengthen or weaken the case for the 

### 13 estrogenicity of these compounds, but the fact that more than 

### 14 one endpoint showed an effect should be helpful in the 

### 15 evaluation and is encouraging. 

### 16 The 2008 SAP indicated that the Tier 1 assays 

### 17 have good coverage for the detection of estrogenic responses 

### 18 mediated through ESR1, and relatively less ability to detect 

### 19 anti-estrogenic effects or effects that might be mediated 

### 20 through other potential mechanisms, such as through ESR2 or 

### 21 membrane receptors, et cetera. This situation remains the same. 

### 22 23 The 2008 SAP had encouraged pursuit of assays 

### 24 for such potential mechanisms, and pursuit of the modification 

### 25 of the uterotrophic assay to improve the ability to detect 

### 26 antiestrogens. It is unclear at this time whether such efforts

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1 are really warranted and in general members of the current panel 

### 2 did not express strong opinions on these issues. 

### 3 For androgens? Three in vivo assays are able 

### 4 to detect the effects on the androgen pathway. The Hershberger 

### 5 assay measured changes in androgen-dependent tissues and is a 

### 6 well-established and reliable assay. The male pubertal assay 

### 7 evaluates sexual development, preputial separation, organ 

### 8 weights and histology, and single point sacrifice serum 

### 9 testosterone. 

### 10 The fish reproduction assay contains five 

### 11 required androgen-sensitive endpoints; testosterone is an 

### 12 optional measurement. Several chemicals altered more than one 

### 13 of these endpoints, although the interpretation of some effects 

### 14 is made difficult by concurrent overt toxicity. 

### 15 As with the estrogen pathway in vivo assays, 

### 16 the summary data presented indicate that multiple endpoints in 

### 17 the male pubertal and FSTRA can be altered by a single chemical, 

### 18 but what role these effects will play in the decision to move 

### 19 forward is unclear until the magnitude and direction of the 

### 20 effects are known and discussed as part of the weight of 

### 21 evidence evaluation. 

### 22 For the thyroid hormone, female and male 

### 23 puberty assays include measurements of T4, TSH, and thyroid 

### 24 gland weight and histopathology as endpoints to evaluate the HPT 

### 25 axis. A variety of chemicals affected one or more of these 

### 26 endpoints with no consistent pattern and with no endpoint

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1 clearly being the most affected, although T4 levels seemed to be 

### 2 the most often altered parameter, followed by thyroid histology. 

### 3 In some cases these effects were sex-specific. 

### 4 The AMA assay contains evaluations of 

### 5 development and thyroid histology and EPA has provided a clear 

### 6 decision logic for evaluating potential thyroid axis activity. 

### 7 For the seven chemicals reported to show effects in the AMA, 

### 8 regardless of solvent interference issues, four showed effects 

### 9 on both development and thyroid histology. So there was some 

### 10 evidence of complimentarity there. And that's all I have to 

### 11 say. 

### 12 **DR. DANIEL SCHLENK:**Thank you. Dr. 

### 13 Horseman. 

### 14 **DR. NELSON HORSEMAN:**I agree. I have nothing 

### 15 more to add at this point. 

### 16 **DR. DANIEL SCHLENK:**Dr. Piersma. 

### 17 **DR. ALDERT PIERSMA:**Just to add to what has 

### 18 been said, I mean, I agree with all that has been said, 

### 19 obviously. But in order to be able to give a little more 

### 20 in-depth response to the questions given here, this both 

### 21 pertains to complementarity as well as to redundancy and battery 

### 22 performance. 

### 23 Dr. Delclos already said that we would have 

### 24 preferred to have a little bit more information. So perhaps 

### 25 it's good to give more specifics about that. We talked about 

### 26 directionality about potency and magnitude of responses. It

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1 would be good to have that kind of information. 

### 2 In addition, talking about in vivo assays with 

### 3 different endpoints, it would be interesting not to just have 

### 4 the dose response of the most critical or most sensitive effect, 

### 5 but all different effects so that the different effects can be 

### 6 compared across the different assays in the battery to look at 

### 7 their relative performances. 

### 8 Another thing, of course, is the situation 

### 9 about 21 chemicals versus 52. I mean, the more the better, of 

### 10 course. 

### 11 And finally, if you want to evaluate assays 

### 12 within a certain realm of activity, it would be good to have 

### 13 compounds that also affect each of these endpoints within the 

### 14 metrics. We know that within the 21, we don't have any 

### 15 compounds that affect estrogen binding or uterotrophic assay. 

### 16 I understand the reasoning for the choice of 

### 17 the chemical at this point, but for considering the questions 

### 18 that we have at hand, it would be good to have a selection of 

### 19 compounds that covers the entire endocrine activity space that 

### 20 is within the metric. Just to have that more specific answer. 

### 21 **DR. DANIEL SCHLENK:**Thank you. Dr. Roby. 

### 22 DR. KATHERINE ROBY: I have nothing in 

### 23 addition. 

### 24 **DR. DANIEL SCHLENK:**And Dr. Scott. 

### 25 **DR. GEOFFREY SCOTT:**Just one or two points 

### 26 real quickly. In terms of complementarity on the androgen assay

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1 with the Hershberger, the male pubertal rat and the fish assay, 

### 2 looking at the complementarity within each of those assays, I 

### 3 was struck by the reported percent complementarity reported in 

### 4 the report by EPA. 

### 5 I think it was 41 percent for the Hershberger, 

### 6 35 percent for the male pubertal rat. Looking at the fish 

### 7 assay, depending upon how many endpoints, it could be as low as 

### 8 six percent agreement for two or six endpoints, where as it's as 

### 9 high as 47 percent for four more endpoints. 

### 10 To me as you look across those different 

### 11 results, I think there is good complementarity exhibited in that 

### 12 particular assay. I think one question that I did not sort out 

### 13 in my analysis was how to deal with the overt toxicity. 

### 14 At this point I'm including it in there for 

### 15 now, but I do think, looking back at that we'll have to sort 

### 16 that out a little bit. That's all of my comment. 

### 17 **DR. DANIEL SCHLENK:**Dr. Selvage. 

### 18 **DR. DANIEL SELVAGE:**I just wanted to make a 

### 19 couple of comments about the mammalian estrogen androgen assays, 

### 20 in terms of the complementarity. 

### 21 **DR. DANIEL SCHLENK:**Can you get a little 

### 22 closer to the mic there? 

### 23 **DR. DANIEL SELVAGE:**So the pubertal assay, 

### 24 you know, I've already made my comments about it regarding 

### 25 potential or HPA axis and stress to alter that and make those 

### 26 interpretations difficult to compare the results from that with

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1 the other assays because of the overlap between HPA and HPG and 

### 2 HPT axes. Those things all work together, especially when 

### 3 you're doing a pubertal assay where they are developing, it's 

### 4 going to be really tough to compare the pubertal assays to the 

### 5 other ones. 

### 6 Also, I did want to say, speaking of estrogens 

### 7 right now, the ER binding assay, where you are using rat cytosol 

### 8 is, in my mind, essentially, an in vivo assay and it has a lot 

### 9 of problems that are associated with that. It did provide 

### 10 complementarity with uterotrophic assay pretty well, but it was 

### 11 a bad assay in particular that is fraught with problems and has 

### 12 a lot of variances with it. If you look at the protocol for the 

### 13 ER binding assay, in terms of taking the cytosol from rats and 

### 14 everything, leading the protocol is probably what curled my 

### 15 hair. 

### 16 And it's been said already that a good idea 

### 17 that they change that assay and there are maybe other ones that 

### 18 will provide better complementarity. Same is pretty much true 

### 19 with the androgen and the pubertal androgen assays in trying to 

### 20 disentangle the different axes. And also, in my mind, the AR 

### 21 binding assay is essentially an in vivo assay that is very 

### 22 elaborate. If that can be changed to something more modern, I 

### 23 think that would be a really good thing. That's it. 

### 24 **DR. DANIEL SCHLENK:**Okay. Dr. Tillitt. 

### 25 **DR. DONALD TILLITT:**I really don't have a 

### 26 whole lot more to say about the complementarity of this other

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1 than agree with what Dr. Delclos said. I will say that upon 

### 2 getting this additional information implied, if it wasn't 

### 3 already stated, that I would also like to see the control 

### 4 information. I guess I agree also with Dr. Piersma's statement 

### 5 about looking at the other scientific relevant information, you 

### 6 know, where we would see maybe positive controls and sort of 

### 7 have a framework. I would also say that we don't have 

### 8 information that indicates that these assays don't operate like 

### 9 the original ought to either. That's about all I have to say. 

### 10 **DR. DANIEL SCHLENK:**Okay. Thank you. 

### 11 Other panel members? Okay. Let me go to the Agency for 7a, any 

### 12 questions or clarification? 

### 13 **DR. THOMAS SEEGER:**No questions. 

### 14 **DR. DANIEL SCHLENK:**All right. Thank 

### 15 you. Let's go ahead and read 7b. 

### 16 **MS. CATHERINE AUBEE:**Charge Question 7b: 

### 17 Please comment on the battery performance with respect to the 

### 18 anticipated redundancy across the 11 assays in the context of 

### 19 the observed responses with the case studies. 

### 20 Please comment separately on the E-, A, and 

### 21 T-related assays and endpoints. 

### 22 **DR. DANIEL SCHLENK:**Dr. Delclos. 

### 23 **DR. BARRY DELCLOS:**Okay. We'll start with 

### 24 estrogens. In general, from the information presented, there 

### 25 appeared to be generally a fairly poor redundancy in the battery 

### 26 of tests used to assess this activity, due to a general lack of

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1 concordance among in vitro and in vivo assays as well as between 

### 2 mammalian and fish in vivo assays. 

### 3 There were no clear ER binders and no test 

### 4 chemicals active in the uterotrophic assay. On the other hand, 

### 5 four of 21 test chemicals gave positive results in the ER 

### 6 transcription assay. It might have been expected that one or 

### 7 more of these compounds would have shown activity in one or both 

### 8 of the other assays; that is, ER binding or uterotrophic. 

### 9 With regard to the in vitro assays, there 

### 10 appears to be little agreement among the tests. Indeed the 

### 11 transactive binding, transactivation and steroidogenesis assays 

### 12 test multiple and diverse aspects of estrogenic properties. 

### 13 Thus, it is not surprising that these assays fail to exhibit 

### 14 significant redundancy. 

### 15 As shown in the Agency presentations, there 

### 16 were no compounds that showed positive only in vitro effects, 

### 17 although in nine of the 10 cases in which both in vitro and in 

### 18 vivo effects were seen, there was a positive in only one in vivo 

### 19 assay, with the FSTRA of a positive assay in eight of those nine 

### 20 cases. 

### 21 There were some potential issues of overt 

### 22 toxicity in several of the positive in vivo studies, so it 

### 23 appeared that there was only one compound, Compound Q, that 

### 24 showed clear positives in two in vivo assays. 

### 25 In short, there is some redundancy across 

### 26 assays for the estrogen pathway endpoints, but it remains to be

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1 determined how clear the interpretation of these redundancies 

### 2 will be. The absence of positive uterotrophic assays in the 

### 3 subset of chemicals on which summary results were reported and 

### 4 performance problems related to toxicity and/or control 

### 5 complications lead to significant uncertainties about E battery 

### 6 redundancy. Also there seems to be a high proportion of 

### 7 equivocals in the aromatase assay and it is not clear how 

### 8 important that assay will be in the overall evaluation. 

### 9 For the androgens, the data presented 

### 10 indicates that there is redundancy across the in vitro and in 

### 11 vivo assays designed to detect effects on the androgen pathway 

### 12 in the sense that 13 of the 17 compounds presented as having 

### 13 been tested in the six androgen pathway assays appear to show 

### 14 effects in multiple assays. 

### 15 As with the estrogen pathways discussed above, 

### 16 the value of these redundancies will be better determined after 

### 17 the weight of evidence evaluation. 

### 18 The thyroid hormone: As indicated in the 2008 

### 19 SAP report and in the current White Paper and EPA presentations, 

### 20 there is less redundancy in the assays for effects on the 

### 21 thyroid axis. The 2008 SAP had suggested exploring additional 

### 22 endpoints, such as thyroid hormone-sensitive gene expression and 

### 23 transcriptional activation assays, but it is not clear if 

### 24 additional endpoints to strengthen the evaluation of this 

### 25 pathway are being considered. 

### 26 Three of the 16 chemicals tested in this

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1 battery showed some thyroid effect in all three thyroid assays, 

### 2 three showed effects in both rat assays but not the frog assay, 

### 3 and three showed effects in the frog but not the rat. Four 

### 4 chemicals showed effects in only one sex in the rat assays. The 

### 5 EPA conclusion that all three assays are necessary to evaluate 

### 6 this axis seems appropriate. I think that's all I have. 

### 7 **DR. DANIEL SCHLENK:**Okay. Dr. Horseman. 

### 8 **DR. NELSON HORSEMAN:**I have nothing specific 

### 9 to add here. I just want to reemphasize, I was aware that this 

### 10 issue of some sort of additional battery controls that Dr. 

### 11 Piersma brought up, and I think to reiterate the importance of 

### 12 this. 

### 13 The inclusion of some sort of a ringer in this 

### 14 battery or something that one has a good prediction by how it 

### 15 ought to perform through this battery of tests, which I think is 

### 16 what Dr. Piersma was alluding to, would be a huge advance, 

### 17 something that you would expect to perform positively in some 

### 18 and negatively in some of the other tests within the battery. 

### 19 I think that that would be a big advance 

### 20 because the controls for the individual assays are not the same 

### 21 as a control carried through the battery, which is what these 

### 22 two questions are asking us about, as the performance of the 

### 23 battery. Thank you. 

### 24 **DR. DANIEL SCHLENK:**And Dr. Piersma. 

### 25 **DR. ALDERT PIERSMA:**Just to follow-up on 

### 26 that, for your information, we just published a paper in

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1 reproductive toxicology where we actually tried that kind of an 

### 2 approach, where we had a battery of different assays tested with 

### 3 a series of compounds. It was like 20 assays with about 12 

### 4 compounds for which we knew that each of the compounds would 

### 5 have to be positive in one of those assays, but negative in the 

### 6 others. 

### 7 You have a positive and a negative control 

### 8 with the same compound and they are active compounds, not the 

### 9 inert compounds like saccharin or penicillin, which is overall 

### 10 negative and should be negative and is rather uninformative. 

### 11 But with this kind of an approach, you can 

### 12 look at specificity of assays within a battery and also look at 

### 13 combined battery performance and identify those caveats that you 

### 14 have in terms of mechanism within the battery. It's has just 

### 15 come out in reproductive toxicology and I'm the first author, so 

### 16 for your information that might be interesting to look at. 

### 17 Thank you. 

### 18 **DR. DANIEL SCHLENK:**Dr. Roby. 

### 19 DR. KATHERINE ROBY: I have nothing new to 

### 20 add, but I would also like to reiterate this idea of having kind 

### 21 of ringer in the battery. It certainly would provide another 

### 22 level of confidence to the Agency that the system and the 

### 23 battery actually are working the way you anticipate it to work. 

### 24 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 25 **DR. GEOFFREY SCOTT:**Just a couple of comments 

### 26 in terms of the redundancy analysis presented by EPA. For the

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1 androgen battery, just a couple of comments here. The results 

### 2 indicated that at least one of the three bioassays, either the 

### 3 Hershberger, the male rat or the fish test detected an effect. 

### 4 If you look at all three assays, 17 of the 17 compounds tested 

### 5 with one of the tests. 

### 6 Now, when you start to look at the multiple 

### 7 tests, when you see co-occurrence we only saw that all three 

### 8 assays detected it, three out of 17, or about 17 percent at the 

### 9 time. Two of three assays, we saw it, it ranged variably among 

### 10 the combinations of the Hershberger or the male rat or the fish 

### 11 test, and those percentages range from as low as five percent to 

### 12 as high as 24 percent with those combination tests. 

### 13 So redundancy was observed in at least two or 

### 14 more of the three bioassays or 12 of the 17 compounds. So about 

### 15 70 percent of the chemicals tested with the male rat and fish 

### 16 test accounting for about two-thirds of that redundancy, and I 

### 17 thought that was sort of interesting to look at it that way. 

### 18 Again, the question of overt toxicity as a 

### 19 confounder will have to be addressed in sorting through some of 

### 20 that. In looking at the thyroid test -- and I'll just make a 

### 21 couple of quick comments. 

### 22 In looking at the overall concordance among 

### 23 the three assays, the AMA and the two female and male rat tests, 

### 24 there was a positive response in all three pathways for 19 

### 25 percent of the chemicals, I think tested. 

### 26 There was a negative response for all three

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1 for 19 percent of the chemicals. A positive response in the 

### 2 male and female rat, but not the AMA in 19 percent of the 

### 3 chemicals; positive response only in the AMA, 19 percent and a 

### 4 positive response in the female rat and the AMA six percent; and 

### 5 then a positive response in only one of the assays, 19 percent 

### 6 of the chemicals. 

### 7 So I think as you look through that, that kind 

### 8 of gives you a little bit of a different look at what you all 

### 9 presented, just slightly analyzing it a little differently 

### 10 perhaps. 

### 11 I think the one thing that I was struck by, 

### 12 that combination of fish assay and those two rat assays 

### 13 specifically -- since we're commenting on the in vivo -- seem to 

### 14 give you broad coverage for most of the endocrine endpoints 

### 15 among all three pathways and it gives you very strong linkages 

### 16 with both an ecotox species and a human health species. 

### 17 I know comments were made yesterday on some of 

### 18 the presentations about only including one of the male rats, but 

### 19 I happen to look at this. I think you're getting more complete 

### 20 coverage with both of those. That's my opinion. You do have to 

### 21 look at cost and other things in looking at redundancy. I'll 

### 22 end with those comments. 

### 23 **DR. DANIEL SCHLENK:**Thanks. Dr. Selvage. 

### 24 **DR. DANIEL SELVAGE:**Yes. I concur with 

### 25 what's been said already. 

### 26 **DR. DANIEL SCHLENK:**Dr. Tillitt.

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1 **DR. DONALD TILLITT:**I agree with the comments 

### 2 that have come before me here on this question. The only thing 

### 3 I would say, maybe, is addressing this idea of a ringer or a 

### 4 positive control. 

### 5 My idea is that we have a lot of that 

### 6 information in the literature and if we could use that maybe to 

### 7 frame these assays and the response to these assays, I don't 

### 8 know and I don't understand how there would be a ringer sent 

### 9 through all these assays to make comparisons with these 

### 10 compounds already, other than what is already out there in the 

### 11 literature, if that makes sense. 

### 12 **DR. DANIEL SCHLENK:**Anybody want to 

### 13 address that? 

### 14 DR. KATHERINE ROBY: I'll chime in a little 

### 15 bit. I'm not sure that it necessarily needs to be a compound 

### 16 that is functional across the battery in total, but something 

### 17 that will allow you to see that the battery is functioning. 

### 18 Like any kind of assay or any dataset that you get, if the 

### 19 answer is nothing was positive, do you know that you could 

### 20 determine anything that was positive? 

### 21 So if everything is a "No," is your assay 

### 22 system robust enough to identify a "Yes?" So I think that's one 

### 23 of the things that having something in the system, and again, it 

### 24 doesn't have to be every test, but something that is kind of -- 

### 25 that you have spiked the system a little bit and can see that 

### 26 your test can identify a positive, especially with the example

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1 of the estrogen-binding or estrogenic type compounds. 

### 2 In this case, there was nothing that was read 

### 3 as a positive. We know that the system can protect the 

### 4 positive, but in the context of how these were run, were we able 

### 5 to detect the positive? 

### 6 **DR. DANIEL SCHLENK:**Any other Panel 

### 7 members want to -- Dr. Schultz. 

### 8 **DR. TERRY SCHULTZ:**It would be nice to see if 

### 9 the chemicals that we use to validate the assays, let's say, the 

### 10 ones we've said have known activity, even though they wouldn't 

### 11 be tested in all 11, would there be chemicals that were tested 

### 12 in all of those that were related to a particular pathway. 

### 13 If that information could be provided, that 

### 14 would allow a better way of looking at the redundancy and 

### 15 overlap that you have with the 11 assays on a pathway-by-pathway 

### 16 basis. 

### 17 So I wouldn't expect that there would be one 

### 18 or more chemicals that was sent through all the pathways, such 

### 19 as what was done in the Dutch study. But I would suspect that 

### 20 you have information from earlier SAPs on this situation that 

### 21 could be pulled out that would assist in making those types of 

### 22 comparisons. And that would add a confidence to what we're 

### 23 seeing. Thank you. 

### 24 **DR. DANIEL SCHLENK:**Dr. Leonard. 

### 25 **DR. JACK LEONARD:**I was taken by the lack of 

### 26 concordance between the two estrogen in vitro assays. The ERTA

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1 is really a functional read out of ER binding. If you look 

### 2 through the panel, there was very little concordance between 

### 3 those two assays. I think there may be an answer to that. 

### 4 Yesterday, during the public comment period, 

### 5 we heard from a laboratory who kind of gave us a historical 

### 6 characterization of the full change in that particular assay, 

### 7 which to my surprise, ranged from 80-fold to four-fold; that's 

### 8 almost a 10-fold difference, in terms of amplification. My 

### 9 laboratory has a lot of experience building these types of 

### 10 Porter assays and if we ever see anything more than two-fold we 

### 11 rebuild the assay. 

### 12 So in part, it's somewhat -- there are some 

### 13 quality control issues in the ERTA assay that seems to be 

### 14 missing. I think that may need to be fixed so you can actually 

### 15 get a much better sense about what happens in vitro. If it 

### 16 binds to the ER, it should impact the ER response element. 

### 17 **DR. DANIEL SCHLENK:**Any other comments? 

### 18 Dr. Scott. 

### 19 **DR. GEOFFREY SCOTT:**Just to follow-up on what 

### 20 Terry said just a second ago, I think it will be really 

### 21 important to know, like looking at the thyroid assay -- just 

### 22 giving you some summary analysis there -- where all three tests 

### 23 found an effect in 19 percent of the chemicals. Are those all 

### 24 in one class? 

### 25 Because it may be highly redundant for one 

### 26 class, but it may not be for another. I really think, as you go

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1 through and do your analysis, that will be very important to 

### 2 evaluate the proficiency and whether it is redundant, there is a 

### 3 balance between being complementarity and the redundancy in 

### 4 complete coverage. 

### 5 So I really think having it analyzed by class, 

### 6 since you do have that in the recordkeeping for the chemicals, 

### 7 and I couldn't tell that from looking at it, that would be 

### 8 important to me. 

### 9 **DR. DANIEL SCHLENK:**Any other comments 

### 10 for 7b? Okay. We'll go to the Agency. Any questions or 

### 11 clarification? 

### 12 **DR. MARY MANIBUSAN:**So the Agency would like 

### 13 some clarification. This Ringer study, it sounds very 

### 14 provocative. In comparison to what has already been done, in 

### 15 terms of validating these individual Tier 1 assays, it was 

### 16 brought before the SAP in 2008, a lot of the information sounds 

### 17 like what Dr. Schultz had described, looking at chemicals by 

### 18 pathway, presenting both strong positive, negative, weak agonist 

### 19 and antagonist and so on, demonstrate the performance of each 

### 20 individual assay along the pathways that were described, were 

### 21 provided in the 2008 SAP. So we can bring that before the panel 

### 22 and provide that information. Is that the type of control that 

### 23 is being described for this Ringer study? 

### 24 **DR. DANIEL SCHLENK:**Who wants to take it? 

### 25 Dr. Schultz. 

### 26 **DR. TERRY SCHULTZ:**That type of information

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1 could be an excellent substitution for the Ringer study. I 

### 2 believe the idea of the ringer study would be to take a compound 

### 3 or a suite of compounds and run through all 11 assays, where you 

### 4 would know it should be negative for a certain pathway. That 

### 5 would be the recommended results. You understand? 

### 6 I think there's a difference between -- I 

### 7 should let those two people talk about the ringer assay. 

### 8 They're much more understanding about what they're talking 

### 9 about. What I was talking about is, yes. 

### 10 I apologize; at my age I don't remember all 

### 11 the information given in 2008, but it's difficult to answer 

### 12 these questions in 2013 without having some of that information 

### 13 on the tip of your tongue or at least brought back to you. 

### 14 The point is the Agency has the information 

### 15 which would make it easier to make those judgments. Thank you. 

### 16 **DR. DANIEL SCHLENK:**Dr. Piersma. 

### 17 **DR. ALDERT PIERSMA:**I recognize that a lot of 

### 18 the information that we're actually heading at has probably been 

### 19 gathered at some point in the past. The point here is that 

### 20 we're talking not just about the validity of individual assays 

### 21 and the response to individual compounds; we're talking about 

### 22 the battery approach. We're talking about complementarity and 

### 23 redundancy. 

### 24 In that respect, it could be -- in my mind, 

### 25 the basic requirement would be that the mechanistic space of the 

### 26 whole battery is covered by the test compounds with which you

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1 actually judge the competence of the battery. 

### 2 So in this particular case, one conclusion 

### 3 could be if these 21 compounds would cover chemical space, there 

### 4 are no estrogens. So we don't need any estrogenic assay, just 

### 5 to take it vary down to earth. 

### 6 And of course, we want estrogenic potency 

### 7 potential to be potentially covered within the battery, but then 

### 8 you need at least a number of examples to show that a particular 

### 9 battery is able to do that and also to judge whether you could 

### 10 actually throw out one of these assays because of redundancy, 

### 11 you would need some positive compounds to be able to make that 

### 12 decision. 

### 13 So it was one of my first surprises when I saw 

### 14 the database that we actually didn't have any estrogen binders 

### 15 or uterotrophic compounds within the group of compounds that 

### 16 were tested, which makes it very difficult for me to make any 

### 17 statement about the group of estrogens that we test in the 

### 18 battery. 

### 19 In other words, you would want to cover the 

### 20 entire endocrine activity space that you would want to cover in 

### 21 the battery, also in terms of the group of compounds that you're 

### 22 testing. 

### 23 **DR. DANIEL SCHLENK:**Does that provide 

### 24 some insight? 

### 25 **DR. STEVEN BRADBURY:**Yes, it does, both 

### 26 taking a look at the historical record, understanding of AOPs,

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1 and maybe taking a look at empirical information. 

### 2 As you write the report up, it might be 

### 3 helpful in some of the considerations in looking at existing 

### 4 literature, as well as opportunities because we know the battery 

### 5 is designed to look at cross-species, cross-sex. So I don't 

### 6 think anybody, you know, the 2008 SAP wasn't expecting 

### 7 everything to be exactly the same across species and taxa, the 

### 8 whole point being taxa metabolize chemicals differently, thing 

### 9 like that. So that's part of the rationale for that. 

### 10 So it would be if you were going to a ringer 

### 11 experiment, how many variables are we trying to cover and how 

### 12 many ringers would you need to be able to say you not only 

### 13 covered activity space, structure space, species space, sex 

### 14 space, metabolic capability space -- and that's also important 

### 15 for researching the literature to see to what extent that 

### 16 multi-variable hyperspace has been evaluated. 

### 17 So as you guys think about that, that would 

### 18 just be helpful to give us some thoughts to think about how we 

### 19 can mine the literature or think about empirical approaches to 

### 20 looking at that. 

### 21 And reflecting on the January SAP where we 

### 22 know, getting back to the E thing, I think that it would be 

### 23 helpful to get some feedback, is that useful to take a look at 

### 24 what we determined there with only two or three percent of that 

### 25 inventory space has the potential to bind to the estrogen 

### 26 receptor.

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1 How can we use some of that information maybe 

### 2 to help formulate a literature evaluation of those empirical -- 

### 3 not disagreeing at all, but just any additional detail you can 

### 4 put in the write up would be helpful. 

### 5 **DR. DANIEL SCHLENK:**Okay. So I will 

### 6 leave that to Dr. Schultz and Dr. Piersma, and Dr. Roby, if you 

### 7 guys can work on that that would be great. Dr. Horseman. 

### 8 **DR. NELSON HORSEMAN:**I just want to come back 

### 9 to this because I think Dr. Piersma's suggestion is very 

### 10 important. It's complicated, but I think it needs to not be 

### 11 over complicated. Well, it needs to not make it more 

### 12 complicated than it might otherwise be. 

### 13 I think coming back to the battery performance 

### 14 as it has been presented to us, each of these tests ends up 

### 15 being basically scored as a binary. Something either scored 

### 16 that did or didn't turn out positive or negative. And when that 

### 17 happens, having something in there that you know ought to be one 

### 18 or the other that goes through the battery. This is different 

### 19 from the individual -- it could be the same compound -- but I 

### 20 think it's really important for getting an answer to these last 

### 21 two questions, which are not about the individual tests. 

### 22 **DR. DANIEL SCHLENK:**All right. With that, 

### 23 we're ready to move forward to Letter C. 

### 24 **DR. MARY MANIBUSAN:**Charge Question 7c: The 

### 25 EPA concluded that the battery has performed as anticipated by 

### 26 the 2008 SAP. Please comment on this conclusion.

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1 **DR. DANIEL SCHLENK:**Dr. Delclos. 

### 2 **DR. BARRY DELCLOS:**This is a relatively short 

### 3 answer. The 2008 SAP concluded that the Tier 1 suite of assays 

### 4 should be able to detect agents that alter E-, A-, and T

### 5 pathways. 

### 6 There can be limited conclusions drawn at this 

### 7 time given the amount of information that has been provided. 

### 8 The assays were able to be performed by several laboratories and 

### 9 showed changes in the assay critical endpoints that will be used 

### 10 to determine if a compound should be required to undergo further 

### 11 testing to establish definitively the ability of the compound to 

### 12 alter the E-, A-, or T- pathways. 

### 13 Again, without information on the direction, 

### 14 magnitude and consistency of the observed effects, it is 

### 15 difficult to fully assess how the assays performed as a group. 

### 16 In any case, the battery of assays did show responses in one or 

### 17 more assays and/or endpoints for estrogen, androgen, and thyroid 

### 18 hormones. 

### 19 As mentioned above and in previous charge 

### 20 questions, overt toxicity overlapping potential hormonal effects 

### 21 is a concern in several of the in vivo assays, particularly the 

### 22 FSTRA and the pubertal assays. Thus, one might question whether 

### 23 the degree of selectivity envisioned by the 2008 SAP was 

### 24 achieved; the weight of evidence analysis will no doubt address 

### 25 this. 

### 26 **DR. DANIEL SCHLENK:**Okay. Dr. Horseman.

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1 **DR. NELSON HORSEMAN:**I have nothing to add. 

### 2 **DR. DANIEL SCHLENK:**Dr. Piersma. 

### 3 **DR. ALDERT PIERSMA:**Nothing to add. 

### 4 **DR. DANIEL SCHLENK:**Dr. Roby. 

### 5 DR. KATHERINE ROBY: Nothing to add. 

### 6 **DR. DANIEL SCHLENK:**Dr. Scott. 

### 7 **DR. GEOFFREY SCOTT:**Nothing to add. 

### 8 **DR. DANIEL SCHLENK:**Dr. Selvage. 

### 9 **DR. DANIEL SELVAGE:**I will add nothing. 

### 10 **DR. DANIEL SCHLENK:**And Dr. Tillitt. 

### 11 **DR. DONALD TILLITT:**I will add nothing. 

### 12 **DR. DANIEL SCHLENK:**Okay. Other Panel 

### 13 members for C? Okay. Let me go to the Agency. Things seem 

### 14 pretty clear? 

### 15 **DR. MARY MANIBUSAN:**No additional comments. 

### 16 Thank you. 

### 17 **DR. DANIEL SCHLENK:**Great. All right. At 

### 18 this point so that we can actually contact our administrative 

### 19 staff, we'll go ahead and take an early lunch I think and get 

### 20 back to Question 8, which should be fairly, relatively quick. 

### 21 Let's be back at 12:15. We'll do an early lunch and get back at 

### 22 12:15. 

### 23 (Whereas a lunch break was taken) 

### 24 **DR. DANIEL SCHLENK:**Everybody take a 

### 25 seat, please. We'll get moving on to Charge Question 8. 

### 26 **DR. MARY MANIBUSAN:**Charge Question Number 8:

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1 The EPA is committed to minimizing animal usage in the 

### 2 screening battery while maintaining the effectiveness of the 

### 3 battery to answer the question of whether a chemical has the 

### 4 "potential" to interact with the endocrine system. 

### 5 Question 8a: Based on the preliminary battery 

### 6 performance evaluation, to what extent can the current Tier 1 

### 7 battery of 11 assays be modified to reduce animal usage and/or 

### 8 lower cost while adequately ensuring the Agency, the EPA's 

### 9 ability to answer the question of whether a chemical has the 

### 10 potential to interact with the endocrine system? 

### 11 Specifically, please comment on whether the 

### 12 uterotrophic and Hershberger assays provide necessary 

### 13 redundancies in the Tier 1 battery based on this preliminary 

### 14 analysis. Please include in your comments what information may 

### 15 be lost and what uncertainties may be introduced by absence of 

### 16 either or both of these assays. 

### 17 **DR. DANIEL SCHLENK:**Okay. Our lead 

### 18 discussant for that is Dr. Delclos. 

### 19 **DR. BARRY DELCLOS:**Okay. Based on the data 

### 20 presented, the majority of the panel indicated that it is not 

### 21 clear that the uterotrophic or Hershberger assays should be 

### 22 discarded from the assay battery at this time given that they 

### 23 are well-established assays that appear to perform well. 

### 24 While the tests are limited in what they can 

### 25 detect, agents with estrogen agonist or anti-androgenic 

### 26 activities have been of particular concern up to this point and

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1 these assays generally give clear answers on these mechanisms. 

### 2 At the very least, a decision to remove these assays from the 

### 3 battery should await the completion of the weight of evidence 

### 4 exercise on these compounds to determine what role these assays 

### 5 play in the decision process. 

### 6 Parenthetically I say that several members of 

### 7 the panel felt strongly that the weight of evidence exercise 

### 8 should include all of the compounds on which data have been 

### 9 collected. In the case of the Hershberger assay, for example, 

### 10 it can be seen from the data presented that a response in the 

### 11 Hershberger assay was accompanied by a response in the male 

### 12 pubertal or the FSTRA assay, but it was not indicated if these 

### 13 effects were in the same direction. 

### 14 In the case of the uterotrophic assay, it is 

### 15 not clear at this point if the negative response for the 21 

### 16 chemicals evaluated in the set of chemicals under review could 

### 17 be factored in to a decision on a compound that showed an effect 

### 18 in the FSTRA assay only in the presence of overt toxicity. Even 

### 19 if they are removed from the battery as absolute requirements, 

### 20 they should be maintained as options if the other assays do not 

### 21 provide clear results. 

### 22 While reduction of animal use and cost are 

### 23 important, it is also important that the screening assays serve 

### 24 their intended function to limit the number of compounds that go 

### 25 to Tier 2 while minimizing false negatives. 

### 26 A potential problem indicated by the Tier 1

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1 results from the first set of 21 compounds is the potential 

### 2 confounding of endocrine effects with overt toxicity in the 

### 3 FSTRA and to a lesser extent, the pubertal assays. There was 

### 4 plenty of discussion of the issue of dose selection in the 

### 5 earlier questions. And that's all I have. 

### 6 **DR. DANIEL SCHLENK:**Dr. Selvage. 

### 7 **DR. DANIEL SELVAGE:**Yeah, I would like to add 

### 8 a couple of things. I think I'm a little bit more enthusiastic 

### 9 about keeping the Hershberger and uterotrophic assays than the 

### 10 other discussants. These assays have the ability to very 

### 11 specifically detect, in particular, uterotrophic estrogenic 

### 12 activity and then Hershberger androgenic activity, and then if 

### 13 they're expanded like Hershberger was, to measuring better 

### 14 anti-androgenic or with the uterotrophic anti-estrogenic 

### 15 activity, I think that they can do that fairly well. 

### 16 What else? Also, since they use gavage, 

### 17 metabolism is involved with those assays. You don't get that, 

### 18 obviously, with the other assays. Let's see. Anyway, that's 

### 19 what I'll say about those. 

### 20 One other thing I wanted to say was that 

### 21 obviously I worry a lot about stress and stress being activated; 

### 22 something I haven't actually said at this meeting is "gavage." 

### 23 I'm sure the labs running these tests, the staff is well 

### 24 training and does an excellent job. Gavage still can be a 

### 25 stress, especially gavage of the compounds. Who knows what 

### 26 exactly you're giving them.

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1 So because of that stress, then you have less 

### 2 of an idea of what's happening at the level of the testes or 

### 3 ovaries in terms of production of testosterone or estrogen, 

### 4 especially with the measures you guys have, the onetime 

### 5 measures. I realize you don't measure estrogen, but 

### 6 testosterone is just a onetime measure. 

### 7 So with these guys, you don't have that 

### 8 parameter there and you don't need to worry so much about 

### 9 whether your testosterone or estrogen levels have been altered, 

### 10 you can really just see what's going on. 

### 11 The other thing I wanted to add was I brought 

### 12 this up with the last question was that the androgen receptor 

### 13 binding and estrogen receptor binding are, well, technically 

### 14 called ex vivo, but essentially, they have an in vivo component. 

### 15 It's obviously been discussed a fair amount, replacing those 

### 16 assays. 

### 17 Those assays, as the email that was read 

### 18 yesterday, said they can use a lot of animals and they're 

### 19 difficult to run. So I would say if you want to stop overly 

### 20 using animals, that's where I would go, initially. I believe 

### 21 that's it for me. 

### 22 **DR. DANIEL SCHLENK:**Okay. Dr. Piersma. 

### 23 **DR. ALDERT PIERSMA:**I agree with what was 

### 24 said by Dr. Delclos, obviously. Perhaps to balance the comments 

### 25 by Dr. Selvage, there is another school of thought which says 

### 26 that uterotrophic and the Hershberger assays are basically in

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1 vivo test tubes which look at the single endpoint, and the 

### 2 questions of that endpoint is whether it's adverse or not. It's 

### 3 a change of weight of an organ. 

### 4 The question is what is the adverse health 

### 5 effect connected to that. Adverse health effects are typically 

### 6 or preferably studied in apical assays, and of course, we have a 

### 7 number of apical assays in the battery. 

### 8 On the other hand, looking at the specificity 

### 9 of an estrogenic response, you could do that in estrogen binding 

### 10 and more specifically, estrogen receptor transactivation assays. 

### 11 And of course also for androgen and thyroid the same holds 

### 12 true. 

### 13 So one could also come to plate, perhaps, 

### 14 changing to a more robust in vitro assays for receptor binding 

### 15 and receptor activation and then just skip the uterotrophic and 

### 16 the Hershberger and go to in vivo immediately. Although, of 

### 17 course, the apical in vivo assays have the issue of isn't that 

### 18 first effect observed really mediated by the hormonal axis or it 

### 19 may be mediated by something else. But that is a general 

### 20 problem that we have with all in vivo assays, I believe. 

### 21 I mean, it's that balance that we have to look 

### 22 for. Actually, the current dataset doesn't give us enough 

### 23 information to make up our minds around those points, but I 

### 24 think these issues need to be considered. 

### 25 **DR. DANIEL SCHLENK:**Okay. Other Panel 

### 26 members. Dr. Green.

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1 **DR. SHERRIL GREEN:**From a different 

### 2 perspective, as a laboratory animal veterinarian who has sat for 

### 3 years on numerous IACUCs, I would like to commend the EPA and 

### 4 the Panel and the public presenters on putting this issue as one 

### 5 of the questions. 

### 6 Certainly, I think everyone here is committed 

### 7 to reduced, replace, refine whenever possible, for a number of 

### 8 reasons: to save money, to make the experiments as efficient as 

### 9 possible and consider animal welfare and reduce the number of 

### 10 animals wherever one can. 

### 11 Speaking specifically to the AMA in Xenopus, 

### 12 although this was not one of the charge questions, I did notice 

### 13 that a couple of the slides presented on the total number of 

### 14 animals used, showed numbers where more than half of the animals 

### 15 used were Xenopus tadpoles. 

### 16 So to put that somewhat in perspective, one 

### 17 breeding pair of Xenopus can produce an awful lot of adults. In 

### 18 the wild, a healthy female at peak production might lay 10 to 

### 19 20,000 eggs. If they are all fertilized -- and there are 

### 20 various papers that predict the outcome of how many will survive 

### 21 into sexually mature adults -- probably less than half of those 

### 22 will survive through the tadpole stage, and probably only a 

### 23 third of those will make it to the froglet stage. 

### 24 So the attrition, by default, in this animal 

### 25 model is huge in the mild. And 300 tadpoles is not a lot of 

### 26 animals from that perspective that we've used in the laboratory.

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1 2 So what would we lose if we turn to a 

### 3 mammalian model to model the thyroid function if we move away 

### 4 from Xenopus? Or for that matter, another aquatic species and 

### 5 other axes such as in the fish studies. 

### 6 Xenopus is a well-established vertebrate model 

### 7 of development. It's highly efficient and very hardy; meaning 

### 8 that a couple of breeding pairs can produce many, many animals. 

### 9 If you move to study this system in a mammalian model, you're 

### 10 going to use a lot more rats. It takes a lot more rats to 

### 11 produce the numbers that you would get with Xenopus. So there 

### 12 are some inherent advantages by using this species that would be 

### 13 lost. 

### 14 The second comment is regarding to the 

### 15 attention paid to minimizing animal models. As I mentioned, I 

### 16 think all groups involved in this are very focused on that for a 

### 17 number of very good reasons. But there are checkpoints along 

### 18 the way that probably should be acknowledged. 

### 19 Every institution has regulatory agencies, 

### 20 animal use and care committees, and in order to do this work in 

### 21 any animal, Xenopus, rats, fish, or mice, you would have to 

### 22 submit an animal protocol to be approved by the institution's 

### 23 animal use and care Committee. And in that you must report the 

### 24 steps you've taken to reduce, replace, and refine. So you would 

### 25 have to justify the number of animals used and you would have to 

### 26 use the statistical test to support the numbers, and that would

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1 have to be approved by the IACUC before the work goes forward. 

### 2 So there is one mechanism which is already 

### 3 built into the system that ensures the minimum numbers of 

### 4 animals that could and should be used. So all in all, I think, 

### 5 again, kudos to all for putting this on one of the question 

### 6 agendas and that due diligence is being done. 

### 7 As the weight of evidence progresses and the 

### 8 EPA moves forward with Tier 1 and Tier 2, there will be 

### 9 continual refinements as science advances and animal modeling 

### 10 advances. But it's good to give attention to it now, and I 

### 11 thank you all for doing that. 

### 12 **DR. DANIEL SCHLENK:**Other comments? Dr. 

### 13 Scott? 

### 14 **DR. GEOFFREY SCOTT:**I would just reiterate 

### 15 the one I made yesterday during the public comment period 

### 16 regarding the cytosol fractions and basically looking at some 

### 17 storage studies to look at each ending, the like use of those 

### 18 products as opposed to having arbitrary time components. 

### 19 We are, according to the lab presenters, doing 

### 20 proficiency as long as those assays are viable. If we can 

### 21 extend the life on those, that means fewer animals and lower 

### 22 cost. I think, to me that would worth pursuing a little 

### 23 further. Thank you. 

### 24 **DR. DANIEL SCHLENK:**Dr. Schultz. 

### 25 **DR. TERRY SCHULTZ:**In the cases of looking at 

### 26 these pathways for endocrine disruption, you're basically

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1 employing the adverse outcome pathway approach in that you 

### 2 identified events at the cellular level, which would be the ER 

### 3 binding and the transcription. And then you have an 

### 4 intermediate effect, which would be the Hershberger or the 

### 5 uterotrophic assay, and then you have an apical outcome that 

### 6 you're looking at in the whole animal or population scenario. 

### 7 The more comfortable you become with that 

### 8 pathway, the more you're willing to spread the distance between 

### 9 the different levels of biological organization. So I think 

### 10 it's very logical that this question be asked because what 

### 11 you're basically saying is we have a lot of experience in ER 

### 12 binding and the transcriptional activation. So we're pretty 

### 13 good at defining the structural space of the activity domain. 

### 14 We have some assays that allow us to do this 

### 15 at the individual population level. In our Tier 1 we have these 

### 16 ones that are intermediate. And you're asking, do we need 

### 17 those? 

### 18 Personally, I think that you need those in the 

### 19 beginning in order to give the confidence to the public that 

### 20 what you're doing is following the pathway. As you become 

### 21 greater experienced with that, it would be more likely that 

### 22 those things will be dropped out or that your ability to skip an 

### 23 intermediate event in your pathway and still have that to be 

### 24 scientifically justified. 

### 25 **DR. DANIEL SCHLENK:**Any other comments 

### 26 from the Panel? Okay. Let's go to the Agency. Any questions

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1 or clarification for the comments made? 

### 2 **DR. MARY MARIBUSAN:**No additional comments. 

### 3 Thank you. 

### 4 **DR. DANIEL SCHLENK:**Okay. Let's go ahead 

### 5 and read (b) into the record, please. 

### 6 **DR. MARY MARIBUSAN:**Charge Question B: 

### 7 Please comment on the scientific criteria the Agency should 

### 8 consider in evaluating necessary redundancies and eliminating 

### 9 assays from the current battery. 

### 10 **DR. DANIEL SCHLENK:**Dr. Delclos. 

### 11 **DR. BARRY DELCLOS:**The decision to remove an 

### 12 assay from the battery could potentially be made after a 

### 13 thorough review of all data from the complete set of compounds 

### 14 that have been run through the Tier 1 screening. If an assay 

### 15 consistently produces results that are not clearly 

### 16 interpretable, removal could be considered. 

### 17 Likewise, if assays produce data that always 

### 18 only confirm the results of other assays and are not helpful in 

### 19 resolving questionable data, they could be considered for 

### 20 removal. 

### 21 Several Panel members recommend a reanalysis 

### 22 of the data using the entire dataset of 52 chemicals to provide 

### 23 an assessment of complementary and redundancy relationships. 

### 24 This more robust dataset would also be amenable to analysis to 

### 25 determine if a particular assay or assays could be eliminated 

### 26 without increasing the probability of finding false negatives.

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1 Further, after completion of this assessment 

### 2 it would be useful to consider splitting the tier 1 assay 

### 3 battery into two sub-tiers using the in vivo assays as a 

### 4 prescreen followed by targeted in vitro tests to explore 

### 5 mechanisms. 

### 6 **DR. DANIEL SCHLENK:**Dr. Selvage. 

### 7 **DR. DANIEL SELVAGE:**Yes. I concur with Dr. 

### 8 Delclos. I would just like to also emphasize that this question 

### 9 would be a lot easier to approach if we had all the data 

### 10 available from the studies. 

### 11 **DR. DANIEL SCHLENK:**Okay. Dr. Piersma. 

### 12 **DR. ALDERT PIERSMA:**Just to follow-up on what 

### 13 was said a little earlier, the scientific criteria should not 

### 14 just depend on the chemicals that happen to be tested, but 

### 15 should be dependent on mechanisms again. 

### 16 The Table 1 in the report is very good there. 

### 17 I mean, if those points really cover certain aspects of the 

### 18 mechanisms of action, you could actually, already on the basis 

### 19 of Table 1 say we need these assays and we can skip those 

### 20 assays, given that those assays are validated and given that the 

### 21 endpoints are valid and can be measured in a decent and robust 

### 22 way. 

### 23 I think that's more important and it gives you 

### 24 a more general idea of how to select assays rather than test 20 

### 25 compounds or whatever you have. 

### 26 **DR. DANIEL SCHLENK:**Other Panel members?

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1 Yes, Dr. Schultz. 

### 2 **DR. TERRY SCHULTZ:**I would just like to refer 

### 3 the Agency to Question 2 that we had yesterday afternoon. There 

### 4 was some question about redundancy among the in vitro assays as 

### 5 it related to the prioritization scheme. That would also fit 

### 6 into the total answer from the Panel on Question Number 8b. 

### 7 **DR. DANIEL SCHLENK:**Anyone else? Okay. 

### 8 Let me go back to the Agency. Any questions or clarification? 

### 9 **DR. MARY MANIBUSAN:**No additional comments. 

### 10 Thank you. 

### 11 **DR. DANIEL SCHLENK:**Okay. As is customary 

### 12 for most panels, what we do, you'll have a chance, at this 

### 13 point, to provide any further comments or inputs that you'd like 

### 14 to provide for the record. We'll just go around the table 

### 15 quickly here. We'll begin with Dr. Klaine. 

### 16 **DR. STEPHEN KLAINE:**Well, I just wanted to 

### 17 thank both the public commenters and EPA for all the hard work 

### 18 and great presentations. I realize that we probably just gave 

### 19 you a bunch more hard work to do. But I think that it's pretty 

### 20 critical that we need the answers and further work in order to 

### 21 consider this in the entire set of issues that started in 

### 22 January and are going to end up with the next two meetings in 

### 23 June and July. 

### 24 So I think it's work well spent. In that 

### 25 regard, I would like to say thanks for the great roadmap that 

### 26 you guys put together for tying these things all together. So,

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1 thanks. 

### 2 **DR. JAMES MCMANAMAN:**I too would like to 

### 3 thank the EPA and the public commenters. They were great 

### 4 presentations. I learned a lot and I hope that we've been able 

### 5 to help you clarify some of the questions that you had to help 

### 6 you move forward. I look forward to the June and July meetings. 

### 7 **DR. BARRY DELCLOS:**I'll second the thanks to 

### 8 the Agency and to the public commenters. They were very useful. 

### 9 And to the SAP staff for a nice meeting. It's very nice to see 

### 10 data coming out of this massive effort, finally. I was happy to 

### 11 be able to participate. Thank you all. 

### 12 **DR. GEOFFREY SCOTT:**I too would like to thank 

### 13 the EPA and the public presenters for diverse presentations. I 

### 14 think we heard on both sides of many issues, ranging from in 

### 15 vivo test, in vitro test. I really appreciate all the foresight 

### 16 and input that we got. It made our job a lot easier with the 

### 17 clarity of the presentations. I really thank you all at EPA for 

### 18 that in helping us understand the data. 

### 19 A couple of things that I'm struck with as you 

### 20 all move forward, we've heard from several of the other Panel 

### 21 members in our concluding remarks, looking ahead to the weight 

### 22 of evidence and next meetings on this. One of the comments I 

### 23 made, and I'll just reiterate it, is knowing your controls. 

### 24 What I mean by that is knowing for each of 

### 25 these assays what is normal. I think we saw a little bit of 

### 26 evidence of that presented yesterday from some of the public

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1 presenters on one of the assays, just with the control data. 

### 2 I really think starting with some of that type 

### 3 of approach and really getting that information is going to be 

### 4 very critical because when you know what is normal, it's much 

### 5 easier to understand what is not normal. And I really employ 

### 6 you to look at that in particularly now that you actually have a 

### 7 lot of different people performing the assay, which is also a 

### 8 little different variable. 

### 9 The other thing that I am struck with these 

### 10 assays is the good balance in the combination of both screening 

### 11 assays, in vitro assays and the live animal models. I think I'm 

### 12 really pleased to see that fish are playing a key role in this, 

### 13 being an aquatic toxicologist. I think they really are one of 

### 14 your real cornerstones. And I think having those tied to 

### 15 mammalian tests along that pathway and then with the frog test 

### 16 is a good combination. 

### 17 As we talked today, the frog test is not 

### 18 without problems. We're going to have to resolve this issue 

### 19 that we talked about today. That's not the first time we've had 

### 20 to do that with an assay. So I think we'll look forward to 

### 21 continuing down that road of seeing where you go with this with 

### 22 the full list of compounds. 

### 23 Finally, in terms of redundancy, I really do 

### 24 think looking across those modes of action for consistency of 

### 25 response across the assays, I'm really looking forward to seeing 

### 26 that information. That's going to really tell us a lot. Again,

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1 thank you for having me here. 

### 2 **DR. DANIEL SELVAGE:**I would just like to 

### 3 reiterate the thanks to the EPA and public as well. I will 

### 4 leave it at that. 

### 5 **DR. TERRY SCHULTZ:**I think this SAP, looking 

### 6 at the 21 chemicals is a wonderful intermediate step towards the 

### 7 weight of evidence. In order for the weight of evidence SAP at 

### 8 the end of July to really be meaningful, we need to look at the 

### 9 information that you have on all 52 chemicals because it will 

### 10 give us a greater confidence and it will eliminate the question 

### 11 of transparency in how the 21 were picked from the list of 52. 

### 12 **DR. DONALD TILLITT:**I guess I would also like 

### 13 to thank the Agency and the public for the comments and 

### 14 compliment them on their presentations. I thought it was very 

### 15 good and very thorough. And reiterate many of things that 

### 16 you've heard already. 

### 17 One of the things, I don't know if I did hear 

### 18 it before was in the presentation of the data for the weight of 

### 19 evidence, I think it might be useful to have some type of 

### 20 statistical power analysis to understand what the expected 

### 21 differences might be, based on the design of these studies and 

### 22 based now on our track record of knowing what the variance is 

### 23 going to be. To understand the power of the test and make those 

### 24 comparisons in the weight of evidence portion of it. 

### 25 DR. KATHERINE ROBY: I'd also like to thank 

### 26 the Agency and the public presenters for really very clear and I

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1 think enlightening information that you presented for us. I'd 

### 2 also like to say, though, that I look forward to some in-depth 

### 3 data that we can really begin to understand the significance of 

### 4 the entire battery and how that will move forward. 

### 5 **DR. ALDERT PIERSMA:**Well, I'm not going to 

### 6 repeat everything that was said. Thank you very much for a lot 

### 7 of very clear presentations and a lot of information that was 

### 8 very useful for the process and also for giving me insight in 

### 9 how this process works in the U.S., being from a different 

### 10 continent. 

### 11 I should also thank Dr. Jenkins and his staff 

### 12 for getting me through the U.S. and EPA bureaucracy to even fill 

### 13 out all these forms that were needed to get me here. I'm really 

### 14 surprised that we go through it, but thank you very much for all 

### 15 your help. 

### 16 **DR. REYNALDO PATINO:**I have nothing much new 

### 17 to say, other than thank you to Fred and Dan for the opportunity 

### 18 to participate in this panel, and to the Agency and the audience 

### 19 for their willingness to listen. I hope the comments were 

### 20 useful. Thank you. 

### 21 **DR. DEBORAH MACLATCHY:**I just would like to, 

### 22 again, than the Agency and the public commentators on flushing 

### 23 out what was in the White Paper and giving us more information 

### 24 to go on as a panel. 

### 25 I would like to also say that as one of the 

### 26 two international Panel members that certainly, I think that the

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1 work that EPA has done on this since the late '90s has been very 

### 2 critical to the science and the regulation around the issue of 

### 3 endocrine disruptors. I think that this is one step that I 

### 4 think in 1998 we didn't think we'd be sitting here talking about 

### 5 this in 2013, but I think it's clear that it's a very complex 

### 6 issue. And what EPA has added to the weight of evidence and 

### 7 volume of information has been critical in moving this field 

### 8 forward. So, thank you. 

### 9 **DR. JACK LEONARD:**I'll just share the 

### 10 gratitude with all my former Panel members to the EPA and to the 

### 11 public presenters. I realize that I was actually one of the 

### 12 original EDSTAC members back in '98. And the assays that you 

### 13 have chosen were actually thought processes at the time, and 

### 14 it's amazing to me how they have morphed into what you've done. 

### 15 And I really appreciate the effort here and I look forward to 

### 16 the future. 

### 17 **DR. NELSON HORSEMAN:**Not to be redundant in 

### 18 thanking the Agency and particularly the outside presenters. I 

### 19 think from my experience on a few of these other panelists, this 

### 20 panel in particular, the information the outside presenters were 

### 21 able to provide really, really did help the Panel. 

### 22 The other thanks will go to the staff here at 

### 23 the EPA for having put together a panel that I think was really 

### 24 well-balanced and brought a lot to the table. 

### 25 The only other comment back to the report I'll 

### 26 make is, this endocrine disruptor area I know is troublesome for

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1 the regulatory agency because you're dealing with a situation 

### 2 which it really does depend on having healthy animals to see 

### 3 effects that are conceptually not normal. 

### 4 So we've dealt over and over again, the 

### 5 problem with this toxicity; and I encourage you to think really 

### 6 hard and maybe come back with some notions about how to deal 

### 7 with this problem of more isn't always better. I think that 

### 8 will help move things forward. Thank you very much. 

### 9 **DR. SHERRIL GREEN:**I too would like to thank 

### 10 everyone; especially, the public commenters provided some really 

### 11 helpful information. I would like to say one last thing about 

### 12 the bent tail. While it was really interesting and somewhat 

### 13 unanticipated finding, I think it truly reflects some lack of 

### 14 experience and husbandry management that those issues are 

### 15 resolvable. And moving forward, I think, with time, the bent 

### 16 tail will be less of an issue. Right now it doesn't seem like 

### 17 that finding alone would be reason to remove the AMA from the 

### 18 battery of tests. And again, thank you. 

### 19 **DR. DANIEL SCHLENK:**Well, thank you all. 

### 20 I would like to thank the Panel, each of you for your hard work, 

### 21 for meeting together, getting everything put in a very logical 

### 22 and articulate form for everyone to listen to. You've done a 

### 23 great job in keeping us on time. You've made my job very easy, 

### 24 which is wonderful. I'd like to thank, again, the Agency, for 

### 25 the presentations, the materials and the steps forward. Again, 

### 26 it's nice to see a path forward and a road map to move forward.

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1 I'd like to thank Fred and his staff for 

### 2 getting us here and hopefully getting us away, and all of the 

### 3 work that goes into that. Getting us coffee, and even cookies 

### 4 this time, which is was awesome. 

### 5 I'd like to thank, again, the public 

### 6 commenters, a very special thanks to you for the input that you 

### 7 had for us. As you heard repeatedly, this was very, very 

### 8 beneficial to us, in terms of putting this report together. 

### 9 So with that, any comments from EPA? Yes? 

### 10 **DR. MARY MANIBUSAN:**Just a few closing 

### 11 remarks, and perhaps as we close out this meeting I can spend a 

### 12 few minutes just kind of setting that roadmap down again in 

### 13 looking ahead. Thank you again to the Panel for all your 

### 14 recommendations, suggestions and ideas. They are very helpful 

### 15 to the Agency as we move forward. 

### 16 As we whet your appetite for the weight of 

### 17 evidence analysis, just a brief reminder that the Agency was 

### 18 very deliberate in the timing and sequencing of the July SAP. 

### 19 We want to take incremental steps forward, building upon the 

### 20 experience and input that we're receiving here at this SAP, into 

### 21 how we continue moving forward to formulating our 

### 22 interpretations and our future approaches. 

### 23 With your input and insights on the assay, 

### 24 performance in particular, the bent tail, we will be continuing 

### 25 that conversation in the next month, SAP, as we embark on 

### 26 completion and validation of the remaining Tier 2 assays. The

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1 LAGDA assay in particular, will have some insights to offer on 

### 2 this bent tail observation and how we can provide some 

### 3 additional advice in terms of test guidance to avoid those 

### 4 particular observations. So we'll continue that conversation in 

### 5 June. 

### 6 Thank you again for your comments on the 

### 7 battery performance. I think it gives us a lot of assurances as 

### 8 we move forward as an agency, and continue to issue test 

### 9 guidance in how to do that more appropriately will be important 

### 10 to receive your information, as well as employing all of your 

### 11 considerations and adjustments to our evaluations in approaching 

### 12 for reviewing and revising our evaluations for the remaining 52 

### 13 compounds for List 1. And certainly with this peer review and 

### 14 input from the panel, it will greatly achieve that scientific 

### 15 soundness that we always talk about as an agency, as a whole. 

### 16 As you've discussed, in terms of the July 

### 17 meeting, I want to emphasize that all the input that we're 

### 18 receiving on the Tier 1 battery and assay performance will be 

### 19 integrated together with the other scientifically relevant 

### 20 information in a weight of evidence assessment to help us build 

### 21 a case and to make a determination of whether a chemical has the 

### 22 potential to interact with the endocrine system. That is what 

### 23 we plan to bring to you. We, again, appreciate that you're 

### 24 allowing us to take this step-wise, slow pace progression in 

### 25 moving forward. 

### 26 It is with that incremental pace that we need

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1 to ensure an anchor, a scientific soundness before making those 

### 2 regulatory decisions because of the impact that we've talked 

### 3 about, in terms of resources, timing, and decision-making. 

### 4 With that, I want to acknowledge the 

### 5 excellence of our EPA team in putting together some complex 

### 6 analyses for this meeting. We are looking at over 100s of assay 

### 7 results, and within a short amount of time they've done an 

### 8 excellent job. I want to acknowledge our team here at the table 

### 9 as well as those in the office and across in our Office in 

### 10 Research Development Program. 

### 11 I want to acknowledge Dr. Fred Jenkins and the 

### 12 SAP staff as a whole. Folks who attend SAP meetings really 

### 13 enjoy it because of the logistics and all the things that the 

### 14 SAP program does to ensure a very smooth transaction with the 

### 15 program. 

### 16 I too would like to reiterate the excellent 

### 17 comments that were provided by the public at large. I think it 

### 18 demonstrates that the endocrine program really reaches out to 

### 19 all the experience within the contract laboratories, to our 

### 20 regulated communities, and to the public to help us ensure that 

### 21 we're moving forward in the right direction. So we definitely 

### 22 appreciate their comments. 

### 23 And then last, but least I want to acknowledge 

### 24 the Chair, Dr. Dan Schlenk, who keeps us on schedule, on time, 

### 25 and on topic. And the rest of the panel, I have never been so 

### 26 impressed with a panel that can speak to such a broad breadth of

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1 issues and across so many different taxa and across the Tier 1 

### 2 assay and battery. 

### 3 The DFO, the SAP team absolutely did an 

### 4 excellent job in picking each and every one of you. And if I 

### 5 can opine a little bit, as we began this meeting we talked so 

### 6 much about the EDSTAC and their conceptual framework and what 

### 7 they thought about this program in putting together this Tier 1 

### 8 and Tier 2 screening and testing approach. 

### 9 I like to believe that as we look back in 

### 10 time, this 2013 year, this SAP meeting will be that reference 

### 11 expert committee that we will call upon as bringing the program 

### 12 up, in terms of the state of science, and really reflect how we 

### 13 can do our jobs better, faster, more efficient. So thank you 

### 14 very much. 

### 15 **DR. DANIEL SCHLENK:**Thank you, Mary. So 

### 16 with that, I will turn it over to Fred to close us out. 

### 17 DR. FRED JENKINS JR.: First, I want to give a 

### 18 special thanks to Dr. Daniel Schlenk for his leadership. It's 

### 19 always a pleasure working with you, Dan. This time was no 

### 20 different. 

### 21 I want to thank the Panel. I really 

### 22 appreciate you all's willingness to serve and your commitment 

### 23 and working really hard throughout this process. I want to give 

### 24 thanks to Dr. David Dix who is our newly appointed office 

### 25 director. Dr. Stephen Bradbury, thank you so much. A special 

### 26 thanks to Mary Manibusan. Mary served as a lead manager in

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1 leading her team to get prepared for this panel. It was a 

### 2 pleasure working with you, Mary, in preparation for this 

### 3 meeting. I thank you for everything. 

### 4 A special thanks to Jess Rowland, another one 

### 5 of the managers involved in preparing for this meeting. A 

### 6 special thanks to Don Bergfelt. He was my lead point-of-contact 

### 7 amongst the staff and he was extremely superb in getting me all 

### 8 the information I needed and helping me and keeping me informed 

### 9 with what was going on in preparation for the meeting. 

### 10 Thank you to Gregory Akerman, John Liccione, 

### 11 Amy Blankinship, Catherine Aubee -- I'm naming all the team 

### 12 members -- Thomas Steeger, Vicki Dellarco, Patience Browne, and 

### 13 Calvin Powell. Thank you to you all. It really was a pleasure 

### 14 to work with you all. 

### 15 Thank you to the public commenters. You all 

### 16 did a great job in providing information and public input 

### 17 through this process, to make this process work even better. 

### 18 Thanks to those who submitted written comments. Thank you very 

### 19 much to the public for attending this meeting. 

### 20 I would be totally remiss if I did not thank 

### 21 my FIFRA SAP staff, under the leadership of Laura Bailey. 

### 22 Thanks to Laura. I wanted to thank Sharlene Matten and Joe 

### 23 Bailey, who are the other DFOs with the FIFRA SAP. Thank you 

### 24 for your support. 

### 25 Thank you to Donald Wood, our administrative 

### 26 team lead and Joyce Coates and Shirley Percival who is also on

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1 our admin staff. And thank you to our contractor for their 

### 2 support throughout this meeting. 

### 3 The last thing I need to say to you all is 

### 4 that the meeting report will be ready no later than 90 days at 

### 5 the close of this meeting. So I want everybody to have a great 

### 6 holiday weekend and thank you all. This meeting is officially 

### 7 adjourned. 

### 8 (Whereas the meeting was adjourned) 

### 9 * * * * * 

### 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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481:4,12 alteration 165:6,8 170:17,26 406:6 434:10 alterations 77:15 78:19 81:5 95:24 103:11 104:1 104:17 167:9 168:9,12,13 169:6,8 170:2 174:1 175:12 276:20 442:4 altered407:17 427:10,11,14 461:12,17 462:2

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265:9 293:22 321:8 401:16 ample 151:12 208:24 331:12 amplification451:22 475:8 amplified422:10 amplify 399:21 450:19 Amy 31:7,19,21 53:24 93:24 94:1 107:13 108:1,8 108:11,17 109:7 110:2,13,16,24 111:11,25 112:14,24 113:11,21 114:3,20 119:22 153:7 164:2,3 172:14 189:16,18 190:8 505:11 analyses 52:4 102:10 239:7 245:14 249:10,14

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278:7 312:25 447:5 455:7 cellular 155:4 398:9 491:2 cell-based 240:8 cell-to-cell 226:9 Celsius 100:15 center 1:13 4:24 5:3,19 7:6 210:19,21 211:9 232:2

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203:24 207:3 236:4 259:26 262:10 284:4 300:8 321:25 328:13,20 339:6 382:15 390:25 418:16 440:15 444:19 453:12 470:21 488:6 498:26 502:13 certainty 14:15 19:6 24:17 Certified 220:2 cervix75:1 cetera 28:15 385:24 387:21 389:20 397:3 460:21 CFR 144:19 chain 34:12 chair 3:23 194:8 288:17 316:26 503:24 chairing 3:23 4:4 challenge 35:24 39:6 53:5 89:25 153:1 199:4

342:21 389:13 391:26 392:20 394:11

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411:6 Cincinnati 6:13 211:26 circled452:3 circles 105:18 circulation401:6 circumstance 192:25 circumstances 215:13 circumvent285:3 cite 126:24 cited 122:12,18,19 291:22 322:7 citing424:21 CLA 313:5 clairvoyant27:4 Clare 305:12,14,18 316:9 317:1 331:20 clarification 35:9 49:25 87:13 88:21 92:17 142:8 190:23 193:15 219:13 263:9 349:21 350:12 380:13 382:10,17 393:26 413:3 417:22 422:12 423:7,10 446:1 450:7 454:25 466:12 476:11,13 492:1 494:8 clarifications 93:22 135:22 282:17 350:7 363:11 clarified 122:13 351:1 390:20 422:21 clarify 88:23 89:5 141:14 189:7 200:6 363:14,18

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445:12 clutches 428:11 Coady 271:15 coalitions 220:9 Coastal 5:3 210:21 Coates 505:26 code 188:1 297:12,14 codes 188:6,11,12

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66:10 67:24,26 68:2,5 95:3 97:26 98:4,5 102:13 106:20,21 109:4,13,15 110:4,17,20 113:9,18 114:2 116:5 118:10 120:3 128:8,26 129:8,9 147:24 152:7 176:4 206:7 224:1 232:3,19 233:20 234:4 237:19 239:2,22 240:5 241:20 242:7,13 251:15 252:24 253:3,13,17 268:22,24 272:1,13 282:23 283:17 326:3 339:11,12,15,25 341:26 342:24 396:4 402:9 406:4 409:21 412:5,7 427:24 459:23 concept 11:18 153:15 154:8 159:25 164:9 167:22 168:1 169:10 291:6 conception 309:23 concepts 25:19 conceptual 18:13 31:16 198:22 199:11 333:5,14,15

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401:21 446:23 461:14 concurrently 73:17 93:14 concurs 374:13 condition 131:15 258:21 259:11,15,21,22 260:6,21

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94:10 96:14 130:1 167:14 175:2 178:17 179:16 227:22 248:14,15 277:19 311:5 347:23,26 348:14 348:19,20 349:25 351:25 conducted 23:19 55:22 64:12,14 66:7 70:24 79:17

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348:17 351:26 352:2 446:22 conducts 23:10 conference 1:13 221:16 408:17

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286:21,22 314:2 337:1 341:18 347:22 435:1 440:19 503:19 contractions 339:19 contractor 506:1 contractors 283:24 contrast 366:22 contribute 108:25 355:3 387:22 412:4 contributing 250:24 356:12 365:6 407:18 439:21 contribution459:20 contributors 31:9 246:20 control 28:1 58:5,6,7,8 59:11 60:18,26 61:1,2,2

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349:14,16 359:24 378:26 controls 46:4 63:12 99:7 119:2 120:16 121:2 123:23 124:20,20 125:10,23 126:10 140:21 141:17,21,25,26 142:8,9 144:12 145:15 162:2 193:21 205:22,22 216:26 253:20 254:2,7 261:16 261:17 262:4,7,14,17,18 269:25 272:20,21,21,23 277:11 299:7 303:9 305:1,2 320:1 339:23 341:13 356:21 360:4 361:23 382:2 391:13 417:26 419:25 423:16 426:6 447:14 448:3 466:6 469:10,20 495:23 convene 27:5 conversation 383:21 422:10 501:25 502:4 conversely 147:23 converts 65:24 convey48:23 convince 303:15 319:5 convinced434:14 convincing 255:16 cookies 501:3 Cooperation 11:23 12:25 Cooperative 6:1 211:14 coordinate7:12 330:6 coordination 6:25 7:9,9 15:19,19 25:1 39:22,23

227:10 Coordinator271:9 296:7 copper432:23 434:7 copy 311:12 corner284:24 cornerstones 496:14 corning 281:17,18,25 Corporation 252:18 321:22 correct59:19 72:4 88:4,18 110:13 113:1,21 143:1 148:19 185:3 189:2 236:13 300:5 303:11,12 312:19 318:17 345:14 353:2 392:22 403:25 412:23 413:17 415:11 corrected 282:22 361:17 correction 236:9 237:1 318:15,17,20 319:16

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314:8 corresponds 145:19 173:16 174:22 corroborate41:23 276:8 corroborated 159:8 161:7 162:1,10 corroborating451:3 corroboration 27:26 49:20 278:26

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www.transcriptionetc.com Page 21 corticocarcinoma 64:7 corticosteroid 423:2 corticosterone 61:2 82:9,17 381:5 Cosmetic 10:6 16:26 cost21:11 198:21 222:2 274:25 279:16,19 288:8

291:25 307:9 308:10 353:12 360:24 367:3 379:16 444:14 472:21 483:8 484:22 490:22 costing 254:24 costly 198:24 222:1,4 costs 178:24 179:2 308:15 349:25 379:23 400:18 cost-effective 238:7 cost-effectively 320:20 cost-savings 349:23 couch 367:7 Counsel 3:3 count61:2 330:18,24 331:7 counterintuitive 125:24 counting 279:20 countries 12:26 313:14 country 309:18,19 couple 2:11 9:25 37:24 42:10 69:18 92:2 120:15 122:9 132:17 134:15 141:3 186:19 198:4 203:1 207:21 208:21 212:14 221:24 224:15 226:18 229:14 256:12 286:19 290:2,11 295:17 297:21 304:15 336:6 337:10 339:7,12 341:18 352:8 353:20 361:19 372:19 382:9 391:18 398:4 403:1 405:6 406:22 422:5,6 437:13 442:1 464:19 470:25 471:1,21 485:8 488:13 489:8 495:19 coupled 120:8 course 19:8 24:18 27:9 31:11 68:22 84:13 89:20 128:9 136:15 145:4 198:9 222:11,24 225:5 227:14 238:15 273:2 299:6 301:1 304:4 335:1 389:6 391:7 427:21 463:8,10 478:6 487:6,11,17 covariance77:20 covariants 82:15 covariate77:21 80:5 113:25 114:1 299:1 350:8 cover 19:9 22:16,17 42:1,25 139:15 187:8 231:11

308:18 310:8 478:3,19,20 479:11 493:17 coverage43:9 460:17 472:14,20 476:4 covered 186:13 256:11 292:9 329:7 427:20 477:26

478:7 479:13 covering 242:8 covers 309:23 378:8 463:19 Cowper's76:12 co-activators 342:2 co-efficient 153:1 co-factor434:7 co-formulated 214:15 co-occur 156:12 co-occurrence471:7 co-repressors 342:2 crash 232:21 CRE 318:15,15 319:19 create 17:5 21:24 332:2,7 created 140:23 creatine 354:3 creating 336:10 CRE's 318:20 crinkled445:17 crippled 323:1,2,3 crippling452:16 criteria 29:1,2 54:6 56:1,3,5,22 58:21,22,25 59:4,7

59:10,25,26 61:4,7,9,17,21 63:2,5,8,9,15,16,18 63:26 64:3,3,23,25 65:5,6,17 66:19,20,21,23,25 66:26 67:10,13 68:22 70:14,22,25 71:2,8,12 72:6 72:16,21,24 74:3 75:8 76:21 77:4 78:23 79:2,15 81:9,10 88:19 93:11 94:9 96:3,4,7,20,23 97:10,11 97:14,18,21 98:17,25 99:22,23 101:1,1,4,4 106:12 111:19 115:6 117:12,13 118:3,9,13,17,19 118:24 119:2,10 120:13,16 125:1 129:16 135:24 141:4 142:12 147:6,14,18,19 148:11 152:12,13 165:9 173:18 187:17 200:3 218:10 223:17,22 237:23 238:3,6,20 243:18,26 246:12,13,19,25 247:1,5,8,10,11,18,21,23 248:3,5,7 252:22 253:3 253:18 271:20,22 272:2,9,10,13,16 273:2,12,17 279:22 284:4,5,6,12 300:11,13 332:24 338:18 340:21 341:3,6,13 344:13,18 345:1 349:12 351:8 351:20,23 356:14,19,20 357:1,15 358:15,23 359:2,2,5,7,10,19,22 360:1,11 361:16,19 362:8 362:12,16 363:19 364:10 365:2,8,9,14,26 370:20 370:21 371:3 373:19,22,25 374:20 375:1,12 376:1,4 378:21 379:18,20,22 380:2,15,17,25 381:8,18,25 382:1 389:25 391:20 393:7,9,18 418:20 429:9 492:7 493:13 criterion 59:12,15 96:13,23 97:1,8 98:11,20 99:4

99:11 100:15 119:7,21 135:13 147:20 148:8 233:24 234:15 235:8 252:23 350:24 392:2,19,22 critical 10:25 17:23 18:10 25:23 27:2 43:21 83:19 147:11 163:26 202:20 224:13 226:26 227:1,22 306:8 362:8 369:20 399:2 411:24 451:12 463:4 481:9 494:20 496:4 499:2,7 critically 331:9,23 criticisms 242:6 319:13 CRO 347:26 355:12 crooked 121:13 441:15 Crop 237:12 CropLife 305:13,14,19,20,22 306:5,6,7,17 310:22

313:5,23 CropScience 258:18 288:16 CROs 348:19 393:22

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451:23 cross-sex479:5 cross-species 479:5 CRYSTAL 1:15 cues45:11 52:24 cull 299:10 culled 326:12,23 culling 324:3 326:14,18 culminated 367:16 culmination 230:14 culture 312:26 cumbersome 231:22 cumulative 361:10 curious 107:8 204:18 curled465:14 current27:14 31:15 49:3 122:20 157:11 225:4

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389:25 390:19 391:3,5 CVs 98:1 109:16 113:14 119:24 120:8 129:2

147:19 152:6 246:19 247:3,10,12,26 359:21,22 386:5,13,14,17 387:8,22 388:26 391:7 392:22 393:14,17,17,20,23 cycle 34:25 78:14 115:25 249:18,18 295:21 353:8

353:19,22 387:13 388:5,14 459:19 cycled 388:9 cycles 249:24 353:7 388:4 cyclicity 77:25 78:20 252:5 386:18,20 394:7 cycling 295:20 386:14,24 387:12 388:1 394:24 CYP19 240:15,16 cytosol 57:15 59:3 62:8 63:6 324:5,6 327:7,15

338:7,24 339:2,5 342:8 355:9,13 371:13 379:16 380:8 465:7,13 490:16 cytosolic 231:23 371:23 cytosols 57:2 64:2 cytotox 235:25 236:12,14,26 cytotoxic 239:17 381:22 cytotoxicity 30:14 61:14 67:22,23 231:19 233:21

233:22 235:24 236:2,6,9,10 237:2,18 239:10,14 239:14 241:12,14 242:5,11 343:1 360:8 372:19 372:25 381:6,8,11,17,20 382:4,6 cytotoxity 380:22 381:13 382:13 cyto-specific 232:18 D d 174:26 daily77:18 80:2 95:15 99:6 348:24 361:1 428:6

438:23 Daland 220:14 Dale434:5 damage 361:14 dams 326:21,26 Dan4:3 5:5 82:6 210:1,7,24 230:22 390:22 399:22

498:17 503:24 504:19 Daniel 3:22,25 5:5 6:22 8:12 15:16 31:26 32:6 33:5

35:7 36:13,24 38:3,10 39:19 49:26 50:13 51:2,25 52:5 53:7,13 68:14 69:14 70:2,11 71:15 72:17 73:12,19,21 82:3,6,12,18,21,23 83:20 84:14 85:20 86:17 87:9 88:6,20 89:11 90:4 91:11,21 92:1 93:21 107:6,21 108:4 109:2,18 113:5 114:8 130:14 132:16 134:7,16 135:6 137:1 138:14 139:4,13 140:17 145:12 147:1 148:21 149:9 151:9 156:20 182:19 184:20,25 185:20 186:17 187:22 190:21 192:8,18 193:3,7,11 194:6,10 195:3 196:12 197:23 199:15 202:7 203:12 204:4 206:14 207:5 210:24 212:4 217:22 219:11 229:16 229:19 230:19 237:7 242:2,22 246:3 252:14 258:3,14 263:6 265:24 266:26 267:24 269:21 270:6,18,23 277:21 278:15 280:17 285:5 287:15 288:13 293:16 294:16 296:1 301:26 303:4,24 304:22 305:4,7,11 315:15 317:18 321:3 323:6 329:25 336:22 345:7,21 347:7,13,16 352:6,8,16 352:18,25 353:3,23,24 354:20,23 356:8,15 362:18,22,26 363:3,9,23 364:14,17,24 365:15 378:5,12 379:3,6,7,9,12,25 380:5,12 382:8 383:14,23 384:2,8,10,11,12,16,20 385:10,12 390:15 391:16 393:12,24 394:17,18 395:2,7 396:7,9 398:19 399:20 401:12 404:12 408:21

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210:18 380:6,7 390:15,16 398:19,20 413:26 459:1,2 462:23 466:1,22,23 481:1,2 483:18,19 486:24 492:10,11 493:8 495:7 deliberate 69:12 208:25 501:18 deliberately 313:24 deliberation 376:22 deliberations 208:10 227:18 delivered 111:14 360:19,22 361:5,6 delivering 111:19 112:7,8 delivery 112:10,11,25 126:4 Dellarco 31:8 505:12 demand 373:18 demonstrate 32:22 89:21 134:12 180:7 199:5

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5:26 6:4,8,12,18,22,24,26,26 8:12,13,15 15:16,16 15:21 16:1,1,1,12 24:12 31:14,18,19,23,26 32:2,3 32:6,8,9,15 33:5,5,6,7,14,15,23,25 34:7 35:7,7,8 35:13,14 36:3,13,13,14,19,24,24,25 37:4 38:3,3,4 38:8,10,10,11,18 39:7,19,23,24,24 49:26,26 50:1 50:2,6,13,14,15,18 51:2,2,3,16,25,25,26 52:1,5,5 52:6,13 53:7,8,13,16 57:1 68:14,14,16,17,20,26 69:14,14,15,20 70:2,2,3,7,11,11,13 71:15,15,16 71:24 72:2,4,5,11,17,19,20,26 73:12,14,19,21,22 73:24 82:3,4,5,6,10,12,13,18,20,21,21,23,23,24 83:1,4,6,8,13,20,20,21,25 84:1,4,14,14,15,20,22 84:25 85:13,20,20,21 86:2,9,13,16,17,17,18,18 87:1,3,5,6,7,9,11,12,17,21,22 88:2,4,5,6,6,7,11 88:16,18,20,22,23,26 89:5,8,11,11,12,19 90:4,4,5 90:9,11,14,22,23 91:1,5,10,11,13,14,19,21 92:1,8 93:21 107:6,7,8,21,21,23 108:4,4,5,8,9,11,14,17 109:2,2,3,7,18,18,19 110:10,14,21 111:8,12 112:5,19 113:2,5,6,7,19,24 114:7,8,17,18,20 115:19 130:14,15,16,19 131:4,7,12 132:4,15,16 132:16,17,23 133:7,9,19,23 134:2,5,7,7,8,14,16 134:17 135:6,10,17 136:1,3,26 137:1,1,3,13,14 137:17 138:4,9,13,14,15,25 139:4,4,5,13,13,14 139:21,22 140:15,17,17,18 142:1,4,7,24 143:1,8 143:17 144:7,9,11 145:11,12,12,13 146:5 147:1,1 147:2 148:16,20,21,21,22 149:9,10,11 150:13,20 150:21,22 151:9,10,24,25 153:21 156:20 157:8 159:26 172:10 178:11 182:4,19,23,24,24 183:3 183:20,26 184:20,20,21,24,25,25,26 185:1,8,18 185:20,22 186:6,16,17,19 187:22,23,24 188:11 188:25 189:2,4,10,15 190:2,18,21,21,23 191:9,22 192:8,8,9,14,18,18,19 193:3,5,7,9,11,13,14,25 194:2,6,6,7,10,14 195:1,3,3,5,5,8 196:12,14,15 196:21 197:23,23,25 198:8 199:15,15,16 200:5 202:7,8,9,9,26 203:12,12,13,14,16,24 204:4,4,5 204:22 205:7,25,25 206:14,14,15,18,19,22 207:5 207:8,22 210:1,10,13,16,18,20,24 211:2,5,8,10 211:13,17,21,25 212:1,4,12,21 214:3 215:23 216:6 217:22,23,24 218:7,17 219:11,23,25,26 220:14 223:10 224:8,9,9 225:10,21 226:14 228:8 228:21 229:16,18,19,25 230:19,21 237:7,8,11 242:2,2,4,12,22 246:3,6 252:14,16 258:3,4,5,8,14 258:17 263:6,6,8,15,24 264:16,19,25 265:17,19 265:23,24,24,25 266:8,26,26 267:1,7,10,13,24,24 267:25 268:9 269:5,7,21,21,22 270:1,6,6,7,14,18 270:20,23 271:4,15 277:21,21,23 278:4,5,8,11,14 278:15,17,21 279:16,18 280:2,5,16,17,19 285:5,6 285:7,13,17 286:4 287:15,15,16,25 288:13,15 293:16,16,18 294:1,16,16,17 295:9,12,15,17,26 296:1,4 301:26 302:3,3,5,9 303:4,5,12,20,21,24 303:24,25 304:8,10,11,21,22,23 305:1,3,4,5,6,7 305:11,14 315:15,16,17 316:9 317:18,22 321:3,4 321:5,17,20,21 322:4 323:6 329:25 330:2 336:22 336:25 345:7,8,9,12,13,15,19,21,21,22 346:1,8 346:14,21,25 347:4,6,7,9,13,15,16,18 352:6,6,8 352:14,16,17,18,19,25 353:1,3,10,23,24,24,25 354:2,10,14,19,20,22,23 355:1 356:8,10,15,16,17 362:18,19,20,22,22,24,26,26 363:1,3,4,5,9,12,14 363:23,23,24 364:13,14,14,16,17,18,24 365:1,15 365:16,17 378:5,5,6,7,11,12,12,13 379:3,3,4,6,6 379:7,9,9,10,12,13,14,25,25,26 380:3,5,6,7,12,14 380:21 381:1,4,7,10,19,23,24 382:5,7,8,9,10,25 383:14,17,23,23,24 384:2,3,4,8,8,9,10,10,11,12 384:12,13,16,19,20,22 385:10,11,12 390:15,15 390:16 391:16,16,17 393:12,12,13,24 394:1,2,17 394:17,18 395:1,2,7,9 396:7,8,9 398:11,19,19,20 399:20,20,21 401:12,12,13 404:12,12,13 408:21 408:21,22 410:15,16,17 411:11,11,12 412:9,9,10 412:11,21,21,23 413:1,4,22,22,24,25,26 414:1,2 414:8,16,18 415:5,5,7,9,12 416:11,12,13,14 417:9,9,10,12,18,18,19 418:12,12,13,22,22,23 419:9,9,10,20,20,21 420:2,2,3,13,15,18,19,21,22 422:1,8,9,9,17,18,19,25,25,26 423:4,11,26 424:23,23,24,24 425:8,10 426:13,14,15 430:19 434:22,22,23,24 438:7 441:13,25,25,26 442:1,2 443:15,26 444:3 445:6,6,7,8,9,10,11,11,25 446:3 446:6,11,13,15,16 447:16,17,18 450:14,14,15,17 450:17,18 451:7,7,8,19,20,21,22 452:10,11,13 454:14,14,15,17,17,18,19,19,20,22 455:1,9,12 456:21,21,22,23,23,24,25,26 457:1,2,4,5,7,9,16 457:19,20,23 458:10,12,26 459:1,2 462:12,12,14 462:16,16,17,23 463:21,21,22,24,24,25 464:17 464:17,18,21,23 465:24,24,25 466:1,4,10,13,14 466:22,22,23 469:7,7,8,10,16,24,24,25 470:18,18 470:19,24,24,25 472:23,23,24,26,26 473:1,12,14

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84:20 85:14,26 97:24 99:2,8 108:25 130:20 138:19,19 143:18 186:20 190:3 205:22 222:3,14 225:22 227:15 266:5,20 272:1,2 273:18 275:4,10 279:5 280:12 287:3,8,26 298:4 332:16 352:11 354:8 381:19 390:5 400:10 403:18 412:15 415:21 416:20 462:18 463:9 487:21 493:17 495:24 meaning 218:1 489:7 meaningful 249:2 250:19,20 252:9 269:20 391:23

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44:10 45:4 47:22 50:22 51:11,23 72:14 110:24 125:8 141:18 151:7 154:13,17 158:18 169:14 170:26 173:19 180:9 187:8,11 190:7 192:4,5,26 193:12 198:6 208:19 212:8 215:23 219:2,12 236:14 242:9,21 243:11 263:19 264:9 267:18 273:9 294:22 321:15 322:8,8 328:9,18 332:12 346:10 352:14 369:18 370:20 371:7,21,26 378:3 380:22 386:15 389:5 397:16 401:1 414:5 431:14 432:4 433:1 449:16 453:2 464:12 465:11 474:12 475:6 478:2,8 483:26 485:11 492:25 499:20 501:24 502:1,4 particularly 6:7 30:5 64:1 78:18 116:22 125:22 163:25 178:17 204:19 218:2 236:2 237:2 245:20 270:11 351:18 352:9 399:2 401:17 402:10 404:23 406:1 431:13 434:25 435:25 437:26 456:6 481:21 496:6 499:18 parties 221:23 partition 152:26 partly 289:1 452:2 parts 30:23 85:14 102:20 403:17 partway 36:2 pass 33:15,23 74:13 75:26 262:10 344:13 413:25 passed 318:18 passes 186:13 pass/fail 71:6 Pat330:15 331:2 path 275:13 286:11 314:1 500:26 pathogen441:8 pathogens 123:26 pathological430:21 pathologies 434:1 pathologist 142:15 446:21 447:3 452:4 pathologists 134:13 450:24 pathologist's 446:24 pathology 138:5 158:15 430:24 446:17 pathology's 449:11 450:4 pathway 11:19 13:11 14:18 17:8 18:6 19:19,20

20:1,7 25:13 26:26 29:15,24,26 30:4 43:11,19,23 44:24 45:18,19 46:23 69:21 115:15 153:7 154:13 154:22,24,26 155:26 156:2,6,10,26 158:6 163:2 163:21 164:4,14,20 165:15,24 166:8,14 169:14 169:15 170:23,26 171:17 172:12,12,16,18,20,23 173:1,7,22 174:8,11,14 176:6,12 177:11 180:9,23 190:12 205:9 251:1 268:23 329:11 334:26 335:13 335:17,19 336:11 342:24 369:16 373:15 375:9 377:15 400:13 410:25 461:4,15 467:26 468:11,13 468:25 474:12 476:18 477:4 491:1,8,20,23 496:15 pathways 5:11 11:18 18:6,7 20:4 23:6 25:14 28:22

29:8 33:18 34:14,15 40:2 41:17 43:10,18 45:10 46:14 48:20 153:9,10,15,20 154:7,15 157:11 163:3,10 164:8,18 166:17 175:8 176:21,23 179:8 179:24 180:12,17,22 181:1,7 183:2 184:2 189:13 190:1,16 191:8 250:25 251:9 334:10 335:21,23 336:3 366:19 367:22 395:15 400:13 458:16 468:15 471:24 472:15 474:18 476:20 481:5,12 490:26 pathway-based 334:6 336:21 pathway-by-pathway474:15 Patience 31:8 505:12 patient295:1 Patino 5:26,26 36:24,25 107:7,8,23 130:15,16,19 131:4,7,12 132:4,15 140:17,18 142:1,4,24 143:8 144:7,11 145:11 149:10,11 150:13,21 203:13,14 211:13,13 265:24,25 269:21,22 293:17,18 295:12 304:22,23 305:3 364:14,16,18 401:12,13 412:9 412:10 415:6,7,12 419:20,21 423:11,26 447:17 447:18 452:11,13 455:1,9,12 457:20,23 498:16 Patino's 216:6 Patricia 323:7,9,10 pattern 123:16 160:16,24 163:4 202:1 251:12

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76:22 77:3 79:14 87:21 90:7,13 93:12 96:5,5,10 96:26 97:26 98:1 99:6,9,12,17,18,25 100:3,4,5,8 100:9 101:21 102:2 103:21 110:22 119:26,26 120:8 123:4,22,23 124:5 148:9 149:1,4 173:24 204:7 214:20 215:3 218:2 230:4 233:17,18 234:6 234:22 235:15,21,26 236:2,6,6,14,23,26 250:15 251:7 253:4 254:6 264:1 265:11 272:9 273:8 301:20 328:4 343:1 345:2,24,25 346:9,10,13,15 347:3 349:7 350:21,24 351:2 359:16,16 371:8,9 376:4,6,10,10 381:20 400:23 405:9,9,10,10,15,15 406:21,24 412:25,26 413:11 415:11,15,20 416:5 416:16 417:1,12,15 418:15,17,21,25 419:3,7,7,11 422:14,15 425:17,22,23 426:16 428:11 429:15 430:15 440:4,23,24 441:21 443:14,16 453:8 464:3,5,6,8,9 471:8,11,12,15,25 472:1,2,3,4,5 475:23 479:24 percentage 33:12 90:26 91:7 407:24 422:11 percentages 471:11 percentile 68:26 359:15 448:5 percentiles 119:5 perception70:9 perchlorate414:21,22 415:2 Percival 505:26 perfect 346:5 389:7 452:10 perfectly 246:12 perform 13:4 32:23 38:22 50:9,11 130:7 199:5

203:19 204:19 339:20 443:17 469:15,17 483:23 performance 1:5 2:8 4:2 7:19,20,25 26:18,23

28:26 29:2 30:11,17 31:22 38:16,21 49:7 51:21 53:19 54:1,3,6 56:1,3,4,5,22 58:22,25 59:4,10,12 59:15,25,26 60:2 61:3,7,16,21 63:2,5,9,15,16,18 63:26 64:3,4,23 65:5,6,15,17 66:19,21,23 67:8,10 67:13 70:4,14,16,25 71:2 72:21,21,24 74:3 75:5,8 75:12,20 76:21,24 77:4,8 78:23 79:2,4,15,18 81:9 81:16,26 88:19 94:9 96:3,5,20,22 98:17 99:22 100:25 106:12 117:13 118:3,7,17 119:2,21 120:12,13,16 129:14,16 135:24 147:5,10 151:24 152:1,5,11,15 153:3,4,11 155:25 156:2,5,23 158:3 159:14 162:26 164:5,18 170:8 172:24 173:6 179:16,18 180:1,1 181:17,23 182:17 196:23,23 204:15 210:4 213:21 215:15 218:10 222:20 223:14,17 231:17 235:6 237:23 238:3,20 243:4 246:11,13,17,19,21,25 247:1,5,8,10,11,18 247:21,23 248:3 251:19 252:22 253:3 266:4 271:20 272:2,8,9 273:2,12 284:4,4,6,12 296:20 297:16,25 300:11,12 301:5,5,18 302:13,16 308:24 337:13,13,21 338:1,18 340:21 344:13,18 345:1 346:11 347:26 349:12,15 351:8,20,23 356:14 357:15 358:22 359:7,10,22 361:16 362:8 362:12,16 365:2,8,9,14,26 366:11 367:25 370:20 370:21,25 371:3,4,6 372:13 373:8,18,19,21,25 374:16,20,25 375:1,5,12 376:4,8 379:18,19,22 380:2,15,17,25 381:8,11,18,25 382:1 391:20 392:2,19 429:9 430:10 432:7 438:9 444:2 458:21 460:5 462:22 466:17 468:4 469:22 470:13 476:19 480:13 483:6 501:24 502:7,18 performances 152:4 463:7 performed 29:5 55:19,21 57:16 64:12 71:5 73:9

75:21 106:2 130:25 217:26 291:13 321:7 325:17 348:24 350:1 373:12 374:10,14 378:16 397:5 480:25 481:8,15 performing 12:14 23:11 24:11 26:25 66:24 120:26 179:17 205:6 252:24 348:12 357:14 431:19 496:7 performs 48:5 65:3 69:26 period 3:7,9 78:10,11 80:24 92:10,14 99:21 116:18

203:6 207:17,26 208:9 219:16 238:10 249:8,20 271:2 305:9,12 356:5 385:19 415:24 417:20 419:5,17 422:15 430:20 475:4 490:15 periodic 123:9 391:14 peripheral 163:16 periphery452:24 455:17 permanent4:14,18,22,26 15:22 permits 236:1 person 182:21,21 396:11

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292:21,23 307:14 308:7 310:13 331:4 368:26 440:10,13 PETA 323:8,12,16 Petri445:15 PGP442:12,15,22 pH 218:4 438:2,3,24 439:3,12,23 pharmacologist296:15 pharmacology4:16 210:14,25 phase 93:14 98:13,13,19,20 99:20 101:12,16,18 159:13 216:7 217:13 244:26 245:25 333:16 phased 243:7 phases 149:24 phenomenon 212:25 215:12 411:20 426:12 433:22

433:25 448:10 phonetic 121:26 232:14 356:2 370:15 photo 94:4 photographs435:7 photomicrographs 433:15 physical 97:3 98:3 148:5 195:24 308:24 312:12

368:25 physical-chemical 54:15 310:26 Physicians 323:17 physicochemical 24:6 26:4 28:9 32:22 36:22 physiochemical 332:14 physiological 6:10 211:23 315:20 Physiology 211:26 phytoestrogen 84:16,17,23 250:4,9 phytoestrogens 85:2 phytoestrogen-free 85:3 pick40:17 44:19 90:3 274:20 342:10 438:18

445:16 picked 191:19,20 255:18 316:7 340:26 342:7 398:4 497:11 Pickford 255:14 292:13 294:3 picking 137:5 387:20,25 504:4 picture 93:18 256:8 pictures 258:25 264:5 piece 19:4 89:17 198:6 396:15 Piersma 5:22,23 36:13,14 51:2,3 52:1 85:20,21

86:9,16 138:14,15 211:10,10 462:16,17 469:11 469:16,24,25 477:16,17 480:6 482:2,3 486:22,23 493:11,12 498:5 Piersma's 466:4 480:9 pigeons 6:15 piling 392:17 pilot223:20 228:6 pipette445:21 Pipiens 261:8 264:23 265:1 pituitary44:7 45:6 47:11 77:16 78:3,26 80:14

81:13 388:11 427:10,12 450:2,3 place 89:18 218:19 274:2 316:17 351:14 355:8

370:23 372:23 379:22 389:6 400:17 411:6 414:3 419:19 placed 198:10 306:26 351:14 placeholder 196:5 places 71:17 357:21,23 plagued 239:2 plan 20:20 156:14 366:16 502:23 plans 320:13 409:15 plasma94:20 95:21 105:13 106:25 168:5,26 169:1 176:2,3 341:26 459:23 plate73:17 265:20 281:17,18,25 346:7,18,19

487:13 plateau 63:11 337:22 plates 64:13 344:23 plating 233:13,16 plausible 23:21 play 121:23 201:10,11 205:18 397:16 431:8 443:5

444:8 461:18 484:5 played 303:19 306:8 player285:20 playing 201:12,18 202:2 496:12 plays 163:18 316:5 pleasant411:10 please 2:2 3:9,14 54:17,26 59:17 73:13 103:14 151:18,21 167:18 170:5 171:19,24 177:18 189:7 208:3,7,11,15,17,19 219:18 237:6 281:10 356:11 365:12,13 385:7,8 395:22,26 405:23 426:4 447:7 457:6 458:20,23 466:17,20 480:26 482:25 483:11 483:14 492:5,7 pleased 8:16 242:26 496:12

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282:13 296:17 304:3 317:24 341:16 398:4 408:12 414:6 437:25 444:1 463:25 487:23 493:17 point-of-contact 505:6 policies 183:15 policy 6:25 7:9 15:19,20 25:1 39:22,23 144:17 183:3 229:21 230:3,4 243:2 252:20 271:2,10 282:11 288:17 290:13 296:2 305:19,21 312:14 313:2 314:11 316:11,26,26 317:6 448:21 pollute439:22 Pollution7:12 8:6 polymer26:5 pondering 114:15 poor 120:9 128:8 235:7 298:17 430:10 460:6

466:25 poorly 128:18 population 117:3 218:21 227:6 240:21 242:18,18

267:2 386:1 403:15 404:18 405:16,18 417:2 429:15 491:6,15 populations 17:16 122:7 139:23 214:20 215:20

262:22 270:4 359:24 425:21 435:14 population-relevant279:2 Porter475:10 portion 33:11 44:15 68:3 121:7 283:13 430:2 497:24 portions 163:10 451:25,25 pose 301:17 309:4 posed 302:17 posing 196:22 positive 21:13 28:1 30:7 41:20 43:6 58:5,5,7 59:11

60:16,18,19,25,26 61:1,23 62:18,19,23 63:12,12 63:17,17 64:15,20,26 66:9,22 75:2,3,6,14 76:17 76:19 78:17 81:3 146:15 156:7 157:25,26 165:17 165:20 166:25 171:8,13 173:18,23 174:1,8 177:19 178:9 189:12 191:7,11,15,16,21 192:4,24 193:2 197:5 198:16 205:21 233:24 234:15,17,23 234:23,25 235:2,8,20 236:8,18,22 243:26 246:16 250:21 252:10 291:11 294:5,5,7 308:3 315:6 329:1,1,3 332:25 337:18 340:6,17,22,22,23 341:24 342:13 346:2,2,9,16,20 351:6 356:21 360:5 369:19 370:17,20,23 380:2,19 382:1 383:7 384:23 390:3 391:6,13,15 395:19 399:4 444:18 444:22,26 458:19 466:6 467:5,16,18,19,22 468:2 470:5,7 471:24 472:1,3,4,5 473:4,19,20,26 474:3 474:4,5 476:18 478:11 480:16 positively 88:17 469:17 positives 61:12 154:2 198:18 225:5,6 232:18

234:18 235:4,9 240:24 245:13 251:22 339:25 467:24 possibility 83:12 191:19 270:5 394:8 448:23

453:17 possible 29:20 48:8 91:12 100:7 116:8 124:23 126:4 132:11,18 135:6 136:17 141:1 146:22 153:18 163:7 186:3 190:2 207:13 221:23 248:16 302:14 331:10 362:13 376:21 399:15 409:22 423:22,23 428:22 429:12 448:19 449:25 488:7,9 possibly 33:13 56:9 121:20 133:24 213:5 276:11

293:8 310:24 317:8 358:1 404:16 406:10 430:3 444:8 post 116:2 196:10 362:1 posted 148:25 poster224:20 376:26 posterior 213:2 259:19 266:24 posters 224:15,18 postnatal 74:17 76:2 77:18 80:2,3 350:9 potassium431:23 439:15 potency 51:9 224:2,6 228:11,11,15 316:15 462:26

478:6 potent227:7 potential 10:9,14 11:12 13:10,20 14:5,22 18:15 19:15 21:16 22:3 24:1 26:10,13 27:16,19 28:21 29:14 30:19 31:1 34:8 35:5 41:11,17 42:26 48:9 48:12 49:22 56:12 60:5 94:14,15 95:26 96:16,19

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73:25 92:4 93:24 94:6 114:10,25 130:11,12 146:9 151:13,18,21 153:21 157:2 158:12 164:2,6 175:9 182:4 185:9 207:9 208:2 212:9,10 219:23 225:10 225:11 226:1,14 229:20 233:25 241:11 242:5,23 252:15 280:18 281:1 285:8 314:4 326:13 379:15 430:19 433:14 497:18 presentations 3:19 31:13 50:25 53:20,22 124:16 151:11,23 152:3,15 153:4 154:15 176:18 179:15 179:26 180:12 181:11,19,22 206:4 207:13 208:5 223:1 229:7,20 230:2 256:12 308:18 312:4 313:4 354:24 430:17 467:15 468:19 472:18 494:18 495:4,13,17 497:14 498:7 500:25 presented 86:19 103:26 115:19 130:26 164:25 166:17 168:7 171:4 172:15 174:14 177:13 181:18 184:14 219:15 255:25 259:3 291:5 294:2 303:6 356:11,23 358:20 362:12 367:8 407:4 426:22,25 430:16 460:4 461:16 466:24 468:9,12 470:26 472:9 480:14 483:20 484:10 488:13 495:26 498:1 presenter 39:21 224:20 237:10 246:4 258:15

288:14 296:2 305:12 317:19 323:7 329:26 336:23 347:17 presenters 6:24 54:17 151:18 186:7 488:4 490:19

495:13 496:1 497:26 499:11,18,20

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333:6 367:10 pre-exposure 98:12,13,19 99:20 101:12,16,18 107:14 111:26 275:20 pre-market 307:24 pre-metamorphosis 401:20 pre-pregnancy 310:4 pre-pubertal 243:23 pre-screen 194:23 pre-screened 308:4 pre-screening 184:22 194:9 196:10 pre-validation 249:23,24 367:13 price 198:21 pride 343:9 primarily 6:3,16 22:15 25:6 38:24 40:7 44:21

47:16 105:3 137:13,14 198:5 257:14 321:24 416:9 primary 23:11 26:22 40:11 54:4 55:19 69:21 76:7

85:24,24 114:21 243:6,22 244:1 401:18 principal 122:11 principle 61:26 principles 25:19 186:21 printouts 297:23 prior 3:8,13 7:6 94:25 98:14 107:14,20 149:21 183:4 184:3 244:18 281:13 290:5 308:4 309:23 322:1 348:13 351:14 352:21 388:4 423:13 priori 302:26 402:17 prioritization 11:8 16:11 18:14 23:1,5 24:5 25:11 26:14 227:26 366:25 371:20 372:1 377:3,9 494:5 prioritize 23:6 24:8 25:12,20 26:1 228:3 prioritized 33:17,21 prioritizing 25:23 226:13 pro429:19 probability492:26 probable431:8 probably 25:3 69:11 72:21 107:15 112:20 113:22 113:22 139:17 149:17 185:1 193:5 216:19 217:15 217:17 220:4 222:15 223:6 240:12 248:2 272:26 276:18 280:9,13 281:8,16 282:5 289:18 296:8,16 296:26 305:22 313:2 319:22 321:9 331:5 342:16 380:20 400:6 407:12 411:14 412:15 415:25 416:4 418:15,17 419:22 435:18 438:2 440:24 441:7 444:11 445:23 450:2 465:14 477:18 488:21,22 489:18 494:18 problem 37:1 69:2 109:26 148:23 215:6 266:6

275:24 281:21 282:1,1 283:24 388:1 392:16 398:14 403:26 406:18 407:3 413:16 415:14 422:16 424:3,14 437:2 438:15,16,20 441:9 444:16 484:26 487:20 500:5,7 problematic 249:2 266:8 347:5 371:11 399:1

408:11 413:7 problems 28:22 83:17 121:5 245:5 257:16 266:3

271:25 276:23 281:7 285:3 287:6 299:14 318:1 319:21,24,25 320:2,5,9 322:22,24 346:24 397:19 406:18,26 443:9,18,19 465:9,11 468:4 496:18 procedure 149:14 283:11,15 298:13 333:21 454:12

455:9,13 457:26 procedures 55:9 126:25 283:6 300:6 314:5 454:9

458:6 proceed 221:14 430:8 451:2 proceeding 387:2 proceeds 314:8 process 8:11,25,25 9:2,9 11:2,7,8 13:2 14:4 27:8,12

32:17 34:1,5,22 35:1 53:4 55:7 72:10 101:15 129:21 150:6 160:15 182:16 196:8 268:16 293:9 320:25 324:3 333:26 336:16,17 362:3 367:18 401:17 429:2 484:5 498:8,9 504:23 505:17,17 processes 8:22 11:22 25:20 39:8 49:16 183:7,13

200:11 427:7 499:13 produce 17:5 78:7 80:21 264:15 316:14 439:25

450:2 488:17 489:8,11 492:17 produced 18:13 60:12 62:15 66:5 99:7 254:23

264:10 produces492:15 producing 62:23 412:14 product307:17 308:23 production 64:14,16,20,22,24 65:12 66:3 206:16

206:21 207:4 238:1 239:12,17 240:4 241:16

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210:13,17,25 211:3,10,18,22,26 212:2 proficiencies 358:18 proficiency 67:16,16,18,19 72:22,25 73:3,5,9,16

73:18 246:21 247:15 343:7,14,17 345:10 356:12 356:19,22,25 357:1,13 358:3,6,8,14 360:11 374:3 374:6 391:14 476:2 490:20 proficient73:2 343:12,14 profile78:6 80:20 84:12 87:23 283:7,26 program 1:4 2:8 4:2 7:14,14 9:16 10:4 13:23 14:17 15:8,11,17 16:3,8,9,10,17 17:1,13,24 18:11,26 22:26 25:3,16 34:24 39:11 40:10 70:15 135:1 184:23 187:6 202:22 210:3 285:16 286:5 287:14 288:2,10 298:11 306:9,20 311:17 313:10,11,11 313:15,18,21 314:6,20 319:12 323:23 329:13 331:2,7,11,14,18,25 366:10 377:7 397:5 503:10 503:14,15,18 504:7,11 programs 8:5,14 16:13 53:15,18 84:26 93:26 114:12 293:12 progress49:5 133:21 195:12 259:11,15 290:9

332:5 417:24 418:6 progressed 121:3 441:11 progresses 490:7 progressing 121:8 progression 121:17 336:20 447:10 502:24 progressively 335:4 project296:23 298:14 307:26 330:6 335:18,21

336:26 projected 23:2 projects 221:8 prolonged 274:5 prominent285:22 promote 118:6 promotion 127:19 pronounce 321:18 pronouncement 301:18 pronouncing 318:13 proper 14:2 112:18 364:21 386:1 properly 10:22 60:23 111:7 203:26 219:19 262:13

262:16 313:20 318:7,19 322:14 409:17 450:9 453:19 properties 24:6 26:4 28:9 32:22 35:21 36:22 54:16

97:4,5 98:3 109:12 195:25 206:20 312:12 368:25 467:12 property 113:14 propionate 283:17 proportion 33:8,10 251:22 405:18 468:6 proportional 149:16 proportions 123:26 proposal 291:4 328:17 367:17 proposals 292:26 propose 293:2 328:7 proposed 13:6 18:4 20:15 49:3 121:20 215:8 257:9

257:13 276:20 288:25 291:7 293:21 355:2 proposing 215:7 293:24 383:26 prostate 62:8 76:10 80:9,9 324:6 355:10,15 protect 16:18 220:7 239:13 321:15 474:3 protection 1:12 10:7 17:26 113:20 237:12 241:17

310:15 323:19 368:8 protective 16:23 22:24 protein 122:17 241:5 263:21 264:7 270:15 339:2 proteins 335:9 400:25 442:12 protocol 21:3 128:21 237:22 245:5,6 281:15 282:5

282:9,10,20 319:10 326:17 330:20 360:21 363:26 364:2 371:12 372:5 376:15 408:26 428:15,17,21 431:20 433:10 451:9 465:12,14 489:22 protocols 5:12 26:20 28:25 128:23 287:17,22

302:7,7 321:24 324:14,21 325:19 330:25 376:12 376:25 proud 271:17 prove 322:15 proven413:7 provide 3:11,13 8:8 16:7,10 27:25 30:25 41:9,24

42:17,20 46:19 47:7 48:24,26 49:20 52:22 56:20 56:24 69:13 87:24 94:7 95:25 102:17 107:1 117:17 118:22 129:26 137:6 150:14 153:9 154:1 154:17 157:9 164:7 171:14 172:16 181:17 208:8 208:11,12 212:7 220:6 222:15 230:23,23 243:3 243:20 278:19 279:11 306:15 349:21 350:2 358:2 358:6 361:6 369:15 393:7 416:25 433:17 447:24 447:24 449:24 453:15 457:20 458:14 460:9 465:9 465:18 470:21 476:22 478:23 483:12 484:21 492:22 494:13,14 499:21 502:2 provided 2:25 11:1,2 16:1 17:23 31:23 60:8 91:15 123:13,20 134:9 143:7 165:12 176:11 178:13 179:25 180:4 200:18 219:23 255:21 305:17 308:20 319:3 350:7 357:17 358:16 360:6 365:20 365:21 368:14 375:7 448:20 449:6 459:6 462:5 474:13 476:21 481:7 500:10 503:17 provides 2:19,21 3:5 94:13 128:15 129:19,23 155:11 157:11 158:20 198:2 245:18 331:15,18 333:7 providing7:9 31:14 48:6 49:6 128:12 151:6 153:8 172:23 179:23 208:2 232:16 285:16 305:23 306:8 368:8 402:1 505:16

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45:24 46:12,24 47:7,13,17 55:2 74:8,8 77:11,12 77:13 78:11 79:22,22,23 80:18,24 81:2 83:15,19 84:18,23 89:3 93:8,8,17 115:19 124:15 157:16,22 158:5,13,19 161:3,12 165:3,25 166:5 167:20 168:2 170:13,15 171:21 172:8 173:3,14 174:6 175:15,18 177:1,4,8 180:14,16,26 181:13 188:1,2 189:1,5,11,11,20 190:15,25,26 191:2,2 225:13 244:23 246:10,13,26 247:8,19 248:1,9,15,24 249:20,25 250:3,14,17,21 251:2,17,20 252:10 255:3,8,19 257:19 276:18 284:6 290:21 291:7,12 292:9 293:23,25 294:21 295:18 300:13 310:9 325:12,12,16,21 328:16 329:3 348:3,4,25 349:8 349:13,19,25 350:7 352:9 353:4 354:15 384:24 385:18 390:23 394:4,5 395:11,11 397:15 427:13 427:15 456:11 459:16,21 460:2,8 461:6,17 464:1 464:6,23 465:3,4,19 481:22 484:12 485:3 pubertals 20:9 47:15 83:2 84:13 87:19 276:11

289:2,4 295:11 324:20 325:8 326:16 puberty 244:18 250:10 295:2 385:19 461:23 public2:16 3:6,7,8,9,11 5:24 8:24 9:8,12 10:25 16:18,24 27:12 33:26 34:5,17 92:3,10 134:3 139:3 149:13 151:18,20 207:16,23,25 208:1,6,8,9 208:11,15,23 214:4 219:16,17 220:7 257:1 271:2 305:9,12 354:24 356:4 368:11 377:3 379:15 380:8 403:3 407:4 430:20 449:6 475:4 488:4 490:15 491:19 494:17 495:3,8,13,26 497:3,13,26 498:22 499:11 500:10 501:5 503:17,20 505:15,16 505:19 publically454:11 publication 234:21 281:13 333:23 publications 238:12 publicly 318:17 published 24:23 135:22 140:20,22 141:2,16 244:10

264:4 290:15 333:23 353:20 404:8 435:3 436:16 437:8,15 438:3 440:9 452:1 469:26 pull 9:24 150:18 202:4 235:19 267:17 304:2

309:14 pulled 230:17 370:12 474:21 pulling 15:10 280:21 pulsatile 295:24 pun264:14 420:14 punctuates 30:22 pups 326:21 purchase 341:19,21 purchasing432:13 purely 195:9 236:11 313:13 purpose 39:26 56:11 57:11 74:2 77:12 79:22 115:2 187:4 198:11 315:3 320:4 322:16 365:23 395:24 402:23 purposefully 263:12 purposely 51:8,19 269:3 purposes 62:25 68:12 124:9 166:8 170:8 307:19

308:1 323:5 361:8 402:19 pursuing 292:11 490:22 pursuit460:23,24 purview 38:14 push 21:20 261:3 286:25 329:14 339:14 pushing236:14 273:21 417:6 put 8:4 9:5,11,19,22 16:22 19:4 22:4,25 23:7 24:23

32:3 33:26 34:5,21 108:2 149:22 202:16 221:2 225:14,16 251:11 261:12 269:18 271:10,16 274:2 274:13 278:2 284:13 287:14 307:26 314:26 338:15 339:17 343:15 367:15 378:1 385:16 397:11 416:21 422:8 423:8 425:3 445:15 480:4 488:16 494:26 499:23 500:21 puts 333:11 putting 113:3 203:10 220:16 266:11 338:11 372:22

414:11 431:24 488:4 490:5 501:8 503:5 504:7 puzzle 19:4 puzzled 32:11 pyrethroids 28:15 35:17,18 p.m 91:25 Q QAQC 112:20 QA/QC 443:26 quad 115:23 qualified 296:14 qualitative 35:5 139:12 143:10,21 425:26 qualitatively 34:7 103:4 140:10 143:24 quality 10:6 71:4 73:17 100:18,22 115:4 117:11

118:12 129:22 217:25 218:4,10,23 264:7 281:12 318:16 322:15 432:8,9,13,15,18 435:11,11 437:2 437:6,21 438:22,25 440:7 441:9 442:5 443:3 444:3,4,9 445:12,22 475:13 quantify 18:23 369:23 374:22 quantitative 34:6 68:19 69:3 143:10 236:10,13

359:9 373:26 407:26 408:1 449:4 453:16 454:6 454:10 456:17 quantitatively 359:13 quantity 121:21 122:8 123:5 quasi 315:20 query 334:25

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492:19 questioning 196:19 questions 31:25 32:1 36:15 39:10 49:24,26 50:13

53:7 68:14 71:19 72:17 73:19 82:3 86:14 87:10 88:20 91:22 92:18 93:21 107:5,6 114:8,14 115:10 130:13,14,17 131:8,21 132:18 137:8,8 145:7 149:17 151:13 156:21 182:18,19 184:20 187:23 193:12 194:20 195:6 197:23 201:21 204:12 207:5 208:10,25 212:6,6,9,10,14 217:23 219:11,12 220:4 223:6 226:4 229:12,16 237:6,7 242:2,23 246:3 252:14 258:3,14,20 260:14 263:6 270:19 277:21,25 278:15 280:17 282:14 284:3 285:5 288:14 289:26 293:15,16 296:1,14 298:5,13,18 298:25 299:9 300:8 301:26 302:1 315:15 317:19 321:2,3 323:3 329:26 336:22 344:2 345:6,7 347:8 352:9 354:20 360:25 363:10 368:5 375:22 379:26 380:13 382:10 390:7 391:22 393:26 403:2,2 407:5 413:2 422:4 423:7 445:26 447:23 450:10 453:20 454:25,26 462:20 463:17 466:12,13 469:22 476:10 477:12 480:21 481:20 485:5 487:2 488:5,12 491:26 494:8 495:5 quick 21:7 25:10,25 207:19,23,23 210:6 213:6

229:26 231:10 237:17 238:17 248:8 304:23 326:19 336:22 354:26 396:12,15 471:21 482:20 quickly 13:22 25:7 27:21 74:6 256:13 298:2 320:20 330:13 333:20 334:15 347:25 354:8 414:24 463:26 494:15 quite 85:14 105:10 106:7 109:9,16 119:15 145:4

222:9 225:10 237:23,26 238:4,24 239:20 240:11 258:22 274:11,23 325:9 414:22 441:2,11 451:25 455:6 quote 30:21 quotes431:25 quoting 318:26 320:15 Q)SAR24:6 382:19 R rabbit261:20 race 261:20 radioactive 337:15 373:4 radioactivity 57:19 radiolabeled 58:17,19 62:12 65:26 radiolabled 57:18,22 rainbow 371:22 377:6,6 382:18 raise 145:7 265:4,9 361:24 372:18 381:12 414:25

435:18 raised 205:26 265:12 361:19 390:5,8 437:26

447:26 raises 298:18 300:6 raising 357:2 435:22,25 Raleigh408:16 ran 92:22 333:22 Rana261:8,8 264:23,26 265:1,18 random 340:26 randomized 98:16 randomizing 83:14 randomly 386:14 range 18:3 20:22 27:26 28:8,10,12 32:19,21,24

36:22 37:14 39:3 41:5 42:2 49:20 54:15 58:9 62:20 67:12 78:24 81:11 83:18 85:14 87:25 92:22 92:26 96:11 97:7 99:9 104:21 105:15 109:16,21 113:26 179:11,12 186:12 206:7,26 232:20 236:21 247:3 274:13 278:3 284:8,9 286:1 309:11 340:24 372:9,24 388:26 393:4 395:14 396:19 403:14 412:2 424:4,5 425:17,22 438:3 458:18 471:11 ranged471:9 475:7 ranges 59:8 60:1,24 61:10,19 67:1 68:25 345:24

349:16 359:14 372:10,12 range-finder 354:4 406:15,21,24 range-finders 274:6 406:12 range-finding 243:15 245:7 277:26 324:2 325:1,5

325:13 326:1 330:18 348:6,17,23 350:1 400:8 406:1 410:12 range-finger 352:3 range-of-finding 387:16 ranging 98:1 495:14

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249:13 268:4 360:18 415:23 417:23,25 418:4 419:7 430:14 440:23 441:10,20 444:16 rates 110:19,23,25 263:21,22 272:14 361:23

407:18 418:8 443:13 rating 236:12 ratings 236:11 ratio 102:2,3 rationale 87:24 107:9,11,24 140:20 144:12,13

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356:9 360:5 366:6 385:14 395:7 408:3 423:23 424:19 426:20 432:17 434:2 439:1 441:14 450:21 457:23 458:11 466:15 474:2 475:1 486:17 492:5 readdressed 371:6 readers438:4 readily 336:17 reading 69:2 324:3 325:3 370:1 438:1 455:13 ready 11:26 39:19 92:2,11 149:10 156:18 205:23

364:26 446:11 480:23 506:4 read-across 35:3 37:8 real 10:1 21:15 46:16 111:23 149:3,5 197:16 210:6

245:5 283:23 300:24,26 354:25 405:25 409:3 430:20 463:26 496:14 realize 223:26 262:20 271:4 390:5 433:20 486:5

494:18 499:11 realized 101:23 305:23 368:2 realizes 368:16 369:6 426:16 realizing 14:6 201:11 really 8:25 12:1 16:7 17:23,24 18:21,24 20:8 21:8

21:13 24:10,14 28:8 30:10 32:26 35:10 36:5 40:9 43:13,15 47:6,7 48:8 52:22 68:22 72:7,21 88:3 89:2 118:21 119:10 123:19 131:25 136:9 138:3 139:26 140:25 144:21 147:22 148:23 153:1 195:12,13 204:10 205:12 207:23 217:8 218:3 223:11 225:6 226:15 228:20 232:14 234:24 236:13 238:12 241:11,20 253:17 257:23 258:25 260:26 264:3 265:10 266:14 267:20 270:10 271:22 273:21,25 274:25 275:5,8,23,24 276:25 277:18 281:3,11,14,19 282:9,20 283:2 284:18 286:7,15,17 287:7,10,13 288:6,11 291:1 295:15 295:24 296:7,14 300:2 301:4,13 302:16 309:4 317:1 322:25 325:3 329:13 330:23 331:24 334:22 341:14 342:3 344:21 347:25 348:19 349:15,26 350:26 351:24 352:3,19 353:21 354:6 364:6,9 378:21 386:8,21 387:9 388:21 389:3 390:1 391:21 394:23 396:22 398:1,2,7,10 400:12,14 401:7,8 405:17,21 407:5,6,22,26 409:2,4 411:24 412:17 413:6 414:23 416:9,18 417:3 419:15 435:23 439:2,22 442:3 443:1,6 444:1 450:21 451:18 456:11 461:1 465:4,23,25 475:1,20,26 476:5 480:20 486:10 487:18 493:17 495:15,17 496:2,3,5,12,13,23,25,26 497:8,26 498:3,13 499:15,21,21,23 500:2,5,10,12 503:12,18 504:12 504:21,23 505:13 realm 143:23 463:12 real-world 24:13 27:11 reanalysis 492:21 reared435:15 rearing437:18,26 reason 22:16 38:25 72:1 193:24 202:26 203:4

222:1 227:11 236:5 238:25 294:25 330:21 341:24 375:14 378:2 402:18 428:25 435:21 442:23 500:17 reasonable 14:15 37:21,21 57:15 200:20,22 413:13

413:20 415:20 416:5 440:24 reasonably 37:11,12,24 reasoning463:16 reasons 14:6 28:8 32:18 145:6 188:19 199:24

297:12 488:8 489:17

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454:1 494:5 schemes 411:9 454:3 Schlenk 3:22,25 4:3 6:22,26 8:12 15:16 31:26 32:6

33:5 35:7 36:13,24 38:3,10 39:19,24 49:26 50:13

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138:9,13 184:20,21 194:7,14 195:5 211:2,2 215:23 242:3,4 287:15,16 302:3,5 365:16,17 378:5,11 380:21 381:10,23 382:5,10,25 410:16 410:17 426:14,15 434:3,23 438:7 441:14 451:7 454:19,20 456:24 474:7,8 476:17,25,26 480:6 490:24,25 494:1,2 497:5 Schultz's 380:3 451:22 science 6:25 7:8,15,17 8:23 9:1 11:5 14:6,8,23 15:19 18:8 25:1 27:15,20 39:23 40:12 49:3 220:5 231:1 335:1 338:14,17 343:6 376:13 428:15 435:23 490:9 499:2 504:12 sciences 4:3 355:12 375:14 science-based 313:18 scientific 1:1,4 2:6,18,19 3:23 4:1 7:10,13 8:3,4,22 9:12 10:22 11:5 16:16 17:20 20:13 25:19 27:13 31:10 37:7 69:18 70:8 129:15 167:6 210:2 239:20 271:9 296:7 312:11 313:26 319:6 354:9 367:10 429:10 466:5 492:7 493:13 502:14 503:1 scientifically 17:3 23:16 54:20 162:7 182:11 192:16 202:14 206:2 233:6 243:10 248:17 491:24 502:19 scientist 301:22 303:21 scientists 9:10 55:20,21 222:7 306:2 scientist-oriented 313:3 scoliosis 121:14 215:25 263:21 scope445:16 score 260:11,12,13 scored 88:17 480:15,15 Scott5:1,1 33:5,6 34:7 35:7,8 68:16,17,26 70:7

87:11,12,12,21 88:2,5 90:4,5,11,23 91:5 109:18 109:19 110:10,14,21 111:8,12 112:5,19 113:2 139:13,14,21 140:15 148:21,22 204:4,5 205:7,25 210:20,20 270:6,7 277:22,23 278:5,11 303:4,5,20 317:20,22,22 321:17,21 322:5 345:8,9,13,19 362:26 363:1,23,24 379:13,14 404:12,13 412:22 412:23 414:8 416:13,14 420:2,3 441:26 442:1 445:6 446:3,6 457:4,5,9 463:24,25 470:24,25 475:18,19 482:6,7 490:13,14 495:12 Scott's 417:12 445:12 screen 17:10,14 18:14 21:12,14,15,21,25 22:20

26:14,17 35:4 40:17 46:13 49:12 52:17 74:13 75:25 143:20 146:16 153:23 157:20 163:23 164:1 176:11 186:13 198:12,15,16,19 199:11 226:22 227:8 233:5 274:26 277:19 312:16 314:22 354:12 428:10 440:12,14 452:4 screened 33:8 40:20 226:19,19 316:5 368:18,20 screening 1:4,5 2:8,8 4:1,2 5:12 7:14 10:26 16:8,12 16:18 17:1,22 18:11 19:1 21:9,26 23:1 25:20 27:6 28:4 29:13 31:15 33:22 40:10,25 41:10 48:16 49:16 73:26 84:7 87:4 89:20,22 94:12 124:10 134:23 135:5 139:23 155:26 156:3,6 187:5 198:2 202:22 210:3 213:24 225:2,20 235:5 254:25 255:16 291:19 299:20 306:9,16,19 311:25 312:1 313:6,25 315:12 318:5,6 319:12 336:1 337:6 366:10,10,16,24 367:20 377:17,20 382:11 384:15 385:6 392:24 393:3 395:24 396:2 398:3 399:3,11 422:12 427:2,13 444:6,7 446:4,4 483:2 484:23 492:14 496:10 504:8 screenings 226:6 322:12 screens 21:10 146:15 185:6 230:13 232:17 307:5,7

315:10 452:8 scrolling 370:10 scrupulous439:26 SDWA 39:3

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se 294:4 426:24 seat482:25 seated 2:2,4 3:10 184:8 second 2:3 7:17 11:9 23:8 24:9,9 26:22 29:4 30:16

54:7 56:5 67:21 78:7 80:21 83:8 87:13 91:8 94:7 98:11 102:12 131:13 161:11 164:8 191:9 198:3 238:22 242:4 248:11 264:20 294:13 310:1 318:3 320:11 332:15 344:6 351:16 361:9 362:10 363:5 367:20 443:8 475:20 489:14 495:7 secondary 23:11 45:15 55:20 95:18 104:23 107:16 107:20 168:24 176:1,13 243:7 409:25 section 17:12 69:3 152:2 156:16,17 250:23 356:11

356:23 357:17 379:15 sections 150:19 248:1 secure49:18 see 3:19 8:16 12:8 13:16,17 18:2 19:19,26 20:1,22

21:18 34:17 35:6 37:18 39:14 49:9 52:24 62:16 63:4 83:17 91:6 93:1 105:26 107:24 117:7 119:14 121:18 122:15,24,25 123:16,17,22 127:8,12 128:25 129:2 132:19 133:24 134:24 136:4 137:19 138:2 140:5,12 141:10,12,17,19,24 144:24 145:25,26 146:12,20 147:24 149:19 150:15 160:3 160:7,26 162:19,24 184:11 188:4 191:4 200:18 206:3 208:3 209:1 216:9,13,21,23 217:3,5 218:13 219:3 221:10 223:3 224:8,23 229:14 234:2,10 247:1,7,12 249:7,8 250:11,15,25 251:3,13,22 259:8,9,21 260:20 261:3 262:6 263:21,22 264:5 266:21 267:23 268:20,20,21,24 269:2 271:26 275:23 276:5 277:8 281:11,13 285:1 286:23 290:21 291:2 294:17 301:14 302:11,19 311:13,16 313:15 317:8 324:14 325:19 328:14 334:1 335:11 337:17,19,22,23 340:4,6,14,26 342:4,26 343:2,3 343:4 344:21,24 346:4,11 353:21 354:4 372:22 383:15 386:21 387:8 389:8 399:14 400:2,22 404:20,24 405:1 408:10 409:9,21 414:26 415:2 416:20 432:2 436:23 437:5,20 443:4,6 444:20 452:1,2 466:3,6 471:7 473:17,25 474:8 475:10 479:15 485:18 486:10 495:9 496:12 500:2,26 SEEGER466:13 seeing 21:20 23:22 24:1 70:21 90:8 91:8 131:26 137:23 152:18 185:25 186:2 201:9,10 202:1 203:6 212:25 213:13 251:7 265:11,15 268:25 275:16 286:24 322:23 329:15 387:23 388:3 394:20 398:2,25 474:23 496:21,25 seek 124:5 213:25 seeks 125:20 215:21 seen41:23 42:4,6 47:21 48:1 56:12,13 61:26 71:18

74:5 125:13 157:6 160:21 161:22,24 162:4,9,22 176:14 177:17 249:16 250:18 276:13 285:23 329:18 355:19,20,24 417:14 436:10 440:3 443:9 467:18 484:10 segue452:11 select 146:24 192:10 246:16 348:6,10 429:22

443:22 493:24 selected 28:7,17 36:17 52:14 54:11 56:23 75:7 76:5

78:4 80:18 92:25 93:5 124:18,22 145:22 155:23 179:10 182:26 185:9 186:21 187:17 206:23 349:3 349:22 351:26 355:25 368:24 369:1 376:24 383:8 432:6,11 selecting 93:20 268:6 311:24 312:15 429:25 selection 32:17 36:18 78:4 80:17,20 87:13,24,24

92:21 102:13 106:17 146:7 180:2 195:1 243:15 311:21 350:4 396:2 405:26 410:9 463:18 485:4 selections 352:2 410:10 selective 7:23 selectivity 481:23 selenium432:23 sell 303:15 Selvage 5:5,5 82:5,6,6,12,18 210:24,24 352:6,8,16

352:18,25 353:3,23 379:6,7 384:10,11 385:11,12 394:2,17,18 396:8,9 399:22 413:22,24 416:12 422:25,26 424:25 464:17,18,23 472:23,24 482:8 482:9 485:6,7 486:25 493:6,7 497:2 semantics 90:16 semi 143:10 seminal 76:10 80:10 semi-quantitative 454:1 semi-terrestrial 264:24 send72:9 sending 320:26 Senior231:1 237:12 305:18 sense 16:15 25:7 37:8 116:15 139:12 143:5 184:8

220:24 228:22 229:2 242:19 281:8 354:3 408:26 440:11 468:12 473:11 475:15 sensitive 18:1 21:5 22:18 78:10 80:24 116:22,26 160:18 215:24 216:2 249:6 251:21 255:24 269:1 275:7 291:8 303:16 316:18,19 317:7,12 404:17 404:18 410:22 455:19 463:4 sensitivity 21:5 29:7 41:6 42:16,18 48:24 74:14

75:5 76:1 124:25 196:18 198:15 223:22,24 257:26 277:7,15 287:12 293:19,25 294:3 453:3 sent71:26 473:8 474:18 sentence 214:3 sentinel 227:1 SEP283:18 separate 119:17 191:1 367:26 separated 239:6 separately 117:10 132:13 158:10 366:3 458:24

466:20 separation 80:3,6,9 81:6,14 274:8 461:7

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357:9 367:20 470:3 serious 319:20 330:24 serum 157:24,25 400:25 401:3,6 461:8 serve 2:12,16 184:1 198:11,14,18 296:6 484:23

504:22 served 153:23 299:19 371:6 504:26 serves 61:2 service 21:12 343:24 session 54:1 133:17 199:25 224:7 225:25 227:19

377:23 set 11:11 22:14 28:6 38:13 39:17 44:24 49:3 55:9

67:15 71:2 150:5 155:14 182:26 185:5,7 186:22 193:21 230:12 253:10 274:1,17,17 315:26 348:26 363:21 390:20 392:22 393:8 399:10 406:1 414:5 430:6 436:23 443:16 484:16 485:1 492:13 494:21 sets 55:24 139:7 setting 33:19 34:10 254:10,10 273:14 276:22

395:13 399:16 406:2 413:14 418:20 501:12 settled 235:1 setup 271:18 274:20 275:4 seven76:26,26 81:21,22 103:24 104:5 138:2 151:1 151:1 154:24 166:12 168:10,20,23 169:3,5,7 172:25 174:10,13 175:25 176:6 177:10,17,21 252:26 255:4 257:20 284:8 437:19,22 460:3 462:7 seventeen 62:3 79:9 99:8 155:26 164:17 severe 141:8 213:5 259:6 266:14 severities 259:17 265:6 severity 125:2,8,23 133:6 141:7 142:10,13 143:10

259:10,22 262:4,11 395:22 405:9,11,12 410:18 413:10 446:19,25,26 447:13 449:2,11 455:26 sex45:15 76:23 95:17,18,23 101:14 102:2 104:3,23 105:8 106:25 107:4,10,16,20 108:7,15 163:10 168:24 169:1 170:17 176:1,13 189:3,5 244:24 275:9 409:25 459:23 469:4 479:13 sexed 101:9,22,26 102:4,8,9,11 106:24 107:10 108:16 sexes 161:19 sexual 175:12 461:7 sexually 351:14 488:21 sex-specific462:3 shades 59:13 65:4 shallow429:5 shame 301:3 shape 125:4 386:2 share 197:1 220:20 222:8 499:9 shared 297:9 449:7 Sharlene 505:22 sharp 339:10,18 SHERILL 83:4 Sherril 6:18,18 52:6 82:24 83:8 132:17,23 133:7,9

133:19,23 134:2,14,17 135:10 136:26 147:2 148:16,20 182:24 183:20 199:16 212:1,1 217:24 218:17 258:5 263:8,24 264:19 265:17,23 285:7 285:17 303:25 304:10,21 321:5,20 322:4 353:25 354:10,19 363:5 417:19 418:23 422:19 434:24 445:11 450:15 454:15 457:1 488:1 500:9 shift 125:7 337:19 shifting 357:7,8 Shirley 505:26 short70:22 71:8 93:6 115:21 181:13 243:16

306:25 385:1 391:23 392:1 396:10 419:17 424:5 467:25 481:2 503:7 shortcomings 385:3 shorter21:10 258:7 shorter-term 367:3 shortly 3:20 17:19 short-term42:14 53:25 55:5 94:2 225:13 360:13

395:12 396:6 shot 193:6,10 200:5 should've 278:6 383:7 show58:2 59:7,8 66:20,21 76:26 128:3 146:9 159:10 160:19 174:12 175:16 177:2 178:1 181:15 191:6 214:5 215:4 245:19 266:2 304:24 308:17 334:20 340:3 342:14 343:13 424:13 426:17 429:17 433:26,26 440:9 462:7 468:13 478:8 481:16 showed 34:21 62:22 65:11 73:6 76:25 79:9 81:17

81:21 93:9 113:26 131:3,17,23 148:1 161:1 175:19,21 176:6,9,12 177:5,9,21,25 224:15,18 233:8 254:16 255:4 267:2 273:24 298:16 304:7 328:4 460:2,14 462:8 467:16,24 469:1,2,3,4 481:9 484:17 488:14 showing 67:5 86:19 146:9 159:23 192:22 251:8

297:1 299:16 391:6 438:19 460:3 shown58:22 62:15,24 63:10,13 64:25 65:25 66:14

66:19 73:1 94:4 95:8 97:9 106:7 129:24 166:15 170:1 173:7 174:7 175:16 176:8 213:19 238:11 296:25 432:7 455:15,19 467:7,15 shows 53:21 54:23 56:19 59:4,16,19 61:11 63:2,14

63:19 64:11,17,19 65:2,9 66:23 103:12 105:12 116:12 121:19 125:18 127:9 166:16 171:1 172:25 175:11,24 176:26 187:26 189:12 216:3 233:15 399:4 455:22 shrimp 214:15 443:18 Shultz 194:6 451:8 456:23

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270:2 392:21 429:5 443:26 sides 186:13 495:14 sign411:2 415:14 signal44:6 342:17 346:3,22 400:13 signaling 28:22 40:8 154:21,23,26 173:10 179:24 181:6 375:9 395:15 458:15 significance 158:21 216:14 498:3 significant29:16 64:21 75:3 103:6 109:13 127:6 127:21 150:11 157:23 159:4 160:1 162:9 166:11 167:9 174:1,4,9 179:19 214:13 216:13,25 217:3,9 234:10 255:9,17 285:20 317:10 350:17 353:21 354:8 355:5 362:6 385:26 391:9 401:25 404:20 405:19 408:7 417:3 467:14 468:5 significantly 108:16,25 257:19,21 398:24 408:5

438:20 signs 56:10 74:25 75:19 76:13 77:2 78:1 79:8 80:13

81:20 82:7 93:12 103:2,6 106:14 117:5 259:8 409:12,24 428:7 429:24 silence451:1 silencing450:25 silent451:26 silico 226:6 371:21 384:15 similar 21:1 22:6 38:12 39:2 42:6 44:24 62:6,11

80:17 83:11 125:18 146:2 172:14,15 212:25 213:17 226:21 257:8,15 264:25 271:18 280:11 291:5 296:26 315:23 335:22 358:23 433:26 443:18 447:1 449:21,21 similarities 42:22 similarly 71:16 81:2 165:6 simple 144:2,4 206:15 297:22 simply 149:21 159:23 215:4 302:26 320:4 366:7

429:22 single 35:4,4 89:6 119:17 124:2 160:16 173:21 175:20 190:8,9,15,17 214:12 240:15,20 249:4 334:7 369:15 461:8,17 487:1 single-generation 21:4 sit270:24 438:26 site 1:8 434:14 sites 40:15 442:18 sitting499:4 situation71:6 151:6 368:21 380:25 392:17 411:5

443:1 460:21 463:8 474:20 500:1 situations 71:7 180:20 369:3 375:1 435:16 453:8 six 19:2 59:1 65:10 74:21 79:6 81:21 96:3,20,22

97:10,11,14,18,21 100:3 101:1 102:1,1 126:13,16 137:4 147:13,18 154:19 164:13 165:10 170:22 245:9 257:20 273:6 339:1 378:2 395:10 464:8,8 468:13 472:4 Sixteen 156:4 size49:13 109:23 151:5,5 214:8 245:9 265:15

269:13 346:19 403:1,14,16,25 405:12 415:25 420:10 sizes 244:11 424:5,6 skeletal 136:10 433:24 skew 120:24 skewed 109:5,6 skip 122:25 370:1 422:3 487:15 491:22 493:19 Slaughter 317:20,22,23 321:17,21 322:5 slices 335:9 slide 22:26 31:12 32:4 43:8 50:3 53:21 54:4 55:7

56:15,19 58:2,13 59:4,16,19 60:8 62:15,24 63:2 64:17 65:2,25 66:23 86:19 97:13 101:25 102:21 102:25 103:26 105:6,12 118:22 121:19 125:17 127:18 135:11 139:26 140:1 147:3 157:6 164:15 166:20 168:7 169:9,24 172:25 174:12,22 175:11 175:22 177:17 180:11 196:13 216:3 229:9 230:1 233:11 248:8 296:25 298:19 331:14 452:5 slides 3:18 42:6 74:6 142:17,19 160:19 174:7 233:1

271:16 297:21 298:20 304:7 488:13 slight 68:18 70:7 90:16 96:14 127:19 133:12 357:4

359:3,5,11 371:5 373:23 374:1 376:3 slightly 59:26 61:9 66:26 67:12 68:25 69:8 147:22 147:25 169:22 250:7 253:9 258:9 262:21 373:22 422:10 436:10 472:9 slop 340:6,7,24 slope 63:11 337:23 403:9,10 424:1,4,6 slops403:14 slow204:6 502:24 slowly455:3,8 small 140:7 228:1,3 244:11 245:10 280:8 337:1,20

337:24 341:5 343:21 344:18 347:3 375:11 376:5 450:22 452:2 smaller 33:11 265:13 400:7 smart275:5 smooth 503:14 snapshot21:7 25:25 261:26 snapshots 262:1 snout417:21 418:3,9,9 428:4 434:15 441:15 snout-vent 117:9,10 120:22 127:19 131:2 149:13 149:20 150:4 151:4 216:10,16,20 217:2,14 298:8 298:15 304:24 403:5 404:1 snout/tail 139:7 Society 323:17 330:1,4 458:2 softer431:22 439:15 softness 439:13 software 133:13 282:19 451:9

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97:4 102:19,23 109:12 111:2 147:22 152:26 232:3 237:18 239:15,17,19 240:10 241:19,21,24 242:9,17,20 273:17 340:11,14 357:6 362:11 382:13 soluble 35:16 63:23 128:18 167:3 344:7 solution 98:6 111:1,3 112:17 340:10 solutions 112:26 410:7 solve 257:16 solved 320:10 solvent95:4,5 98:8,24,25 100:13 110:26 116:5,6,8 120:10 126:3,9,9,11,12,12,14,15,17,21,22 127:2 127:2,8,20,23,24 144:14,15,21,25 145:5,9 160:8 161:24,25 162:2 167:13 174:26 240:9 241:21 255:10,12 272:20,22,24 277:11 329:2 363:15 364:20,22 446:8 462:8 solvents 126:4,6 272:18 344:7 solves 320:2 solving407:3 somatic 95:19 somebody 37:14 185:1 292:11 320:1 327:23

370:15 404:7 435:24 436:20 445:15 something's 69:8 somewhat44:18 136:6 142:4 143:21 186:25 252:8

266:8 278:18 303:15 426:26 475:12 488:16 500:12 soon 284:24 338:2 376:19,20 428:22 429:4 sooner 318:8 320:23 SOP232:13,14 sophisticated 245:4 451:16 sorry 91:23 170:19 193:14 215:26 347:9,13 390:6

415:12 422:26 sort9:21 10:19 12:16,25 13:1,12 34:20,21 38:11

39:15 92:12 109:1 110:4,7,25 111:6,26 112:1 113:13,17 131:24 132:1 133:21 140:24 142:19 146:8,18 149:8 150:3,6 186:2 189:23 192:21 200:4 201:6,10,12 204:26 205:14,23 212:5 216:20 240:10 258:24 259:17 261:2,6 262:16 265:2 266:11 268:17,23,26 270:14,15 273:24 277:5,7 287:12 296:9 297:3 301:10,16 312:6 315:23 317:11 328:23 330:26 332:17 333:16,25 337:11,26 344:1 363:21 367:13 368:21 373:16 379:21 386:1 400:3 407:2 411:17 414:9 416:3 422:2 423:14 431:8 459:8 464:12,15 466:6 469:10,13 471:17 sorting 101:11 415:1 471:19 sorts 225:26 272:15 315:22 sought25:18 182:1 sound7:10 10:22 70:8 183:14 243:10 307:5 330:22 459:3 soundness 129:15 502:15 503:1 sounds 196:12,13 452:10 476:13,16 source 57:3 62:9 66:12 109:26 111:16 112:9

360:20 361:26 362:7 393:15,16 400:24 435:8 sources 17:15 383:3 400:24 SOUTH 1:14,15 so-call 327:22 space 54:12 155:22 192:3,6 463:19 477:25 478:3

478:20 479:13,13,13,14,14,25 491:13 spaced78:7 80:21 spaces 166:21 space-wise 140:1 span 155:3 spawn98:23 432:9 spawning98:14 273:10 412:13,14 spawns 214:13 speak43:19 46:18 82:22 136:1 137:21 142:26 150:20 189:17 192:5 194:1 208:17,19 219:18 234:20 237:14 322:6 503:26 speaker 219:18,20 228:22 229:22 230:20 271:3 speakers 224:11 229:24 270:24 311:16 324:8 speaking 3:7 31:10 214:4 224:10 237:13 309:16

403:8 446:7 465:6 488:11 speaks 308:18 special 133:1 501:6 504:18,25 505:4,6 specialize 5:7 210:26 211:15 Specially 375:18 species 22:19 94:5,22 115:22,24 212:24,25 213:15

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402:3 466:4 478:17

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276:12 459:24 Steve4:11 8:13 190:23 195:5 200:5 202:26 210:10

230:20,21 231:1 338:4 STEVEN 8:15 33:25 35:13 37:4 38:8 478:25 Steve's 347:1 stick 269:5 302:1 sticks 219:5 stimulate 389:14 394:12 stimulating 76:16 157:21 389:3 stimulation 345:24 450:1 stock 111:3 112:22,26 360:26 361:8 435:8 436:15

437:24 stocks 112:15 stomach 352:13,22 stop 16:19 225:15 287:13 486:19 stopped 275:21 317:16 429:7 storage 338:22 379:20 490:17 store 338:24,26 story 159:17 straight 199:17 330:13 422:4 straightforward 125:6 strain 214:9 260:20,21,21,22 strains 82:25 260:19 419:8 443:22 strange 203:7 strategically 24:8 26:1 strategies 333:1 strategy 32:12 225:2 244:6 245:23 333:1 334:20 Strauss457:26 458:3 stream429:4 streamline 199:23 302:21 422:2 451:18 strength46:16 78:9 257:11 409:4 strengthen460:12 468:24 strengths 80:23 255:22 256:4 275:26 stress 8:19 82:10,13 186:9 239:14 252:4 312:24

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226:17 313:7,9 314:10 315:6,22 348:9 397:1 427:3 substantial 17:16 29:3 297:5 substitution477:1 substrain 390:25 substrate 60:12 65:26 subtle 22:9 subtract227:20 sub-chronic 309:11 sub-legal 112:6 sub-lethal 395:17 408:8 sub-q 83:23 sub-sampling 262:21 268:14,26 270:4 sub-tiers 493:3 success 95:15 99:5,5 168:24 176:1 371:14 successes 71:13 222:10 successful 8:9 118:7 205:5 209:1

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444:26 Syngenic422:20 Syngenta237:12 321:21 322:5 422:20 synonyms456:25 syntheses43:13 synthesis 43:12 46:11 276:12 449:19 synthesize 64:8

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