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### FIFRA SCIENTIFIC ADVISORY PANEL (SAP)

OPEN MEETING

SCIENTIFIC REVIEW OF ISSUES ASSOCIATED WITH THE PROPOSED ENDOCRINE DISRUPTOR SCREENING PROGRAM (EDSP) TIER **2** ECOTOXICITY TESTS

DOCKET NUMBER: EPA-HQ-OPP-2013-0182

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

JUNE25-28 , 2013

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1 **DR. SHARLENE MATTEN:**Good morning. My name 

### 2 is Sharlene Matten. I am the designated federal official for 

### 3 this FIFRA Scientific Advisory Panel Meeting. It will be a 

### 4 four-day meeting to consider and review a set of scientific 

### 5 issues associated with validation of the Endocrine Disruptors 

### 6 Screening Program Tier 2 Ecotoxicity test. 

### 7 As the DFO for this meeting, I am the liaison 

### 8 between the Panel and Agency and am responsible for ensuring the 

### 9 provisions of the Federal Advisory Committee Act are met. I 

### 10 want to thank Dr. Martha Sandy to my left for serving as the 

### 11 Session Chair for this SAP meeting. I also want to thank all of 

### 12 the members of the Panel who have traveled near and far to 

### 13 participate in this very important meeting. And I want to thank 

### 14 the public for attending this meeting and offering public 

### 15 comment. I would also like to thank the entire EPA team from 

### 16 all of its locations for their hard work in preparing for this 

### 17 FIFRA SAP meeting. 

### 18 The FIFRA Scientific Advisory Panel is a 

### 19 legally mandated Federal Advisory Committee. It provides 

### 20 independent scientific peer review and advice to the agency on 

### 21 pesticides and pesticide related issues regarding the impact of 

### 22 proposed regulatory actions on human health and the environment. 

### 23 24 Only the EPA makes policy and regulatory 

### 25 decisions. The FIFRA SAP's peer review of the science enhances 

### 26 the scientific integrity, credibility and transparency of the

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1 Agency's policy and regulatory decisions. 

### 2 As a designated federal official for this 

### 3 meeting, a critical responsibility is to work with appropriate 

### 4 agency officials to ensure that all appropriate ethic 

### 5 regulations are satisfied. In that capacity, Panel members are 

### 6 briefed, take ethics training on the provisions of the federal 

### 7 conflict of interest laws and other ethics regulations. In 

### 8 addition, each participant has filed a government confidential 

### 9 financial disclosure report. 

### 10 I, along with our Deputy Ethics Officer for 

### 11 the Office of Science Coordination and Policy and in 

### 12 consultation with the Office of General Council, Ethics Advisor 

### 13 have reviewed these reports to ensure all ethics requirements 

### 14 are met. 

### 15 Members of the public are offered an 

### 16 opportunity to make oral comments during this meeting. Please 

### 17 limit your comments to five to ten minutes, unless prior 

### 18 arrangements have been made. 

### 19 At this time, I have five public commenters in 

### 20 total, each representing the Endocrine Policy Forum. Each 

### 21 presenter is scheduled to speak for approximately 15 minutes. 

### 22 For those who have not preregistered and would like to make 

### 23 public comments, please contact me or one my colleagues on the 

### 24 FIFRA Scientific Advisory Panel staff. 

### 25 The charge questions and all background 

### 26 documents related to this meeting are available by going to the

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1 public e-docket located at http://regulations.gov and entering 

### 2 the docket number for this meeting; EPA-HQ-OPP-2013-0182. That 

### 3 number is found on the top of your roster and agenda. 

### 4 EPA's presentations and any public comments 

### 5 made during this meeting will be available in the public 

### 6 e-docket within approximately three to five business days 

### 7 following the conclusion of the meeting. After the meeting, the 

### 8 Scientific Advisory Panel will prepare a written report of their 

### 9 responses to the Agency's charge questions. Only those comments 

### 10 or specific issues made during the meeting may be included in 

### 11 the report as part of the open and transparent process 

### 12 requirement under the Federal Advisory Committee Act. 

### 13 The report serves as the meeting minutes and 

### 14 will be completed within 90 days following the conclusion of the 

### 15 meeting. The meeting report is not a verbatim transcript 

### 16 although a transcript will be made available in approximately 

### 17 two weeks following the meeting. 

### 18 In closing, I again would like to thank all 

### 19 the members of the Panel, the public and the members of the EPA 

### 20 staff who have participated in preparing for this meeting, as 

### 21 well as my colleagues on the FIFRA Scientific Advisory Panel for 

### 22 their preparations in this meeting. I am looking forward to a 

### 23 very challenging and interesting meeting in the next four days. 

### 24 25 Now I will turn the microphone over to Dr. 

### 26 Martha Sandy, who will introduce herself and the rest of the

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1 members of the Scientific Advisory Panel. Thank you very much. 

### 2 **DR. MARTHA SANDY:**Thank you Sharlene, and 

### 3 good morning. As Session Chair of this meeting, I will start by 

### 4 saying my name is Martha Sandy. I am a Toxicologist. I am 

### 5 Chief of the Reproductive and Cancer Hazardous Assessment Branch 

### 6 of the Office of Environmental Health Hazard Assessment, which 

### 7 is part of the California EPA. 

### 8 **DR. BARRY DELCLOS:**I am Barry Delclos from the 

### 9 FDA's National Center for Toxicological Research. I am a member 

### 10 of the permanent Panel. I work in Reproductive and General 

### 11 Toxicology. 

### 12 **DR. JAMES MCMANAMAN:**Good morning. I am Jim 

### 13 McManaman. I am a Professor at the University of Colorado, 

### 14 Reproductive Biologist and a member of the permanent Panel. 

### 15 **DR. DANIEL SCHLENK:**My name is Dan Schlenk. 

### 16 I am the current SAP Chair and a Professor of Environmental 

### 17 Toxicology from the University of California, Riverside and a 

### 18 member of the permanent Panel. 

### 19 **DR. STEPHEN KLAINE:**Hi. I am Steve Klaine. 

### 20 I am a Professor at Clemson University and I direct the 

### 21 Institute of Environmental Toxicology and I am a member of the 

### 22 permanent Panel. 

### 23 **DR. MARION EHRICH:**Marion Ehrich from 

### 24 Virginia Tech, Teacher of Pharmacology and Toxicology and I am a 

### 25 member of the permanent Panel. 

### 26 **DR. GEOFFREY SCOTT:**Geoff Scott, Director of

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1 the National Oceanic and Atmospheric Administration Center for 

### 2 Coastal Environmental Health, Bimolecular Research. I am a 

### 3 Marine Ecotoxicologist. 

### 4 **DR. VICKI BLAZER:**Vicki Blazer. I am with 

### 5 the U.S. Geological Survey, Leetown Science Center. I do 

### 6 primary fish pathology work. 

### 7 **DR. REYNALDO PATINO:**I am Reynaldo Patino. I 

### 8 am also with the U.S. Geological Survey with the Texas 

### 9 Cooperative Fish and Wildlife Research Unit at Texas Tech 

### 10 University. And I am an Aquatic Toxicologist Comparative 

### 11 Endocrinologist. 

### 12 **DR. CATHERINE PROPPER:**I am Cathy Propper. I 

### 13 am from Northern Arizona University in Flagstaff. I am an 

### 14 Environmental Endocrinologist and Comparative Endocrinologist, 

### 15 and I am an ad hoc member of the Panel. 

### 16 **DR. EDWARD PERKINS:**I am Ed Perkins. I am 

### 17 Senior Scientist with the U.S. Army Corps of Engineers. I am a 

### 18 Molecular Toxicologist. I am an ad hoc member of the Panel. 

### 19 **DR. ROBERT DENVER:**Good morning. I am Bob 

### 20 Denver. I am Professor of Molecular Cellular Developmental 

### 21 Biology from the University of Michigan and I am an 

### 22 Endocrinologist. 

### 23 **DR. JOHN MCCARTY:**I am John McCarty. I am 

### 24 Professor of Biology and Director of Environmental Studies at 

### 25 University of Nebraska at Omaha, and I am an Avian Ecologist. 

### 26 **DR. DALLAS JOHNSON:**My name is Dallas Johnson

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1 and I am Professor Emeritus from Kansas State University. I am 

### 2 a Statistician. 

### 3 **DR. COLIN SCANES:**Colin Scanes. I am a 

### 4 Professor of Biological Science at the University of Wisconsin, 

### 5 Milwaukee with expertise in Avian Endocrinology. 

### 6 **DR. SETH KULLMAN:**Good morning. I am Seth 

### 7 Kullman, North Carolina State University and Environmental and 

### 8 Molecular Toxicology, ad hoc member of the Panel, expertise in 

### 9 Molecular Endocrinology Developmental Biology. 

### 10 **DR. SHEA TUBERTY:**I am Shea Tuberty, 

### 11 Associate Professor of Biology at Appalachian State University. 

### 12 I have expertise in Invertebrate Zoology and Reproductive 

### 13 Physiology and Ecotoxicology, ad hoc member. 

### 14 **DR. JENNIFER FREEMAN:**I am Jennifer Freeman. 

### 15 I am from the School of Health Sciences at Purdue University. I 

### 16 am Assistant Professor of Toxicology and I am an ad hoc member 

### 17 of the Panel. 

### 18 **DR. MARY CHRISTMAN:**Hello. I am Mary 

### 19 Christman. I am a private statistical consultant and courtesy 

### 20 professor at University of Florida. My area of expertise is 

### 21 environmental and ecological statistics. 

### 22 **DR. GEORGE BENTLEY:**Good morning. I am 

### 23 George Bentley. I am a Professor at U.C. Berkeley in the 

### 24 departments of Integrated Biology and Neuroscience, and I am an 

### 25 Avian Endocrinologist. 

### 26 **DR. MARTHA SANDY:**Thank you. And if I could

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1 ask the members of the Panel to pull your microphones closer to 

### 2 you and talk directly into the microphone. The audio, we are 

### 3 having a little bit of breakup in the sound. 

### 4 **DR. SHARLENE MATTEN:**Just one other note. 

### 5 Dr. Ken Portier is on the Panel. He had a sudden death in his 

### 6 family and is at a funeral today. But his comments have been 

### 7 sent to Dallas Johnson and Mary Christman, and those comments 

### 8 will be summarized and synthesized for the variety of questions 

### 9 and at a variety of points before the meeting is closed. Ken 

### 10 has agreed and has wanted to review some of the summary 

### 11 comments, so he will be included in the meeting and reviewing 

### 12 the report. Thank you. 

### 13 **DR. MARTHA SANDY:**So now I would like to turn 

### 14 it over to Dr. Steven Knott, who is Deputy Director of the 

### 15 Office of Science Coordination and Policy and Dr. Steven 

### 16 Bradbury, who is the Director of the Office of Pesticide 

### 17 Programs. 

### 18 **STEVEN KNOTT:**Okay. Thank you, Dr. Sandy. 

### 19 And it is actually just Steve Knott, no doctor associated. So 

### 20 my name is Steve Knott. I am the Deputy Director of the Office 

### 21 of Science Coordination and Policy within EPA's Office of 

### 22 Chemical Safety and Pollution Prevention. Our Acting Office 

### 23 Director, Dr. David Dix had hoped to be here today to welcome 

### 24 the Panel. He had a conflicting engagement and asked me to make 

### 25 some remarks on his behalf. 

### 26 I want to begin by thanking the Panel for your

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1 work in preparation for this meeting. As a former DFO with the 

### 2 SAP, I know first-hand the work that is required to prepare for 

### 3 these meetings and to participate in them and work on the report 

### 4 afterwards, so we really appreciate your efforts. 

### 5 We are very much looking forward to the 

### 6 discussions that will take place over the next several days and 

### 7 to the advice that we will receive from the Panel. In addition, 

### 8 I would like to thank the public for participating in this 

### 9 important meeting and for providing comments for the Panel's 

### 10 consideration. I also want to thank Dr. Sharlene Matten, the 

### 11 Designated Federal Official for this meeting, and all the SAP 

### 12 staff who make these meetings possible. 

### 13 There is a tremendous amount of work that goes 

### 14 into organizing an SAP meeting. Much of it takes place behind 

### 15 the scenes. And the SAP staff does an outstanding job of 

### 16 putting together a seamless meeting process so that the Panel, 

### 17 the public and the Agency can fully participate in the 

### 18 proceedings and focus on topics under consideration. 

### 19 This week's SAP addresses the third Endocrine 

### 20 Disrupters Screen Program related topic in this calendar year. 

### 21 It is focused on the validation of proposed Tier 2 ecotoxicity 

### 22 test. This is an important milestone for the EDSP building on 

### 23 advice from previous advisors in representing years of 

### 24 collaborative efforts from several offices across the Agency and 

### 25 with our international partners. 

### 26 The role of the Scientific Advisory Panel in

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1 terms of providing a publicly transparent peer review is key to 

### 2 ensuring that we will move forward using the best available 

### 3 science. The Office of Science Coordination and Policy provides 

### 4 leadership and coordination on scientific issues such as 

### 5 endocrine disruptors and has had a lead role in the validation 

### 6 of the test systems that will be under review this week. 

### 7 In a few minutes, we will move into the 

### 8 scientific presentations beginning with an overview provided by 

### 9 Mary Manibusan, the Director of our Exposure Assessment 

### 10 Coordination and Policy Division. 

### 11 I want to acknowledge the efforts of Mary, the 

### 12 scientist in her division and across OCSPP and also a scientist 

### 13 in the Office of Research and Development and others who have 

### 14 worked tirelessly to move the EDSP forward and bring us to the 

### 15 point where we have reached today. 

### 16 So in closing, I want to thank you again for 

### 17 your efforts in reviewing this important topic and we look 

### 18 forward to the discussions over the next several days. And I 

### 19 will turn the mic over to Dr. Steven Bradbury, the Office of 

### 20 Pesticide Programs. 

### 21 **DR. STEVEN BRADBURY:**Thank you and good 

### 22 morning. Just a couple of brief statements. I definitely want 

### 23 to echo Steve's acknowledgements of the DFO and the staff that 

### 24 helps prepare for the meetings, and thank all of you on the 

### 25 Panel for your hard work in providing us input and advice as we 

### 26 move forward, as well as the public and the comments that we

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1 will hear later on today. All that information is critical for 

### 2 giving the advice to the Agency as we implement the Endocrine 

### 3 Disruptor Screening Program. 

### 4 Just to backup again, just want to stress how 

### 5 important the Scientific Advisory Panel is to not only the 

### 6 business of the Office of Pesticide Programs, but more broadly 

### 7 across the Office of Chemical Safety and Pollution Prevention. 

### 8 As you may know, within FIFRA, the statue that 

### 9 sets our business path forward, the Scientific Advisory Panel 

### 10 was put by congress right into the day-to-day operations of our 

### 11 organization. I think it really underscores the importance of 

### 12 scientific peer review and an open and transparent process as we 

### 13 go forward on many fronts. And clearly, the endocrine program 

### 14 is a very challenging area. And from our meeting in January, we 

### 15 were talking about 21st Century toxicology techniques and 

### 16 genomics and expert systems and QSAR to understand what kind of 

### 17 methods could be used to help prioritize chemicals for 

### 18 investigation. 

### 19 Then the meeting we had very recently looking 

### 20 at the Tier 1 battery and getting some insights on what kind of 

### 21 methods and the data that will come from those methods to give 

### 22 us insights into the potential for a chemical to interact with 

### 23 the estrogen, androgen or thyroid system, and then coming to 

### 24 this week's meeting to get feedback on the methodology that can 

### 25 be used to provide additional insights on the mechanistic 

### 26 attributes of the chemical and to get that dose-response

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1 information that could inform an ecological risk assessment, 

### 2 incremental steps that are important to build the foundation and 

### 3 to build the building of our program of screening chemicals for 

### 4 their potential to interact with the endocrine system, and if 

### 5 they do, to have the kind of information necessary to inform our 

### 6 risk assessments. Going through this incremental process and 

### 7 getting the feedback step-by-step we think is really critical so 

### 8 that we can get feedback and input as we move forward. 

### 9 So I want to thank the Panel, the permanent 

### 10 members, especially, since this has been quite a path we have 

### 11 been going down over the last several months, and one more to go 

### 12 next month. But we think it is really important to have these 

### 13 incremental steps so that we can get feedback as we go and as we 

### 14 move forward. So again, I just want to thank all of you for 

### 15 your time and effort in preparing for the meeting and during the 

### 16 efforts this week and as you prepare the report. I want to 

### 17 thank the public for the comments we will be hearing later on 

### 18 today, and we are really looking forward to a very in-depth set 

### 19 of discussions this week and valuable advice from the Panel. So 

### 20 with that, I will turn it back over to the Chair. Thanks. 

### 21 **DR. MARTHA SANDY:**And thank you Dr. Bradbury 

### 22 and Mr. Knott. And now I believe we are ready to have a 

### 23 presentation from Mary Manibusan. 

### 24 **MS. MARY MANIBUSAN:**Thank you. Good morning 

### 25 everybody. And let me start by thanking Mr. Knott and Dr. 

### 26 Bradbury for their opening remarks. Thank the permanent Panel

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1 as well as the ad hoc Panel for all the work that you have 

### 2 already done in preparation for today's meeting, this week's 

### 3 meeting, and over the course of this week, engaging with the 

### 4 agency as we move this very important program forward, and then 

### 5 after this meeting, to help prepare the final report for the 

### 6 Agency to consider. 

### 7 I want to especially thank the DFO, Dr. 

### 8 Sharlene Matten for all of the work that she has put in to 

### 9 making this meeting happen. Certainly a meeting such as this is 

### 10 not something easy to pull together, so logically and 

### 11 content-wise, Dr. Matten has been at the forefront of 

### 12 leadership. 

### 13 So my job this morning and the next few 

### 14 minutes is to introduce you to the Endocrine Disrupters 

### 15 Screening Program; to give you a feel and sense of the program 

### 16 of where we are going, where we are headed, where we have been 

### 17 and where we would like to be. 

### 18 As Mr. Knott had indicated, the Endocrine 

### 19 Disrupters Screening Program is housed in the Office of Science 

### 20 Coordination and Policy, and that is in fact what we do. We 

### 21 coordinate across our sister offices and the Office of 

### 22 Pesticide, Pollution and Prevention and our Office of Pesticide 

### 23 Programs reaching out to Office of Water and certainly engaging 

### 24 with our colleagues in the Office of Research and Development to 

### 25 bring to you and to the public advanced technologies to push our 

### 26 program forward.

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1 With that in mind, with my overview slide 

### 2 here, what I would like to address are a few components, 

### 3 building the house as Dr. Bradbury had indicated. Starting from 

### 4 the top laying down what our mission and vision for the program 

### 5 is, following the congressionally mandate statutes of the 

### 6 Federal Food, Drug and Cosmetic Act as well as the Safe Drinking 

### 7 Water Act and how that informs our universe of chemicals that we 

### 8 have to screen and test. 

### 9 We will also point back to the FACA that was 

### 10 put together focusing on the Endocrine Disrupters Screen Program 

### 11 back in 1998 and what they cast forward as their conceptual 

### 12 framework of what the program should look like in terms of a 

### 13 Tier 1 and Tier 2 screening that we are here to talk about. 

### 14 And then I would like to go through and just 

### 15 talk briefly with you our validation process that we have 

### 16 followed for not only the Tier 1 test methodology, but in 

### 17 particular, for the Tier 2 and how we have walked through each 

### 18 of those processes very carefully. And what we bring today to 

### 19 you for consideration is an incremental amount of work that 

### 20 everyone has put together for your review. 

### 21 And lastly, I have been asked many times in 

### 22 past SAPs to put together a roadmap. And I hope what I can do 

### 23 within our talk today is just to talk you through where we have 

### 24 been, and in doing so, laying down that roadmap for you to begin 

### 25 to understand where we have been, how we have incrementally laid 

### 26 down these SAPs to help inform the Agency, and then closing out

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1 with our last SAP that is coming in the next month. But I would 

### 2 like to begin with the end in mind. 

### 3 Here is our mission statement. It is to 

### 4 protect public health and wildlife by screening and testing 

### 5 chemicals and, more importantly, taking appropriate action for 

### 6 those chemicals that are found to have endocrine effects. So we 

### 7 are not in the business of just identifying which chemicals have 

### 8 endocrine effects and which do not, but where they do have 

### 9 effects we learn on our risk assessment paradigm to fold that 

### 10 into our risk assessment so that we can assure that we are being 

### 11 public health and wildlife protective of chemicals with those 

### 12 effects. So we are indeed a risk-based program. 

### 13 We operate under two specific legislative 

### 14 mandates. We are congressionally mandated under the 1996 FFDCA 

### 15 under 408P. I point you to this particular language because it 

### 16 helps guide the program. It requires the Agency to put together 

### 17 the EDSP focusing on utilization of validated test systems and 

### 18 other scientifically relevant methods to in particular focus on 

### 19 chemicals that have similar effects to naturally occurring 

### 20 estrogens in the human population and opens the door for the 

### 21 administrator to expand that coverage to include other pathways. 

### 22 And we will talk a little bit about that. 

### 23 Under 408P we are required to screen all 

### 24 pesticide chemicals actives and inert ingredients. Very 

### 25 different from the 1996 Safe Drinking Water Act Amendment that 

### 26 was issued; under Section 1457 it stipulates that the Agency can

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1 test chemicals only if we find them in sources of drinking water 

### 2 to which a substantial human population may be exposed. So 

### 3 there is an exposure component there that we need to meet before 

### 4 we screen those chemicals. 

### 5 Shortly after in 1996, we formed a Federal 

### 6 Advisory Committee, the EDSTAC Committee. This EDSTAC Committee 

### 7 had a wide range of representation across federal agencies, 

### 8 state agencies as well as experts in their environmental fields 

### 9 in the endocrine area as well as research scientists. This 

### 10 committee deliberated between 1996 and 1998. They had issued 

### 11 their final report which really helped inform the Agency of what 

### 12 it looks like today. 

### 13 So here are some of their key recommendations; 

### 14 one of which is to expand the protection to not only include 

### 15 human health but also to wildlife. This was in recognition that 

### 16 wildlife was inherently valuable in the element of the 

### 17 ecosystems and is a good indicator of the overall health of the 

### 18 environment, which humans live in. It also expanded the 

### 19 coverage beyond estrogenic chemicals to be inclusive of the 

### 20 androgen and thyroid pathways, because those were the most 

### 21 common pathways at that time. But even so, the EDSTAC had 

### 22 recognized that science was advancing very rapidly and soon 

### 23 there would be additional validated methods coming online, 

### 24 additional information in terms of other endocrine pathways that 

### 25 we will need to cover. And that recognition is evident in the 

### 26 Tier 1 as well as in the Tier 2 assays.

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1 Perhaps the most critical recommendation was 

### 2 the formation of this two tiered screening and testing program, 

### 3 and that was their conceptual framework. The Tier 1 screen is 

### 4 comprised of about 11 assays, five in vitro assays and six in 

### 5 vivo assays. And it sought to answer the question of whether 

### 6 that chemical has a potential to interact with regards to the 

### 7 estrogen, androgen and thyroid hormone pathways. If chemicals 

### 8 are deemed to interact, have the potential to do so, and if 

### 9 weight of evidence considering all of its biological 

### 10 information, that we pull together a weight of evidence that 

### 11 warrants additional testing and it be the Tier 2 test design, 

### 12 those Tier 2 tests have a higher expectation in terms of 

### 13 answering that question of whether that chemical does interact, 

### 14 and if it does, then at what dose. What is that dose-response 

### 15 relationship on a quantitative level, and that will help inform 

### 16 the risk assessment. So that is the conceptual framework. 

### 17 Here is a visual depiction of the conceptual 

### 18 framework translated into today's language, what we call 

### 19 strategic testing approach. We think about this in terms of a 

### 20 big funnel. What we have from the Safe Drinking Water Act as 

### 21 well as from FFDCA is a universe of 10,341 unique chemicals. 

### 22 That is the chemical list that we brought to the SAP in January 

### 23 for consideration of prioritization. So when we have 

### 24 prioritized that list of chemicals and should they be ranked to 

### 25 have a higher probability of having potential to interact with 

### 26 the endocrine system, we would advance those forward for Tier 1

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1 screening. 

### 2 And again, after Tier 1 screening, we build 

### 3 the weight of evidence assessment alongside with other 

### 4 scientifically relevant information and move that through if the 

### 5 data warrants Tier 2 testing. Not all chemicals will 

### 6 automatically go forward for Tier 2 testing. There could be 

### 7 other opportunities, other interim studies that could be done to 

### 8 address a particular pathway. But if those chemicals warrant 

### 9 additional multigenerational studies, they would be advanced for 

### 10 our Tier 2 testing. 

### 11 Alongside on the arrow to the left, I 

### 12 highlight that we are not only interested in human health 

### 13 protection but also ensuring that we are protecting against 

### 14 ecological impacts for chemicals that may have the ability to 

### 15 interact with the endocrine system. 

### 16 Here is just a quick timeline flash we moved 

### 17 from vision. Here is a slide that highlights operationally how 

### 18 we have put together that vision. 

### 19 So starting from 1999, the EPA had established 

### 20 the endocrine program at that time. We had spent about a decade 

### 21 validating 11 Tier 1 assays, and I will show you a list of those 

### 22 in a few minutes. But surely after validation of those assays, 

### 23 we moved very quickly in putting forward our initial list of 

### 24 pesticide chemicals for test order issuance, and that was 

### 25 comprised of 67 pesticide chemicals. 58 were active ingredients 

### 26 and nine were high production volume inert ingredients.

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1 In 2009, what we also brought before the SAP 

### 2 was our attempt at putting together a solid method based on the 

### 3 QSAR validation principles, and that was the estrogen receptor 

### 4 expert QSAR system that at that time was focused on food use 

### 5 inert ingredients as that was our universe of focus. 

### 6 Shortly after in 2013, we brought back that ER 

### 7 expert system but expanded that domain of coverage to be 

### 8 inclusive of non-pesticide inert ingredients. What we also 

### 9 brought for the first time is some consideration of P-chem 

### 10 properties, such as whether a chemical is too acidic or too 

### 11 basic or too large in terms of being a polymer that would not 

### 12 interact with the endocrine system, certainly would not become 

### 13 bio-available enough to trigger any endocrine effects, and so 

### 14 therefore would be excluded from Tier 1 testing. 

### 15 We also brought forward for the first time 

### 16 high throughput technology to help expand that ER expert system 

### 17 in terms of its chemical domain of coverage. And that certainly 

### 18 was a big step for the Agency in order to do so. 

### 19 As Dr. Bradbury and Mr. Knott had indicated, 

### 20 it was just a month ago that we brought to the Panel our 

### 21 evaluation of the Tier 1 assays in battery performance bringing 

### 22 forward 21 of the 52 chemicals that submitted Tier 1 data to the 

### 23 Agency, and we really appreciate those comments and 

### 24 recommendations. 

### 25 And here we are today moving forward in that 

### 26 completion of the conceptual framework and bringing forward our

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1 ecological Tier 2 test methods for validation. And we will talk 

### 2 with you in a lot more detail over the course of today with our 

### 3 experts here with regards to each of those assays. 

### 4 And lastly, we have an SAP focused on weight 

### 5 of evidence analysis bringing forward the Agency's case studies 

### 6 on the Tier 1 data and how we have melted that information 

### 7 together with other scientifically relevant information to 

### 8 determine whether a chemical interacts with E, A or T. 

### 9 Here is another look at our 2013 year for 

### 10 scientific advisory panels. This is my attempt at putting 

### 11 together a roadmap, so we look to the four discrete steps that 

### 12 are laid out here. The first step for a chemical as it moves 

### 13 through the endocrine program for evaluation is the 

### 14 determination that it meets the prioritization goals of the 

### 15 program so it does not have even a higher probability of 

### 16 interacting with E, A or T. The second step would be initiating 

### 17 the test order issuance for Tier 1 assay data review, and then 

### 18 conducting that full weight of evidence determination. And we 

### 19 have our guidance as we had issued on our public website in 

### 20 September of 2011 to help illustrate the guidance in which we 

### 21 will be following to do so. 

### 22 And what we are here today to do is to 

### 23 complete the validation effort for the Tier 2 assays. And these 

### 24 are, again, longer term reproduction studies across multiple 

### 25 taxa and consistent with the EDSTAC recommendations to provide 

### 26 that larger coverage.

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1 Before I begin talking in a little bit more 

### 2 detail about the Tier 2 assays, I wanted to remind the Panel of 

### 3 the 11 Tier 1 screening battery that was brought to the SAP in 

### 4 May for review. Again, it includes five in vitro assays and six 

### 5 in vivo assays. 

### 6 The in vitro assays are specific to an AOP 

### 7 concept in adverse outcome pathway and looking at some earlier 

### 8 molecular initiating events to help secure our understanding of 

### 9 whether that chemical has a specificity to interact with the 

### 10 endocrine system. 

### 11 The in vivo studies are intended to give us a 

### 12 sense of what the toxico-dynamics, as well as the toxicokinetics 

### 13 in combination would do in a whole animal system. Here, the 

### 14 coverage is larger than those specific AOPs that the in vitro 

### 15 can cover, but it can cover other modes of action that are not 

### 16 yet know or identified. 

### 17 Here is a look at the proposed Tier 2 test 

### 18 methods. We have coverage for the mammalian system, the 

### 19 Mammalian Two-Generation Reproduction Study. And we provide as 

### 20 an alternative, the Extended One-Generation Reproduction Study. 

### 21 And this will help fit that data into the human health risk 

### 22 assessment. For the ecological risk assessment, we have four 

### 23 taxa here represented and we will talk a little bit more in 

### 24 detail about that. 

### 25 I just want to use this slide to remind 

### 26 everyone of the comparison between Tier 1 and Tier 2. These are

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1 very different assays answering very different questions with 

### 2 different levels of acceptable uncertainty. 

### 3 So for the Tier 1 screening on the left, these 

### 4 are of course shorter termed lower cost assays. They are 

### 5 intended to screen thousands of chemicals. So our acceptance of 

### 6 false negatives is going to be a lot lower, but our 

### 7 understanding and acceptance of false positive is equally as 

### 8 high. So we need to make sure that that is at an acceptable 

### 9 level, or else it does not serve the impact of what a screen 

### 10 should be. 

### 11 The question we are answering again is whether 

### 12 that chemical interacts with the endocrine system, so it is a 

### 13 binary answer; it is a yes/no. We are looking at limited doses 

### 14 because these are short-term studies we are going to push the 

### 15 kinetics high. So you will see oftentimes in Tier 1 screening, 

### 16 we will have high doses for the animal because we don't have a 

### 17 lot of animals per dose, so the power is not there. 

### 18 And again, the combination is looking at the 

### 19 balance between the in vitro and in vivo assays and clicking 

### 20 them back together like a puzzle piece and giving us a sense of 

### 21 what that totality of information is telling us in terms of its 

### 22 interaction. And the battery stand as a whole. It is 11 assays 

### 23 large, and it maintains its structure because of the ability to 

### 24 inform us across the adverse outcome pathway and across 

### 25 different levels of biological organization. Very different from 

### 26 the Tier 2 testing, here we are answering a question of whether

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1 that chemical interacts, so a lot more certainty. We need a lot 

### 2 more refinement, and that means a lot more animals. 

### 3 There are longer term studies, repeat doses, 

### 4 we have a wider coverage of dose response because we want to get 

### 5 at what the NOEL or LOEL value is and what the NOEC and LOEC 

### 6 values are. We are also not restricted from looking at 

### 7 particular modes of action. This is an in vivo system so we are 

### 8 going to get everything from compensatory mechanisms to adaptive 

### 9 effects to apical endpoints and that really is the bases for 

### 10 risk assessment. 

### 11 These are in vivo studies and we are cutting 

### 12 across multiple taxa, all the way from rat to frog to fish and 

### 13 invertebrates, so we will cover that over the course of today. 

### 14 And different from Tier 1, these are not 11 assays built 

### 15 together as a battery. We are going to go and ask for Tier 2 

### 16 testing if we need that information, and if so, we will be 

### 17 selective about which Tier 2 that we will be issuing test orders 

### 18 for. 

### 19 So starting from the mammalian side, we have 

### 20 already two validated test methods, the two-generation 

### 21 reproduction study and then the extended F1 generation. The F1 

### 22 generation is a mere streamlining of the multi-generation study. 

### 23 It includes a direct exposure to post weanling of F1 offspring 

### 24 after lactation until termination, but there is no mating of the 

### 25 F1 animals to produce the F2 offspring. 

### 26 A distinction here is that there is still the

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1 flexibility of going forward with the second generation, if data 

### 2 from the first generation indicates that you need to do so. 

### 3 We moved from the two-generation to extended 

### 4 one-generation based on a retrospective analysis of a rich 

### 5 dataset of 300 chemicals, in which it informed the Agency that 

### 6 without that second generation study, we would not be making 

### 7 changes in our risk assessment. That is, we would not be less 

### 8 protective. That case is a case that we will be looking to make 

### 9 across remaining Tier 2 test methods, as you will see in a few 

### 10 minutes, but here is our baseline. 

### 11 Our baseline is this; that these are very 

### 12 complex studies. They are not to be taken lightly. So even for 

### 13 the mammalian reproduction study, our advice to those who are 

### 14 initiating study is to come in early to the agency, ask for 

### 15 consultation, and along the way as you move forward in that test 

### 16 design, make sure you are staying connected with the Agency so 

### 17 that if something comes up, some incident happens, we are able 

### 18 to move quickly and address the issue. Because these are very 

### 19 expensive studies, we want to make sure that we are doing them 

### 20 right and we are doing them in a way that can inform the Agency 

### 21 to be health protective. 

### 22 So there is that tight connection already 

### 23 build-in to the extended one-generation as well as the 

### 24 two-generation study, and we will look for that close connection 

### 25 as we move forward with the remaining ecological toxicity test 

### 26 guidelines.

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1 The Avian Tier 2 test method, again, this is 

### 2 proposed for evaluation and for inclusion in our tool box. The 

### 3 Japanese quail was selected for Tier 2 testing based on its 

### 4 ability, its robustness in a laboratory setting as well as the 

### 5 rate in which it matures. So it really fits what we want it to 

### 6 do. It is fit for purpose. 

### 7 This is designed to detect chemical disruption 

### 8 of hormonally controlled sexual differentiating birds. And 

### 9 specific to the study, it enables us to determine the longer 

### 10 term effects of maternal transfer and in ovo exposure. 

### 11 Like many of the studies that you will hear 

### 12 about today, we have not done these validation efforts alone. 

### 13 For this particular test, we collaborate very closely with the 

### 14 U.S. Army as well as USGS. My next speaker Dr. Touart will be 

### 15 providing a lot more detail on this test method. 

### 16 The Fish Tier 2 test method, the medaka 

### 17 species was selected again because they are smaller, easier to 

### 18 handle and has been deemed robust for this particular assay and 

### 19 for this particular purpose. What we provide in the ISR are two 

### 20 methods; the Medaka multi-generation toxicity test as well as 

### 21 the streamline Medaka reproduction test. And that MRT is our 

### 22 preferred test method as we move forward because it enables us 

### 23 to get at the answer that we are seeking to ask, and that is 

### 24 whether that chemical has the ability to cause adverse outcome 

### 25 in this particular species across many doses. 

### 26 It also allows us more specifically to

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1 determine the genetic sex by obtaining fish samples. So again, 

### 2 this species was picked, selected for a particular reason. Like 

### 3 the previous study, this particular validation effort could not 

### 4 have happened without the collaboration with the US Japan 

### 5 Bilateral. The US and the Japanese Ministry of Environmental 

### 6 really work together to really refine this particular test 

### 7 method and has demonstrated its ability to perform. 

### 8 The amphibian Tier 2 test method, we selected 

### 9 the xenopus laevis, and this was selected in particular to 

### 10 correspond back to the Tier 1 AMA assay. And this again has the 

### 11 ability to be genetically sexed, which is an advancement in 

### 12 technology. It was designed to characterize perturbations of 

### 13 normal development and growth, especially as we focus on the 

### 14 larval stage that is the sensitive stage for the amphibian. 

### 15 And this inter-laboratory collaboration effort 

### 16 was also done under the US Japanese Bilateral, and you will hear 

### 17 more from Dr. Sigmund Degitz later on today about the validation 

### 18 effort. 

### 19 And lastly, the Mysid. The Mysid is a very 

### 20 important species for ecological risk assessment. It certainly 

### 21 has been used for quite a number of decades. This particular 

### 22 test method is a mere adaptation from the existing 850 test 

### 23 method, life-cycle test that is already on the books for 158. 

### 24 This particular test method was selected with this species 

### 25 because of the assumption that chemicals that affect 

### 26 invertebrate hormones may indeed affect invertebrate hormones

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1 resulting in altered reproduction development and growth. 

### 2 And again, we have collaborated with NOAA on 

### 3 this particular validation effort, and you will hear more from 

### 4 Dr. Touart as we close the day with the EPA presentations. 

### 5 Of course, all these test methods could not be 

### 6 incorporated into the endocrine program without it being 

### 7 validated. And here is a definition that I pulled from ICCVAM 

### 8 that a validation is a scientific process for which the 

### 9 reliability and relevance of an assay method are evaluated for 

### 10 the purpose of supporting a specific use; so does it fit the 

### 11 purpose of its intention. 

### 12 Reliability as defined as the reproducibility 

### 13 of results from assays within and between laboratories. The 

### 14 relevance is whether a test is meaningful and useful for a 

### 15 particular purpose. 

### 16 Here is a five-step validation process that we 

### 17 have followed. The first step involves a lot of published 

### 18 literature research and looking at what has been already done so 

### 19 that we can glean and build from that. That is a method 

### 20 development and preparation of this detailed review paper that 

### 21 lays out the structure of the study protocol. The second step 

### 22 is the pre-validation step that is really the intra-laboratory 

### 23 testing in which we look to demonstrate relevance to the 

### 24 question that we are asking as well as looking to optimize the 

### 25 protocols. So we have selected a number of key reference 

### 26 compounds for each particular assay to really challenge that

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1 protocol so that we can learn and we can address those issues 

### 2 and further improve the protocol as we move forward through the 

### 3 validation process. 

### 4 We are also in this step looking to determine 

### 5 its readiness for the inter-lab validation effort, which is 

### 6 right after we have demonstrated the reliability across labs. 

### 7 But we have done this inter-lab seeking to, again, learn as we 

### 8 go. And I hope that is the message you received when you read 

### 9 the ISRs, is that as we move through this validation process, we 

### 10 have highlighted areas of deficiencies and we have noted those. 

### 11 And we have learned from them and we are looking to build upon 

### 12 all of that intelligence and all of that experience in putting 

### 13 together the final test guideline, because that is where the 

### 14 information will be really, really important. And that 

### 15 assurance is what our speakers here are to provide today, as 

### 16 well as in the ISRs. 

### 17 The ISRs are the subject of today's review and 

### 18 we look for your recommendations and comments on how those 

### 19 efforts have been and whether they have demonstrated that 

### 20 relevance and reliability that we seek to answer before we 

### 21 accept these test methods as regulatory protocols. 

### 22 So with that, I would like to end my 

### 23 presentation with where it all began. And where it began was in 

### 24 a little retreat called Wingspread in Racine, Wisconsin. And 

### 25 this was in the summer, July 1991 that a group of 

### 26 multidisciplinary scientists, much like yourselves sitting here

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1 today, had talked through the issue of endocrine disrupting 

### 2 chemicals and its prevalence in the environment as well as for 

### 3 human health exposure. 

### 4 And I pull out this particular comment because 

### 5 as I read through the consensus statement that was signed in 

### 6 1991, it read very much as if it had been written today; so here 

### 7 is the quote: "The testing of products for regulatory purposes 

### 8 should be brought in to include hormonal activity in vivo." And 

### 9 it was recognition 20 years ago that we were lacking these test 

### 10 methods in order to answer the question that we have before us. 

### 11 And I hope with this meeting that we will come away with 

### 12 utilization of test methods so that we can get back to the 

### 13 mission and to the vision that we stated earlier and end the 

### 14 program with all the advancements of test methodology so that we 

### 15 can answer the question. 

### 16 This slide is just to acknowledge the hard 

### 17 work as Dr. Matten had indicated. This is a cross-Agency 

### 18 effort, so we have our representatives from OSCP as well as from 

### 19 our Office of Research and Development here today to provide 

### 20 their presentations. We will hear from Dr. Touart on the 

### 21 Japanese Quail Two-Gen. Following behind is Dr. Johnson for the 

### 22 Medaka assay; Dr. Degitz for the LAGDA assay. And then finally 

### 23 Dr. Touart will close us out with the Mysid Two-Generation 

### 24 Study. With that, I thank you for your attention and I am open 

### 25 to any questions you might have. 

### 26 **DR. MARTHA SANDY:**Thank you very much, Ms.

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1 Manibusan. And I will ask the Panel if you have any questions 

### 2 of clarification? 

### 3 **DR. DANIEL SCHLENK:**Hi Mary. It is nice to 

### 4 be down on this end of the table for a change. I have got a 

### 5 bunch of questions for Rodney later but I thought I would hit 

### 6 you with this one first. 

### 7 So as I understand this, the Tier 2 test is 

### 8 supposed to demonstrate a dose response relationship and that is 

### 9 very important in the processes, and some of the assays, that is 

### 10 what we are hoping to see. 

### 11 I am curious how the Agency is defining dose 

### 12 response relationship. Is that going to be a monotonic 

### 13 dose-response relationship? And particular, with the LOEC 

### 14 measurements, if that is a single point on a dose-response 

### 15 relationship that is statically significant, would that still be 

### 16 good enough in a risk assessment for the next step? Does that 

### 17 question make sense? Do you know what I am saying? 

### 18 **MS. MARY MANIBUSAN:**So I can start and pass it 

### 19 to others who might want to comment. That is a really relevant 

### 20 question because, as stated, the Tier 2 test is to inform us of 

### 21 whether we can identify the apical endpoint, and more 

### 22 importantly, at what dose should we be regulating, so not 

### 23 necessarily a dose-response but a dose that we understand to be 

### 24 protective of endocrine activity. 

### 25 Having said that, I think it is important to 

### 26 delineate the issues of non-monotonic dose-response curves and

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1 monotonic dose-response curves. And it is very publicly made 

### 2 aware that the Agency had put forward our Save the Science 

### 3 document on non-monotonic dose-response curves for the NAS 

### 4 review this summer over the course of this year. 

### 5 Having said that, whether it be monotonic or 

### 6 non-monotonic, the two-generation or multi-generation studies 

### 7 within the Tier 2 test toolbox should inform the Agency of 

### 8 indeed what dose we should be regulating, if we are not already 

### 9 regulating at a lower dose to be protective of endocrine 

### 10 activity. And so, to that extent, different from Tier 1, the 

### 11 Tier 2 test are inclusive of a larger range of doses for 

### 12 testing, and additionally, more animals per dose for testing, so 

### 13 to increase the statistical power to find those subtle effects 

### 14 and then to regulate a dose that is protective, but is also 

### 15 environments reflective of what the current exposure is. 

### 16 **DR. DANIEL SCHLENK:**Yes. I guess one 

### 17 specific question I had was does the Agency consider a 

### 18 dose-response relationship more than a single dose that is 

### 19 statistically significant from the previous dose or does that 

### 20 dose -- for example, there are many data in the documentation, 

### 21 there is a lot of data that presents an LOEC, but it is the 

### 22 highest dose that is statistically significant. But it is not 

### 23 different from the previous dose, statistically, and it is not 

### 24 different from the dose above that. So the sequential doses are 

### 25 not statistically significant. There is a trend sometimes 

### 26 towards dose dependence but at least there is not -- that I can

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1 see a lot of times -- a statistically significant dose-response 

### 2 relationship. So would an LOEC of a single dose count as a 

### 3 valid Tier 2 test? I guess that is what I am after. 

### 4 **DR. STEVE BRADBURY:**Good line of questioning. 

### 5 I think as we go through the different assays, y'all can kind 

### 6 of delve into that a bit more. I think, just standing back in 

### 7 terms it is the risk assessment process and information that 

### 8 informs a risk assessment. Certainly we look at all the doses 

### 9 and try to discern what kind of trends we are seeing. Obviously 

### 10 some designs are setup for different kinds of statistical 

### 11 approaches, but a risk assessment does not operate in isolation 

### 12 from other information that is available. 

### 13 So I foresee if chemical X had gone through 

### 14 the prioritization, had gone through Tier 1, had gone into Tier 

### 15 2, we are going to be looking not only at the Tier 2 

### 16 information, we will be taking a look at the Tier 1 information, 

### 17 there may be mechanistic information, there may be other 

### 18 information along different parts of the adverse outcome pathway 

### 19 in understanding what those dose relationships look like. As 

### 20 Mary said, the Tier 1 does not give you a full dose-response but 

### 21 it gives you a sense of what could be happening at different 

### 22 doses and different tissues or different levels of biological 

### 23 organization. So I think it is looking at the totality of 

### 24 information. 

### 25 I think as you go forward, and perhaps on a 

### 26 case-specific situation where you may know something about the

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1 mechanism and some of the toxico-kinetics and dynamics, that may 

### 2 influence some of the dose selection for, say, a Tier 2 assay 

### 3 and gets back to the point -- you know, with mammalian studies, 

### 4 we encourage the registrants to come in, let's talk about all 

### 5 the information we know about that chemical so that we set the 

### 6 doses and hopefully get a well informed set of data that comes 

### 7 out. 

### 8 I think another aspect of ecological risk 

### 9 assessments is, if not quantitatively, at least qualitatively. 

### 10 And I think more and more, moving towards quantitative based on 

### 11 the NAS report on ESA, Endangered Species Act and consultation 

### 12 with the services, how do you integrate dose-response curves, 

### 13 say for reproductive effects in the population model? So I 

### 14 think all of that information will help come together to sort of 

### 15 think about the dose selection and dose interpretation. 

### 16 So I am not giving you a specific answer 

### 17 because when you move into a risk assessment process you are 

### 18 doing more of a case-specific analysis and you are not going to 

### 19 be going in blind 99 times out of a hundred. You are going to 

### 20 have a lot of information to help guide you in that dose 

### 21 selection and interpretation of dose-response curve. 

### 22 **DR. DANIEL SCHLENK:**Okay. So I define 

### 23 dose-dependence for this particular SAP as being a single point 

### 24 that is statistically different from the rest of the doses. 

### 25 That is how dose-dependence is defined here. Is that correct? 

### 26 **DR. STEVE BRADBURY:**Yes.

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1 **DR. DANIEL SCHLENK:**And is sounds like a 

### 2 weight of evidence will probably be utilized in this after Tier 

### 3 2 as well, in terms of putting this together as well, right? 

### 4 **DR. STEVE BRADBURY:**Correct. 

### 5 **DR. DANIEL SCHLENK:**Okay. 

### 6 **DR. MARTHA SANDY:**Any other questions from 

### 7 the Panel? Yes? 

### 8 **DR. ROBERT DENVER:**I am just curious how the 

### 9 Tier 2 assays will be selected from the hits from the Tier 1 

### 10 assay? You said that the Tier 2 is not a battery, but there is 

### 11 overlap in the information that you can get from the different 

### 12 Tier 2 assays. So has that been thought about? Is there any 

### 13 guidelines for that? 

### 14 **MS. MARY MANIBUSAN:**So the Agency has been 

### 15 very clear in the Tier 1 assays being -- to answer the question 

### 16 of whether that chemical has the potential. Once we declared 

### 17 whether a chemical has the potential and needs to advance 

### 18 forward, it's not an automatic that we move forward to Tier 2. 

### 19 And as we have stated the Tier 2 to be a targeted testing 

### 20 approach -- meaning that it would be judged on a case-by-case 

### 21 basis, as Dr. Bradbury indicated -- when we do a risk 

### 22 assessment, we would bring together the totality of the 

### 23 information not only looking at the hazard but looking at 

### 24 exposure, exposure pathways to help inform us of whether we need 

### 25 additional Tier 2 data, and if so, which among the taxa that we 

### 26 are presenting today do we need that information in order to

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1 complete our risk assessment. 

### 2 But the focus of this particular SAP is not on 

### 3 the policy determinations of how the Agency plans to select 

### 4 among the Tier 2, but whether the Tier 2 can be performed 

### 5 adequately and replicated across the laboratories and can be 

### 6 used in a regulatory setting. 

### 7 **DR. ROBERT DENVER:**Right. But just to 

### 8 follow-up on that, our evaluation of the Tier 2's will be 

### 9 evaluating what they can tell you also. And so I presume that 

### 10 you are going to use that information in deciding which Tier 2's 

### 11 to move to. 

### 12 **DR. LESLIE TOUART:**I think one of the context 

### 13 that we are looking for is that the Tier 2's will be able to 

### 14 identify adverse consequence. So if we have identified a 

### 15 potential endocrine activity, we want to understand what is the 

### 16 adverse consequence of that. And so if the determination based 

### 17 upon the available information that we seek, that we would need 

### 18 to address that adverse consequence in the particular taxa, then 

### 19 that particular Tier 2 test would be needed and required. And 

### 20 so we would evaluate that on the context of what the consequence 

### 21 was and maybe what the point of departure or effect 

### 22 concentration would be what we would need to be concerned about, 

### 23 and that gets more into the risk assessment realm. 

### 24 **DR. MARTHA SANDY:**Dr. Christman? 

### 25 **DR. MARY CHRISTMAN:**I have a point of 

### 26 clarification. We were talking about the dose-response earlier,

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1 and the question that I have is, is the LOEC -- I thought the 

### 2 LOEC was the lowest concentration of the test substance that was 

### 3 statistically different from the control, not from all other 

### 4 levels, which is what I thought I heard. But what we mean is it 

### 5 is the lowest level that is statistically different than the 

### 6 control state, correct? It does not need to be different from 

### 7 the others, it just needs to be the first concentration, the 

### 8 smallest concentration. 

### 9 **DR. LESLIE TOUART:**Yes, that is correct. I 

### 10 mean the lowest observed effect concentration would be the 

### 11 highest concentration above a no-observed effect concentration 

### 12 compared to controlled. 

### 13 **DR. MARY CHRISTMAN:**That's correct. 

### 14 **DR. MARTHA SANDY:**Dr. Scanes. 

### 15 **DR. COLIN SCANES:**I want to follow-up on Bob 

### 16 Denver's question. If we look at the Tier 1 test, they are 

### 17 predominantly rodent test. Now does that mean when we are 

### 18 considering Tier 2, the need to look at a variety of taxa come 

### 19 into play? 

### 20 **MS. MARY MANIBUSAN:**I will start and I will 

### 21 pass it to Dr. Touart. So the Tier 1 assays are 11 assays 

### 22 large. The question again is whether that chemical interacts 

### 23 for E, A or T by pathway. So they are not distinguished by 

### 24 species, they are fitted together because they are informative 

### 25 to answering that question for an AOP. So that is very 

### 26 different from the Tier 2, which are different across taxa.

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1 **DR. COLIN SCANES:**By reasonably there are 

### 2 going to be differences in pathways across taxa. And so the 

### 3 question I am raising is, when one has the Tier 1 battery, which 

### 4 is predominantly a rat battery, if there are differences with, 

### 5 first of all, across taxa buds, amphibians, et cetera -- that is 

### 6 where the Tier 2 comes into play -- but there is no test early 

### 7 on across taxa. Am I correct? 

### 8 **DR. LESLIE TOUART:**Let me try to explain in 

### 9 the context of the battery. Again, there are in vitro assays in 

### 10 which are identified to look at some mostly initiating events 

### 11 like receptor binding or transcriptional activation or effects 

### 12 in something like steroidogenesis. There are the mammalian 

### 13 assays that are included but we also have fish, and there is an 

### 14 amphibian that is represented on the thyroid side. And these 

### 15 assays work in concert, in a sense the battery is an integrated 

### 16 concept and the general thinking is that we are covering 

### 17 somewhat bookends within the vertebrate taxa where we have the 

### 18 rats as well as the fish kind of looking at both of those. 

### 19 But the context is that there is conservation 

### 20 across the, and in the validation effort of the Tier 1, we look 

### 21 at a variety of at least the known or at least the suspected 

### 22 endocrine active materials to determine if we were able to 

### 23 detect all known mechanisms at a time. And so we looked across 

### 24 a variety of estrogenic/androgenic and thyroid active mechanisms 

### 25 to determine, "Okay, we can identify those." But the outcome of 

### 26 those interactions is not going to be determined until we get

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1 into more of Tier 2 where we are able to characterize whether 

### 2 there is an adverse effect. But the intent was that Tier 1 

### 3 would be able to identify somewhat comprehensively the potential 

### 4 to interact with estrogen/androgen or thyroid activities, not a 

### 5 particular life-stage or not just that the hormonal action would 

### 6 be interacted. 

### 7 **DR. COLIN SCANES:**If I could just follow it 

### 8 up. And that is even at receptive binding level, you can get 

### 9 mark species differences. 

### 10 **DR. MARTHA SANDY:**Any other questions from 

### 11 the Panel? I see none. I think it is time for us to take a 

### 12 break. It is just about 10 o'clock. Sharlene, how about we 

### 13 come back at 10 after 10? Thank you. 

### 14 (Whereupon a break was taken) 

### 15 **DR. MARTHA SANDY:**Coming back. If we can get 

### 16 the Panel members to take their seats. 

### 17 So I have been asked to remind everyone to 

### 18 speak clearly and into the microphone. Get as close as you can 

### 19 because they are having trouble hearing the speakers, Panel 

### 20 members and the speakers making presentations for the recording. 

### 21 So I believe we are now going to hear from Dr. 

### 22 Touart. Is that right? 

### 23 **DR. LESLIE TOUART:**Again, good morning to the 

### 24 Panel and thank you for the time and effort having been involved 

### 25 in putting together the integrated summary reports and a lot of 

### 26 the supporting material. I know that there is a lot to try to

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1 look through and I do not envy your task at all. And hopefully 

### 2 I can be helpful in trying to walk through right now the 

### 3 Japanese Quail Two-Generation test and some of the information 

### 4 to try to touch on some of the highlights that we have with this 

### 5 particular test methodology. 

### 6 The outline of my talk is basically to 

### 7 consider the purpose and basis of an Avian Two-Generation test 

### 8 and what is it intended to try to do. Then I want to go into a 

### 9 little bit of the history of the validation effort -- the things 

### 10 that we did -- and try to accomplish as part of demonstrating 

### 11 the utility of this methodology. 

### 12 Give a brief summary of the protocol in terms 

### 13 of the general design and some of the endpoints that are 

### 14 included. I will talk briefly about some egg injection studies 

### 15 that were done as part of the effort to gain some insights -- 

### 16 not really part of that test itself, but just in terms of some 

### 17 other associated information -- then discuss some of the 

### 18 demonstration and inter-laboratory studies, some of the results 

### 19 that we saw, and then talk advantages/limitations that were 

### 20 evident and then end up with kind of the value added of a 

### 21 multi-generation test in the context of the birds. 

### 22 Briefly, the purpose of the test is to 

### 23 characterize adverse effects and to define the concentration 

### 24 response to the extent, if possible, for any potential endocrine 

### 25 disrupting compound for birds. 

### 26 We want the method to be responsive and

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1 sensitive to these endocrine effects in birds. The method being 

### 2 comprehensive would evaluate adverse effects through other 

### 3 mechanisms, but we are interested in ensuring that any endocrine 

### 4 mechanisms or effects that may manifest would be covered by the 

### 5 test itself. The test is to provide a multi-generation of 

### 6 protocol that contains basically all phases of lifecycle so that 

### 7 we can somewhat isolate the exposure within those phases to 

### 8 understand which life-stages are sensitive and what cumulative 

### 9 effect may be occurring. 

### 10 In terms of, you know, look at avians and what 

### 11 is the basis here, hormonal control and sexual differentiation 

### 12 in birds differs from that of mammals and other vertebrates. 

### 13 The hormonalcomedic sex in birds is the male, which is different 

### 14 from others. Oviparity in birds is quite large in terms of the 

### 15 size of the eggs compared to the body size of the bird itself. 

### 16 And this affects retention and transfer of compounds both in 

### 17 terms of detoxifying material by moving it into the egg but also 

### 18 for what the embryo may see. And the context here too is the 

### 19 embryo may see actual transfer of test material but also of 

### 20 maternal factors that could carry over. 

### 21 Existing protocols that we have, primarily the 

### 22 avian reproduction test that is part of the established 

### 23 guidelines in the OCSPP, these are not considered sufficiently 

### 24 robust to define all reproductive and development effects for 

### 25 EDCs. They are not designed to determine the long-term effects 

### 26 of an in ovo exposure and fitness to the offspring. These tests

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1 are just exposure of the parents and production of eggs to 

### 2 14-day chicks. So it is not carrying forward in terms of the 

### 3 development or in terms of the maturation phases that may occur 

### 4 or indications of effects due to transfer. 

### 5 So they do not assess effects on all four 

### 6 critical life-stages that could be sensitive to endocrine 

### 7 disruption. So the concept of using a two-generation test would 

### 8 ensure that all life-stages have been included and any adverse 

### 9 consequence manifest within the context of the test itself. 

### 10 Each portion of a multi-generation test 

### 11 protocol then provides a unique treatment in that the F0 or 

### 12 parental generation, their maturation and exposure as adults. 

### 13 The F1s are exposed embryonically and through a full lifelong 

### 14 development process. And then their offsprings in the F2s would 

### 15 be solely an embryonic exposure. Together, all of these 

### 16 life-stages are considered and would be expected to express any 

### 17 potential adverse consequence. 

### 18 So the other context is there is suspicion 

### 19 that some chemicals may exert effects that would have long-term 

### 20 impacts on populations over multiple generations without 

### 21 continued EDC exposure. This is still somewhat hypothetical but 

### 22 the consideration is that if this is indeed the case, we would 

### 23 need some type of test methodology to evaluate that potential 

### 24 outcome. 

### 25 The history of the effort goes back to the 

### 26 adoption of the Endocrine Disrupters Screening Program and

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1 recommendations from EDSTAC. One of the initial evaluations 

### 2 that were done was a species comparison study using a 

### 3 two-generation methodology, somewhat the forwarner of the 

### 4 protocol that we are using today. And this included both 

### 5 bobwhite and Japanese quail, was a test done with methoxychlor. 

### 6 And the outcome of that really demonstrated 

### 7 bobwhite quails an impractical species that carried forward 

### 8 through two generations because of its extended life-cycle in 

### 9 terms of its development and growth and the reproductive phase, 

### 10 whereas Japanese quail, given its much shorter maturation 

### 11 development and more consistent reproduction in terms of the 

### 12 egg-laying was a more practical choice. 

### 13 A detailed review paper considering avian 

### 14 toxicology and the available methodologies and recommendations 

### 15 for testing was completed in 2005, and really formed somewhat of 

### 16 an outline for the protocol that we ultimately adopted. 

### 17 In the same time period there was what we 

### 18 refer to as the avian dosing study. This was done with 

### 19 estradiol. And the purpose of this test was to evaluate some of 

### 20 the differences in terms of exposure, whether one has continuous 

### 21 exposure or whether one just exposes the parental generation, 

### 22 leaves the offspring unexposed and then exposes later. And the 

### 23 outcome of that really was to determine that really continuous 

### 24 exposure would be the most appropriate because all stages would 

### 25 have some level of exposure to be evaluated. 

### 26 There were also some egg injection studies

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1 that were done. These were done because they are somewhat 

### 2 shorter term and I will talk a little bit more about these 

### 3 later. But the context was that this was to provide some 

### 4 additional information in terms of how a particular 

### 5 endocrine-active material when exposed at an embryo stage may 

### 6 manifest an effect later in life. 

### 7 In terms of demonstrating the protocol that we 

### 8 have now, there were two studies done sequentially at the Army, 

### 9 and this was their Public Health Command out at Aberdeen, 

### 10 Maryland. And they did an endosulfan test followed by 

### 11 trenbolone. 

### 12 We also had collaboration with USGS where they 

### 13 also did a trenbolone study in roughly the same time period as 

### 14 the Army but somewhat following onto what the Army had done. 

### 15 And then we entered an inter-laboratory trial where we had three 

### 16 contract laboratories involved in testing a compound, 

### 17 vinclozolin. 

### 18 In giving kind of a brief summary of the 

### 19 protocol itself, again, the recommended species that we 

### 20 supported was Japanese quail. This seemed to be the only 

### 21 practical species that we could carry through a multi-generation 

### 22 test paradigm. Based on this avian dosing study results, a 

### 23 pre-egglaying treatment continued through with treatment to 

### 24 chicks was considered the appropriate dosing regimen that would 

### 25 be utilized as part of these multiple generations. 

### 26 The test is to be initiated with sufficient

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1 pairs to ensure a minimum of 12 in the parental generation 

### 2 control group at the end of treatment. In practice, this 

### 3 usually works in 14, maybe up to 16 pairs being utilized. With 

### 4 these, it is intended that one set a minimum of six eggs per 

### 5 pair, that is 40 per level, from the 6th and 7th weeks of 

### 6 exposure, and these would be hatched and those offspring would 

### 7 be characterized later. And from these, one would develop a 

### 8 minimum of 12 pairs per test level to establish the F1 

### 9 generation. 

### 10 The one context here is that, although eggs 

### 11 are laid and counted and maybe the fertility and the embryo 

### 12 viability is identified through the egg-laying regime. Only 

### 13 eggs from the 6th and 7th weeks are collected to be set and 

### 14 hatched, so the test is terminated with the 14-day survival 

### 15 observation of these 6th or 7th week offspring in the F2. There 

### 16 is option to continue these to interpret potential maturation 

### 17 effects if there are indications that there would be some 

### 18 subsequent effect that may occur. 

### 19 I should mention that as part of the 

### 20 validation exercise, the laboratories did carry these further to 

### 21 evaluate what additional information might be there. But from 

### 22 our viewpoint, the amount of information collected post this 

### 23 time period in terms of the 14-day survival would be minimal in 

### 24 context. 

### 25 In terms of the kinds of endpoints that are 

### 26 included in the method, I have lumped these into what I am

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1 referring to as fitness endpoints. These are somewhat the usual 

### 2 characters or endpoints that are present in, say, existing avian 

### 3 reproduction test or egg production, fertility, the eggshell 

### 4 integrity of both the thickness and strength are measured, 

### 5 embryo viability, the hatchability in terms of a hatching 

### 6 success, sex ratio in terms of the offspring outcome, chick 

### 7 health in terms of any signs of toxicity, and so these would be 

### 8 identified. 

### 9 In terms of another group of endpoints, which 

### 10 I have lumped as kind of endocrine endpoints, these would be 

### 11 ones that would be somewhat more susceptible or indicative of 

### 12 some kind of an endocrine disruption or activity. And these are 

### 13 within the morphology/histopathology organ/gland weights, the 

### 14 histology of these glands, spermatid counts, morphology and 

### 15 other gross anomalies that one might identify in organs, 

### 16 especially if they are related to, say, the sex organs. 

### 17 Developmental landmarks, feather dimorphism, 

### 18 which are generally under hormonal control as well as the 

### 19 cloacal foam gland in the Japanese quail. Sexual maturation and 

### 20 behavior are also included. And then actual hormone measures, 

### 21 both plasma and fecal/urate were steroid and thyroid hormones. 

### 22 For histopathology there are a number of 

### 23 tissues that have been looked at in terms of tissues that are 

### 24 collected, preserved and then processed through Histotech and 

### 25 then read by a pathologist. That will be one of the charge 

### 26 questions for the Panel later in terms of is this list overly

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1 comprehensive and are there ways of maybe economizing. 

### 2 But primarily, we are looking at brain 

### 3 kidneys; liver, adrenal glands, the reproductive tissues, the 

### 4 cloacal gland, bursa. And we use the right thyroid gland for 

### 5 histopath. The left is taken for actual hormonal measures and 

### 6 things. But the context is that looking at the pathology is 

### 7 considered to be perhaps indicative of any disruption that may 

### 8 be occurring in the particular organism as indicators of the 

### 9 overall health and integrity of that organism. 

### 10 This is kind of a diagrammatic schematic. It 

### 11 is not exactly to scale, but diagrammatically it tries to 

### 12 represent what is being done in the different generations. And 

### 13 treatment begins at approximately six weeks of age in terms of 

### 14 post-hatch time. And from that six-week time period the egg 

### 15 laying is anticipated to really begin in earnest within about 

### 16 two weeks. 

### 17 For Japanese quail, there may actually be some 

### 18 birds that are producing eggs even at six weeks, but the context 

### 19 is that they are expected to be all pairs producing by the 8th 

### 20 to 9th week. And then in terms of progression, the egg-laying 

### 21 would be observed and collected for a six-week time period and 

### 22 those sixth and seventh week eggs would then be transferred, 

### 23 set, hatched. And these would form the bases for the F1 

### 24 generation and these would then grow, you know, the egg laying 

### 25 produces. And then at their six-week of egg laying, eggs 

### 26 collected to form the F2 generation, in terms of those eggs set

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1 and hatched, and these F2 birds would be untreated. 

### 2 So these later birds are not treated with diet 

### 3 and they are going to be observed for at least 14 days. And 

### 4 this potentially could be extended to evaluate some maturation 

### 5 levels, if it is indicated by effects that may have been seen 

### 6 in, say, F1 generation maturation. 

### 7 Moving now to a little bit of discussion of 

### 8 these egg injection studies that were summarized within the 

### 9 integrated summary report. 

### 10 These tests were designed to simulate maternal 

### 11 deposition of EDCs into eggs prior to the oviposition by the 

### 12 female. These were done because it is relatively short term and 

### 13 we would be evaluating the compound effect on the embryo stages 

### 14 and the outcomes post-hatch. It targets at embryo stage. And 

### 15 the thinking at the time was that maybe these could be 

### 16 potentially used in range-finding so one would not have to do a 

### 17 several-week reproduction study to try to establish what doses 

### 18 to set in this multi-generation test. So the context was we 

### 19 want to try and see if that was going to be possible. 

### 20 In terms of the kind of results, this chart is 

### 21 to kind of give an indication of some of the things that we are 

### 22 seeing. This is comparing some studies that were done with 

### 23 estradiol and trenbolone, an estrogen and androgen. 

### 24 The context here, in terms of those arrows 

### 25 that are up or down would indicate an increase or decrease. If 

### 26 they are not parentheses, those were significant increase or

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1 decrease. If there is a parentheses around that, that means 

### 2 that there may be a trend but it was not considered significant, 

### 3 or that if the parentheses around double arrows, that means a 

### 4 non-linear dose-response was observed. So the ED represents the 

### 5 embryo day. 

### 6 So the context here is that the exposure at a 

### 7 particular time point -- the embryo day-4 -- may have some 

### 8 effects seen in estradiol up here where you do not have a 

### 9 response if the exposure is at embryo day 11. So there would be 

### 10 some sensitivity at that particular point. But then you have 

### 11 some effects seen in embryo day 11 that you do not see manifest 

### 12 at embryo day 4. And same on the trenbolone side; you see some 

### 13 differences although the embryo day 4 and embryo day 11 seem to 

### 14 match pretty well, except kind of contradictory in the sense of 

### 15 the bursa mass being decreased at embryo day 4 treatment, but 

### 16 with an embryo day 11 treatment, it increases. 

### 17 So really the main point here is just this is 

### 18 an example of the kinds of information that one can glean to 

### 19 understand that in endocrine activity, the actual embryo stage 

### 20 would be important, and so what factors that embryo sees would 

### 21 be an effect. 

### 22 In general, I think one of the things that we 

### 23 determined was that there is not an easy transfer of the egg 

### 24 injection data in terms of the concentration injected into the 

### 25 egg to translate that into a dietary exposure. So the utility 

### 26 of egg injection or establishing range finding information is

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1 really somewhat limited or suspect. 

### 2 The information that one can glean more from a 

### 3 research standpoint, I think it probably would be helpful but 

### 4 not as part of the actual test methodology that we take forward. 

### 5 In the actual two-generation test itself, one 

### 6 of the first iterations that we did was the endosulfan that the 

### 7 Army had done. 

### 8 Endosulfan was chosen for a couple of reasons. 

### 9 One was that the Army had an interest since endosulfan was 

### 10 being used on lots of bases and they needed to understand the 

### 11 potential risk that it posed on their responsible areas. 

### 12 But the other context was that there is 

### 13 literature on endosulfan that suggested it may have some week 

### 14 estrogenic activity, but also suspect in terms of some other 

### 15 androgenic thyroid type level. So the context was that this was 

### 16 somewhat a win-win for both Army and us to evaluate this 

### 17 particular compound. 

### 18 In the actual conduct of the test there really 

### 19 were not very many real effects seen. They were sporadic 

### 20 effects across all the endpoints but nothing that was consistent 

### 21 or considered dose-related. 

### 22 In terms of the table here, just looking at 

### 23 reproduction, which would be one of our apical summary endpoints 

### 24 for determining an adverse effect. In terms of a risk 

### 25 assessment, the Agency generally considers survival, growth and 

### 26 reproduction as key elements for adverse consequence, so trying

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1 to translate particular endpoints into one of these is 

### 2 important. 

### 3 But in this case for the egg production 

### 4 itself, there were no statistical differences. And in looking 

### 5 at the average number of eggs produced per week by groups, there 

### 6 were really no marked significant differences either between 

### 7 concentrations or across the generations that were concluded. 

### 8 So endosulfan was re-demonstrated. There were very little or no 

### 9 adverse effects within the test as we proceeded. 

### 10 Now this is the next compound that was used, 

### 11 and this test was one of the trenbolone studies. This was done 

### 12 at USGS. And this was presented because -- and it may be a 

### 13 little difficult to read but I wanted to make sure that I had 

### 14 all three generations here to look at eggs laid per day. 

### 15 The different colors would be with increasing 

### 16 concentrations. And what is evident in the parental generation 

### 17 is their highest concentration, the 40 ppm, really shutdown 

### 18 reproduction so there was not sufficient production to even make 

### 19 individuals into the F1 generation, so F1 generation only goes 

### 20 up to the 20 ppm level. And while there is some reduction at 

### 21 the 20 ppm level in the F1 generation, there is more reduction 

### 22 across in that concentration. In terms of the other 

### 23 concentration, there is somewhat of a trend in terms of 

### 24 decreasing production. 

### 25 When we get to the F2s, these are again 

### 26 untreated, and most of the concentrations they are no different

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1 from control, but the 20 ppm still is reduced here, in terms of 

### 2 some carryover response. 

### 3 When we look at the trenbolone study that the 

### 4 Army did, it has similar results, although I do not have a 

### 5 graphic of their levels. But the 20 ppm had significant effects 

### 6 as well as the 10 ppm in the parental generation. In the F1s, 

### 7 the 20 ppm virtually shut down reproduction so there were no 

### 8 individuals carrying over to the F2s. But again, the 10 ppm 

### 9 would be an effect concentration which would be consistent with 

### 10 what was seen at USGS in terms of an effect. But in F2, there 

### 11 are really no differences in terms of the production. 

### 12 One thing to note here with the F2s is these 

### 13 were not carried as long in terms because this is averaged over 

### 14 the course of the egg production in both the F0 and the F1. But 

### 15 in the F2, it is just the intimal seven weeks. So part of the 

### 16 reason, the production here is lower is the initial weeks of egg 

### 17 laying is somewhat less than when the animals reached their 

### 18 stride. With the Japanese quail it is not uncommon for a 

### 19 productive pair to be producing six or even seven eggs on a 

### 20 weekly basis. 

### 21 This is another response that was seen in the 

### 22 trenbolone. This is, again, back at the USGS where they were 

### 23 monitoring some of the hormone levels. And with this, you see 

### 24 with the blue, this is the parental generation. The red is the 

### 25 F1 generation, and then this kind of green color is the F2. But 

### 26 with this model androgen, there is an increase in estradiol

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1 level with eggs with increasing concentrations. 

### 2 Within the F1, there is an increase also. The 

### 3 line is a little bit less, but with the variations that we have 

### 4 here, they are probably close to the same. But with the F2s 

### 5 where there is no more exposure, then they fall back and compare 

### 6 them closely to controls. So this is a curious indication of 

### 7 what may be going on, but the indication was in terms of the 

### 8 adverse effects. Remember, reproduction was really affected at 

### 9 the 10- and 20-part per million but the indications of some 

### 10 hormonal deviations were starting to show at somewhat lower 

### 11 concentration. 

### 12 Now in terms of the vinclozolin inter-lab 

### 13 where we had three different laboratories, and again, looking at 

### 14 average number of eggs laid per hen per day, the context here 

### 15 across all three laboratories and in the parental and the F1 

### 16 generations, the production was consistent that there was no 

### 17 significant difference that we saw with vinclozolin in this 

### 18 particular study. There were little significant effects seen in 

### 19 this inter-laboratory exercise with the vinclozolin, which is 

### 20 considered an androgen antagonist. 

### 21 The egg production is fairly consistent and 

### 22 between laboratories. And this is a key point, is that there is 

### 23 consistency in terms of the egg production that is made 

### 24 generally close to .9 eggs a day, so that is almost an egg a day 

### 25 in all cases. They may trail here or there but not 

### 26 statistically different. So the context is that, in terms of

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1 reproducibility, the Japanese quail is very consistent in its 

### 2 ability to produce eggs. 

### 3 And the next slide, I think this gets into 

### 4 vinclozolin in terms of the effect that was observed, which was 

### 5 based on histopathology. And there were potential lesions 

### 6 attributed to vinclozolin that included lesions in the 

### 7 epididymides of the parental and F1 generation adult male quail 

### 8 in all three laboratories. So this was seen in all three, 

### 9 although the histopathology that was observed did not manifest 

### 10 into any other adverse effects in the other endpoints that were 

### 11 evaluated. 

### 12 Background lesions, particularly in liver and 

### 13 ovary, were identified in different tissues but these lesions 

### 14 were unrelated to vinclozolin exposure so there is no 

### 15 consistency in terms of what those might have been. And clearly 

### 16 there were no vinclozolin related lesions in any of the other 

### 17 tissues that were evaluated in the adult male/females or 

### 18 hatchlings. 

### 19 This is just to show some of the differences. 

### 20 I do not think this slide shows up as well when it is projected 

### 21 but the context is that there were clear effects, in terms of 

### 22 actualization and the tissues between control and treatment. 

### 23 Now in terms of the inter-lab, we did some 

### 24 further statistical analyses to look at the range of the CVs 

### 25 across laboratories by category. And the context here is that, 

### 26 really there is fair consistency within the Japanese quail where

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1 our CVs are predominantly less than 20 percent in most cases, 

### 2 and 20 percent is considered pretty good. And in terms of the 

### 3 reproduction, which is one of our key endpoints of concern, the 

### 4 median CV was close to 10 percent in the parental generation and 

### 5 5 percent in the F1s, which would indicate there are consistency 

### 6 between generations as well. 

### 7 When we come down to mating behavior that was 

### 8 observed, this is pretty high. So mating behavior as an 

### 9 endpoint was highly variable. I would believe this is due 

### 10 somewhat to the actual technique or method for identifying. In 

### 11 the Japanese quail, the mating behavior was observed primarily 

### 12 by evaluating the onset of the population when naive pairs are 

### 13 brought together, exposed naive pairs. So the time to first 

### 14 copulation and the number amount of attempts that are made, 

### 15 these were used to quantify mating behavior. 

### 16 And again, this was a fairly highly variable 

### 17 in terms of the measure itself. So it raises the question as to 

### 18 how useful that would be. Although we feel behavior should be 

### 19 an appropriate methodology, but at least in Japanese quail we 

### 20 did not identify a more appropriate method to utilize. So 

### 21 again, any recommendations the Panel may have on how better to 

### 22 do that or how better to improve the measure itself would be 

### 23 useful. 

### 24 This slide which is fairly busy, again, this 

### 25 one was done to look at the ability to -- at least the power of 

### 26 the test -- to have 80 percent power of detecting a difference

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1 from control. And the point here again -- and I just 

### 2 highlighted it in green for reproduction -- we feel that we 

### 3 would have sufficient power at 22 percent change difference to 

### 4 observe in the reproduction. 

### 5 But again, coming down with mating behavior as 

### 6 the extreme, this does not give us a lot of confidence that we 

### 7 would be able to detect an effect unless it was very gross in 

### 8 context. But overall, for other effects like in development 

### 9 growth, these are very good in terms of our ability to determine 

### 10 some differences in context. 

### 11 So trying to summarize a little bit of the 

### 12 inter-lab comparison, I think one thing we also recognize is 

### 13 there were some clear differences in strains that were used, 

### 14 basically in terms of size. 

### 15 Japanese quail are somewhat semi-domesticated 

### 16 and there are strains that are produced for different reasons. 

### 17 Some for, say, the meat-market, which would be a larger sized 

### 18 body mass would be selected for, and those that are selected 

### 19 more for, say, the egg market where egg production, and so body 

### 20 size is not as important. But there is a range in size. There 

### 21 are some different strains that do exist. 

### 22 And strain differences may be a factor in rate 

### 23 of maturation in some reproductive characteristics, and some 

### 24 variables also show some variability and it is not clear whether 

### 25 that is necessarily indicative of an unreliable measurement of 

### 26 the endpoint or just the method of measurement that requires

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1 definement; behavior being one of the context in terms of is 

### 2 there a better way of trying to measure that, that would be a 

### 3 little bit more consistent. 

### 4 The one thing with the differences in strains 

### 5 though, across the laboratories, there was sufficient 

### 6 consistency as seen in our power to detect that we would be able 

### 7 to observe effects if they were to occur. 

### 8 Again, the primary measures indicative of 

### 9 endocrine disruption would required somewhat a protocol that 

### 10 would be able to discern the vulnerability across the different 

### 11 stages. We feel all of that is consistent here and that the 

### 12 laboratories were able to carry fourth these measures in their 

### 13 tests. 

### 14 This is just to try to summarize some of 

### 15 advantages that Japanese quail bring in context. And, you know, 

### 16 Japanese quail precocial model matures rapidly and chicks can be 

### 17 treated by dietary administration. 

### 18 The Japanese quail is relatively well 

### 19 characterized with a great deal of information on the particular 

### 20 species. A multi-generation test with this species can be 

### 21 completed in seven months for the in-life portion of the test, 

### 22 and that is just not possible with other bird species that we 

### 23 commonly would utilize within the laboratory setting. 

### 24 A potential to develop straight-forward tests 

### 25 that elucidate mechanisms is prominent for this species. It 

### 26 appears to be moderately sensitive to EDCs that would be

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1 somewhat more broadly applicable to other avian species. A wild 

### 2 type strain retains the photoperiodicity and other 

### 3 characteristics of wild populations, and so we recommend that 

### 4 one steer more towards wild type individuals. 

### 5 The other point is just that the immune, 

### 6 adrenal and thyroid system were also pretty well understood in 

### 7 the Japanese quail. There are limitations though, is that being 

### 8 precocial, they differ from altricial birds in pattern. And 

### 9 Song Birds are of interest in how well one can translate from 

### 10 the Japanese quail to these is somewhat suspect, but we 

### 11 currently do that in context of the avian tests that are 

### 12 currently utilized by the Agency would use precocial birds. 

### 13 They are initiatives to try to develop some altricial birds, but 

### 14 they pose many challenges to doing any kind of long-term study 

### 15 since there is a lot of parental care for the hatchlings that 

### 16 would be necessary. 

### 17 Japanese quail do vary by strains and there 

### 18 are some differences there and so it may need to be a minimal 

### 19 set of strains recommended in terms of protocol for moving 

### 20 forward. 

### 21 Finally, I guess Japanese quail provide a 

### 22 model species that does not completely emulate all birds, 

### 23 especially long-lived birds, but they are a practical species to 

### 24 utilize. 

### 25 For multi-generation exposures, the data 

### 26 indicate that adult birds are generally less vulnerable to

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1 adverse effects of EDCs whereas the embryonic exposure or the 

### 2 offspring might be more susceptible. 

### 3 It is felt that it is critical to have an F1 

### 4 generation and to observe the capability of those F1s to 

### 5 successfully reproduce. So exposure outcomes maternal transfer 

### 6 that may take place, both of chemical as well as other modified 

### 7 backers being deposited into the egg, one would want to carry 

### 8 that forward to determine what is the consequence in terms of 

### 9 those individuals to mature and reproduce successfully. 

### 10 In a multi-generation test, each life-stage is 

### 11 included and can be evaluated. And it is considered important 

### 12 to incorporate some measures of these endocrine perturbations or 

### 13 behavioral perturbations that may be in place. Including, an F2 

### 14 generation provides a test for assessing the possible 

### 15 trans-generational effects if these were to occur, and that is 

### 16 another consideration in terms of an advantage of maintaining 

### 17 something of a multi-generational test paradigm. 

### 18 So to summarize, I think the Japanese quail 

### 19 two-generation tests, the protocol that we utilized was 

### 20 successfully performed in five laboratories, meaning they were 

### 21 able to meet the performance criteria and collect information to 

### 22 be utilize. The CVs and statistical power relevant for avian 

### 23 fitness and endocrine integrity have been determined in terms of 

### 24 how well certain endpoints play out. And it would appear then 

### 25 that the test is reasonably reliable for its intended purpose to 

### 26 be able to characterize potential adverse effects when

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1 challenged by a compound that would affect one or more of these 

### 2 endpoints. 

### 3 And with that, I am finished with my 

### 4 presentation and would take any clarifying questions or comments 

### 5 the Panel might have at this time. 

### 6 **DR. MARTHA SANDY:**Thank you very much for 

### 7 that summary of the Japanese quail two-generation toxicity test. 

### 8 Do we have comments, questions of clarification from the Panel? 

### 9 Yes, Dr. Patino? 

### 10 **DR. REYNALDO PATINO:**I am just curious if you 

### 11 could provide the rationale for the inter-laboratory validation, 

### 12 the rationale for using the compound that you chose, 

### 13 vinclozolin. Why was that particular one chosen for this test? 

### 14 **DR. LESLIE TOUART:**I think in hindsight we 

### 15 might wanted to have used something different. But the thinking 

### 16 at the time in terms of trying to use vinclozolin, there was 

### 17 some information from egg injection studies that indicated that 

### 18 vinclozolin would potentially have activity. We knew that it 

### 19 was an androgen antagonist and so there was some consideration. 

### 20 Well we have already looked at an estrogen and a model of 

### 21 androgen of sorts. And given the cost and time associated with 

### 22 these tests, we did not really have a lot of time to do multiple 

### 23 chemicals, so the context of what might be an appropriate one to 

### 24 utilize. 

### 25 We looked at the vinclozolin and recognized 

### 26 that, well, we were also using vinclozolin in terms of some of

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1 the other Tier 2 tests, so we felt that would be useful in terms 

### 2 of comparison. It was really somewhat unknown how this would 

### 3 play out in the test. We knew that it potentially would be 

### 4 challenging compound because vinclozolin also requires a 

### 5 metabolic step to go through, since there are some metabolites 

### 6 that are somewhat more active as the anti-androgen type 

### 7 responses. So we felt that would be useful too, since the birds 

### 8 have somewhat different metabolism, or at least they have a 

### 9 higher metabolic rate than other species. So we felt, well this 

### 10 one should be useful. 

### 11 And in the context of the test, based upon 

### 12 evaluating the trenbolone and ensulfan results was we felt that 

### 13 the test could be performed fairly easily in terms of being 

### 14 successful. And we felt that any effects that would be seen, we 

### 15 would be able to quantify those, and so we selected that 

### 16 compound on that kind of bases and the results were what we got. 

### 17 I think that in comparing across controls 

### 18 gives us indication that the method is reasonably reproducible 

### 19 in that context in terms of the effects seen, we cannot say so 

### 20 much about that in context since vinclozolin -- the only 

### 21 consistent response was this histo-effect in the epididymides, 

### 22 so that was consistent in that context. 

### 23 So in terms of quantitative comparison for 

### 24 this particular method and how the, say, a NOEC might be 

### 25 observed, we would rely upon the trenbolone results to 

### 26 demonstrate that with the USGS and the Army, that the NOEC level

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1 would have been identified at roughly the same concentration in 

### 2 that context. 

### 3 **DR. MARTHA SANDY:**Dr. Ehrich? 

### 4 **DR. MARION EHRICH:**You mentioned the egg 

### 5 injection test and sort of like you dropped it. And I am just 

### 6 asking for clarification. That is just out of the picture, 

### 7 right? 

### 8 **DR. LESLIE TOUART:**Yes, that is correct. I 

### 9 mean the egg injection was done early on because we felt we 

### 10 could look at some different endocrine activities more quickly. 

### 11 But the other driver in terms of why we entertained it at all 

### 12 was that, potentially, this could be useful as a range-finding 

### 13 if we were able to figure out a way to convert those injection 

### 14 concentrations into some dietary level so that that would save 

### 15 time and animals in doing range-finding to establish the 

### 16 concentrations that would go into a two-generation test later 

### 17 on. 

### 18 But we did not realize that particular 

### 19 advantage so the egg injection studies were dropped in terms of 

### 20 any continuation, so they are not part of the guideline in terms 

### 21 of potential range-finding exercise. And they really returned 

### 22 more towards an academic level test to evaluate different 

### 23 effects and how they might manifest later. And there are public 

### 24 studies that are referenced within the document to demonstrate 

### 25 some of those kinds of outcomes. 

### 26 **DR. MARTHA SANDY:**Dr. Johnson?

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1 **DR. DALLAS JOHNSON:**You have indicated that 

### 2 there were differences in the strains of Japanese quail that 

### 3 were used, and I am just wondering how those strains varied 

### 4 across laboratories. Did different laboratories use a 

### 5 particular strain or were the strains sort of selected and 

### 6 assigned to different laboratories, or questions about how 

### 7 randomization took place and so on, things that we statisticians 

### 8 worry about? 

### 9 **DR. LESLIE TOUART:**Right, just to respond, 

### 10 there were no specific directions given to the laboratories in 

### 11 terms of selecting strains. That was left to the particular 

### 12 laboratories, and so they would collect the animals that were 

### 13 available from whatever supplier that they may have. 

### 14 I believe one of our contract laboratories 

### 15 maintains somewhat of an in-house culture, so they maintained an 

### 16 in-stock crew. But for the others, they were going outside and 

### 17 it was just what was available at the time providing some 

### 18 different strains. I do not recollect specifically which labs 

### 19 had what particular source of birds that were utilized. 

### 20 I am also trying to remember -- and I can find 

### 21 this information out for later -- whether like for the Army they 

### 22 had done the endosulfan and the trenbolone test, whether for 

### 23 both of those tests, it was the same strain utilized or whether 

### 24 they had a different strain. I do not recall at current. But I 

### 25 believe there were, again, strain differences through the 

### 26 laboratories, but they were not specified or they were not

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1 established in any kind of context to evaluate what those were. 

### 2 In looking at the different strains and the 

### 3 differences in terms of the actual size of the individuals, some 

### 4 of the responses endpoints in terms of what the actual measure 

### 5 was, but in terms of comparisons and especially in terms of, 

### 6 say, the egg production that was seen. Those still were 

### 7 comparable between the different strains to indicate that they 

### 8 did not make that much of a difference in context. But how much 

### 9 that strain difference might make, understanding how strain 

### 10 differences affect toxicity in other taxa groups, one would 

### 11 anticipate, yeah, that is a potential factor that could affect 

### 12 the outcomes and one would have to pay attention. But there is 

### 13 no information to suggest one strain being more sensitive than 

### 14 the other or more susceptible for certain kinds of activities. 

### 15 **DR. MARTHA SANDY:**Dr. Colin Scanes? 

### 16 **DR. COLIN SCANES:**A couple of questions for 

### 17 you. First of all, at the beginning you mentioned that the 

### 18 current avian protocol is not sufficiently robust. Could you 

### 19 provide a little bit of clarification about that? And secondly, 

### 20 when you look at the trenbolone studies and the vinclozolin 

### 21 studies, there is a different bases for the dose-response scope. 

### 22 One is logged to the base 2, one is to the base 2.5. And I 

### 23 think the third study in the report, which is logged to the base 

### 24 3. What is the thinking as to what log-base should the 

### 25 dose-response curve be done at? 

### 26 **DR. LESLIE TOUART:**Could you repeat the first

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1 question again? 

### 2 **DR. COLIN SCANES:**The first question was 

### 3 about the current avian protocol is not sufficiently robust, and 

### 4 then you did not really give any more information. So if you 

### 5 could provide a little bit more clarity on that. 

### 6 **DR. LESLIE TOUART:**Right now the existing 

### 7 avian product tests that are done, long-term test are bobwhite 

### 8 quail or mallard duck reproduction test and those tests are 

### 9 exposed prior to the onset of egg laying. 

### 10 So the test has continued treatment through 

### 11 egg-laying for several weeks while the eggs are collected and 

### 12 the hatchlings are produced, and they carry these two 14-day 

### 13 hatchlings. So there is just that single partial generation of 

### 14 a maturing bird and adult reproduction, the egg and embryo 

### 15 survival in 14-day chicks, there is no histopathology, there is 

### 16 no biochemical that is measured as a routine course, so it is a 

### 17 subset, I think, of the range of life-stages that would exist. 

### 18 So that is the context of why it is felt that it is not 

### 19 necessarily as comprehensive in terms of what is looked at. 

### 20 Now to respond to your second question, which 

### 21 I am not sure I can at the present, in terms of why there were a 

### 22 different log-based that were utilized for the comparisons, I am 

### 23 not sure of that. It is something that I would have to go back 

### 24 and look at. It may just be the way the data were presented but 

### 25 I would have to investigate to determine why that may be the 

### 26 same.

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1 I do know that in terms of how different 

### 2 laboratories recorded information, they sometimes use some 

### 3 different reference point or how the time course for when the 

### 4 eggs were collected are counted. But in terms of the log-bases 

### 5 for the evaluation, I have to check and see what that was. 

### 6 **DR. MARTHA SANDY:**Dr. Bentley? 

### 7 **DR. GEORGE BENTLEY:**You mentioned large 

### 8 variations in mating behavior. I wonder if you could clarify 

### 9 which aspects of behavior were measured, because measuring 

### 10 behavior is quite complex. 

### 11 **DR. LESLIE TOUART:**And what I said was that 

### 12 the mating in the Japanese quail in terms of the information 

### 13 that was collected, it was based on mating behavior, meaning the 

### 14 time to (inaudible) birds are brought together, how soon the 

### 15 male mounts the female; so that time period would be measured as 

### 16 one indication. And then the number of mounts over a time 

### 17 period would be identified, so how often the male may mount the 

### 18 female would be observed. 

### 19 So that is pretty much the extent of what 

### 20 would be evaluated. And across the different laboratories in 

### 21 terms of how the birds were handled before being brought into 

### 22 that mating condition in terms of the birds being brought 

### 23 together for the first time and how they had been kept separate 

### 24 prior to that and what socialization that may impact how well 

### 25 they have done in context. 

### 26 I think in the reports, if I am not mistaken,

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1 I believe it was the Army's trenbolone study or the mating 

### 2 behavior was evaluated, there was sufficient observation to be 

### 3 able to determine an actual effect occurring there, but the 

### 4 other laboratories that attempted to make these evaluations were 

### 5 not able to measure anything because of the high variability 

### 6 across those, so there was no ability to discern what might be 

### 7 going on. 

### 8 So the concern is that, well, behavior is 

### 9 something that may be affected by the endocrine, especially in 

### 10 birds, because that is a critical element. And so to the extent 

### 11 that we can exploit some aspect of Japanese quail would be 

### 12 useful in trying to do so. So that is the context. 

### 13 **DR. GEORGE BENTLEY:**So the focus was on 

### 14 consumatory mating behavior versus repetitive. And also the 

### 15 focus was on male sexual behavior, and there was no measure of 

### 16 female interest in the male? 

### 17 **DR. LESLIE TOUART:**That is correct. There 

### 18 may be some aggression shown. Sometimes the female would be 

### 19 more aggressive to the male, depending upon the situation, but 

### 20 most often the measures here would be somewhat more male 

### 21 specific. 

### 22 **DR. MARTHA SANDY:**Dr. Propper? 

### 23 **DR. CATHERINE PROPPER:**So along those lines 

### 24 one of the other areas where you identified a lot of variability 

### 25 across the control groups especially was in reproduction in the 

### 26 table on slide 44. And I am wondering what went into that value

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1 as reproduction. 

### 2 **DR. LESLIE TOUART:**In terms of the 

### 3 reproduction value in terms of the measure, that was based on 

### 4 the number of egg laid per hen, so that reproduction measure is 

### 5 eggs laid. It did not include like fertility measures 

### 6 associated with that. It was primarily the variability around 

### 7 the eggs laid per hen. 

### 8 **DR. CATHERINE PROPPER:**Okay. And as I recall 

### 9 with the vinclozolin study, there was no difference across 

### 10 treatments in the number eggs laid but there was a huge amount 

### 11 of variability then across labs in the control groups? I am 

### 12 trying to put together the summary data for eggs laid and the 

### 13 variability and the controls that we are seeing in slide 44. 

### 14 **DR. LESLIE TOUART:**Let me see if I can go 

### 15 back. I am just going to try and go back to slide 44 so I can 

### 16 see. Now which were you identifying? 

### 17 **DR. CATHERINE PROPPER:**Under Reproduction, 

### 18 which is number of eggs laid. 

### 19 **DR. LESLIE TOUART:**Right. The power to 

### 20 detect that effect was 22 -- the level of effect that one is 

### 21 able to differentiate was at 80 percent power from control, is 

### 22 22 percent here. So I am not sure of what your question is in 

### 23 terms of the variability across the species. Like, 17 percent 

### 24 in the initial generation in 22 here -- and this is across all 

### 25 three laboratories that were part of the inter-lab evaluation, 

### 26 so the variability would have been fairly small.

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1 And I go back to the previous slide; this is 

### 2 the one that looks at the actual CV percent with the range and 

### 3 the median. And for reproduction, the median is 10 percent and 

### 4 9 percent. The range was what was large from .54 to 103 across 

### 5 those. But again, the median was fairly small, so there were 

### 6 outliers, meaning there were likely a pair that was 

### 7 nonproductive or minimally productive in context. So one pair, 

### 8 given that we are only dealing with maybe 12 pairs optimally, 

### 9 that has a big influence on the ultimate CV value that one might 

### 10 have from that. But in terms of the median response across 

### 11 those, it is somewhat narrow. 

### 12 **DR. CATHERINE PROPPER:**Okay. Thank you. 

### 13 **DR. MARTHA SANDY:**Dr. Denver? 

### 14 **DR. ROBERT DENVER:**Thank you for the 

### 15 presentation. In the interest of understanding endocrine modes 

### 16 of action using the Tier 2 assays predicted from the Tier 1 

### 17 assays, can you comment on whether any of the data allow for a 

### 18 clear diagnoses of an endocrine mode of action that fits within 

### 19 the known or predicted modes of action? For example, the 

### 20 trenbolone clearly affected plasma estradiol in the males, but I 

### 21 think it had no affect on aromatase. So I mean how do we 

### 22 interpret that kind of result? 

### 23 **DR. LESLIE TOUART:**Thanks for that question, 

### 24 I think. I think it is a difficult kind of thing when you get 

### 25 into the Tier 2 because these are designed to be comprehensive 

### 26 and we are including apical level endpoints that are -- being

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1 apical, you know, they are a summary of a lot of other things 

### 2 that are going on. 

### 3 In terms of our ability to interpret what 

### 4 endocrine manifestations there are, there would be some 

### 5 endpoints that are a little bit more, I guess you would say 

### 6 diagnostic or at least endocrine-relevant than others, meaning 

### 7 like the hormone measures that one would have or some 

### 8 histopathology that one would see. 

### 9 But clearly, other endpoints, which would be 

### 10 the growth or reproductive which are the important apical 

### 11 endpoints from a risk assessment context, these do not tell us 

### 12 very much in terms of whether that is strictly through an 

### 13 endocrine mechanism or secondary to some other systemic response 

### 14 that may be occurring. So we would be evaluating the additional 

### 15 information that we have based upon the Tier 1 outcomes in terms 

### 16 of any initiating event or indications that it may be operating 

### 17 through an estrogen pathway more than an androgen pathway or 

### 18 not. So I think that is probably the best I can try to 

### 19 characterize. 

### 20 Because of the number of endpoints that we 

### 21 have, there is somewhat of a pattern that would be evaluated and 

### 22 you would want to try to interpret that in context. But I do 

### 23 not want to suggest that there are easy clear-cut answers that 

### 24 this is indeed the activity or that it is definitively 

### 25 androgen-active or not. 

### 26 For a lot of these materials, there can be

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1 multiple areas it can go in and there can be somewhat 

### 2 conflicting information. And that is going to be a challenge in 

### 3 interpretation because, given the large number of endpoints and 

### 4 that there likely could be some that are statistically 

### 5 significant just by chance, trying to interpret is that "by 

### 6 chance" or is there some way of connecting that to real outcome 

### 7 is a consideration. So hopefully you can provide some 

### 8 additional context that we can use in assisting in the 

### 9 interpretation. 

### 10 But our ability to identify kind of a 

### 11 point-of-departure or like a no-observed or low-observed 

### 12 concentration or some adverse effect is really the context that 

### 13 would be carried forward in terms of interpreting what the 

### 14 effect of a material might be. 

### 15 **DR. MARTHA SANDY:**Dr. McManaman? 

### 16 **DR. JAMES MCMANAMAN:**Thank you for the 

### 17 presentation. So I have a couple of questions just for 

### 18 clarification. On slide 37, I thought you said that there was 

### 19 no statistical difference in egg production in the F2 generation 

### 20 at 20 ppm on trenbolone. But is that true, because it looks 

### 21 like there should be -- in the F2s? So on the bottom panel, the 

### 22 light blue bar, it looks like it is much lower than the other, 

### 23 the F2 generation. 

### 24 **DR. LESLIE TOUART:**Yes. In the F2, the 20 

### 25 ppm still had a significant effect. 

### 26 **DR. JAMES MCMANAMAN:**It was significant.

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1 Okay. 

### 2 **DR. LESLIE TOUART:**I think what I was 

### 3 mentioning was that that seemed to sustained into the F2. You 

### 4 know these are untreated. I think what might have been 

### 5 confusing is I was talking about the other effects. These, you 

### 6 know, there appear to be no trend associated with these other 

### 7 concentrations, whereas in the parental and the F1 generations, 

### 8 there appears to be (inaudible); these were not statistically 

### 9 significant, but at least looking at the bars, there is a trend. 

### 10 But in the F2s, there is not. But the 20 ppm, there was 

### 11 effect. 

### 12 **DR. JAMES MCMANAMAN:**There was at 20 ppm, 

### 13 okay. And then the next slide, just a question about 

### 14 interpretation here. If you look at the effect on egg 

### 15 production at the 20 ppm and compare that at the 10 -- so for 

### 16 the F0 and the F1, it looks like there is statistical 

### 17 differences at the 10 and 20 ppm doses for the F0 and the F1. 

### 18 But the magnitude of the effect at the 20 ppm looks to be much 

### 19 greater than one would expect from a simple dose relationship. 

### 20 I mean it looks like there might be 

### 21 sensitization going on and I just had a question about what is 

### 22 your interpretation of that and has that question been built 

### 23 into the apical endpoint analyses? 

### 24 **DR. LESLIE TOUART:**I will try to explain. I 

### 25 do not know that it is necessarily built-in. And this was a 

### 26 question in terms of the F1. The magnitude of that difference

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1 was much larger there and whether that is due to maternal 

### 2 transfer of the trenbolone and the effect that that might have 

### 3 on the individual later is possible. And it is not just within 

### 4 the bird studies, but I think it is what is seen with the other 

### 5 taxa as well. And it is something that we would want to be 

### 6 careful in our interpretations of is that, oftentimes where 

### 7 there are effects -- especially if you have a major effect in 

### 8 terms of something like reproduction, and you have got fewer 

### 9 individuals that are carried into the next generation, those 

### 10 individuals might be less sensitive than the original generation 

### 11 because they are the ones that have demonstrated -- they have 

### 12 survived enough to be able to get there. So we have to be 

### 13 careful for those situations where you see a reduced effect in 

### 14 the subsequent generation as much as if you have an enhanced 

### 15 effect in the next generation. 

### 16 So trying to evaluate to determine is that a 

### 17 simple linear additive response into the next generation or 

### 18 whether there is something that is modified may be more 

### 19 exponentially different between one and the other would need to 

### 20 be considered. And one can evaluate generational effects that 

### 21 may occur. 

### 22 In the case of the birds, there was not enough 

### 23 chemicals to be able to evaluate in context of the generational 

### 24 effects that might occur. I think later today when we talk 

### 25 about the invertebrate side where we did have many more of those 

### 26 kinds of situations, the generational effects were evaluated

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1 statistically to determine how much one generation differs from 

### 2 the other generation kinds of thing. 

### 3 But here that was not the case, and it is 

### 4 something that one does have to pay attention to and consider in 

### 5 terms of the interpretation, you know, whether it is really a 

### 6 generational effect or whether there is no difference between 

### 7 what might have occurred or not. And I think when you are 

### 8 dealing with the oviparous animals because of the embryo 

### 9 exposure is so dependent upon the material exposure and 

### 10 transfer, it is important to recognize how that then ultimately 

### 11 manifests. 

### 12 **DR. JAMES MCMANAMAN:**And then the final 

### 13 question is what will be evaluated on the F2 -- I was not clear 

### 14 about that -- on the F2 generation? And with that, are you 

### 15 planning on including positive controls for reproductive effects 

### 16 in the assays themselves? 

### 17 **DR. LESLIE TOUART:**To that last question, 

### 18 there would be no positive controls added to these tests. They 

### 19 are logically difficult as they are just with the compounds that 

### 20 would be evaluated. But in terms of the F2s, the way the 

### 21 guideline as we are proposing it, it really just takes the F2 

### 22 chicks to 14 days with an option to continue if you have 

### 23 identified some maturation effects that you may want to carry 

### 24 forward. 

### 25 As I mentioned for the validation exercise, 

### 26 the F2s were carried longer into maturation and actual

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1 reproduction so we do get some numbers from those. And that 

### 2 becomes a question of, well, should the test continue to that 

### 3 point for those potentials to occur, or in terms of our 

### 4 interpretation, the level of effect that had been seen in, say, 

### 5 the F1s would be sufficient to identify what your level of 

### 6 concern of point of departure would be in terms of how that 

### 7 would feed into somewhat of a risk assessment. 

### 8 But just to reiterate, the F2s are intended 

### 9 only to evaluate the chicks and the F2s are untreated at this 

### 10 point. So we are just looking at the hatching success and 

### 11 viability of the chicks with the possible option of carrying 

### 12 that into maturation observation. 

### 13 **DR. JAMES MCMANAMAN:**Okay. So based on the 

### 14 USGS data showing an F2 effect on egg-laying, you think that you 

### 15 will be able to discern potential effects? Because you will not 

### 16 be looking at F2, as I understand what you have just explained 

### 17 to me about the F2 assay. So doing it that way would eliminate 

### 18 what the USGS found with theirs? 

### 19 **DR. LESLIE TOUART:**Right. I think it was 

### 20 based on those observations that we felt that the concentration 

### 21 that pose an effect were seen in the F1s, so we would be able to 

### 22 identify the NOEC in terms of what that would carry on. 

### 23 So whether that would be additional 

### 24 information that would be of value and worth the additional 

### 25 effort in terms of length of test and collecting that additional 

### 26 information in terms of the reproduction of those individuals,

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1 if that is necessary for something like a population model, that 

### 2 would be in question. But the general sense was we felt that 

### 3 there was enough information to be able to have confidence that 

### 4 the level of concern based upon the lowest observed effect 

### 5 concentration would not have changed from the F2 versus the F1 

### 6 generation. 

### 7 **DR. MARTHA SANDY:**I think we are ready to 

### 8 move on. Thank you very much. We will move from quail to fish, 

### 9 so we will now hear from Dr. Rodney Johnson. 

### 10 **DR. RODNEY JOHNSON:**Hello. I am Rodney 

### 11 Johnson and I am pleased to be here to present this information. 

### 12 And I think for expedience, we will just get started. I am 

### 13 with the Mid-Continent Ecology Division of ORD of EPA and I will 

### 14 be presenting the fish multi-generation toxicity test 

### 15 information. 

### 16 First of all, just to give you a real quick 

### 17 overview of what the presentation is intended to do, I will 

### 18 first of all introduce the Research Medaka Multigeneration Test 

### 19 protocol. And then I am going to go through and review some of 

### 20 the Medaka multigeneration test data describing some of the 

### 21 control outcomes, chemical control data and some inter-lab 

### 22 outcomes. And then from there I am going to introduce a test 

### 23 that we are proposing as a recommended Tier 2 test, fish test 

### 24 which is a subset of the Medaka multigeneration test. We call 

### 25 it the Medaka reproduction test. 

### 26 And after I present that information, I will

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1 present some conclusions and then we will go into conversation 

### 2 about implementation of a test protocol. 

### 3 First of all, I would like to review the 

### 4 purpose of the Tier 2 fish test. I am going to just very 

### 5 quickly read it. We have all read this if we read the summary 

### 6 reports, but in any case, it is to determine whether a test 

### 7 substance adversely affects a test organism through 

### 8 endocrine-mediated pathways. It is to quantify those effects 

### 9 with respect to the estrogen, androgen and thyroid systems. It 

### 10 is to include exposure during potential life-stages that may be 

### 11 more sensitive than those that were tested in Tier 1. It is to 

### 12 characterize the dose-response relationship and it is intended 

### 13 to include exposure during gametogenesis to allow for potential 

### 14 effect of parental transfer of chemical and other endogenous 

### 15 factors across generations. 

### 16 I would like to discuss why was Medaka chose 

### 17 as the test species. First of all, it is a small fish. In 

### 18 fact, it tends to be quite a bit smaller than, often, some of 

### 19 the fish that are used in environmental testing paradigms. It 

### 20 is generally about 300 milligrams or up to 500 milligrams in 

### 21 adult size. Small size allows them to be exposed and tested in 

### 22 relatively small aquaria which then, because of that, also have 

### 23 relatively smaller amounts of test chemical to use in the test 

### 24 protocol. 

### 25 They are sexually dimorphic, so the phenotype 

### 26 is expressed dimorphically fairly easily in young adult through

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1 adult fish. 

### 2 One important aspect that we chose for Medaka 

### 3 was that the sex-determining gene for Medaka has been identified 

### 4 and was identified some years ago by the Japanese, and we use 

### 5 that to our advantage insofar as identifying the genetic gender 

### 6 of each fish in the test protocol, so that we can look at 

### 7 relating what would be the expressed phenotype with the gender 

### 8 of each organism. 

### 9 It has a relatively short life-cycle. That 

### 10 short life-cycle allows for keeping the test reasonably shorter 

### 11 than it would be if it had fish with longer life-cycles. That 

### 12 being said, it is not exactly really short either. 

### 13 The fish is cultured easily. It is fairly 

### 14 hardy, it is fairly disease-resistant, and its basic biology has 

### 15 been well characterized, especially by Japanese scientists. It 

### 16 is a fairly well understood fish. 

### 17 Some of the work that we have done is in 

### 18 cooperation with the Japanese. The fish is a Southeast Asian 

### 19 fish. The Japanese has extensive experience understanding its 

### 20 biology, studying its biology, studying its culture, and we have 

### 21 capitalized on that information and cooperated with them to try 

### 22 and develop protocols for testing EDCs that would be harmonized 

### 23 at least across the two countries, but also more globally than 

### 24 that. 

### 25 And we did not start this work without some 

### 26 previous work having been done by the Japanese. The Japanese

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1 had, at the beginning of our work, had already developed a full 

### 2 life-cycle fish test with the Medaka. And that full life-cycle 

### 3 test did not allow one to test for transgenerational effects, so 

### 4 we used that as the beginning point but we added other 

### 5 generations so that we could look for effects across 

### 6 generations. 

### 7 But the Japanese had developed all the 

### 8 baseline endpoints or most of the baseline endpoints that are 

### 9 employed. They certainly had developed good methods for 

### 10 breeding and culture, and information on how to optimize growth, 

### 11 and they also had good information regarding gonadal development 

### 12 timelines. So that gave us a good kick-start when we began this 

### 13 work. 

### 14 I am going to discuss what we are calling the 

### 15 Research MMT Protocol. The Research MMT Protocol was intended 

### 16 to be a protocol from which Tier 2 test could be derived. It 

### 17 includes three generations but not three complete generations. 

### 18 It starts out with an F0 generation of adults that are exposed. 

### 19 The embryos from that are used to begin the F1 generation. The 

### 20 F1 generation then goes through hatch development and the 

### 21 adulthood. And then from that, the F2 generation is derived, 

### 22 and that too then is taken, at least in the original MMT 

### 23 protocols, all the way through reproduction. 

### 24 The test took 29 weeks in most of the 

### 25 iterations that we did. Some of this was variable. This is 

### 26 like what the very first research protocol was. As we learned

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1 things and as we worked with the Japanese, these protocols are 

### 2 not exactly identical between tests because we took the 

### 3 information derived to change the protocol to try and improve 

### 4 it. 

### 5 So for instance, you will see here that -- 

### 6 well F1 and F2 are in fact what we would call rubber stamps of 

### 7 each other. They are intended to be, as much as possible, 

### 8 identical, or all the endpoints are measured at the same time in 

### 9 both generations. And both generations were exposed for their 

### 10 full life history. 

### 11 The generations overlapped by three weeks. 

### 12 That was intended to try and reduce the total duration of the 

### 13 test. One of the consequences of that is that it took quite a 

### 14 bit more infrastructure to hold one generation while you were 

### 15 starting the next generation. We worked with the Japanese on 

### 16 how to do that. They did not like that, so we have some of the 

### 17 later tests; the overlap was not three weeks, it was one week, 

### 18 so that you did not have to have two full exposure systems going 

### 19 at one time to keep the two generations going. 

### 20 So what I am really getting at is this is our 

### 21 research protocol for which we intended to try and derive a Tier 

### 22 2 protocol. It measures lots and lots of endpoints across all 

### 23 generations. So we can see that in reproduction, I just want to 

### 24 emphasize, was a five-week assessment in each generation where 

### 25 the egg production -- the fecundity in this case -- that is what 

### 26 we are calling fecundity -- and the fertility were evaluated

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1 four days per week across five weeks for 20 measurements. So 

### 2 from Tuesday through Friday of each week, it was assessed. 

### 3 So there was pathology assessed in each 

### 4 generation, and the other AOP endpoints like vitellogenin, the 

### 5 secondary sex characters, were all again all measured at each 

### 6 generation with the intention to make inter-generational 

### 7 comparisons. 

### 8 I want to mention this one part about the test 

### 9 protocol that we think is a very, very strong attribute for the 

### 10 protocol, and that is that the genetic sex of each fish is used 

### 11 in the protocol to set up breeding pairs. 

### 12 As we know, when animals grow up in an 

### 13 androgen or an estrogen fish, a certain concentration sex 

### 14 reversal can often occur, and one cannot identify which fish was 

### 15 reversed unless you know the genotype. 

### 16 So to set up, breeding pairs and all those 

### 17 sorts of aspects of the actual conducting the test, as well as 

### 18 to evaluate the endpoints of the test, the genetic sex of the 

### 19 fish is determined in real time during the test. So the 

### 20 animals' genetic sex is determine nondestructively by taking a 

### 21 small fin clip -- anesthetizing the fish -- taking a small fin 

### 22 clip and then returning the fish to the tank and then doing PCR 

### 23 to identify the presence of the gene called DMY, which is the 

### 24 sex determining gene for Medaka. 

### 25 So all the fish that have DMY are genetic 

### 26 males. All the fish that do not have DMY are genetic females.

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1 So all outcomes in the test are measured against that particular 

### 2 individual's genetic sex. Because it is so important to the 

### 3 test, we feel any test laboratory has to be capable of doing 

### 4 that part of the test to adequately perform the test. 

### 5 So now I am going to just review the endpoints 

### 6 in the multi-generation test. I have organized them by what we 

### 7 would consider apical endpoints, reproduction. And fecundity, 

### 8 which consists of fecundity and fertility -- in this case 

### 9 fecundity again is total eggs laid by the female. And then of 

### 10 course fertility is the number of fertile eggs laid by the pair, 

### 11 embryos produced. 

### 12 We also histologically determined sex 

### 13 reversal, again, comparing against the genetic sex of the 

### 14 animals, and intersex conditions. 

### 15 Now I did not say it here but obviously growth 

### 16 is an important endpoint, but I don't consider it in general to 

### 17 be an endpoint that we would consider necessarily to be involved 

### 18 with endocrine action. I mean I know that is a generalization 

### 19 that may not be true, but the most important thing is we 

### 20 consider growth effects to be most important in the context of 

### 21 toxic effects. 

### 22 So the idea is that we evaluate whether or not 

### 23 some kind of an impact is perhaps due to a non-endocrine 

### 24 mediated AOP that results in growth impairment, and it sort of 

### 25 raises a flag that there is toxicity overlaid perhaps on 

### 26 endocrine activities or reductions in fecundity, for instance,

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1 or would be due to a toxic AOP independent of an endocrine AOP. 

### 2 We also have what we would call biomarker AOP 

### 3 endpoints. Almost all of these, although not entirely all of 

### 4 these, are taken at the sub-adult stage of the bioassay at the 

### 5 same time that we would prepare or set up breeding pairs. So 

### 6 that when we set up breeding pairs, we setup the breeding pairs 

### 7 and then the animals that are left are sampled for these AOP 

### 8 biomarker endpoints, which includes secondary sex characters, 

### 9 which is primarily in this case, the anal fin of the fish, which 

### 10 in the male has papillary processes. So in the phenotype of the 

### 11 male in the secondary sex is to have papillae on the anal fin 

### 12 that can be counted. You can just simply count how many there 

### 13 are on the anal fin. 

### 14 We also measure the level of liver 

### 15 vitellogenin mRNA there is in each fish, and that is done at a 

### 16 PCR reaction. And then we also do gonadal pathology in this 

### 17 MMT. And in this case, the primary gonadal pathology was 

### 18 isolated to the kidney and to the gonad itself. And it was 

### 19 primarily looking for gonadal phenotypes, things like 

### 20 sex-reversals, intersex conditions, but for instance, not other 

### 21 tissue pathology. So it did not include liver in all cases, 

### 22 although in some cases it did. It does not include spleen. I 

### 23 mean lots of other tissues are not involved in that. 

### 24 Then the last endpoints I want to discuss will 

### 25 be these toxicity endpoints where we have hatching success, 

### 26 growth as I mentioned earlier, survival at several points of the

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1 test, but the most important survival endpoints actually occur 

### 2 in our experience very early in the test, so primarily early 

### 3 life-stage survival. 

### 4 Then liver pathology; and liver pathology in 

### 5 this case is intended primarily to indicate, as I said, toxicity 

### 6 as distinct from an endocrine type endpoint. So for instance, 

### 7 if vitellogenin goes down and there is liver pathology and there 

### 8 was an effect on growth, you would be very suspicious that this 

### 9 particular toxicant was acting through a toxic mode of action 

### 10 and not an endocrine mode of action. 

### 11 I will just review the histology endpoints -- 

### 12 well not really the endpoints very much. But what I really want 

### 13 to say is that we have also developed guidance for doing 

### 14 histopathology, and this guidance is still being developed in 

### 15 the online form of it. There are paper versions of this, but we 

### 16 feel an online version is very, very useful, amongst other 

### 17 things, because it provides guidance to any pathologist that 

### 18 wishes to be engaged in this process. It also provides an 

### 19 opportunity for consistency in pathology evaluation in different 

### 20 pathology laboratories. 

### 21 So this guidance provides SOPs for doing 

### 22 necropsy, histotec work, data structure, literally how to handle 

### 23 data and organize data as pathology is being done. So it allows 

### 24 efficient statistical analysis. There are some diagnoses of the 

### 25 pathology that you would expect and then there are examples of 

### 26 images that are annotated that show normal histology of tissues.

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1 2 For instance, this is a normal testis in a 

### 3 controlled medaka. And this for instance is a testis that has 

### 4 some testicular oocytes in it. And this would be what we would 

### 5 consider a severity score of two, and this is the same diagnoses 

### 6 with a severity score of three. So the point being that you can 

### 7 develop consistency in how you would score a pathology outcome 

### 8 by using this tool. First of all, it gives you the diagnostic 

### 9 character, and then it also provides you with a uniformed 

### 10 scorning tool. 

### 11 The intention in this case also is that this 

### 12 be able to be updated online so that as information accrues on 

### 13 effects, that it can be entered into this guidance and the 

### 14 guidance can become more mature. 

### 15 And I mentioned about liver pathology as an 

### 16 indication of toxicity. This is just a panel that shows what 

### 17 happens to the liver exposed to a chemical called dichloroacetic 

### 18 acid, which in this case did reduce the fecundity and the growth 

### 19 of the fish that were exposed to it. We did not measure 

### 20 vitellogenin in this case, but as you go from control to higher 

### 21 concentrations, the liver gets increasingly damaged due to the 

### 22 exposures; another example then of toxicity and not endocrine 

### 23 activity. 

### 24 One can identify limited endocrine activity in 

### 25 the liver also, but it tends to be not as sensitive as some of 

### 26 the other endpoints.

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1 So the diagnostic endpoints that we have found 

### 2 useful in the MMT -- so what we would call primary EDC diagnoses 

### 3 in the testis would be the intersex condition and sex reversal, 

### 4 so in this case intersex being what we would call the testicular 

### 5 oocytes with various severity scores. And then the worse 

### 6 condition are, let's say, once when the exposure is high enough 

### 7 and it is done early enough in the developmental stages of the 

### 8 animal, instead of getting an intersex condition, you end up 

### 9 with a sex reversed animals. 

### 10 In the ovary, the same. Sex reversal is an 

### 11 endpoint that we identify. We also see increases in oocyte 

### 12 atresia, decreases in the yolk formation within each egg, and 

### 13 then we also see what is called perifollicular cell hypertrophy 

### 14 in response to this steroidogenesis inhibitors. And then -- I 

### 15 am beating a dead horse here -- we also look at liver, 

### 16 especially as a diagnostic endpoint for toxicity. 

### 17 I want to go through now some of the typical 

### 18 control values that we get in an MMT, and this data is compiled 

### 19 from a number of MMTs, and it was also compiled in some cases 

### 20 with fish that were sub-adults. And as I mentioned earlier, the 

### 21 tests that we are comparing, in some cases, the sub-adult 

### 22 sampling stage occurred at different times within their -- 

### 23 sometimes it was seven weeks, sometimes it was eight weeks, 

### 24 sometimes it was nine weeks as we were developing these tests. 

### 25 Consequently, some of the variance that you 

### 26 see is not necessarily as high as it looks because of the time

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1 course difference in when they were measured. But I just want 

### 2 to have some representative information for you to look at that 

### 3 allows you to evaluate as the test is progressing in some cases 

### 4 whether the test is on track. So let's just go through a few 

### 5 things. 

### 6 The typical fecundity output for adults is 

### 7 around 25. It certainly in some cases is higher than that. 

### 8 This is by 25 eggs per pair per day. And we consider an 

### 9 acceptable range to be something greater than 20 at this stage. 

### 10 Fertilities, control values tend to be 80 

### 11 percent and higher. We consider values of 75 percent and higher 

### 12 as acceptable. 

### 13 Sex reversal also happens in control animals, 

### 14 and it happens in what would be the XX genotype. And in the XX 

### 15 genotype, there is a relatively low prevalence, but it seems to 

### 16 be relatively constant of less than four percent in I would say 

### 17 most cultures of fish we see at being less than two percent, but 

### 18 it gets pretty hard to evaluate what that percentage really is 

### 19 when it is down at that level. It takes a lot of animals to 

### 20 actually put a good number on that. 

### 21 So what we are basically saying is we would 

### 22 prefer that labs actually check their cultures and be sure that 

### 23 the prevalence of this in their cultures is less than five 

### 24 percent. 

### 25 And then the intersex condition seems to be 

### 26 actually less prevalent. And I would say, in most cases, we see

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1 it fairly rarely and put our proportion of that at less than two 

### 2 percent. So these biomarker endpoints, we have secondary sex 

### 3 characters, which I mentioned before. In a sub-adult fish, 

### 4 meaning before it is like 10 weeks old, typical numbers are 70. 

### 5 We have seen them in some cases to be quite a bit lower than 

### 6 that in other laboratories, so to be conservative, we feel like, 

### 7 in this case an acceptable range is greater than 20. That would 

### 8 be number of papillae on the anal fin of a male at seven weeks 

### 9 and older. But we like to measure it now. We are proposing 

### 10 that it be measured at nine weeks, in which case, I think in 

### 11 most cases it tends to be quite a bit larger than 20. 

### 12 Vitellogenin mRNA numbers: These are measured 

### 13 in both the males and in the females. And the thing to note is 

### 14 that this number to some extent is defined by your PCR method. 

### 15 And to tell you what an absolute value of what that ought to be 

### 16 is, we feel probably not appropriate, but we certainly can tell 

### 17 you that in the males there is vitellogenin mRNA, so it is not 

### 18 zero. And in the females, it is quite a bit higher; three 

### 19 orders of magnitude tends to be. And probably more importantly, 

### 20 within any particular study, the ratio of male to female mRNA 

### 21 copy number in your control should be several hundred fold. 

### 22 So then this would talk about toxicity hatch 

### 23 success. In the embryos, we feel it tends to be around 80 

### 24 percent or greater, and we feel that an acceptable range is 

### 25 about 70 percent as kind of a minimum. Growth, once again, 

### 26 depending on the time exactly, but we do feel that growth is

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1 actually very important to getting good fecundity measures. So 

### 2 we actually encourage very strict feeding regimens that will 

### 3 result in the fish growing to an acceptable and optimal size for 

### 4 reproduction by the time that starts in the assay. 

### 5 We feel that control males of 170 milligrams 

### 6 at the sub-adult stage is about where we like to see it, 95 in 

### 7 the females. However, anything greater than 100 in the males 

### 8 and greater than 150 in the females will likely produce good 

### 9 test results. 

### 10 Once again, early life-stage survival, 80 

### 11 percent tends to be an acceptable control value or a control 

### 12 value that we find, and greater than 65 is acceptable. Once 

### 13 again, liver pathology in adults tends to be very low in control 

### 14 animals, and that will be the prevalence of pathology in the 

### 15 liver. Once in a while you get liver pathology due to some 

### 16 infectious diseases and that is a good hallmark to tell you that 

### 17 your test might be in trouble or have been in trouble. 

### 18 I think I have gone through this enough that 

### 19 we are all on the same page. So unless there are some 

### 20 questions, I am just going to move on. 

### 21 I want to talk to you a little bit about the 

### 22 statistics that we have used for evaluating the data. We have 

### 23 been using a program that is called StatCHARRMS that was 

### 24 developed for us to use to be able to allow us to systematically 

### 25 and effectively test statistical effects in a systematic way 

### 26 across all the tests that were run. It is a SAS-based program

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1 that was developed for us. And it is our version currently in 

### 2 development in beta form that we would expect to be available 

### 3 shortly for anyone who wish to have it. 

### 4 It basically has three modules. There is an 

### 5 open module that is used for the data that continuous response 

### 6 is measured once, so most of the data that was taken at the 

### 7 sub-adult stage can be evaluated in this program. And it 

### 8 basically tests for abnormality, tests for homogeneity and the 

### 9 variances, and then depending on the outcomes, it goes through 

### 10 different protocols, if it passes or fails. What you will see 

### 11 is different statistical tests that are performed, given that 

### 12 the meeting acceptance criteria are met. 

### 13 There is another module that is for the 

### 14 reproductive data which is, in this case, a repeated measures 

### 15 ANOVA. And it is of course for a continuous response measured 

### 16 multiple times in the same pair of animals. So I am not going 

### 17 to go through reading all of these details. 

### 18 And for the pathology, the severity score, 

### 19 which is an ordinal response, is used in a statistical fashion 

### 20 using a test that was developed by Springer, Green and Saulnier, 

### 21 and it is a variation on a Rao-Scott/Cochran-Armitage 

### 22 nonparametric test protocol. And the slices what was added by 

### 23 these folks, where basically it goes through a step-down 

### 24 procedure and looks for significance in a particular level 

### 25 compared to a control response. And not only does it look for 

### 26 the differences in the prevalence of the observation, but also

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1 the severity score of the observation. So any significance that 

### 2 is highlighted is highlighted so that you know at what severity 

### 3 that particular outcome is significant at, at what treatment. 

### 4 And that is what is probably the most unique of this StatCHARRMS 

### 5 program. 

### 6 Now we probably are all aware of the errata 

### 7 regarding the statistical analysis of the data, and I will go 

### 8 through that quickly here. 

### 9 As I mentioned, StatCHARRMS consist of three 

### 10 modules. There is a one-way ANOVA module, and that is the one 

### 11 that we had the error in, and the error was in how we 

### 12 implemented the one-way ANOVA module. And that is that the 

### 13 replication data was not considered in the analysis. The 

### 14 statistical unit of the analysis was the individual and it was 

### 15 not the tank. 

### 16 So there were erroneous estimates for 

### 17 biological variance, treatment variance and variance estimates 

### 18 of the biological means in that data analysis within the MMT. 

### 19 So it entirely involved the sub-adult AOP biomarker endpoints, 

### 20 vitellogenin secondary sex characteristics and growth. 

### 21 Turns out that when we recognized this error, 

### 22 which was last week, we went through and we reanalyzed the data, 

### 23 but we did not have the option to reanalyze the Japanese data. 

### 24 And when they provided us with the data in the first place, they 

### 25 provided us with their own significant testing outcomes, so all 

### 26 the data that we present of the Japanese data following,

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1 basically substituted what would have been the StatCHARRMS 

### 2 outcome with the Japanese data outcome. So all the data 

### 3 presented from Japan is with their analysis of the AOP biomarker 

### 4 endpoints. 

### 5 The adult reproduction data, fecundity and 

### 6 fertility were not affected and the pathology analysis was not 

### 7 affected. 

### 8 The power analysis which I will be discussing 

### 9 later, which discusses the fecundity by number of reproductive 

### 10 pairs was also not affected. 

### 11 And lastly, I just want to state that "R" 

### 12 version of StatCHARRMS correctly uses replicate data to identify 

### 13 the statistical sampling unit as the tank. So let's move on. 

### 14 I want to just review some data expectations. 

### 15 This information is primarily derived from literature and Tier 1 

### 16 test with fish. The point being, we don't have a lot of 

### 17 experience with these outcomes in the MMT because we do not have 

### 18 all of these modes of action in the studies that we are 

### 19 presenting. 

### 20 But real quickly, an ER agonist in the males 

### 21 should show an increase in vitellogenin, a decrease in the 

### 22 secondary sex score and tends to produce sex reversals and 

### 23 intersex. And the gonads tends not to have an effect on growth. 

### 24 25 And the females, those arrows that have 

### 26 parenthesis basically indicate that the vitellogenin levels in

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1 the females may go up but it is not a defining character. There 

### 2 is no effect on the secondary sex of the females because they 

### 3 don't have any, no effect on the gonad to speak of unless the 

### 4 doses are very high. But the outcome is a reduction in 

### 5 fecundity by ER agonist and no apparent effect on fertility. 

### 6 AR-agonist cause increase in the male's 

### 7 secondary sex characters, but not necessarily a large one and it 

### 8 may not be real heavily noticed. But in the female it 

### 9 definitely causes a female which does not have any sex 

### 10 characters to show anal fin papillae, and typically a reduction 

### 11 also in vitellogenin in the female. 

### 12 Once again, we also see sex reversal and we 

### 13 see perifollicular cell hypertrophy and hyperplasia. And the 

### 14 most important thing I wanted to mention there, with the 

### 15 AR-agonist, we do not see intersex conditions. So basically it 

### 16 is either sex reversal or nothing, and once again, reduced 

### 17 reproduction. 

### 18 The AR-agonist, we don't have a very good 

### 19 record here on what to expect there. Steroidogenesis 

### 20 inhibitors, we definitely know that we have effects and 

### 21 reductions in vitellogenin. We also get the perifollicular cell 

### 22 hyperplasia and hypertrophy and again, reductions in fecundity. 

### 23 Now with the toxicity, we see reductions in 

### 24 vitellogenin. Oftentimes we see a poorly developed gonad in 

### 25 both the male and the female, reduced growth. And then other 

### 26 kinds of pathology would be liver pathology and once again,

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1 reductions in fecundity and perhaps fertility but almost always 

### 2 fecundity. So the idea being that at least we can try and 

### 3 identify the difference between toxicity and other endocrine 

### 4 modes of action. 

### 5 So I am going to just review some of the 

### 6 summaries from the multi-gen test. These are the data that I 

### 7 can summarize. And what we have here are tests from our 

### 8 Japanese colleagues, tests at the EPA lab in Duluth, and we have 

### 9 a set of inter-lab tests, three. 

### 10 11 And also, I mentioned earlier that some of the 

### 12 protocols were full protocols. However, as we moved towards the 

### 13 end of this process we, along with the Japanese, discussed ways 

### 14 to abbreviate the protocols so that later on we did not go 

### 15 through what would be full MMTs. We reduced the F2 generation 

### 16 to sub-adult sampling for some of these tests. So in some 

### 17 cases, we do not have F2 reproduction that we can compare to the 

### 18 F1 reproduction. But in any case, this is the series of test 

### 19 chemicals that we have to evaluate. 

### 20 What I have here, I have highlighted the 

### 21 places where the data changed due to the reanalysis. Anytime 

### 22 there is a yellow highlight, it is explaining that the LOEC that 

### 23 we had reported in the Integrated Summary Report -- the ISR -- 

### 24 if it changed what was in the ISR or in the supporting 

### 25 appendixes -- we did of course change it in those, but I am 

### 26 highlighting what changed here in what we are discussing. So

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1 what am I getting at? 

### 2 The Japanese data, for instance, at one time, 

### 3 the LOEC and the biomarker endpoint for 17-beta estradiol was 

### 4 2.8 nanograms per liter, and the sub-adult XY fish where there 

### 5 was a reduction in the secondary sex characters. 

### 6 Well after the reanalysis, that was no longer 

### 7 statistically significantly different than the controls and this 

### 8 became the LOEC for the biomarker endpoint which is 8.9 

### 9 nanograms per liter, or in this case, the sub-adult XX fish 

### 10 where there was an increase in vitellogenin. 

### 11 And what I am trying to show you basically is 

### 12 there were some changes and what the nature of the changes was. 

### 13 But probably more importantly what we really should be looking 

### 14 at would be the LOECs in this case. So the LOEC for fecundity 

### 15 for 17-beta estradiol was 28 nanograms per liter. It was in the 

### 16 F1 generation. 

### 17 The biomarker LOEC was 8.9 and that was in the 

### 18 sub-adult females with increased vitellogenin in the F2 

### 19 generation. And the published literature data that supports 

### 20 these as being appropriate and reasonable or comparable is that 

### 21 fecundity was reduced in Japanese Medaka and was 17-beta 

### 22 estradiol at 28 nanograms per liter, 27.9 published. 

### 23 Octylphenol; reporting octylphenol outcomes. 

### 24 One study done in the EPA laboratory in Duluth and the other one 

### 25 done by the NIES laboratory in Japan. We can see that the 

### 26 fecundity LOEC in the Duluth lab was 51 micrograms per liter in

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1 the F2 generation and was 100 micrograms per liter in the F2/F1 

### 2 generation in the Japan study. 

### 3 Once again, supporting biomarker endpoints in 

### 4 the EPA study, 13 micrograms per liter caused a reduction in the 

### 5 male fish's secondary sex characteristics in the F2 generation; 

### 6 13 micrograms per liter adult XY intersex in the F2 generation. 

### 7 And once again, reported information for 4-t-Octylphenol is 

### 8 reduction in vitellogenin at 11.4 micrograms per liter. 

### 9 The Japanese study showed again, as I said, 

### 10 100 micrograms per liter is the LOEC and three different 

### 11 biomarkers all at the 6.5 microgram per liter in one case F1 

### 12 generation and in another case the F2 generation. Once again, 

### 13 similar concentrations in the literature published. 

### 14 DDT study performed in Japan; the LOEC for 

### 15 fecundity 1.9 micrograms per liter in the F2 generation, and the 

### 16 biomarker endpoints, .07 micrograms per liter for both the 

### 17 sub-adult females with an increase in vitellogenin and adult 

### 18 females with an increase of vitellogenin. 

### 19 And in this increase of vitellogenin in the 

### 20 sub-adults was both in the F1 and the F2 generation whereas the 

### 21 increase observed in the adult females was in the F2 generation. 

### 22 And the published literature shows vitellogenin response at the 

### 23 .1 microgram per liter for DDT, so similar again. 

### 24 This is 4-chloro-3-methylphenol. This is a 

### 25 study that we did at the EPA lab in Duluth and we ran that 

### 26 chemical because an in vitro assessment indicated that that

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1 chemical bound to the estrogen receptor. And we felt that it 

### 2 would be a very interesting chemical to see what the 

### 3 relationship was between the in vitro response, so it would have 

### 4 been one of the screening assays in Tier 1 and compare it to 

### 5 what would be an outcome in Tier 2. 

### 6 And the most important thing I think to show 

### 7 is that at our highest concentration there was no effect on 

### 8 fecundity or fertility, but in any case I want to go back and 

### 9 then show that there were slight increase in female vitellogenin 

### 10 expression, liver vitellogenin mRNA at the 88 microgram per 

### 11 liter level, and there is no data with respect to this in the 

### 12 literature, but I think it is a good example of a negative 

### 13 control, to some extent. We know that it is an estrogen binder 

### 14 and we know that with pretty good information. 

### 15 We also know that what we did just to try and 

### 16 confirm that our result made sense was that we ran a study to 

### 17 look for what might happen at or near the LC-50 of that 

### 18 chemical, and then we measured the effect on vitellogenin 

### 19 production. And as you reach the LC-50, vitellogenin production 

### 20 goes to zero. You can't induce it. You get toxic effects 

### 21 before you get EDC effects. So I guess what I am suggesting is 

### 22 that for CMP, while it may potentially have been an endocrine 

### 23 disruptor in vivo, it is toxic before it is an endocrine 

### 24 disruptor. 

### 25 Tamoxifen outcome; we have a study done in 

### 26 Japan and the fecundity LOEC was 20 micrograms per liter and the

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1 biomarkers were 1.3 micrograms per liter. And they were effects 

### 2 on vitellogenin, so it caused an increase in vitellogenin in the 

### 3 male sub-adult. It caused a decrease in vitellogenin in the 

### 4 female in F1 and F2. And in the sub-adult males, it caused 

### 5 decreases in the secondary sex characters of the F2. 

### 6 Published information again, 625 micrograms 

### 7 per liter affecting fecundity. So the point being that we are 

### 8 showing in this case quite a bit greater sensitivity with the 

### 9 MMT than the published literature. 

### 10 17-beta trenbolone; 32 nanograms is the LOEC 

### 11 for reproduction or for fecundity in the F1 generation. The 

### 12 same level for the biomarkers, 32 nanograms per liter for 

### 13 sub-adult female increases in secondary sex characters, adult 

### 14 decreases in males for vitellogenin and, once again, 

### 15 sex-reversal in adult females. And once again very similar 

### 16 concentrations listed in the literature for vitellogenin 

### 17 effects. 

### 18 Prochloraz, LOEC fecundity 25 micrograms per 

### 19 liter, F2 generation. We see nine micrograms per liter as a 

### 20 sub-adult concentration reducing secondary sex characters and 

### 21 this became statistically significant in the -- this is the same 

### 22 as listed by the Japanese in their statistical analysis and once 

### 23 again, 30 micrograms per liter for fecundity in the literature 

### 24 comparable to 25 in the MMT. 

### 25 Vinclozolin; the EPA vinclozolin study shows 

### 26 that the fecundity was at the greater than 136 microgram per

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1 liter level and no information available at this time for the 

### 2 generation. We also are showing that the evaluation of 

### 3 secondary sex characters was changed by one treatment level; it 

### 4 went from 17 to 33 and that was in the reduction of the number 

### 5 of papolary processes in XY males in the F1 generation. And 

### 6 that moved up one level, so it went from 17 to 33. And this 

### 7 particular observation became significant and that was at the 33 

### 8 microgram level for these sub-adult females with an increase in 

### 9 vitellogenin. 

### 10 The published information regarding effects on 

### 11 spermatogenesis is very, very high, as you can see. It is like 

### 12 2.5 at 250 milligrams per liter. It is very high. And we think 

### 13 that this study was running vinclozolin at higher than 

### 14 solubility. 

### 15 And then just real quickly, the one study 

### 16 where we increased the replication in the reproductive part, the 

### 17 MRT, and that was at 4-t-Octylphenol, once again, the same 

### 18 chemical we present here. And in that case, we show the 

### 19 fecundity LOEC at 23 micrograms per liter in both the F1 and the 

### 20 F2 generation. And the published literature shows effects in 

### 21 vitellogenin at 11 micrograms per liter. That was a long slide 

### 22 but it does in fact truly, I think, summarize the essential data 

### 23 that we have for looking at this test protocol. 

### 24 Summary Control Data. Now this is the one 

### 25 where I wanted to mention to you that -- I did mention to you -- 

### 26 that there is quite a bit of variance or differences in the

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1 values in the sub-adult biomarker data partly because, as I 

### 2 mentioned, these studies were changing between the beginning 

### 3 ones and the end ones. And the changes included not only the 

### 4 sampling time for the sub-adults but also the feeding regimens 

### 5 and the temperature regimens that the studies were run at. And 

### 6 that was intended, as I've mentioned, to try and optimize the 

### 7 test for fecundity, especially for the fecundity endpoints. 

### 8 So let me just real quickly go through and 

### 9 just show some of the data with respect to reproduction. 

### 10 17-beta estradiol, controlled data in fecundity 27 eggs per day, 

### 11 fertility 26 eggs. I am going to just go down. This is all 

### 12 going to be fecundity first. So 27, 17, 29, 16, 39, 23. 

### 13 The lowest is 14 on the prochloraz and that is 

### 14 probably lower than what we would consider to be acceptable 

### 15 today. You can see that in the interlabs most of them were 

### 16 quite good. This one was quite bad, but this fish also were 

### 17 very, very small. They were not adequately sized for good 

### 18 reproduction. 

### 19 You can see that in most cases, the control 

### 20 fertility is very high. Growth; you know, we have in some cases 

### 21 with the Japanese data they did not discriminate between males 

### 22 and females in the sub-adult, so that is combined. So this is 

### 23 just simply a fish weight rather than a male weight and female 

### 24 weight. You can see that the Japanese had tended to have larger 

### 25 fish early on. We learned a lot from the Japanese. This is our 

### 26 first full MMT and our fish were much smaller.

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1 As we got further on, we were able to develop, 

### 2 first of all, better fish and plus we also sampled later. This 

### 3 was sampled in seven weeks and most of these others were sampled 

### 4 in nine weeks. The fish are in their linear growth phase at 

### 5 that time, so a difference in about a week makes a huge 

### 6 difference in the size in the sub-adult weights. 

### 7 Anyhow, let us get back to talk about 

### 8 secondary sex characters tend to be related to how big the fish 

### 9 are during this sub-adult sampling. You can see that -- I 

### 10 mentioned the MED, the Duluth octylphenol study, small fish, low 

### 11 secondary sex numbers. As we started sampling later, they tend 

### 12 to be larger values. 

### 13 And once again, vitellogenin reported in 

### 14 different ways. We have been reporting vitellogenin as mRNA 

### 15 copy number. And the Japanese data is measuring vitellogenin 

### 16 through ELISA techniques and reporting the amount of 

### 17 vitellogenin per unit weights. So they are not necessarily real 

### 18 comparable but I just wanted to literally put the data in so 

### 19 that you can see it. 

### 20 Now I want to look at a little bit about the 

### 21 reproducibility in the MMT by chemicals. We do not have a lot 

### 22 of comparisons because there were not a lot of chemicals that 

### 23 were run multiple times. But octylphenol was run three 

### 24 different times; twice in the EPA lab in Duluth. In the EPA, 

### 25 this was very, very early in the study protocol and this was 

### 26 recent, so these two studies are separated by about four years.

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1 2 And the Japan study was done in the middle and 

### 3 we can see that the fecundity outcome is comparable in terms of 

### 4 the LOEC in the Japan and the U.S. study. And then you get to 

### 5 the last U.S. study with increased replication and the LOEC 

### 6 dropped to two treatment levels, and that is primarily I think 

### 7 because of statistical power not because of actual changes in 

### 8 the LOEC. Fertility, very similar, and sort of the same story, 

### 9 I think. 

### 10 Growth; let us look at those values. The LOEC 

### 11 for growth was 102 micrograms per liter in the EPA study and in 

### 12 the NIES study. 

### 13 Secondary sex, 51 micrograms per liter at the 

### 14 EPA study, substantially lower at the NIES/Japan study with 

### 15 octylphenol. 

### 16 Vitellogenin, once again, the same at the 51 

### 17 micrograms per liter level, and with the Japan study, 25 

### 18 micrograms per liter, so a difference of one treatment level 

### 19 again. 

### 20 Vinclozolin; this chemical was run both at EPA 

### 21 and in three inter-lab studies. And we see that the LOEC for 

### 22 fecundity was, in most cases, the highest treatment level, so it 

### 23 was greater than the highest treatment level. The point is they 

### 24 are all comparably high in terms of their LOECs. And the 

### 25 fertility LOECs are essentially the same as the fecundity LOECs. 

### 26 And let us look at effects on growth; what we

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1 can see is that the LOEC for growth is, once again, very high in 

### 2 the EPA study and somewhat lower in the two inter-lab studies, 

### 3 and then in the third inter-lab study, very high again, the 

### 4 highest treatment level. 

### 5 Effects on secondary sex 17 micrograms per 

### 6 liter EPA study, 197 micrograms per liter in the lab one of the 

### 7 inter-lab and higher than the highest dose in the -- so greater 

### 8 than the highest dose in lab two and not measurable in lab three 

### 9 because of primarily problems in genetic sexing and other 

### 10 problems. 

### 11 Let us go to vitellogenin; once again, the 

### 12 high treatment level of vitellogenin, no change. In other 

### 13 words, not statistically significant at the highest treatment 

### 14 level, same here and 360 in this one. So the point was 

### 15 vitellogenin was not reactive to vinclozolin in all the 

### 16 laboratories. 

### 17 I want to just summarize the inter-lab data. 

### 18 We have basically -- I mean this is a very broad summary -- the 

### 19 control data from two of the three interlabs. The studies were 

### 20 very similar. Fish from laboratory number two were very small 

### 21 compared to the other labs and we have tried to understand some 

### 22 of the reasons why and we don't know why. So basically, the 

### 23 fish in lab 2 were very small. 

### 24 And the consequence of that is their 

### 25 reproduction was low and lots of other things were affected. A 

### 26 lot of data was affected because of the small fish. In all

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1 cases, there were no effects on reproduction of vinclozolin, in 

### 2 all labs. 

### 3 The treatment related decreased in secondary 

### 4 sex characteristics were observed in one of the three 

### 5 laboratories and that was consistent with the same biomarker 

### 6 effect that we observed in the EPA study with vinclozolin. So 

### 7 in other words, there was a treatment related decrease in the 

### 8 anal fin papillae of male fish. 

### 9 Vitellogenin was not affected by treatment and 

### 10 essentially the effect outcomes are consistent between the 

### 11 inter-lab and the EPA study with vinclozolin and there were 

### 12 problems determining genetic sex across all the inter-lab 

### 13 laboratories. So genetic sexing was a problem in the inter-lab 

### 14 studies. 

### 15 So what are our lesions learned in the 

### 16 inter-lab? We feel like we did not adequately transfer the 

### 17 information, the techniques to allow successful execution of the 

### 18 MMT protocol, the labs with limited experience with testing 

### 19 Medaka. 

### 20 Examples of this inadequacy include some 

### 21 issues with Medaka culture and growth. At least one of the labs 

### 22 clearly did not rear fish in a way that led to fish that were 

### 23 adequate to study the endpoints in. Genetic sex determination 

### 24 was problematic across all the labs. 

### 25 There were problems in sample archiving for 

### 26 the secondary sex characters and for vitellogenin evaluation and

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1 there were also problems with sample tracking and QA/QC. And I 

### 2 feel like there are, for instance, there were some things that 

### 3 one would consider very simple, and that is naming conventions 

### 4 for samples across generations cannot be the same. You just 

### 5 simply cannot do that because you cannot tell which generation 

### 6 you are looking at when you evaluate the outcome. 

### 7 And because some of the labs that were doing 

### 8 these kinds of tests had not experienced those requirements in 

### 9 QA in multigeneration tests they just simply overlook some of 

### 10 the things that would seem obvious. So I am just saying, 

### 11 something that would seem as obvious with sample tracking turned 

### 12 out to be problematic and we just have to work on developing 

### 13 better tools for the labs to use. 

### 14 More explicit guidelines; training to provide 

### 15 information to the labs interested in performing a Tier 2 test 

### 16 protocol. Make sure that they can do it and make sure that they 

### 17 can do it before they start the test, genetic sexing being 

### 18 essential. And I believe that as labs gain experience, that we 

### 19 can expect that they will be able to perform a Tier 2 test 

### 20 successfully. 

### 21 I am going to move on now real quickly I hope 

### 22 to some comparisons of the F1 and F2 generations in the MMT. 

### 23 **DR. MARTHA SANDY:**Excuse me, Dr. Johnson. 

### 24 Maybe, given the late time, we take a break here for lunch and 

### 25 come back. 

### 26 **DR. RODNEY JOHNSON:**That is just fine for me.

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1 I am sure it is fine for everybody else too. 

### 2 **DR. MARTHA SANDY:**Sure. If we can come back 

### 3 at 1:30 promptly, then we will start again. And that will give 

### 4 you a little bit of a break. Thank you so much. 

### 5 (Whereupon a break was taken) 

### 6 **DR. MARTHA SANDY:**So good afternoon. I think 

### 7 we are going to try to get the Panel members in their seats. I 

### 8 think we are missing just a few and then we will get started. 

### 9 Good afternoon. This is Martha Sandy and we are about ready to 

### 10 kick off where we left off with Dr. Johnson. Are you on slide 

### 11 72? We are. Please go ahead. 

### 12 **DR. RODNEY JOHNSON:**All right. Everybody is 

### 13 nourished. So I wanted to discuss with you some information 

### 14 regarding comparing the reproductive output in F1 compared to F2 

### 15 and just very quickly look in the context of the lowest observed 

### 16 effect concentration measured in F1 and F2 within the same test 

### 17 where we had that kind of data. 

### 18 So here we have got estradiol where we showed 

### 19 in the study in Japan that the LOEC for fecundity was 28 

### 20 nanograms per liter in F1 and it was greater than 28 nanograms 

### 21 per liter in F2. And because of reproductive failure, that is 

### 22 the highest concentration that was in F2. So does that make 

### 23 sense? So reproductive failure in F1 eliminates the higher dose 

### 24 in F2, so the LOEC automatically has to be there or higher, 

### 25 unless it is lower, of course, but the point is you cannot go up 

### 26 higher than what you have tested.

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1 The octylphenol study at the Duluth lab or the 

### 2 EPA lab showed 102 as being the LOEC in F1, and the next lower 

### 3 dose of 51 micrograms per liter in F2. And the NIES study show 

### 4 100 micrograms per liter as the LOEC in F1, and then once again, 

### 5 same as in the estradiol study, the highest dose with which 

### 6 there was information is not the hundred anymore so the LOEC is 

### 7 greater than 50 micrograms per liter. 

### 8 The EPA octylphenol study in the last one 

### 9 where we increased the replication where it was 12 replicates, 

### 10 we show 23 micrograms per liter as the LOEC in both F1 and F2. 

### 11 DDT, the Japan study, shows the LOEC in F1 as 

### 12 being greater than 1.9 micrograms per liter and in F2 it was 1.9 

### 13 micrograms per liter. In the trenbolone EPA study it shows 32 

### 14 nanograms per liter being the LOEC for fecundity in both F1 and 

### 15 F2. 

### 16 Now this is the biomarker comparisons. In 

### 17 this case, I have been through this first study with estradiol 

### 18 once before. Basically, we show the LOEC in F1 as in papillae 

### 19 goes away. In other words, it is not the lowest LOEC anymore. 

### 20 And the lowest LOEC is in fact liver vitellogenin, and it is at 

### 21 28 nanograms per liter is the LOEC in F1, 8.9 nanograms per 

### 22 liter in F2, so it is lower in F2 than in F1. 

### 23 Octylphenol shows 51 micrograms per liter in 

### 24 F1 and a lower LOEC of 13 milligrams per liter in F2, and that 

### 25 is for secondary sex or papillae. 

### 26 Octylphenol in Japan shows equal LOECs for

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1 liver vitellogenin endpoints of 6.5 milligrams per liter in both 

### 2 F1 and F2. 

### 3 DDT, same, F1/F2 again for vitellogenin. 

### 4 Tamoxifen, same LOEC again for liver vitellogenin and in F1 and 

### 5 F2. For trenbolone, the same LOEC for the biomarker of both 

### 6 papillae and liver vitellogenin at 32 nanograms per liter. 

### 7 So I am going to try and summarize these 

### 8 outcomes in the context of just simply that LOECs F1/F2 by 

### 9 reproductive endpoints. And simply F1 was more sensitive than 

### 10 F2 that has a lower LOEC for 17-beta estradiol, and F2 has a 

### 11 lower LOEC for octylphenol at EPA and DDT for Japan, and they 

### 12 are equal for three different studies, octylphenol Japan, 

### 13 trenbolone and octylphenol at EPA. 

### 14 Biomarkers F1, lower biomarker LOEC than F2 in 

### 15 none of the studies whereas F2 is lower than F1 in two of the 

### 16 studies, and they are equal in four of the studies. 

### 17 These outcomes with the research Medaka 

### 18 multigeneration Test suggest the following to us and that is 

### 19 that the F2 generation does not provide sufficient value added 

### 20 to warrant inclusion in the Tier 2 test protocol. 

### 21 And to realize that the test that we are 

### 22 proposing is called the Medaka Reproduction Test rather than the 

### 23 multigeneration test because it does not span multiple 

### 24 generations anymore and it is a subset. The timeline of the MRT 

### 25 is a subset of the MMT, so it is not an entirely different study 

### 26 or test protocol, it is a test protocol that is derived from the

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1 research MMT. 

### 2 The differences that we are proposing are 

### 3 primarily that the exposure would stop at F2 hatch and so the 

### 4 study duration would be 19 weeks. Endpoints are measured 

### 5 primarily in F1 and pathology is measured in F1 adults only. 

### 6 And we are recommending an increase in the replication in F1 

### 7 reproduction for the fecundity and the fertility endpoints. 

### 8 So this is the protocol. You have seen these 

### 9 types of diagrams before. It starts with exposure to F0. The 

### 10 embryos are used to populate F1. We take the F1 all the way 

### 11 through reproduction. We take embryos into F2 through hatch and 

### 12 the study is terminated. 

### 13 The endpoints are measured are hatched in F1, 

### 14 survival F1, biomarker endpoints, week nine of F1. There 

### 15 survival, growth, vitellogenin and secondary sex characters. 

### 16 Reproduction now is three weeks long continuous measuring 

### 17 reproduction, fecundity and fertility for 21 days in succession. 

### 18 19 We measure hatch with pathology then as 

### 20 occurring at the end of reproduction and survival growth 

### 21 vitellogenin is not measured. Vitellogenin is only coming at 

### 22 the sub-adult point, and then we measure hatch in F2. So it is 

### 23 a 19-week study, reproduction, 21 days continuous. The 

### 24 generations do not overlap, and that is primarily for 

### 25 infrastructure reasons. Basically allows you to do the study 

### 26 with the same number of tanks throughout the full study.

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1 I want to just review a little bit about how 

### 2 the experimental design works here. What we have is three 

### 3 different generations; F0, F1 and F2. I want to show that this 

### 4 is sort of the replication structure that we are recommending. 

### 5 There are six replicate pairs in F0. We collect eggs from those 

### 6 six pairs. We pull them and we distribute them into six 

### 7 separate incubators, each in a tank by itself. 

### 8 After the hatch, data is collected. All the 

### 9 hatchlings are pulled into one tank and they are all 

### 10 redistributed into six separate tanks, 12 fish per tank. 

### 11 As they are reared and grow up, we reach 

### 12 finally week nine. At week nine all the fish are genetically 

### 13 sexed. Four males and four females from each replicate are 

### 14 pooled into this set of tanks; one tank is male, one tank is 

### 15 female. All the remaining fish are sampled for the biomarker 

### 16 endpoints, and their information with respect to replicate is 

### 17 retained. So all of the information regarding the biomarker 

### 18 endpoints are analyzed and understood in the context of the 

### 19 replicate from which they came. 

### 20 These pooled males and females then are paired 

### 21 up randomly into 12 replicate breading pairs and reproduction is 

### 22 evaluated and we set up F2 incubators and they are sampled for 

### 23 pathology and for secondary sex, and that is the design. 

### 24 So we have the endpoints across the top hatch, 

### 25 survival, growth, vitellogenin, secondary sex, reproduction, 

### 26 pathology, secondary sex and hatched. So this is F1 hatch data;

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1 this is F2 hatch data. Survival, growth, vitellogenin, 

### 2 secondary sex all taken on six replicate pairs, reproduction, 

### 3 pathology secondary sex all taken on 12 replicate pairs. 

### 4 So that is the experimental design overview. 

### 5 The rationale for going to 12 replicate pairs for reproduction 

### 6 follows. This is representing a Monte Carlo power analysis 

### 7 derived from the control reproductive data that we got in our 

### 8 previous last two MMT studies. And what these lines represent 

### 9 are basically different levels of reduction in fecundity 

### 10 compared to the control values. Each line represents a 

### 11 different reduction in fecundity, starting with -- 70 percent 

### 12 reduction is this bottom line, 70 percent all the way down to 20 

### 13 percent. 

### 14 The point being that if this is true, that 

### 15 amount of reduction, the probability that you will detect that 

### 16 is affected by the number of pairs that you have in your test 

### 17 design. So at the highest level of reduction, there is not a 

### 18 whole lot of difference between the power of detecting an effect 

### 19 with 12 pairs compared to six pairs. 

### 20 If you go to that particular line, which is 

### 21 the 30 percent reduction in fecundity, you can see that the 

### 22 power changes very dramatically by the number of pairs and goes 

### 23 all the way from about .45 with six pairs to .83 with 12 pairs. 

### 24 Basically, I think what we feel is that this 

### 25 line here, which represents the 40 percent reduction in 

### 26 fecundity is a fairly reasonable amount to be able to detect.

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1 And with six replicate pairs, you can see that the probability 

### 2 of detecting a 40 percent reduction in fecundity is about 75 

### 3 percent. 

### 4 And the other thing to recognize is that 

### 5 replicate loss has a fairly serious consequence in the power. 

### 6 All the way from up to 12 coming down to six goes from nearly 

### 7 one to .75. 

### 8 With 12 treatment pairs, the probability 

### 9 increases to nearly one. The replicate loss does not affect 

### 10 power seriously. So in other words if you lose replicates here 

### 11 you are in this part of the curve. If you lose replicates here, 

### 12 you are in this part of the curve. And it is not unusual to 

### 13 lose replicates during this study. 

### 14 So I guess what I am arguing is that if we 

### 15 specified 12 replicate pairs as the recommended amount and you 

### 16 lose some, you still have quite a bit of power to detect effects 

### 17 on your apical endpoint. 

### 18 Recognize also that this is what we are 

### 19 calling the two/one experimental design, and that experimental 

### 20 design is one where there are two times the number of control 

### 21 replicates compared to each treatment. So for each treatment, 

### 22 there are 12 replicate pairs. And for the controls, there are 

### 23 24 replicate pairs for a total number of cells in this study of 

### 24 84 cells. 

### 25 This just demonstrates the difference in two 

### 26 different octylphenol studies that we did; one of them with 12

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1 replicate pairs, the other with 6 replicate pairs. And I think 

### 2 the most important thing was that, compared in the 12 replicate 

### 3 set, first of all, the LOEC for fecundity is 23 in F1, it is 23 

### 4 in F2. And with six replicate pairs in another octylphenol 

### 5 study, this treatment LOEC is the highest treatment in the 

### 6 study, which is 100. And in this case it is 51. So I guess 

### 7 what I am saying is that increasing the replication really 

### 8 substantially improved the ability to detect effects with 

### 9 confidence. 

### 10 So in conclusion, EPA asserts that the 

### 11 proposed MRT protocol fulfills the EDSTAC-defined purpose of a 

### 12 Tier 2 test. It can determine whether a test substance 

### 13 adversely affects a test organism through endocrine-mediated 

### 14 pathways. It can quantify those effects with respect to the 

### 15 estrogen and the androgen systems. It includes exposure during 

### 16 potential life stages that may be more sensitive than those 

### 17 tested in Tier 1. It characterizes the dose response and it 

### 18 includes exposures during gametogenesis to allow for potential 

### 19 effects of parental transfer of chemical and other endogenous 

### 20 factors. 

### 21 And I have one more slide and I want to just 

### 22 go through some conversation regarding implementation. 

### 23 We have learned a lot as we have tried to 

### 24 develop these tests and transfer the information to other people 

### 25 so that they can do these tests. We feel that support and the 

### 26 improved guidance in pathology and for data reporting is an

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1 important part of tech transfer that we feel can be improved and 

### 2 that we are dedicated to working on. We feel that there needs 

### 3 to be training in genetic sex and other molecular methods. 

### 4 Now, I want to sort of emphasize that 

### 5 laboratories that do these kinds of tests will have to use 

### 6 modern molecular biology tools and they will have to have them 

### 7 in their laboratories or they cannot do the test. If they 

### 8 expect to do these tests by shipping samples and getting data 

### 9 back by which to make decisions on what they are doing, the time 

### 10 turn-around and the QA problem is enormous. They simply have to 

### 11 have the infrastructure within their laboratories to form these 

### 12 tests. And we have been working with tech transfer of these 

### 13 methods and I think they are very doable. 

### 14 One of the things I have not mentioned at all 

### 15 is that, one of the most challenging parts of doing a study like 

### 16 I have described is the exposure and the exposure chemistry 

### 17 part. And all of these studies are done with flow-thru exposure 

### 18 systems. And in order to keep the concentration of chemicals 

### 19 that you are intending to dose real, you have to make sure that 

### 20 your exposure systems are clean. You have to go through 

### 21 prophylactic cleaning of your test systems while they are on 

### 22 tests. We have methods for doing that and we have trained 

### 23 people on how to do that and we have transferred methods to do 

### 24 that, but it is essential. 

### 25 Hederaltrophic growth within tubing systems, 

### 26 aquarial systems will absolutely destroy the chemical that you

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1 expect to be testing if you do not ever clean the system during 

### 2 the test. It has to be done. 

### 3 The other thing is that we have methods to 

### 4 expose and delivery what we think we are. We have very good 

### 5 quality diluter system and dilution delivery systems. And labs 

### 6 that do these tests should have good systems. Once again, we 

### 7 are wiling to provide all the information necessary for these 

### 8 apparatuses to be used by test labs. 

### 9 We would provide statistic programs based at 

### 10 "R" for people who wish to use them. We have lots of guidance 

### 11 and specimen tracking, QA/QC, which we are also willing to 

### 12 share. We even have something that seems as trivial as 

### 13 calendars on how to do daily protocols, but they are very, very 

### 14 helpful. They help us out and they will be helpful for anybody 

### 15 else. 

### 16 I guess I am trying to emphasize that I do 

### 17 think these tests are runable by other labs besides us and 

### 18 Japan, but it will take diligence and a lot of training to pull 

### 19 it off. I think in time, the labs we will get, we will be able 

### 20 to. And I appreciate your time and I am sorry I was so 

### 21 long-winded. 

### 22 **DR. MARTHA SANDY:**Thank you very much, Dr. 

### 23 Johnson. So now is the time for the Panel to ask any questions 

### 24 of clarification, and I would like to go first. Could you go 

### 25 back to slide 69 for us? 69 is tilted MMT Reproducibility by 

### 26 Chemical.

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1 So I had a question because it looks like here 

### 2 for the 4-t-octylphenol on the fecundity for the F1s, the EPA 

### 3 values 102 micrograms per liter and the NIES is 100 micrograms 

### 4 per liter. If you go back two more slides, if we look at the 

### 5 fecundity for the EPA 4-t-octylphenol, it looks to me like it is 

### 6 51 micrograms per liter. Am I misreading these slides? The 

### 7 previous slide was 100. 

### 8 **DR. RODNEY JOHNSON:**And it is between F1 and 

### 9 F2, that is why. 

### 10 **DR. MARTHA SANDY:**So you are only presenting 

### 11 the F2. 

### 12 **DR. RODNEY JOHNSON:**Yeah, yeah. 

### 13 **DR. MARTHA SANDY:**Okay. Thank you. And Dr. 

### 14 Schlenk? 

### 15 **DR. DANIEL SCHLENK:**Okay. I have got a 

### 16 couple of questions, just a couple. 

### 17 So again, going back to sort of Tier 2 and 

### 18 what you would really like to have in a Tier 2 model. You would 

### 19 really like something that has a population metric or endpoint. 

### 20 And I was just curious. I did some literature search just to 

### 21 see, but is there an equivalent model like you have with the 

### 22 Miller model with fathead minnows? Is there an equivalent model 

### 23 for Japanese Medaka that actually has an estimate of population 

### 24 change connecting reproduction to population that you know of? 

### 25 I could not find anything. 

### 26 **DR. RODNEY JOHNSON:**No. I don't know of any.

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1 We have not pursued anything like that recently with using the 

### 2 Miller type models. At the same time, we are working with a 

### 3 different group within EPA to try and develop what we would 

### 4 call, instead of population level models, what we are calling 

### 5 toxicity translator models where we are trying to get what would 

### 6 be a way to translate the data that comes out of an MRT or an 

### 7 MMT, literally growth data, fecundity data, and get those into 

### 8 what would be models that could go into iterative population 

### 9 models where you literally have -- and in this case, it would be 

### 10 not even a population model as much as a growth and fecundity 

### 11 model for a year class, and its probability of surviving into 

### 12 the next year to reproduce. 

### 13 But they are not what you would think of as 

### 14 classical population models. We are trying to put one set of 

### 15 models in between them and that is where we are working right 

### 16 now. And actually we are working with fathead minnows right now 

### 17 to develop those models, but we think that they are applicable 

### 18 to the Medaka. And we have not been working them yet into the 

### 19 endocrine disruptor pathways. So far we have been working with 

### 20 them in the growth toxicity pathways. 

### 21 **DR. DANIEL SCHLENK:**Okay. Thanks. One of 

### 22 the things, as you are aware of, is water quality is a very 

### 23 unique issue between laboratories. And I noticed in the 

### 24 document that the Lake Superior waters which you guys use, the 

### 25 MED -- I am curious. In Japan, any comparison of water quality 

### 26 parameters between what you have there and what is in Japan and

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1 any recommendations for that, in particularly sort of nutrient 

### 2 level type of things, micronnutrients, any sort of movement in 

### 3 that direction at all? 

### 4 **DR. RODNEY JOHNSON:**Well we will just send 

### 5 that Lake Superior water to everybody and they can have as much 

### 6 as they want. No, that is maybe not true. 

### 7 So we have not done that in particular. We 

### 8 know that Medaka are fairly capable of reproducing through a 

### 9 major range of water quality. They tend to be more tolerant of 

### 10 your (inaudible) conditions than probably most fish. In fact, I 

### 11 think when you get into really, really soft waters, that is when 

### 12 they tend to be stressed. 

### 13 So I guess I would just off-the cuff, I mean 

### 14 there is nothing formal put together yet in this case, recommend 

### 15 using, if at all possible, harder waters. And I would recommend 

### 16 in most cases, if at all possible, de-gased well waters and not 

### 17 surface waters. We use Lake Superior water because it is 

### 18 available to us and we can sterilize it and degas it and things 

### 19 like that. But I think in most cases, it would be simpler for 

### 20 any laboratory to use degassed well water that is reasonably 

### 21 hard, not super soft. 

### 22 The Japanese as well as us have never seemed 

### 23 to have see big differences in our outcomes, and I honestly 

### 24 cannot tell you how similar our waters are. I don't know. 

### 25 **DR. DANIEL SCHLENK:**I am curious also. I 

### 26 mean the other three labs that ran those as well, there was not

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1 any information regarding what their water was. Because I do 

### 2 know from personal experience that even well water can be quite 

### 3 soft and it took us about six months to figure out that we had 

### 4 to add Instant Ocean to get the two parts per thousand just to 

### 5 get reproduction to take place. I mean those are issues -- I 

### 6 don't know if those are going to be in the guidance documents 

### 7 but it's something to be working on. 

### 8 **DR. RODNEY JOHNSON:**Well I would certainly 

### 9 want you to recommend something to that effect and we can 

### 10 clearly look into it more carefully. It is just simply 

### 11 something that we have not studied carefully. 

### 12 **DR. DANIEL SCHLENK:**Sure. Also, on your 

### 13 slide 60 which compares all of the control values for gender, 

### 14 the gender change to me was quite interesting with the 

### 15 temperature. I did not know that actually. That was quite 

### 16 interesting. I am wondering did the Japanese have the same 

### 17 issue with regard to gender change and temperatures that has 

### 18 been sort of well characterized there? 

### 19 **DR. RODNEY JOHNSON:**That is well 

### 20 characterized. It is well characterized by temperature studies. 

### 21 I think we have done the most extensive temperature study but 

### 22 we have also compared our results with the Japanese results, and 

### 23 they are very similar. 

### 24 So it does turn out to be apparently a strain 

### 25 dependent and we are recommending a specific strain of fish to 

### 26 use. You know, it is the derivative of what they call the

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1 carbio, which is probably what you have and most of us in the US 

### 2 have. But they are not like the bread strains that are 

### 3 available in Japan. In fact, we exchange fish with Japan with 

### 4 an intention to keep, if there is such as a thing, as an 

### 5 out-bread strain. I mean it is an oxymoron in a sense, but we 

### 6 try and make sure that our strain is consistent and not 

### 7 changing. 

### 8 **DR. DANIEL SCHLENK:**Okay. Moving to your 

### 9 steroidogenesis AOP with prochloraz; so in the fathead minnow 

### 10 Tier 1, their steroid measurements in those particular assays, 

### 11 any reason why steroid measurement is not a part of the Tier 2? 

### 12 **DR. RODNEY JOHNSON:**The fish are really 

### 13 small. 

### 14 **DR. DANIEL SCHLENK:**Yes, I know. 

### 15 **DR. RODNEY JOHNSON:**They are very difficult 

### 16 -- we tried to get blood out of them and it is consistently 

### 17 difficult for us to get blood -- and felt that we could give 

### 18 that up. I mean to be honest, just not capable of getting that 

### 19 information, the steroid information. 

### 20 **DR. DANIEL SCHLENK:**So you have tried it and 

### 21 the QA/QC is such that your signal to noise is low enough that 

### 22 you do not feel that is minimal to a guidance, sort of; is that 

### 23 right? 

### 24 **DR. RODNEY JOHNSON:**The most important thing 

### 25 is we seriously tried to get consistent amounts of bloods. 

### 26 **DR. DANIEL SCHLENK:**Well what about a whole

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1 tissue though? 

### 2 **DR. RODNEY JOHNSON:**We never tried it with 

### 3 whole tissue, never did. We just have not. And if we did do it 

### 4 with whole tissue, it would probably have to be with heads, 

### 5 because that is probably the only tissue we have available to 

### 6 work with after the secondary sex sample. 

### 7 **DR. DANIEL SCHLENK:**Yes, unless you allocate 

### 8 it. 

### 9 **DR. RODNEY JOHNSON:**Unless we allocated it 

### 10 certain numbers of fish for that. And we have been pretty 

### 11 careful in streamlining the design as much as possible to not 

### 12 have excess animals, and that has been part of -- part of the 

### 13 calculus is sort of how can we do this and keep animal use low 

### 14 but still get the information that we felt we needed. And that 

### 15 was one that -- it was something that fell to the side quite a 

### 16 while ago and we have not reconsidered that in the last several 

### 17 years. 

### 18 **DR. DANIEL SCHLENK:**Okay. And you mentioned 

### 19 the flow-thru system. One of the things that I was a little 

### 20 unclear about was, you take measured values periodically 

### 21 obviously throughout this process that the egg chambers that you 

### 22 are exposing the animals to, is that still flow-thru or is that 

### 23 a static system? It appeared from the documentation that was a 

### 24 static system but I was not sure if you have that submerged with 

### 25 a flow-thru component that allows that to maintain the flow-thru 

### 26 characteristic.

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1 **DR. RODNEY JOHNSON:**The incubator is a small 

### 2 test tub that we cut off and put a screen in it. And then that 

### 3 is suspended in the aquarium that the water is delivered to. 

### 4 And then it is agitated with air so that there is exchange with 

### 5 the tank through the screen system. 

### 6 **DR. DANIEL SCHLENK:**Okay. So it is 

### 7 flow-thru, it is just not -- 

### 8 **DR. RODNEY JOHNSON:**Yeah, yeah. 

### 9 **DR. DANIEL SCHLENK:**Okay. I just wanted to 

### 10 make sure that was there. So with the vinclozolin studies in 

### 11 lab two with the low weight, it was recommended that you 

### 12 normalize according to weight. Was that just for that one lab 

### 13 or is that something that you are proposing to do across the 

### 14 board? 

### 15 **DR. RODNEY JOHNSON:**We do not recommend 

### 16 normalizing data by weight. We think there is no really good 

### 17 justifiable biological well defined relationship between weight 

### 18 and fecundity. We know that they are correlated but we 

### 19 certainly do not have an understanding of the correlation at 

### 20 all. 

### 21 **DR. DANIEL SCHLENK:**Okay. I just want to 

### 22 make sure that was not in the guidelines or anything. And it 

### 23 was stated -- and I kind of got this impression in reading -- 

### 24 that some of those labs did not follow particular guidelines 

### 25 that you had given to them. And I think you may have alluded to 

### 26 a few things in terms of feeding and other issue. Were those

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1 primarily the main things, temperature and feeding where the 

### 2 things that were not followed or -- were guidelines provided 

### 3 that everybody had? I guess I am just kind of curious as to why 

### 4 would one lab not do what you told them to do and the other two 

### 5 do it? I mean is that essentially what happened? 

### 6 **DR. RODNEY JOHNSON:**We've asked ourselves 

### 7 that. I am not sure why there were deviations from the 

### 8 recommendations. I do believe that our recommendations were not 

### 9 as clear-cut emphatic as they need to be and should have been. 

### 10 I mean I think there are certain places where you say, there is 

### 11 no discretion here unless you can show that whatever it is that 

### 12 you propose meets at least the minimum standard. And I honestly 

### 13 do not care if they deviate but they have to demonstrate that 

### 14 they can meet some minimum. And for starts, you would think at 

### 15 least they would maybe try the guideline and then deviate, but 

### 16 they did not. 

### 17 And the other thing I had emphasized early on 

### 18 in the talk was that demonstrated capacity of genetic sexing 

### 19 should be a priori; do not even start the test until you can do 

### 20 that. 

### 21 **DR. DANIEL SCHLENK:**Next question actually. 

### 22 So the DMY method, is this the gel run basically where you look 

### 23 for the ban? I mean it was not clear in the documentation which 

### 24 method you are actually proposing, if it is the gel 

### 25 visualization method or is it actually just qPCR. 

### 26 **DR. RODNEY JOHNSON:**qPCR method.

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1 **DR. DANIEL SCHLENK:**So whether it is there or 

### 2 not. 

### 3 **DR. RODNEY JOHNSON:**Yeah. 

### 4 **DR. DANIEL SCHLENK:**So a fairly 

### 5 straight-forward method, I mean just in terms of just the 

### 6 thermocycler essentially, and sterile techniques. 

### 7 **DR. RODNEY JOHNSON:**Thermocycler and good 

### 8 probs. 

### 9 **DR. DANIEL SCHLENK:**Okay. And I am curious 

### 10 too, just noticing your paper that just came out not too long 

### 11 ago, how would you compare the DMY versus the (inaudible) or any 

### 12 of the other probes that you guys saw in that particular study? 

### 13 I mean what gave you the best -- I know that the DMY is probably 

### 14 more robust. 

### 15 **DR. RODNEY JOHNSON:**It is definitive. I mean 

### 16 it does in fact tell you which one is a male and which one is a 

### 17 female genetically. Now the other markers are more phenotypic 

### 18 actually, and they are not genotypic. Those would be the 

### 19 expression phenotype of a male or a female independent of DMY. 

### 20 And we are quite certain that some chemicals can trigger that 

### 21 expression pattern of a male or a female gonad independent of 

### 22 DMY. And chemicals can do it and temperature can do it and some 

### 23 things that we do not understand can do it. And it is the 

### 24 things that we don't understand that we want to keep below two 

### 25 percent in our controls. 

### 26 **DR. DANIEL SCHLENK:**So I am just curious.

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1 Would you be proposing those as particular endpoints that might 

### 2 function in that capacity? 

### 3 **DR. RODNEY JOHNSON:**Absolutely. They very 

### 4 much could be, yes. 

### 5 **DR. DANIEL SCHLENK:**Okay. And on slide 65, 

### 6 which is going to be the Data Expectation slide -- it kind of 

### 7 segways into the last question I have here. So you have for 

### 8 males in toxicity in the bottom line, that toxicity, you would 

### 9 see a decrease in male vitellogenin. Wouldn't you also see that 

### 10 in female vitellogenin as well? 

### 11 **DR. RODNEY JOHNSON:**Yes. Absolutely. 

### 12 **DR. DANIEL SCHLENK:**Okay. I wanted to check 

### 13 that, which gets to the next question. So in the MRT, you are 

### 14 proposing essentially to eliminate any sort of growth or 

### 15 measurements in F0, is that correct? 

### 16 **DR. RODNEY JOHNSON:**Well it is certainly 

### 17 prudent to measure growth anytime you can. I mean I would just 

### 18 simply say that. 

### 19 **DR. DANIEL SCHLENK:**So growth is still going 

### 20 to be measured in F0. 

### 21 **DR. RODNEY JOHNSON:**I think it should be, but 

### 22 we don't have very much time in their adults in the first place, 

### 23 so you could see probably some toxicity reductions perhaps. 

### 24 **DR. DANIEL SCHLENK:**Because you are not going 

### 25 to be doing histopath in those, correct? 

### 26 **DR. RODNEY JOHNSON:**No histopath in F0, no.

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1 **DR. DANIEL SCHLENK:**So the only metric you 

### 2 would actually have to determine an acute non-endocrine based 

### 3 toxicity would be growth if you are not going to be doing 

### 4 histopath on those guys, right? 

### 5 **DR. RODNEY JOHNSON:**You would have growth. 

### 6 And more than likely, if it is serious toxicity, you would see 

### 7 pathology. But if we are not doing pathology in that group, 

### 8 which we are not, growth would be the best one for F0 toxicity. 

### 9 **DR. DANIEL SCHLENK:**And you think that is 

### 10 strong enough to give you toxicity maternal -- 

### 11 **DR. RODNEY JOHNSON:**I think we will get it in 

### 12 F1. I think we will get toxicity understanding in F1 even 

### 13 though we may not have it in F0. 

### 14 **DR. DANIEL SCHLENK:**So the F2s that would be 

### 15 resulting from F1 as a result of F1 exposure -- I guess what I 

### 16 am concerned about is maternal effects that would manifest 

### 17 themselves in developmental abnormalities but it would be 

### 18 indirect. It would be because of acute toxicity. 

### 19 So for example, if you had hepatic toxicity 

### 20 with some agent that targets the liver that has no endocrine 

### 21 response whatsoever, but you are not going to be making 

### 22 vitellogenin for example. If you are not making vitellogenin, 

### 23 you are going to have reduced fecundity. And then that may lead 

### 24 itself to things in F1. 

### 25 **DR. RODNEY JOHNSON:**And at the end of F1, 

### 26 there is histopathology.

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1 **DR. DANIEL SCHLENK:**Right. But I guess at 

### 2 that point, the F1s may not have any endocrine response, per se, 

### 3 but it is all because of what happened at F0. Does that make 

### 4 sense? It is the maternal effects that happened at F0 that 

### 5 could lend themselves to something that is not really there, I 

### 6 guess. 

### 7 **DR. RODNEY JOHNSON:**Perhaps. I guess I am 

### 8 not sure. 

### 9 **DR. MARTHA SANDY:**Dr. Schlenk, I think maybe 

### 10 you are bordering a little bit into getting into the charge 

### 11 question. 

### 12 **DR. DANIEL SCHLENK:**No, I am not. 

### 13 **DR. MARTHA SANDY:**No, you are not, okay. 

### 14 **DR. DANIEL SCHLENK:**I am asking if you think 

### 15 that the maternal effects at F0 basically can manifest 

### 16 themselves or -- the bottom line is, what you are saying is you 

### 17 think the assay as it stands, MRT, would pick up any effects at 

### 18 F0 in the F1s. That is what you are saying. Is that what I 

### 19 hear? 

### 20 **DR. RODNEY JOHNSON:**I think that if there is 

### 21 maternal transfer of contaminant of chemical or maternal factors 

### 22 that are caused by exposures in F0 that they will be expressed 

### 23 in F1 and they will see them in F1. 

### 24 **DR. DANIEL SCHLENK:**Okay. And then last, 

### 25 this gets back to the Jonckheere-Terpstra for the stats. So can 

### 26 you just help me out with that? That is a stepwise measure. So

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1 when I see a JT in the figures, what does that mean? Does that 

### 2 mean that that point is different from the point preceding it or 

### 3 it is different from -- 

### 4 **DR. RODNEY JOHNSON:**It is the one that is 

### 5 statistically different from the controls. 

### 6 **DR. DANIEL SCHLENK:**So it is the same thing 

### 7 as a Dunnett. 

### 8 **DR. RODNEY JOHNSON:**Yes. It is a different 

### 9 procedure, but the JT -- 

### 10 **DR. DANIEL SCHLENK:**And Dunnett's are 

### 11 basically the same thing. 

### 12 **DR. RODNEY JOHNSON:**Right, right. Well 

### 13 again, different procedure, and it is a trend test which means 

### 14 that -- 

### 15 **DR. DANIEL SCHLENK:**Is it a trend for 

### 16 dose-dependents is really what I am after. 

### 17 **DR. RODNEY JOHNSON:**There are several 

### 18 requirements for it to be a legitimate statistical test. One of 

### 19 them is that the dose-response has to be monotonic. And then 

### 20 secondly, what the procedure does if it is doing it is that you 

### 21 look for the statistically significant dose, take off the high 

### 22 dose, run it again and look for the lowest treatment that is 

### 23 statistically different from the controls as you take off the 

### 24 higher steps, and that becomes the treatment at which there are 

### 25 significant difference from the control. 

### 26 **DR. DANIEL SCHLENK:**So is it a test to

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1 determine dose-dependence? 

### 2 **DR. RODNEY JOHNSON:**Yes. 

### 3 **DR. DANIEL SCHLENK:**It is? 

### 4 **DR. RODNEY JOHNSON:**Yes. 

### 5 **DR. DANIEL SCHLENK:**Okay. I am done. 

### 6 **DR. MARTHA SANDY:**Dr. Patino? 

### 7 **DR. REYNALDO PATINO:**I have some follow-up 

### 8 questions to the DMY or genetic sex determination of fish. You 

### 9 have shown that you can have some, was it XY female in these 

### 10 matches with temperatures, is that right? 

### 11 **DR. RODNEY JOHNSON:**XX males. 

### 12 **DR. REYNALDO PATINO:**And that that was 

### 13 strain-dependent? 

### 14 **DR. RODNEY JOHNSON:**The prevalence of that 

### 15 within a population is strain-dependent. 

### 16 **DR. REYNALDO PATINO:**Yes. Do you have 

### 17 similar information just generally speaking on whether there may 

### 18 be some strain differences in the background mismatches of XX 

### 19 males and XY females using in any population or any strains of 

### 20 medaka? 

### 21 **DR. RODNEY JOHNSON:**There is a very low 

### 22 prevalence -- in the studies that have been done with what would 

### 23 be XY females, there are a few studies that have been done with 

### 24 wild fishes in Japan where they have looked at the relationship 

### 25 between DMY and the expressed gender, and they have shown that 

### 26 in about -- it is very, very low prevalence. One in a thousand

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1 kinds of numbers that they get what would be XY females. 

### 2 And then what they have done is they have 

### 3 sequenced the DMY gene, and they have shown that in those fishes 

### 4 that express that phenotype that there is mutations in the DMY 

### 5 gene preventing it from being expressed. So it is like a stop 

### 6 code on them. And then those fish, instead of becoming males, 

### 7 become females. So it is an XY female that is very rare, XX 

### 8 males is not as rare and seems to show a strain dependency. 

### 9 And so the prevalence of XX males is 

### 10 detectable. It is likely to show up once in a while in the 

### 11 controls of an MRT study or an MMT study. Any study that is 

### 12 looking for genotype/phenotype matching, it is not going to be 

### 13 unusual. It is going to be low but it is not unusual to find XX 

### 14 males. 

### 15 **DR. REYNALDO PATINO:**Okay. In your study 

### 16 with vinclozolin, if I read correctly the report, you did find 

### 17 some XY female mismatches and you only found them in the 

### 18 treatments, is that correct, not in the controls? 

### 19 **DR. RODNEY JOHNSON:**I think they were also in 

### 20 controls but I am not certain. I would have to do some checking 

### 21 on that. I did not study that data as carefully because of some 

### 22 reasons where -- I think the great majority of problems that we 

### 23 found in the inter laboratory studies had to do with QA/QC 

### 24 problems. So we tended to take that data with a major grain of 

### 25 salt. 

### 26 **DR. REYNALDO PATINO:**Yes. So I am more

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1 interested in the XY female mismatches, and I remember in the 

### 2 report you only found them in the treatments. But please do 

### 3 check and correct me if I am wrong. But my question is, you 

### 4 know, the Japanese study -- they did a similar study. They did 

### 5 not do the vinclozolin, did they? 

### 6 **DR. RODNEY JOHNSON:**No. 

### 7 **DR. REYNALDO PATINO:**I guess you already 

### 8 answered that question in part. But in the controls of any of 

### 9 the studies, other than the inter-laboratory validations, the 

### 10 incidence of XY females is very rare or rarely found? 

### 11 **DR. RODNEY JOHNSON:**Can I ask a question? 

### 12 Have we ever seen that, XY females in our study? No. 

### 13 **DR. REYNALDO PATINO:**All right. Thank you. 

### 14 **DR. MARTHA SANDY:**Dr. Johnson, can you tell 

### 15 us who you were conversing with? 

### 16 **DR. RODNEY JOHNSON:**Oh. I spoke with Kevin 

### 17 Flynn. Kevin Flynn is the coauthor on the talk and Kevin has 

### 18 done a gazillion genetic sexing studies compared to me. 

### 19 **DR. REYNALDO PATINO:**Thanks. Follow-up 

### 20 questions. So in your assessment of endpoints in the EPA study, 

### 21 you used a genotype to determine males/females and then do the 

### 22 calculations on the endpoints, is that correct? 

### 23 **DR. RODNEY JOHNSON:**Yes. 

### 24 **DR. REYNALDO PATINO:**Yes. But if I read the 

### 25 report correctly, in at least one of the laboratories because of 

### 26 the problems of incorrect sexing genotypically, it appears that

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1 they used either the gonadal sex or the secondary sex. 

### 2 **DR. RODNEY JOHNSON:**Right. 

### 3 **DR. REYNALDO PATINO:**I guess my question was 

### 4 what is the rationale for using the secondary sex to sex the 

### 5 fish as opposed to the gonadal sex and why either or? 

### 6 **DR. RODNEY JOHNSON:**I think it was what was 

### 7 most readily available to the laboratory to use and what seemed 

### 8 to jive. 

### 9 I mean because there were so many problems 

### 10 with the genetic sexing, there was an attempt to try and 

### 11 understand the data with what would be concordance amongst the 

### 12 phenotypic sexes that they had. And they had secondary sex and 

### 13 they had gonadal sex, and then tried to see if they were 

### 14 concordant. And then if they were, try and say well then that 

### 15 probably was the genetic sex of the animals, especially if it 

### 16 was a control. 

### 17 Now if you start asking that in the context of 

### 18 the treatments, then you start getting into trouble because then 

### 19 you have to start looking at population responses and the sample 

### 20 sizes are not big enough to do that kind of analysis of effects 

### 21 on the gender phenotype. It just simply does not have the power 

### 22 to do that. So quite frankly, I think those parts of those 

### 23 studies were flawed sufficiently that to take home a real 

### 24 serious story of what did vinclozolin do to these fish is 

### 25 probably not possible. 

### 26 Except for the things that I did mention in my

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1 summary -- which was that there were sort of some consistencies 

### 2 in some growth effects -- the one thing that we saw that looked 

### 3 to be a treatment related effect was an effect on the secondary 

### 4 sex characters of the male, and they did show some tendencies 

### 5 towards having that same response. And we did not show any 

### 6 other effect in our study either and we were pretty careful for 

### 7 at least the exposure part of that study. I mean we obviously 

### 8 were careful with the whole study. 

### 9 The most important thing I want to say is we 

### 10 tried to be really careful with keeping our flow dynamics of our 

### 11 exposure system relatively brisk so that we were testing parent 

### 12 and not degradence because vinclozolin is a very, very quickly 

### 13 degrading chemical in aqueous solution, and not only that, some 

### 14 of the degradence are apparently endocrine-active. So in order 

### 15 to test the parent and not the degradence, you have to try and 

### 16 turn over your tank volumes very quickly. And we did measure 

### 17 parent and degradence in our studies, and in the inter-lab 

### 18 studies, the degradence were not measured. 

### 19 I mean some of the differences I cannot 

### 20 account for. I do think there were similarities to say well, we 

### 21 did kind of show somewhat the same result. 

### 22 **DR. REYNALDO PATINO:**Yes. So that leads me 

### 23 to another question on the test chemical used for the 

### 24 inter-laboratory validation. Why vinclozolin? I ask that 

### 25 question in regards to the avian studies, but why did you pick 

### 26 that one for the study?

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1 **DR. RODNEY JOHNSON:**Well I think the primary 

### 2 reason was to see the response in the fish study to an 

### 3 antiandrogen, extensively an antiandrogen. If we could do it 

### 4 again, I think we would certainly use a chemical that is much, 

### 5 much more simple to deal with, especially as it relates to the 

### 6 dynamics of chemistry. We just wouldn't pick a chemical like 

### 7 that. At the same time, I understand the interest in looking 

### 8 for another mode of action, but I do not think it was the right 

### 9 place for that. So it was something that is a mistake, should 

### 10 not have done that chemical. 

### 11 **DR. REYNALDO PATINO:**Okay, thank you. And 

### 12 one last simple question. In your results -- I think it was 

### 13 trenbolone -- what is your interpretation of the finding that 

### 14 you found that would use vitellogenin expression in females? 

### 15 **DR. MARTHA SANDY:**Can you repeat the name of 

### 16 the chemical? 

### 17 **DR. REYNALDO PATINO:**Trenbolone. I was 

### 18 trying to match the expected outcome versus what you found. 

### 19 **DR. RODNEY JOHNSON:**I do not have at the top 

### 20 of my head a good AOP explanation, and certainly I can converse 

### 21 with my colleagues and we can talk about that, but I honestly do 

### 22 not know for sure if that is derived from the data. 

### 23 I mentioned when I showed that slide that some 

### 24 of that data has been shown in the Tier 1 test with the fish and 

### 25 I honestly cannot tell you for sure whether that is the 

### 26 rationale for some of those arrows on that or whether it was our

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1 test explicitly, so I just would have to talk to my people about 

### 2 that. 

### 3 **DR. REYNALDO PATINO:**Well thank you. 

### 4 **DR. MARTHA SANDY:**Before I take anymore, I 

### 5 just want to remind, it is very important that the Panel get a 

### 6 chance to ask the clarifying questions, but remember it is 2:30 

### 7 and we are a little behind. So if we can keep the questions and 

### 8 the answers short, it would be great. Jim, I think you are 

### 9 next. 

### 10 **DR. JAMES MCMANAMAN:**I think the answer is it 

### 11 acts as an androgen. 

### 12 **DR. RODNEY JOHNSON:**Well it sure should. 

### 13 Good. 

### 14 **DR. JAMES MCMANAMAN:**So on slide 66 you show 

### 15 a summary of several EDR compounds and prochloraz appears to be 

### 16 the only steroidogenic inhibitor that you tested, and it appears 

### 17 to be primarily acting as an aromatase inhibitor. 

### 18 So my question is, is this a concern for 

### 19 evaluating effectiveness of the MMT versus the MRT assays as a 

### 20 T2 because you really did not take that steroidogenesis 

### 21 inhibitor all the way through the F2s and you did not look at 

### 22 its effectiveness as well as you did some of the anti-estrogens 

### 23 or anti-androgens. 

### 24 **DR. RODNEY JOHNSON:**I guess I do not know 

### 25 what I would have expected at the end of an F2 with an aromatase 

### 26 inhibitor. I certainly do think we got the kind of response

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1 that we would have expected out of an aromatase inhibitor in the 

### 2 context of its effect on the pathology and effect on the 

### 3 vitellogenin production. And it also did have effect on 

### 4 fecundity. And it is not a real strong indicator of the mode of 

### 5 action or the AOP, and I do not think the MRT or the MMT are 

### 6 strong supporters of modes of action, per se. 

### 7 I think they do a good job of identifying 

### 8 estrogen agonist. I think they do a really good job of showing 

### 9 androgen receptor agonist and I do not think we have a large 

### 10 dataset to tell you how it behaves with other modes of action. 

### 11 And I just simply do not think we can use our data to give you 

### 12 strong expectations of those types of modes of action. 

### 13 **DR. JAMES MCMANAMAN:**That answer partially 

### 14 addresses my question, but I was also thinking about compounds 

### 15 that could have more of a global steroidogenic disrupting effect 

### 16 rather than just an anti-aromatase. 

### 17 **DR. RODNEY JOHNSON:**I do not know what they 

### 18 might be. 

### 19 **DR. JAMES MCMANAMAN:**Maybe it would be assay 

### 20 pick up, something like that; that is my question. 

### 21 **DR. RODNEY JOHNSON:**Well I do think it would, 

### 22 yes. Yes, I think it would. 

### 23 **DR. LESLIE TOUART:**I think though that with 

### 24 prochloraz, is it not just an aromatase inhibitor. It does 

### 25 affect other enzymes within the steroidogenesis pathway, so 

### 26 maybe not as much as the aromatase but there are some of the

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1 enzymes that are affected. So I think the context is that the 

### 2 test method would still detect adverse consequences. The 

### 3 specifics of what it has interfered or modulated through a given 

### 4 AOP or steroidogenesis pathway may not be as elucidated but you 

### 5 could still pick up the adverse effects. 

### 6 **DR. JAMES MCMANAMAN:**Well I was thinking 

### 7 about multigenerations having an effect on F1 that could be 

### 8 magnified at F2 if it had a larger global effect, but just 

### 9 whether it has been considered or not. 

### 10 **DR. RODNEY JOHNSON:**I do not know of any 

### 11 evidence that I have read in the literature -- and certainly we 

### 12 have not done it -- where we show exacerbation of effects in F2 

### 13 compared to F1. 

### 14 **DR. JAMES MCMANAMAN:**Right. So the last 

### 15 question is what pathology assays are you proposing, or is it 

### 16 just standard histopath? And given the problems with getting 

### 17 labs to do simple PCR assays correctly, what plans do you have 

### 18 to train people to do the histopath assays? 

### 19 **DR. RODNEY JOHNSON:**Well it actually turns 

### 20 out that there are several histopathology labs in the US that do 

### 21 a good jobs on fish, and they have been in fact part of sort of 

### 22 our training programs already and they have been working with us 

### 23 on guidance documents. And they have also been involved in the 

### 24 OECD guidance documents that have been developed. So I think, 

### 25 actually we are doing better with the pathology than we are with 

### 26 some of the other parts of the tech transfer. So I think

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1 actually pathology tech transfer is reasonably on track. 

### 2 **DR. JAMES MCMANAMAN:**Is there a biochemical 

### 3 assay for patotoxicity that you could use, like in mammals use 

### 4 ALT? 

### 5 **DR. RODNEY JOHNSON:**Perhaps. 

### 6 **DR. JAMES MCMANAMAN:**I mean I am just 

### 7 wondering if that might simplify it. 

### 8 **DR. RODNEY JOHNSON:**Simplify it, yes. 

### 9 **DR. MARTHA SANDY:**I think next we have Dr. 

### 10 Christman and then Kullman. 

### 11 **DR. MARY CHRISTMAN:**I will try to make this 

### 12 quick. I want to ask questions about the statistics, so 

### 13 StatCHARMMS. You sent out the information saying that the 

### 14 one-way ANOVA had been corrected. Could you explain 

### 15 specifically how it was corrected; I did not quite get that. 

### 16 **DR. RODNEY JOHNSON:**Okay. 

### 17 **DR. MARY CHRISTMAN:**I mean, to me there is 

### 18 one of two ways to go. One is you simply average to the tank 

### 19 and use those means; and the other ways is you added a random 

### 20 effect for the nesting. 

### 21 **DR. RODNEY JOHNSON:**And what we ended up 

### 22 doing was we reran the statistics in another part of the module, 

### 23 in fact, were ran it in the repeated measures module with one 

### 24 measurement rather than a series of measurements. And then that 

### 25 particular module incorporates the replication component and 

### 26 there is a standard ANOVA with a Dunnett. So we did not correct

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1 that particular module. 

### 2 Now I know that, instead -- I mean in other 

### 3 words -- how do I say this? The repeated measures module of 

### 4 StatCHARRMS takes the replicate information into account 

### 5 properly, so we used that part of StatCHARRMS which has an ANOVA 

### 6 which is an ANOVA, and instead of having repeated values, it was 

### 7 one value. 

### 8 **DR. MARY CHRISTMAN:**At one point in time. 

### 9 **DR. RODNEY JOHNSON:**Yes, one point in time. 

### 10 And that was how that analysis was rerun. 

### 11 **DR. MARY CHRISTMAN:**Okay. Which leads to the 

### 12 question about the repeated measures analysis or the fecundity 

### 13 and fertility. What was the covariant structure used for the 

### 14 repeated observations on the same pair? 

### 15 **DR. RODNEY JOHNSON:**I cannot answer that. I 

### 16 do not know. 

### 17 **DR. MARY CHRISTMAN:**Okay. And finally, for 

### 18 the power analysis, did you do pair-wise T tests or did you 

### 19 actually do the power analysis based on the idea that it would 

### 20 be a step-down procedure for the LOEC? 

### 21 **DR. RODNEY JOHNSON:**It was based on the 

### 22 step-down procedure. It used the repeated measures module of 

### 23 StatCHARRMS procedure. 

### 24 **DR. MARY CHRISTMAN:**No, not the repeated 

### 25 measures but the testing of where the LOEC was, not the time 

### 26 problem. Because a description that I saw in another study said

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1 that the power analysis was done based on a T test; and so I am 

### 2 wondering whether this was that same power analysis or whether 

### 3 this used the JT procedure and we would have had to have done a 

### 4 Monte Carlo type analysis. 

### 5 **DR. RODNEY JOHNSON:**And I will have to check 

### 6 on that too. 

### 7 **DR. MARY CHRISTMAN:**Okay, thank you. 

### 8 **DR. MARTHA SANDY:**Dr. Kullman? 

### 9 **DR. SETH KULLMAN:**Okay Rodney, last but not 

### 10 least. So a couple of things concerning your QA/QC protocol in 

### 11 the SOP in general. I think it has been alluded to that it 

### 12 appeared that there were several aspects of the SOP that did not 

### 13 appear to be present. And I was wondering if that might have 

### 14 some relation to some of the variability of some of the data 

### 15 that you saw. 

### 16 So for example, just simple PCR; I know you 

### 17 have a protocol for sex determination. But also things like 

### 18 vitellogenin, you know, the amount of RNA, the storage of the 

### 19 liver, how the liver is. The RNA is extracted, primers used, 

### 20 all those can have really large impacts on an output of a qPCR. 

### 21 22 Do you guys have an SOP for that? And I guess 

### 23 the question sort of extends beyond that as well, is, are there 

### 24 SOPs that you could establish similar guidance that you put 

### 25 together for the histology that would sort of allow the 

### 26 laboratories to sort of coordinate their processes in these

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1 endpoints? 

### 2 **DR. RODNEY JOHNSON:**We have internal SOPs. 

### 3 We have not put them into the guidelines explicitly and we 

### 4 certainly can. And obviously Kevin would be willing to and with 

### 5 no problem, absolutely. 

### 6 **DR. SETH KULLMAN:**Yes. I mean the simple 

### 7 thing is primary design for vitellogenin, so it is it not clear 

### 8 if you are going for vitellogenin 1, vitellogenin 2, you know, 

### 9 which vitellogenin you are after in that. 

### 10 Okay. So we have touched on a couple of these 

### 11 but I want to get a little bit more clarification. You also 

### 12 have shown that you are using a genetic gender to identify 

### 13 breeding pairs, and I think that is a great way to go about 

### 14 doing that. But it seems like, in many of the assays, that you 

### 15 are not fully exploiting the capability of making ratios for sex 

### 16 determination, so I did not see in any of the data numbers of 

### 17 males, numbers of females, and certainly no ratios between how 

### 18 well the genetic sex correlates to phenotypic sex. 

### 19 And it seems like it might be a very 

### 20 interesting output to have where you are looking at your 

### 21 secondary sex characteristics such a papillae or even a split in 

### 22 the anal fin for the males when comparing that to your genetic 

### 23 sex. Is there any reason why that type of comparison is not 

### 24 done in the assay? 

### 25 **DR. RODNEY JOHNSON:**I think every case, I 

### 26 think -- if this true, I may be wrong -- but the gender

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1 phenotype as expressed by the gonad has been, I think in all 

### 2 cases, the same as the gender phenotype expressed by the anal 

### 3 fin. So it does not give you an independent different answer 

### 4 than comparing the outcome of the gonad phenotype with the 

### 5 genotype. 

### 6 **DR. SETH KULLMAN:**Okay. In the introduction 

### 7 of the ISR, you talk about organizational versus activational 

### 8 effects. Can you clarify that a little bit more on what exactly 

### 9 do you mean about activational versus organizational and how the 

### 10 modifications in the MRT might sort of in essence not allow you 

### 11 to really make those determinations for those differences? 

### 12 **DR. RODNEY JOHNSON:**Well I think in general 

### 13 the interpretation of activational versus organizational effects 

### 14 is generally thought of in the context of a developmental effect 

### 15 being what we would consider an organizational effect as 

### 16 compared to activation where we are primarily talking about the 

### 17 endocrine functions that lead towards the development of actual 

### 18 reproductive process in behavior and the outcome of production 

### 19 of viable embryos. 

### 20 So think of that as being primarily an 

### 21 activational process. It is clear though that those 

### 22 distinctions are somewhat fuzzy. I mean to imply that 

### 23 activation does not result in some kind of an organizational 

### 24 effect in some tissue I think is naive. 

### 25 But I think, in general, the idea of being 

### 26 that there is sort of a developmental effect, which we think of

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1 as organizational, activational being sort of those kinds of 

### 2 events that are occurring later on in development that lead to 

### 3 the actual successful reproduction. 

### 4 Like for instance, the Tier 1 fathead minnow 

### 5 reproductive test primarily is measuring effects on activation, 

### 6 for the most part, because those animals that are being used in 

### 7 that study have already developed all of their gonadal 

### 8 structures, all of their secondary sex characters. Everything 

### 9 about them that makes the male and female has already happened 

### 10 before they start the study. So when you treat those fish with 

### 11 a chemical to try and look for an impact, what you are really 

### 12 looking for is what I think would be an activational effect. 

### 13 When we go into the MMT or the MRT, what we 

### 14 are doing is measuring both. We are literally looking for 

### 15 effects on embryo development and early stage development on 

### 16 gonad structure, gonad function, and ultimately its capacity to 

### 17 be activated also, and of course brain involvements through that 

### 18 whole process. 

### 19 So to some extent, I think it is simplistic to 

### 20 say we are studying organization and activation, and then 

### 21 instead it is sort of a continuum; and in the MRT and in the MMT 

### 22 what you are really doing is looking at the whole progression of 

### 23 development in the context of reproductive competence. 

### 24 **DR. SETH KULLMAN:**Right, I agree with that. 

### 25 And I am curious though, if you are looking at for 

### 26 organizational, why you do not include any sort of developmental

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1 checkpoints within that. 

### 2 **DR. RODNEY JOHNSON:**Right. I mean we truly 

### 3 are looking for what would be an apical outcome of ostensibly an 

### 4 organizational process that is disrupted. Yes. 

### 5 **DR. SETH KULLMAN:**Okay, let's see, just a 

### 6 few more things. Go to slide 76, if you will. It is your 

### 7 illustration of the new MRT protocol. Okay. 

### 8 So my question here is, if you look at the 

### 9 nine-week in the F1 generation, you are not conducting any 

### 10 histopathology. And if you look at the 18-week of the same 

### 11 animals, you are not conducting any vitellogenin. I am curious 

### 12 of why you have not coordinated histopathology and vitellogenin. 

### 13 So I imagine, in the F1 generation, having histopathology sort 

### 14 of at the same time that the animals are vitellogenic might 

### 15 produce a nice concordance of any potential effects on 

### 16 vitellogenin with gonad morphology. 

### 17 And then vice-versa, you are doing 

### 18 histopathology in the F1 at 18 weeks but you are not doing any 

### 19 vitellogenin. I would imagine that if you coordinated both your 

### 20 histopathology and your vitellogenin you are going to sort of 

### 21 maximize your output for that data by correlating histopath in 

### 22 the gonad and/or liver with vitellogenin production. 

### 23 **DR. RODNEY JOHNSON:**I agree in concept. I 

### 24 mean I do not disagree with you at all. What we have done is we 

### 25 have sort of made a choice to correlate the pathology with the 

### 26 fecundity endpoint rather than correlate the pathology directly

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1 with the biomarker vitellogenin endpoint. So as it truly is a 

### 2 decision as to what would be the best use of the tissue because 

### 3 of the fact that all of the pathology is done with all the 

### 4 tissues in situ, so it is a whole animal histopathology 

### 5 analysis. 

### 6 And to do the vitellogenin, you remove some of 

### 7 the liver or part of the liver, and we have done that. In fact, 

### 8 that is what we have done at the nine-week sacrifice. We have 

### 9 also taken some fish for vitellogenin and some fish for 

### 10 histopathology. 

### 11 But we felt especially in the MRT where we 

### 12 have reduced the histopathology effort to what we consider to be 

### 13 a bare minimum, which is 12 animals of each gender per 

### 14 treatment, and then tried to correlate it directly with the 

### 15 reproductive output of those animals. So we know that this pair 

### 16 had this reproductive output and this is the pathology 

### 17 associated, we feel like we are better able to correlated 

### 18 pathology with reproduction and essentially give it up, if you 

### 19 will, for the biomarker endpoint of vitellogenin, which I do 

### 20 believe is an important one, and I am not denying that. 

### 21 **DR. SETH KULLMAN:**Yes. I actually do not 

### 22 know if it needs to be exclusive. I mean we personally run 

### 23 studies where we cut the liver in half and use half for histo 

### 24 and half for RNA. 

### 25 **DR. RODNEY JOHNSON:**We did. We have done 

### 26 that too, but the difficulty has been with the histological

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1 method that we have chosen to use, which in this case, we used 

### 2 to do a method where we did a transfer sort of a frontal type 

### 3 sectioning with the sub-adult fish, which pretty much leaves you 

### 4 working with the gonad and the kidney, and I did mention that. 

### 5 Because we wanted to have comprehensive 

### 6 histopathology across all possibilities of tissue, we are doing 

### 7 sagittal sectioning now at the end of the reproductive endpoint, 

### 8 and that allows us to get gonad, liver, pituitary, thyroid, all 

### 9 the other tissues that may or may not be associated with an 

### 10 explanation of responses. 

### 11 So it is a more comprehensive histopathology 

### 12 in that it samples more tissues, fewer animals but animals that 

### 13 we have better case data for. So I think it works in the end to 

### 14 the advantage of the assay. 

### 15 **DR. MARTHA SANDY:**I do not want to keep Dr. 

### 16 Johnson here too long. I see we have two more people here, and 

### 17 realize we are running an hour behind, so why don't we just move 

### 18 up the table. So that is Dr. Scanes. 

### 19 **DR. COLIN SCANES:**Continuing on the 

### 20 histopath, a number of questions. Did you take sample in a 

### 21 random way if you are looking at the liver or the gonads or was 

### 22 this representative samples or how did you define -- what were 

### 23 you sampling? 

### 24 **DR. RODNEY JOHNSON:**It varied between 

### 25 studies. In most of the studies that we did at EPA, we took all 

### 26 the samples we had. And on occasion, I think the Japanese

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1 subsampled, and they subsampled a prescribed number per 

### 2 treatment of males and females and did some histopathology on 

### 3 those. 

### 4 **DR. COLIN SCANES:**I am thinking of the 

### 5 sections. Did you do that in a systematic way or random way or 

### 6 a judgment based? 

### 7 **DR. RODNEY JOHNSON:**We do that in a judgment 

### 8 way. The SOP is specific in the sense that you cut, for 

### 9 instance, certain place into the specimen and then take four 

### 10 sections, 200 microns apart and mount them on one slide and read 

### 11 all slides, all sections. 

### 12 So it is a systematic with a not highly 

### 13 specified start point, but it is a recommended approximation of 

### 14 where to start. 

### 15 So the point, I think, is that if there is a 

### 16 big animal or a small animal, there is some variance in that 

### 17 it's like exactly what part of the gonad are you looking. But 

### 18 we do step sections through the tissue and we start with 

### 19 landmarks -- on the sagittal sectioning, we start with landmarks 

### 20 within the animal. 

### 21 So on the medial margin of the eye is where 

### 22 the first section comes, and we know that we are going to get 

### 23 liver and spleen and the different tissues there; then take two 

### 24 sections at the midline and they are 200 microns a part and then 

### 25 another one at the other side of the lateral median of the other 

### 26 eye, so there are five samples taken for every specimen at the

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1 adult stage. 

### 2 **DR. COLIN SCANES:**And a follow-up to that; 

### 3 and that is what is basically your thinking to do this simply by 

### 4 a scoring system rather than say a morphometric analysis where 

### 5 you could look at number of oocytes per unit area, for instance, 

### 6 or number of vacuoles per unit area in the liver? 

### 7 **DR. RODNEY JOHNSON:**The morphometric methods 

### 8 are arduous in terms of time, and they are difficult to do 

### 9 consistently across laboratories. It is possible and it is 

### 10 possible to do it with image analysis help. I think, if we 

### 11 review the methods with the pathologists, they tend to reject 

### 12 those notions and would prefer to do -- Vicky will tell me I am 

### 13 wrong maybe, I do not know. But all that to say, I think having 

### 14 good references does a good job. 

### 15 **DR. COLIN SCANES:**On the other hand, you 

### 16 could make the point that if you are using a morphometric 

### 17 analysis, image analysis, you do not have to get into very 

### 18 different ways of doing statistics. You have got a much more 

### 19 normal distribution. 

### 20 **DR. RODNEY JOHNSON:**Yes, I agree. 

### 21 **DR. COLIN SCANES:**And finally, what is the 

### 22 basis for the histopath doing this simply by hematoxylin and 

### 23 eosin staining as opposed to say in situ or immunasotic 

### 24 chemistry? 

### 25 **DR. RODNEY JOHNSON:**The primary rationale is 

### 26 that we do not have a good set of particular things that we

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1 would want to look for. I mean it is a very generalized but 

### 2 powerful method to detect lots of general outcomes, but 

### 3 obviously it does not give you the capacity to pursue a 

### 4 particular mode of action in great detail. And it could 

### 5 probably be done almost for sure, but in a sense that is out of 

### 6 the range of the capacity of the test system to -- I mean it is 

### 7 sort of out of its design to some extent. I agree with you 

### 8 completely in the context of the research agenda and mode of 

### 9 action, I agree. I agree, yeah. 

### 10 **DR. MARTHA SANDY:**We will go to Dr. Dallas 

### 11 Johnson and then Dr. Scott. 

### 12 **DR. DALLAS JOHNSON:**On the same slide that is 

### 13 showing up there on the screen right now, the words that you did 

### 14 not talk about on the right, experimental design consist of 

### 15 seven blocks, five blocks for chemical treatment and two blocks 

### 16 for control treatments. And I have the feeling that you are 

### 17 using blocks differently than I use blocks, and I wondered if I 

### 18 just could have you clarify your use of the word blocks there. 

### 19 **DR. RODNEY JOHNSON:**Almost for sure I am 

### 20 using the words differently than you do and I probably should be 

### 21 careful. So what I do mean? What I mean is that, essentially, 

### 22 the experimental design is parsed out into seven blocks where 

### 23 two of them -- and each block, if you will -- boy, I don't know 

### 24 how to say this -- group of replicates, let's say, which are 

### 25 multiples of six, so seven groups of replicates that are 

### 26 multiples of six.

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1 **DR. DALLAS JOHNSON:**For each dose group? 

### 2 **DR. RODNEY JOHNSON:**And there are six of 

### 3 those allocated to each dose group, except the controls which 

### 4 have 12 or two blocks. So if there is a block of six, it would 

### 5 be six for the high treatment, six for the next one until you 

### 6 get down to the controls, and then in that case it is two blocks 

### 7 of six each or a total of 12. 

### 8 **DR. DALLAS JOHNSON:**Okay, thank you. And 

### 9 then on the next slide after that one, your experimental design 

### 10 overview, I want to make sure I understand. So on the left you 

### 11 have six here, but this would generally be 12, is that right? 

### 12 **DR. RODNEY JOHNSON:**On the left side, it is 

### 13 six, and it is six replicate pairs of fish, okay? F0; this is 

### 14 describing what I called in the previous slide a block. 

### 15 **DR. DALLAS JOHNSON:**Okay. So this is one 

### 16 block? 

### 17 **DR. RODNEY JOHNSON:**This is one block. I am 

### 18 sorry. It is not clear enough. This is one block, one of 7. 

### 19 **DR. DALLAS JOHNSON:**Okay. So there would be 

### 20 seven of these. Two of them would be control and five would be 

### 21 for each dose level? 

### 22 **DR. RODNEY JOHNSON:**One for each dose level. 

### 23 24 **DR. DALLAS JOHNSON:**Okay. And then as I look 

### 25 at this, you take all the eggs that are produced by the first 

### 26 column. And do I understand that you are taking all of those

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1 eggs and you are putting them in -- I'll call it a basket, I am 

### 2 sure it is not a basket -- you throw them in the basket and you 

### 3 are mixing them all up, and then you are taking 20 eggs out and 

### 4 putting them in this first little deal at the top and then 20 in 

### 5 the next little deal and so on. 

### 6 **DR. RODNEY JOHNSON:**Yes. 

### 7 **DR. DALLAS JOHNSON:**And then they go in the 

### 8 incubators then? 

### 9 **DR. RODNEY JOHNSON:**Yes. 

### 10 **DR. DALLAS JOHNSON:**And then they come out 

### 11 with hatchlings. And then again you do the same thing. You put 

### 12 all the hatchlings in a bigger basket. 

### 13 **DR. RODNEY JOHNSON:**A bigger basket, right. 

### 14 **DR. DALLAS JOHNSON:**And you mix them all up 

### 15 again and then you again redistribute them. Okay. And is this 

### 16 the type of thing that was done in the studies that are reported 

### 17 in here, or is this just the thing that you are planning to do 

### 18 in the future? 

### 19 **DR. RODNEY JOHNSON:**This was done in most, if 

### 20 not all the studies that we report, and it is what we recommend 

### 21 for the future. 

### 22 **DR. DALLAS JOHNSON:**Okay, thank you. 

### 23 **DR. MARTHA SANDY:**Dr. Scott? 

### 24 **DR. GEOFFREY SCOTT:**Yes. Slide 69, just a 

### 25 quick comment and just want to make sure I understand where EPA 

### 26 is coming from in terms of expectations on multiple lab testing

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1 for the vinclozolin. 

### 2 If we look at the fecundity and fertility 

### 3 results there, the range of effects between the labs, the 

### 4 variation appears to be somewhere between a factor of two or 

### 5 three, somewhere in that range. 

### 6 And I guess I would just go back to a lot of 

### 7 inter-lab calibration exercises with a lot less eloquent test 

### 8 just looking at LC-50s. Seems like to me the range of variation 

### 9 was three to four, four to five. 

### 10 And I guess what I am trying to find out is 

### 11 how do you set your expectations on what you expect out of 

### 12 inter-lab comparisons? And particularly, since you are looking 

### 13 at a LOEC that is at the bottom of the curve towards the tail as 

### 14 opposed to the mid point where, like on an LC-50 where you are 

### 15 going to be in the center of the data. The point I am trying to 

### 16 make is, it would seem there should be some value in going back 

### 17 and looking -- and this is not just true for Medaka but for any 

### 18 species -- historical inter-lab testing to set some of that. 

### 19 Are you all doing that, or can you enlighten us a little bit as 

### 20 to what is your standard? 

### 21 **DR. RODNEY JOHNSON:**I do not have very much 

### 22 experience with inter-lab studies but -- all that to say, so I 

### 23 have not, and perhaps our colleagues in OSCP have. So I do not 

### 24 have an answer with respect to a standard to expect because it 

### 25 is somewhat new to me. We can talk about that and try and come 

### 26 up with some answers.

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1 **DR. GEOFFREY SCOTT:**I am only saying it could 

### 2 be inherent to the species. 

### 3 **DR. RODNEY JOHNSON:**Yes. 

### 4 **DR. GEOFFREY SCOTT:**And I am saying it could 

### 5 be redundant across endpoints, so it may be worth looking back 

### 6 at, was my suggestion to you to do as you said that. 

### 7 And then the last one and you do not have to 

### 8 look at the slide, this is really going back and looking at the 

### 9 new proposed test. You mentioned your collaborations with the 

### 10 Japanese in trying to do this concordant. I just was curious, 

### 11 are they moving to modify their test or is this the first 

### 12 attempt to modify the test? I just was kind of curious for 

### 13 standardization. 

### 14 **DR. RODNEY JOHNSON:**I sent them copies of the 

### 15 ISR and asked them to comment and they did comment. And 

### 16 basically what they said was that they agreed with some parts of 

### 17 it and they were not sure about some other parts and that they 

### 18 wanted to discuss it further. And the parts that are concerning 

### 19 to them honestly was the replication structure in the sense that 

### 20 they were concerned of the infrastructure required to run a 

### 21 test, and they said it takes a large lab. And I agree in a 

### 22 sense, but I also believe that there are labs in the U.S. that 

### 23 can take this on and have in fact considered their 

### 24 infrastructure in a way that they probably can take this on. 

### 25 **DR. GEOFFREY SCOTT:**One last clarification. 

### 26 What were their comments regarding shortening the F2 endpoint?

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1 I just was curious of that. 

### 2 **DR. RODNEY JOHNSON:**They were circumspect on 

### 3 that. They did not come down either way. 

### 4 **DR. GEOFFREY SCOTT:**Fair enough, thank you. 

### 5 **DR. MARTHA SANDY:**I would like to propose 

### 6 that we move on. So we are going to take a 10-minute break. We 

### 7 have two more presentations by EPA staff, so please be back at 

### 8 3:15 by this clock. Thank you. 

### 9 (Whereupon a break was taken) 

### 10 **DR. MARTHA SANDY:**Okay. So as the Panel 

### 11 takes their seats, I believe we have one comment or question 

### 12 from Dr. Christman. 

### 13 **DR. MARY CHRISTMAN:**Hi Rodney, this is Mary 

### 14 Christman again. I just won't let StatCHARRMS go away. 

### 15 The specific questions that I would like to 

### 16 have answers to, because you had said you would look into some 

### 17 things for me. 

### 18 In the one-way ANOVA, is the experimental unit 

### 19 the individual with a random effect per tank to acknowledge the 

### 20 clustering or is it in fact the individual observations are 

### 21 averaged over the tank and then the tank becomes the 

### 22 experimental unit with no random effect? So that is one 

### 23 question. 

### 24 And then the second question was, in the 

### 25 repeated measures design, what that means is if you have 

### 26 multiple time points, you are observing the same data on the

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1 same experimental units through time. So what I would like to 

### 2 know is how did they adjust for the covariance of the fact that 

### 3 I am measuring it from the same pair of individuals? 

### 4 So if you can find out that covariance, how 

### 5 that was accounted for, that would be great. And in the absence 

### 6 of all that, I will take the SAS code, so thank you. 

### 7 **DR. MARTHA SANDY:**Thank you. It is 3:20. I 

### 8 believe today we are only going to accomplish running through 

### 9 the last two presentations from the EPA. I think we will be 

### 10 starting the public comments tomorrow morning. So that everyone 

### 11 knows that. Unless we get surprised and move through this 

### 12 really fast. So I would now like to turn it over to Dr. Degitz 

### 13 where he will be talking about the larval amphibian growth and 

### 14 development assay. Thank you. 

### 15 **DR. SIGMUND DEGITZ:**Now this is not the first 

### 16 time I have come to D.C. and followed Rodney's presentations. I 

### 17 had one occasion I came to give a presentation which I never 

### 18 gave. So I came all the way to Washington, D.C. to never give 

### 19 that presentation because Rodney has consumed most of the day. 

### 20 So I am kind of use to this in trying to catch up and that sort 

### 21 of stuff. 

### 22 So what we are here to do is, I am going to do 

### 23 a walk-thru the integrated summary report. There are a lot of 

### 24 details in the slides. I wrote those slides such so that they 

### 25 would be a guide to you in the absence of my narration so that 

### 26 you could take them back to your hotel room, go through them.

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1 And I hope I have provided enough information there that the 

### 2 story is told completely. Thank goodness I did because now we 

### 3 might be able to jump the rocks across the river and get their 

### 4 quicker. 

### 5 I will start out by saying the outline. We 

### 6 are going to talk about rationale of the test, test species, 

### 7 test duration, test overview. I will spend a majority of the 

### 8 time talking about the inter-laboratory and then lessons 

### 9 learned. You will never go through an inter-laboratory and not 

### 10 learn things. 

### 11 So the purpose of the test is to characterize 

### 12 the adverse consequences of aquatic exposures in an amphibian 

### 13 species. The intent is to cover development maturation. And 

### 14 the initial intent was to cover reproduction, although that is 

### 15 not currently possible given the state of science for these 

### 16 frogs. 

### 17 There is only one other test method that has 

### 18 been validated for amphibians and that is the amphibian 

### 19 metamorphosis assay, which was developed in Duluth about nine or 

### 20 10 years ago and went through. We have heard some discussion 

### 21 about that already, but there is no standardized test for 

### 22 evaluating chronic effects of chemicals on amphibians. 

### 23 So ideally, the test would be capable of 

### 24 genetically sexing animals and capable of encompassing all the 

### 25 period of development through reproduction. We will get into it 

### 26 more. We fell short of that.

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1 So ideally, the test species should be robust 

### 2 in a laboratory setting, capable of determining genetic sex and 

### 3 must develop to maturity in a reasonable amount of time. Given 

### 4 those criteria, we are limited to two amphibian species 

### 5 currently in our minds. That is xenopus laevis and xenopus 

### 6 tropicalis. 

### 7 Both species are widely cultured now, although 

### 8 the tropicalis has not been in the lab nowhere near as long as 

### 9 the laevis; and laevis is considerably more robust in the 

### 10 laboratory in general. 

### 11 Both species, we have the ability to 

### 12 genetically sex both. One is with a W which is analogous to the 

### 13 Medaka DMY. And then out of the Duluth lab we developed a 

### 14 polymorphism, a marker that is linked to the female phenotype, 

### 15 but unfortunately that came from a single individual female and 

### 16 it was not possible to develop the strain up enough to be able 

### 17 to transfer that strain broadly for testing, although we have 

### 18 done that at this point. 

### 19 Although they are closely related, the one 

### 20 thing that is a very important difference is tropicalis reach 

### 21 sexual maturity much sooner, so it would be the ideal species 

### 22 over laevis, which is one and a half to two years. 

### 23 But given the lack of the animals, we were 

### 24 forced to chose xenopus laevis to move forward into 

### 25 inter-laboratory evaluation. So we had basically developed this 

### 26 test with both species, but when it was identified that we would

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1 not have the animals, we made the move for laevis. 

### 2 Test duration; due to the lack of ability to 

### 3 measure fecundity in the laboratory, basically the husbandry and 

### 4 the inability of these animals to reproduce in a laboratory 

### 5 without intervention with hormone injections, we do not have the 

### 6 luxury they do in fathead minnows in medaka. 

### 7 So then we move to the point where the intent 

### 8 was to go to surrogate measures to look at sperm count, oocyte 

### 9 numbers, things like that. We embarked on several studies; one 

### 10 was published in 2009 by my Olmstead, and those endpoints were 

### 11 incredibly variable. 

### 12 And it was clear to us after doing some 

### 13 simulations that we could not build a study large enough to 

### 14 robustly test toxicants, given the variability that existed in 

### 15 these populations in the laboratory. 

### 16 To prove that to ourselves, we conducted our 

### 17 40-week octylphenol study and basically, again, proved to 

### 18 ourselves that there was very little value in running that type 

### 19 of a study. And then as we have moved to laevis, we were 

### 20 talking more in terms of 78-week study. I do not think anybody 

### 21 really would want to do that study. 

### 22 So since it is impractical to go to sexual 

### 23 maturation, it was decided we would start with freshly 

### 24 fertilized eggs or stage eight eggs just about as soon as you 

### 25 can get them on test, given when they are laid and what you 

### 26 would have to do to get them prepared to go on test, to when

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1 significant hallmarks of development have occurred in the 

### 2 organism, superficially in the gonads. 

### 3 So here I show you two images of cross 

### 4 sections of an ovary. And on the left is a female gonad at 

### 5 stage 66 which is just at the completion of metamorphosis. We 

### 6 explored the possibility of ending the study here. Then we did 

### 7 a developmental series study from zero through 12 weeks post 

### 8 metamorphosis and here we are at 10 weeks post metamorphosis 

### 9 with basically complete ovary development and pre-vitellogenic 

### 10 oocytes. 

### 11 And then the male, we have the male stage 66 

### 12 testis on the left and the 10-week on the right. We have cystis 

### 13 formation, spermatogonia, Sertoli cells, so development is 

### 14 fairly well advanced. 

### 15 Based on these studies, we concluded that the 

### 16 stage 66 gonads were not differentiated enough for us to be able 

### 17 to make confident histological analysis of perturbation of the 

### 18 germs cells so we chose 10 weeks post, and then here is a 

### 19 summary of things that we identified as having completed 

### 20 development. 

### 21 I am going to pass over this because this 

### 22 figure it much better. The previous slide gives you more of the 

### 23 details, but the figure here -- so if I narrate this, on our 

### 24 left, this is when the study is initiated. This study proceeds 

### 25 for approximately 16 weeks. So at stage 62, the subset of 

### 26 animals are sampled and measured for thyroid endpoints

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1 histology, T4 measurements and time two stage 62. And then 

### 2 basically, these are rough numbers. It takes about another week 

### 3 to two weeks to get to stage 66. 

### 4 So what we have done is we have added eight 

### 5 weeks or 10 weeks onto 62, eight weeks onto post metamorphosis, 

### 6 and this corresponds to those histological sections that I 

### 7 showed you in terms of the development. 

### 8 To remind everyone, this is the period of 

### 9 thyroid hormone mediated development here. In the early phase, 

### 10 we have gonad differentiation in this period, and then this is 

### 11 post metamorphosis when the maturation process starts. 

### 12 So here we have the endpoints. I have divided 

### 13 it out. It is very important for us to have a suite of measures 

### 14 of overt toxicity. Specifically if we are interested in 

### 15 labeling chemicals an endocrine disruptors; we want to be 

### 16 certain that that would be occurring at concentrations where 

### 17 overt toxicity is not occurring. 

### 18 So for that, we basically observed the animals 

### 19 daily, look for mortalities, look for signs of toxicity, 

### 20 disease, those types of things. At stage 62, we make assessment 

### 21 at growth. At test termination we also make an assessment of 

### 22 growth. And then at test termination we also look at liver 

### 23 somatic index, liver histology and kidney pathology. 

### 24 So then we have thyroid endpoints, and these 

### 25 are fairly straight-forward and I already mentioned them; time 

### 26 to NF stage 62; thyroid histopathology and T4 measurements. And

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1 at the end of the test, in addition to gonad histopathology, we 

### 2 gave a determination of genetic sex, compared that to the 

### 3 determination of phenotypic sex as well as look at vitellogenin 

### 4 levels. 

### 5 So we conducted an inter-laboratory study 

### 6 utilizing ourselves, the U.S. Army Center for Environmental 

### 7 Health and Research. That was interagency agreement. 

### 8 IDEA is a consulting company in Japan that is 

### 9 actually funded by the Japanese Ministry of Environment to do 

### 10 this work. And then we contracted to Battelle who then 

### 11 subcontracted to two laboratories, one was ABC and the other was 

### 12 Fort Environmental Laboratories. 

### 13 So I want to point out here that these 

### 14 laboratories were provided a protocol. It was not quite as 

### 15 complete as the protocol in the integrated summary report that 

### 16 you have, although we did participate in a number of conference 

### 17 calls with them and provided them all the protocols that you see 

### 18 in there, the SOPs, as the process was unfolding. 

### 19 So as everyone already knows, all the 

### 20 inter-laboratory data was analyzed by the EPA. There was an 

### 21 error discovered and it was a way in which there was a 

### 22 misunderstanding in how StatCHARRMS was handling the data input. 

### 23 24 We had a replicate column. We had assigned 

### 25 replicates; control 1, 2, whatever. It was our understanding 

### 26 that StatCHARRMS was taking that and generating a tank mean and

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1 moving forward with it. We discovered that it was not. 

### 2 So what we did to solve this problem is we 

### 3 generated tank means, taking into account the individuals per 

### 4 tank. And the ANOVA was run weighted and the end was the tank. 

### 5 So that was the solution. So what happened was, we got to the 

### 6 point of tank means and all of that prior to going into the 

### 7 ANOVA module for StatCHARRMS. 

### 8 To give you a sense of the endpoints that may 

### 9 have been impacted by this era, mainly growth, that would be 

### 10 length and weight and LSI, time to stage 62 and vitellogenin 

### 11 measurements. All these other measurements, the histopathology, 

### 12 the thyroid hormone measurements were not impact by this 

### 13 data-entry error. 

### 14 So in general, there were only minor changes 

### 15 from my perspective, and the reported LOECs in the original 

### 16 integrated summary report. In most but not all cases, the 

### 17 difference was we observed an increase in the LOEC. And in two 

### 18 cases we observed a decrease, and in one other case we saw a 

### 19 non-monatomic response popping up, but it did not have a 

### 20 significant impact on the overall analysis of the dataset. 

### 21 So here is the four test chemicals that we 

### 22 tested, prochloraz, octylphenol, trenbolone. Rodney has 

### 23 mentioned these as well as I believe Les as well. We tested in 

### 24 collaboration with the Japanese a different chemical called 

### 25 benzophenone-2. That was a very interesting chemical. 

### 26 So this table is to try to give a sense of the

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1 scope of the inter-laboratory. So in the case of prochloraz, we 

### 2 had four labs participating; octylphenol, three. In the case of 

### 3 trenbolone, NBP-2 where only one lab ran the studies. 

### 4 I guess the one point I will make is, 

### 5 prochloraz is also known to be an antagonistic androgen 

### 6 receptor, and in mammals it produces abnormalities in rat 

### 7 reproductive tract that are consistent with an anti-androgenic 

### 8 effect. 

### 9 Okay. I am going to take a minute to talk 

### 10 about this because all my tables are like this. I do not plan 

### 11 to go through all this data, trying to show more patterns here 

### 12 rather than data. 

### 13 So down here, the highlight, it says if it is 

### 14 yellow it means it changed when we redid the statistical 

### 15 analysis. And a dash line is -- did they come out on your copy, 

### 16 because here we do not see them? There are dashes. All those 

### 17 blank areas should have dashes and maybe I just cannot see them. 

### 18 I cannot see them. Okay. 

### 19 So that meant it was not statistic in either 

### 20 of the first analysis or the second analysis. And then there 

### 21 are situations where we have highlighted a dash, and that means 

### 22 that we had got a value in the first analysis which would have 

### 23 been, in this case, 180. And if we go over to lab C and we look 

### 24 at LSI, what we are saying here is in the reanalysis, we lost a 

### 25 concentration that showed significance, so all the tables are 

### 26 going to be oriented this way.

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1 The red arrows have nothing to do with 

### 2 statistics but the directionality of the change. Did it go up 

### 3 or did it go down? So a red arrow is down and a green arrow is 

### 4 it went up. 

### 5 Here, what we observed with prochloraz is -- 

### 6 and these are the growth parameters for the most part, larval as 

### 7 well as juvenile males and females. And lab D showed an impact 

### 8 on growth as well as lab B. And there was an impact on LSI in 

### 9 lab A. And then lab C did not show very much. But what I will 

### 10 talk about later in the presentation is that lab C had a number 

### 11 of issues, we could say. 

### 12 And then we here have, in all laboratories, we 

### 13 saw effects on male and female kidney and liver. So basically 

### 14 prochloraz was pretty toxic in these studies. 

### 15 And I think many of the studies that have been 

### 16 published previously with prochloraz have not had a whole lot of 

### 17 data that looked at overt toxicity. So some of these chemicals 

### 18 that everyone is reporting the endocrine effects, they are not 

### 19 looking at the underlying toxicity that is occurring as well. 

### 20 And I think that, as we do these studies, we are starting to 

### 21 realize that some of these endocrine disruptors have their 

### 22 endocrine activity about where they are also overtly toxic. 

### 23 Thyroid endpoints, in this case we saw effect 

### 24 on T4 and pathology, and we saw effects on pathology in three of 

### 25 the four labs. And what that means is there was glandular 

### 26 change consistent with the disruption of the thyroid axes.

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1 Here we are on the reproductive endpoints. 

### 2 And I guess this is a good a time as any to bring this up. It 

### 3 is a charge question and we have pulled it out. Basically 

### 4 vitellogenin is not present in males or females at this stage of 

### 5 development because estrogen is not present at this stage of 

### 6 development. So it is only an endpoint that we expect to be 

### 7 observed when there is an estrogen involved. 

### 8 So then here we are going to the gonad 

### 9 pathologies where pathologies reported across all four 

### 10 laboratories for males and females. There were duct pathologies 

### 11 reported in the males across all laboratories, although the LOEC 

### 12 varied from lab to lab. And several labs showed duct changes in 

### 13 females. 

### 14 This is an example of the high treatment. 

### 15 What I am showing you here is a control test that is on the top, 

### 16 and I just showed these to give you an indication of how 

### 17 dramatic these effects were. So these are the same treatment 

### 18 level, and this is very severe. Probably the section is fraught 

### 19 with artifact due to the germinal epithelium breaking down and 

### 20 being filled with macrophages and infiltrate that is there to 

### 21 try to repair the tissue. 

### 22 So then we also tested octylphenol, and again, 

### 23 I do not think octylphenol needs much of an introduction other 

### 24 than to say it has probably been tested in every aquatic model 

### 25 there is, at this point. 

### 26 The one thing I guess would be noteworthy is

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1 Kloas et al. (1999) reported sex reversal with OP, and in that 

### 2 he was using sex ratios. And we are fortunate enough now to 

### 3 have the newly discovered sex determining gene and have the 

### 4 ability to do genetic sex and do not have to rely on ratios. 

### 5 And we did a study in tropicalis where we could not reproduce 

### 6 that, so just to point that out. 

### 7 You know octylphenol was fairly -- and I do 

### 8 not recall to what extent we pulled this out in the integrated 

### 9 summary report -- octylphenol was fairly toxic to the organisms 

### 10 during our organogenesis. 

### 11 So concentration, we tested 50. We tested 60 

### 12 in a range-finder and the animals did not make it for longer 

### 13 than five days. So this test concentration is much lower than 

### 14 what you will commonly see tested in adult fish where those 

### 15 organisms are not developing and are much more capable of 

### 16 handling high test concentrations, so it had very little effects 

### 17 on growth. 

### 18 Lab C, again, showed some effect on LSI but 

### 19 they had some feeding issues. Their animals were really small 

### 20 but we will talk about that later. Not really sure what to make 

### 21 of lab C's finding when it comes to the growth parameters. Here 

### 22 we did see a consistent impact on the female kidney in all three 

### 23 laboratories. 

### 24 Here is more what we are looking at stage 62; 

### 25 lab A, who I know did really good work found a thyroid effect in 

### 26 all three parameters. Lab B and lab C did not. I do not know

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1 how to explain that. Lab C did see something but it was 

### 2 actually an acceleration in the time to get to stage 62 as 

### 3 opposed to a delay in the time to get -- I am sorry. I said 

### 4 that wrong. They saw an acceleration of metamorphosis and we 

### 5 saw a delay because green means it took more time to get there. 

### 6 7 So then T4, throughout these studies the 

### 8 contractors were to make an effort to optimize procedures for 

### 9 measuring T4. They attempted, were not successful. So as a 

### 10 result we went back through -- we tend to use this ion coupled 

### 11 plasma mass spect approach, but it does require a fairly 

### 12 sophisticated piece of equipment. 

### 13 We went back to one of the commercially 

### 14 available kits and developed an ethanol extraction procedure, 

### 15 and I was even accused of ginning this up. Here is the two 

### 16 different methods of analysis side-by-side. Granted it is on 

### 17 the same samples but it is remarkably consistent. And this 

### 18 method is now in the guidance document. 

### 19 Reproductive endpoints; I guess the main 

### 20 consistency here was, we saw an impact in the ducts of females 

### 21 which is consistent with advancement, which would be an 

### 22 estrogenic response. Here we summarized those effects and you 

### 23 guys can take a look at that. I would never be able to read it 

### 24 anyway. 

### 25 Okay. So then we turned our attention to 

### 26 trenbolone and, again, that was only run in one laboratory. We

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1 chose this chemical because this chemical had been tested 

### 2 previously in multiple fish species as well as tested by 

### 3 ourselves in xenopus tropicalis. And the test concentrations 

### 4 that we used were based on the test concentrations from the 

### 5 Olmstead 2012 paper. 

### 6 So here we have observed some degree of 

### 7 effects on male and female liver and kidney pathologies. And 

### 8 here we are looking at thyroid endpoints. It was an observed 

### 9 effect on thyroid gland pathology. Now the reproductive effects 

### 10 were not terribly dramatic. What we had learned is that we had 

### 11 to keep the trenbolone concentrations at 100 or less because in 

### 12 the tropicalis study we killed the animals by advancing their 

### 13 larynx development and shutting off their airway prior to 

### 14 metamorphosis. So we held the concentration in the high to 100. 

### 15 16 We saw some pathology in the male gonad. We 

### 17 had duct pathology in the males. The most striking thing was 

### 18 duct pathology in the females. 

### 19 So here is an oviduct. This is the duct that 

### 20 will carry the oocyte from the ovary to the external 

### 21 environment. And this is a female and this is a trenbolone 

### 22 treated female. So this structure forms and then regresses to 

### 23 this. And in this instance, trenbolone results in the 

### 24 regression of this duct. So in this case there would be no 

### 25 ability for an oocyte to pass to the external environment for 

### 26 fertilization.

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1 Benzophenone-2 may not be as well known. It 

### 2 is a UV filter used in cosmetic suntan lotions, products of that 

### 3 nature. It has been shown to be estrogenic as well as has been 

### 4 shown to be a thyroid peroxidase inhibitor, in essence, 

### 5 preventing the synthesis of thyroid hormone. 

### 6 We thought it was a unique opportunity to look 

### 7 at two modes of action at once. And I will just go ahead and 

### 8 say that this was done by our Japanese collaborators. They were 

### 9 limited by the exposure systems that they had, so we allowed for 

### 10 a modification to the experimental design. And as recommended 

### 11 in the guideline, you have eight controls replicates. You have 

### 12 four replicates for treatments with four treatments. Let me say 

### 13 that again; eight control replicates. You have four replicates 

### 14 per treatment at four treatments. In the case of the Japanese 

### 15 study, they reduced the control from eight to four. And here 

### 16 are the findings. 

### 17 So these are the test concentrations, 1.5, 3 

### 18 and 6 milligrams per liter. And basically the high treatment 

### 19 causes a significant amount of mortality at the larval phase, 

### 20 and then an additional amount of mortality from the larval phase 

### 21 to the juvenile phase, so that was a fairly hard-hitting test 

### 22 concentration. 

### 23 There were effects obviously at the high and 

### 24 the growth parameters in both males and females. Yes, there 

### 25 were effects on LSI in females. 

### 26 In the case of liver pathologies, it is not

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1 surprising we saw effects on both the liver and kidney at 

### 2 basically three and above. But in the case females, we saw 

### 3 effects in the kidney at 1.5. I want to point out that that is 

### 4 not necessarily overt toxicity. These kidneys were swimming in 

### 5 vitellogenin with no place to put it because they did not have 

### 6 vitellogenic oocytes. You will see later that the vitellogenin 

### 7 levels are screaming high and it is not uncommon to see kidney 

### 8 pathologies in fish when vitellogenin levels get elevated. 

### 9 In this case, you will see that there was an 

### 10 effect on Time to NF62 and there was dramatic effects on thyroid 

### 11 gland pathology. 

### 12 Here are the thyroid glands of a control and 

### 13 this is not a camera trick. So this cavity right here is where 

### 14 they reside, so this is lower mg than this. But you see the 

### 15 same cavity the glands have now basically taken it over, so this 

### 16 is extreme hyperplasia. 

### 17 In the case of vitellogenin, we saw an 

### 18 increase at all the test concentrations. We see effects on male 

### 19 gonads at all test concentrations. There were effects on duct 

### 20 pathologies and likewise, there were effects on all the 

### 21 reproductive endpoints in females. 

### 22 At the 1.5 we saw some intersects. At the 3 

### 23 and the 6, they were all completely sex-reversed and we could 

### 24 make that determination based on genetic sex. And we also had 

### 25 at the 1.5, we had both sex-reversal and intersex. 

### 26 So here is an example of control male testis

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1 right here, and this is an example of an intersex male testis. 

### 2 And you see potentially a lumen, you see oocytes but then there 

### 3 is also cystis formation over here, so it is a mix-sexed. 

### 4 So what are the take-home messages? And you 

### 5 are going to have to look at this. So this table attempts to 

### 6 compare where there were consistencies observed across the 

### 7 laboratories. I indicate here the number of laboratories that 

### 8 participated in each of the studies, so any inter-laboratory 

### 9 comparisons are only limited to prochloraz and octylphenol. 

### 10 In the case of prochloraz, we saw growth 

### 11 effects in liver pathology and kidney pathology in all labs, so 

### 12 the high concentration was fairly toxic. We saw thyroid effects 

### 13 in three of the four labs and we saw reproductive effects in 

### 14 three of the four labs. 

### 15 In terms of octylphenol, we had thyroid 

### 16 effects and delays only in one laboratory and we had impacts on 

### 17 vitellogenin induction in two laboratories. And in terms of 

### 18 gonad and reproductive ducts, we had effects in all 

### 19 laboratories. That is mainly what that table has been 

### 20 constructed for. 

### 21 So diet formulation and feeding rates have 

### 22 been shown to be correlated with organismal growth and 

### 23 development rates in the LAGDA. 

### 24 What we had was a laboratory that chose to 

### 25 increase their feeding by 30 percent. So they participated in 

### 26 both studies, prochloraz and octylphenol.

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1 Prochloraz was their first study here, and 

### 2 they fed the formulation and the quantity that we recommended. 

### 3 Here they were concerned about potential for early mortality 

### 4 during earlier organigenesis and they added a few extra 

### 5 organisms, I think it was five. And they increased their food 

### 6 by 30 percent, and that increase in 30 percent resulted in it 

### 7 doubling in the mean weight for their larvae. This is not that 

### 8 uncommon. Feeding rates are more dictated by tank flow rates 

### 9 than the number of organisms that are in the tank. We were not 

### 10 consulted about this and actually it did not come to our 

### 11 attention until the end. And then here is an overall mean at 

### 12 the bottom of 1. 

### 13 The other issue was that these animals also 

### 14 reported a high frequency of scoliosis or bent-tail or spinal 

### 15 curvature. We know from work in our laboratory that over-feeding 

### 16 commonly produces this effect, so these are images of scoliosis 

### 17 in severity. 

### 18 I was asked to bring some attention to this 

### 19 because the previous SAP, in terms of the AMA, recognized this 

### 20 and brought attention to it, so I am here to kind of tell you 

### 21 what I know. I know it is a controllable phenomenon. 

### 22 Now is this not laevis, it is tropicalis. Now 

### 23 we have never done any work with laevis and scoliosis because we 

### 24 do not get it. We have a diet, we use it and we do not get 

### 25 scoliosis with xenopus laevis. 

### 26 With tropicalis, we get a lot of scoliosis,

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1 because that diet that worked for laevis did not work for 

### 2 tropicalis. So we have developed a diet that works for 

### 3 tropicalis as you can see here. But I want to make the point 

### 4 that we have had some fairly high frequencies with certain types 

### 5 of diets. It is a controllable phenomenon and it can be 

### 6 controlled by diet. 

### 7 We now have a diet for this species that 

### 8 produces zero scoliosis and provides for good growth and 

### 9 development. And the diet recommended for the LAGDA did not 

### 10 produce scoliosis in our hands. To our knowledge, it only 

### 11 produced scoliosis in one laboratory and that is when they 

### 12 increased the feeding rate. 

### 13 Lab C; remember I kept saying lab C? There is 

### 14 a problem with lab C. This is the problem with lab C. They fed 

### 15 their own formulation and that was against my insistence, but I 

### 16 lost that argument. And if you see their mean weight -- I mean, 

### 17 basically, in the octylphenol study, their juveniles were half 

### 18 the size of the mean weights of everyone else's. We also saw 

### 19 some pathologies in the livers that were unique for this 

### 20 laboratory, and it is in the integrated summary report. We do 

### 21 not know if this laboratory was fighting disease issues, and in 

### 22 conjunction with diet was providing for some fairly poor growth 

### 23 rates. And they also had the smallest juveniles as well. 

### 24 So our take-home is this lab fed a feed 

### 25 formulation -- its own -- and that it can be concluded that that 

### 26 is inadequate, and that we recommend that anyone who performs

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1 this assay adhere fairly strictly to the feeding recommendations 

### 2 that we have, because we do know that they are flow-rate 

### 3 dependent, not necessarily individuals in the tank dependent, 

### 4 which is kind of what you would think intuitively, the more 

### 5 individuals, more food. 

### 6 The simple diet; Trout Starter, 

### 7 algae/TetraFin, you probably feed your fish at home that; brine 

### 8 shrimp, which is readily obtainable and easy to culture. It 

### 9 takes 24 hours. 

### 10 Just to point out the level of detail at which 

### 11 we provided in the protocol, we were telling them on a biweekly 

### 12 basis how much to increase the food in this. So we have to 

### 13 understand that -- and even in light of this level of guidance, 

### 14 contract labs will give you what we got. 

### 15 So lessons learned; overall, the vitellogenin 

### 16 was not really measurable in the control males and females, and 

### 17 it is questionable what was really going on in terms of the 

### 18 prochloraz and the octylphenol studies, were those measures 

### 19 actually real? 

### 20 In the case of BP-2 there was no question that 

### 21 there was clearcut induction of vitellogenin. Yes, so BP-2 

### 22 shows you what could happen when you are actually working with 

### 23 an estrogen. 

### 24 Inconsistencies in pathology findings; I am 

### 25 not going to read this directly. Because of contract situations 

### 26 and resources, in the midst of the prochloraz study, the

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1 pathology contract was changed. And a two of the studies were 

### 2 read by different -- so we had two studies read by one, two 

### 3 studies read by the other, and there were inconsistencies in the 

### 4 findings. 

### 5 I will be honest with you. The glass coming 

### 6 out of one of the contract laboratories was in my opinion 

### 7 inferior to the other laboratory and probably substandard. I am 

### 8 not sure I could have read anything in those slides. 

### 9 So we have to ensure that -- and in the same 

### 10 case with Rodney who keeps harping that it is going to be 

### 11 implementation of these things that are going to be the 

### 12 challenge to instill in these folks that they have to make thin 

### 13 sections and we have to have diagnostic criteria. And Rodney's 

### 14 group has developed some of these tools for fish and we are also 

### 15 working on developing them for the frog as well. Hopefully we 

### 16 can move through these types of inconsistencies we see. 

### 17 Serum T4; the take-home on serum T4 is, in 

### 18 terms of having inter-laboratory comparisons, it was a bust. 

### 19 The two contract labs did not have a successful technique for 

### 20 measuring T4, and as a result, the only place that T4 

### 21 measurements were made were at our laboratory. We used two 

### 22 different techniques, one that we published on; the ICP-MS and 

### 23 then the ethanol extraction. They were very comparable. And we 

### 24 also know that that is very comparable to some work coming out 

### 25 of Dow using MSMS analysis to measure T4. So the absolute 

### 26 amounts at stage 62 are very similar across the three methods.

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1 So when the guidelines are adhered to, the 

### 2 contributing laboratories were able to conduct the test and the 

### 3 controlled performance was consistent across all three 

### 4 laboratories. 

### 5 Stage 62 is optimal stage for evaluating 

### 6 thyroid-related endpoints. And I guess this is a question. It 

### 7 is not necessarily a charge question but it is a question that I 

### 8 have, and that is, currently we have three thyroid endpoints. 

### 9 We have time to metamorphosis, pathology and T4 measurements. 

### 10 Given the robustness of the thyroid histopathology, do we really 

### 11 need T4? That is a good question, I think. 

### 12 Ten weeks post-stage 62; I think the gonads 

### 13 were developed well enough and we got a very good indication of 

### 14 chemical-induced abnormalities in the gonads. 

### 15 The ducts, I really had not paid all that much 

### 16 attention to the ducts prior to this inter-laboratory. And then 

### 17 it turns out that the ducts seem to be one of the more sensitive 

### 18 endpoints in terms of their reproductive system. And you know 

### 19 the ducts are an incredibly important structure given that 

### 20 failure in their development basically would result in no 

### 21 reproduction. 

### 22 And histopathology tended to be inconsistent 

### 23 between the two contract laboratories that we utilized. And 

### 24 again, we need to dig into why that was that way and provide 

### 25 guidance that corrects that issue. 

### 26 So control vitellogenin levels are below

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1 detection limits and measurable only when induction occurs. And 

### 2 to be honest with you, I think that is okay. You may decide if 

### 3 vitellogenin is useful or not, but do not let the fact that it 

### 4 is not measurable in controls steer you away from the fact that 

### 5 it could be valuable. And with that, I am done. I will take 

### 6 questions. 

### 7 **DR. MARTHA SANDY:**Thank you very much. Mary? 

### 8 **DR. MARY CHRISTMAN:**To your very last slide, 

### 9 the reason that you say you think it is okay is because of your 

### 10 earlier statement that it should not be expressed. 

### 11 **DR. SIGMUND DEGITZ:**Yes. 

### 12 **DR. MARY CHRISTMAN:**And therefore any 

### 13 expression is evident. 

### 14 **DR. SIGMUND DEGITZ:**Yes. Right. 

### 15 **DR. MARY CHRISTMAN:**Okay. I agree with you. 

### 16 I was just wondering. 

### 17 **DR. SIGMUND DEGITZ:**Yes. We struggled with 

### 18 this data in terms of statistics. We make an estimate, and then 

### 19 that gives you a number that really is not real. 

### 20 **DR. MARY CHRISTMAN:**You mean when it is below 

### 21 the detection limit? 

### 22 **DR. SIGMUND DEGITZ:**Yes. And I don't really 

### 23 know how to handle when you have 10 individuals and eight are 

### 24 not there and two are. You know, do we need statistics? 

### 25 **DR. MARY CHRISTMAN:**Thank you. 

### 26 **DR. SIGMUND DEGITZ:**Yes.

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1 **DR. DALLAS JOHNSON:**Yes, but we need 

### 2 statisticians here. 

### 3 **DR. MARTHA SANDY:**Yeah. I have a follow-up 

### 4 on that. So benzophenone-2, is that a strong estrogen agonist 

### 5 or a weak or a moderate compared to estradiol? Can you give us 

### 6 a sense? 

### 7 **DR. SIGMUND DEGITZ:**I think if you look at 

### 8 the test concentrations, you know we are pretty high up there. 

### 9 The Japanese -- the low is 1.5 milligrams per liter. Estradiol 

### 10 you would test at about three orders of magnitude less than that 

### 11 to produce a similar type of effect. Does that answer your 

### 12 question? 

### 13 **DR. MARTHA SANDY:**Yes, thank you. So Dr. 

### 14 Scanes? 

### 15 **DR. COLIN SCANES:**Can we flip to slide 125? 

### 16 **DR. SIGMUND DEGITZ:**Thank you. You will have 

### 17 to tell me when we get there. 

### 18 **DR. COLIN SCANES:**The one with the oviduct. 

### 19 Yes. You talk about this as a histopathology endpoint. It is 

### 20 either a score 2 or a 1. Well the one is very small. And if 

### 21 you decided to actually use some quantitative measures, you 

### 22 could get an area for the duct and you may have much finer 

### 23 discrimination. 

### 24 **DR. SIGMUND DEGITZ:**I agree, yes. 

### 25 **DR. COLIN SCANES:**And I think the same 

### 26 applies to the thyroid where if you simply look at the area of

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1 the thyroid gland, you are going to get some very -- as long as 

### 2 you are systemically section, you are going to get much more 

### 3 fine ways of looking at a potential endocrine disruption. Would 

### 4 you like to comment on that? 

### 5 **DR. SIGMUND DEGITZ:**Well for the sake of 

### 6 responding, you know this staging system is something that is an 

### 7 inherited thing. You know we have not put much time and effort 

### 8 into thinking about a staging system that would have better 

### 9 resolution as what you are saying, and I agree. 

### 10 **DR. COLIN SCANES:**But you can simply take the 

### 11 area of the oviduct here. 

### 12 **DR. SIGMUND DEGITZ:**You know and the oviduct 

### 13 is probably amenable to that as well. 

### 14 **DR. COLIN SCANES:**So it is the thyroid. 

### 15 **DR. SIGMUND DEGITZ:**Well the problem with the 

### 16 thyroid, the thyroid is shaped like a football in the frog. It 

### 17 tends to be football shaped. And you get on the ends and the 

### 18 diameter is much smaller. 

### 19 **DR. COLIN SCANES:**But that is why systematic 

### 20 sectioning comes in. And you could equally well look at the 

### 21 size of the total area of the colloid. 

### 22 **DR. SIGMUND DEGITZ:**Yes. I would hate to pay 

### 23 the histo bill on that. 

### 24 **DR. COLIN SCANES:**It might be done much more 

### 25 with using images. 

### 26 **DR. SIGMUND DEGITZ:**Yes, yes.

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1 **DR. MARTHA SANDY:**I see many hands. Vicki 

### 2 Blazer? 

### 3 **DR. VICKI BLAZER:**I just wanted some 

### 4 clarification on the contract labs. So were they both actually 

### 5 pathology contract labs? 

### 6 **DR. SIGMUND DEGITZ:**Yes, they were. 

### 7 **DR. VICKI BLAZER:**And you said that, in one 

### 8 case the slides were really pale. So it wasn't necessarily the 

### 9 pathologist? 

### 10 **DR. SIGMUND DEGITZ:**No. That is what I am 

### 11 saying, is the glass was not very good. So we were provided 

### 12 images of the sections. And if you have experience with this, 

### 13 you would know when you are seeing nice fine detail and when you 

### 14 are seeing basically a purple something. 

### 15 **DR. VICKI BLAZER:**So you read the slides. 

### 16 **DR. SIGMUND DEGITZ:**I did not read the 

### 17 slides. What I did was I reviewed images that we were provided 

### 18 by the histopath contractor. 

### 19 **DR. VICKI BLAZER:**Okay. Thanks. 

### 20 **DR. MARTHA SANDY:**Dallas Johnson? 

### 21 **DR. DALLAS JOHNSON:**Just a question. Both 

### 22 you and the previous presenter talked about correcting the 

### 23 analyses for the statistical program that you are using, but you 

### 24 both corrected it different ways. 

### 25 **DR. SIGMUND DEGITZ:**Yes. 

### 26 **DR. DALLAS JOHNSON:**One of you corrected it

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1 using your repeated option; one of you corrected it by using 

### 2 weights. 

### 3 **DR. SIGMUND DEGITZ:**Weights? 

### 4 **DR. DALLAS JOHNSON:**Well I thought you said 

### 5 you did a weighted analysis. 

### 6 **DR. SIGMUND DEGITZ:**We did a weighted 

### 7 analyses, yes. I think that the way we did it is appropriate. 

### 8 **DR. DALLAS JOHNSON:**And that previous author 

### 9 thinks the way they did it is appropriate? Or maybe you do not 

### 10 want to speak for them. 

### 11 **DR. SIGMUND DEGITZ:**I do not want to speak 

### 12 for them, no. I think what we did was appropriate. 

### 13 **DR. MARTHA SANDY:**Dr. Bentley? 

### 14 **DR. GEORGE BENTLEY:**Yes. Just a quick 

### 15 follow-up from Dr. Scanes' comment on the thyroid 

### 16 histopathology; in measurement of the song control system areas 

### 17 of the brain in the songbirds, you can routinely measure 

### 18 strangely shape of the brain and measure them relative to the 

### 19 rest of the brain, so you could quite easily measure the volume 

### 20 of the thyroid gland, for example, relative to the rest of them 

### 21 for that part of the body. 

### 22 **DR. SIGMUND DEGITZ:**Okay. 

### 23 **DR. MARTHA SANDY:**Dr. Propper? 

### 24 **DR. CATHERINE PROPPER:**I will try not to 

### 25 monopolize the conversation here, but with regards to the 

### 26 imaging, I do agree. There is, in fact, some really new and

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1 cool technology where they can just slide slides by and get 

### 2 images very quickly, and then the program automatically can 

### 3 measure things like area of the individual slides and then 

### 4 compile it into volume data, and it involves little or less, 

### 5 let's say, human contact. 

### 6 So there are technologies out there that are 

### 7 available and are being upgraded probably nearly on a daily 

### 8 basis at this point to start getting some really nice histo data 

### 9 that could be useful for a number of these studies. 

### 10 And now I wanted to just ask for the T4 and 

### 11 the vitellogenin assays, did you go into anymore debt with the 

### 12 validation? Was there any parallelism to a standard done, and 

### 13 did you do any evaluation of inter and intra-assay coefficients 

### 14 of variation? 

### 15 **DR. SIGMUND DEGITZ:**The vitellogenin actually 

### 16 has already been published. That approach was published in the 

### 17 Olmstead et al. It was not the '12 paper because that was 

### 18 trenbolone. I think it was the '09 or '10 paper, so we covered 

### 19 that there. 

### 20 **DR. CATHERINE PROPPER:**Okay. 

### 21 **DR. SIGMUND DEGITZ:**The T4 is literally weeks 

### 22 old and we are working through getting that nailed down, so no 

### 23 other lab had an opportunity to use it. And we have been trying 

### 24 to as quickly as we can run some studies to produce samples for 

### 25 perturbations so that we can confirm its utility. The hard part 

### 26 is in running the 21-say assays to get samples, so we are

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1 actually trying to validate it ourselves by running studies and 

### 2 confirming that we get dose response. 

### 3 **DR. CATHERINE PROPPER:**Okay. And it is not 

### 4 so much dose response I am looking for but just even dilution of 

### 5 your samples and see that it is running parallel to the curve. 

### 6 **DR. SIGMUND DEGITZ:**Yes. 

### 7 **DR. CATHERINE PROPPER:**Yes. Okay. So that 

### 8 is in progress? 

### 9 **DR. SIGMUND DEGITZ:**Yes. Well in terms of 

### 10 the T4, because of the ethanol perception, so you could have a 

### 11 -- let me state this correctly. It is a matrix issue, is why 

### 12 the contract labs could not make it work. 

### 13 As you move to the ethanol precipitation, you 

### 14 take the matrix effect out of it. And we are testing to what 

### 15 extent we do that by looking for a change in the slope with 

### 16 these that occur. That is where you can see it. 

### 17 **DR. CATHERINE PROPPER:**Okay. Thank you. And 

### 18 then I have got a few others here. 

### 19 So this gets back to the stats. And for both 

### 20 studies, I get a little concerned about just the loss of power 

### 21 with going to the tank effects. And I am not a statistician so 

### 22 please step in statisticians if I am going down a different path 

### 23 or an invalid path. But is there a way in with those tank 

### 24 effects can be taken into account without losing the power that 

### 25 you get with the individual animals? 

### 26 **DR. SIGMUND DEGITZ:**You did not get power

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1 with the individual animals; you were using an inappropriate 

### 2 statistic. I mean you only get power when you use the same 

### 3 statistic and get the result you want. 

### 4 **DR. CATHERINE PROPPER:**Neither one of us 

### 5 might be wording this correctly. 

### 6 **DR. SIGMUND DEGITZ:**Right. But the point is, 

### 7 do not be mislead; there wasn't power in the first analysis. It 

### 8 was an incorrect analysis. 

### 9 **DR. CATHERINE PROPPER:**Oh, that I understand, 

### 10 so I do understand that. 

### 11 **DR. SIGMUND DEGITZ:**So I would not say that 

### 12 we loss power; I think we found the right power. 

### 13 **DR. CATHERINE PROPPER:**So I understand why 

### 14 you have to include the tank effect in there. I do worry when 

### 15 you start going down to -- and maybe it is because I do not 

### 16 understand quite how you switched your stats when you start 

### 17 going to the tank mean. 

### 18 **DR. SIGMUND DEGITZ:**Okay. First off, we did 

### 19 not want to do our statistics the way we did it. We made an 

### 20 assumption that we were loading columns. The SAS code had been 

### 21 written to take those and turn them into tank means. That had 

### 22 not been done. So using the individuals as a treatment would 

### 23 not be appropriate to run ANOVA that way, so we cannot even go 

### 24 down that road of talking about what happened when we did do 

### 25 that. 

### 26 **DR. CATHERINE PROPPER:**Okay. All right.

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1 Well I will go with that for the moment. I have a few other 

### 2 questions but I will let other folks jump in while I organize my 

### 3 thoughts. 

### 4 **DR. MARTHA SANDY:**So I have three other 

### 5 people, I see their hands; Dr. Kullman, Scott and Denver. Dr. 

### 6 Kullman? 

### 7 **DR. SETH KULLMAN:**So just a couple of quick 

### 8 questions. So if you look through the literature there is a 

### 9 number of other sort of messenger RNAs or proteins that seem to 

### 10 be mediated by the thyroid hormone receptor. So I am curious 

### 11 why maybe those are not incorporated into the assay, you know, 

### 12 things like looking at thyroid releasing hormone, thyroid 

### 13 stimulating hormone, in particular TPO activity, as such. Those 

### 14 are all well-known and well reported throughout the literature 

### 15 to respond, and maybe pretty decent biomarkers for having 

### 16 effect. 

### 17 **DR. SIGMUND DEGITZ:**We do all that. So when 

### 18 we do TPO assays we have approaches for measuring TSH. The 

### 19 question becomes what is appropriate for contract testing and 

### 20 what are we going to do with those endpoints in a risk 

### 21 assessment if we have them. That really is what it comes down 

### 22 to. 

### 23 I mean it probably would be best served to 

### 24 hand that off to my colleagues to speak to but, you know, they 

### 25 are still questioning whether we are going to risk assess off 

### 26 the histopath, let alone gene expression.

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1 **DR. SETH KULLMAN:**But we tend to use 

### 2 vitellogenin as a pretty prominent biomarker for endocrine 

### 3 disrupting effects. I do not see a whole lot of difference 

### 4 using mRNA expression using a vitellogenin versus an mRNA 

### 5 expression of the transporter, like in one of the other thyroid 

### 6 hormone biomarkers. 

### 7 **DR. SIGMUND DEGITZ:**It is a biomarker for -- 

### 8 agreed, it tends not to be the kind of endpoint that we would 

### 9 make regulatory decisions on. 

### 10 **DR. LESLIE TOUART:**Just to try to explain or 

### 11 to follow-on. I mean biomarker, a physiological bio-chemical, 

### 12 even pathological type effects, are not on their own a regulated 

### 13 endpoint in terms of something that we would determine to be an 

### 14 adverse consequence needing some type of regulatory 

### 15 intervention. 

### 16 But the context is that these things are 

### 17 indicative of what may be adverse consequences. So following 

### 18 adverse outcome pathways and these things, one might determine 

### 19 that the end result of a biomarker like Vtg may give an early 

### 20 indication that reproduction may be affected later. And if your 

### 21 concentration response study that you have demonstrates a 

### 22 reproductive effect at a higher concentration and a Vtg change 

### 23 at a lower concentration but concomitant at that higher 

### 24 concentration, one might then start to determine is the level of 

### 25 the departure a concern level, should that be based on that 

### 26 lower level; recognizing on the ecological side which is

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1 different from the human health side where health of organisms 

### 2 are a driver. On the eco side, it is more population level 

### 3 effects. 

### 4 So unless we identify that particular adverse 

### 5 effect may have some consequence to population or to maybe like 

### 6 an endangered species in terms of its survival or ability to 

### 7 continue, those could be questions. 

### 8 But the risk assessment component part, it is 

### 9 a little bit further out of line. And I think Sig's comment 

### 10 that what is practical from contract laboratory, what the cost 

### 11 effectiveness of some of these measures, these are some things 

### 12 to be considered. But I think any advice that you have or 

### 13 others on the Panel in terms of endpoints that we might want to 

### 14 try to add in without toppling the Christmas tree, so to speak, 

### 15 those are things to be considered, but again, recognizing that 

### 16 with each of these the interpretations might become a little bit 

### 17 more complex. 

### 18 **DR. SETH KULLMAN:**I hear what you are saying. 

### 19 I do not see a true distinction between the vitellogenin as a 

### 20 marker of reproduction effects other than its estrogenic 

### 21 mediator. 

### 22 In the same way, I sort of do not see these as 

### 23 possibly impacting reproduction other than that it is a thyroid 

### 24 effect and it will impact growth and development, so on and so 

### 25 forth. 

### 26 So I am having a hard time in making the

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1 distinction of why use a biomarker like vitellogenin in one 

### 2 instance and maybe not a biomarker in another instance. 

### 3 Certainly do not have to discuss it in detail now but it is 

### 4 something that should be discussed. 

### 5 **DR. SIGMUND DEGITZ:**Well I could give you a 

### 6 very quick answer to that, and that is, in terms of the thyroid, 

### 7 we have so much better diagnostic capabilities there. We have 

### 8 T4 measurement. We have glandular change. I mean we are really 

### 9 strong there. It is actually in the reproductive end where we 

### 10 are a little softer. So that is why you are reaching to 

### 11 vitellogenin to pull it in, in terms of a weight of evidence. 

### 12 **DR. MARTHA SANDY:**Next we have Dr. Scott. 

### 13 **DR. GEOFFREY SCOTT:**On slide 148 you proposed 

### 14 a question about T4 and I just was trying to come back and 

### 15 understand that a little bit. In the Tier 1 screen with the 

### 16 amphibian assay, we measured T4, right? 

### 17 **DR. SIGMUND DEGITZ:**No, we do not. 

### 18 **DR. GEOFFREY SCOTT:**We do not? 

### 19 **DR. SIGMUND DEGITZ:**We do not. 

### 20 **DR. GEOFFREY SCOTT:**Okay. I was just trying 

### 21 to remind myself. 

### 22 **DR. SIGMUND DEGITZ:**At the time that that 

### 23 went forward, we did not have a readily transferrable 

### 24 methodology. And plus, that assay stops a little sooner so we 

### 25 have not reached the peek that works to our advantage in this 

### 26 case to make the measurement.

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1 **DR. GEOFFREY SCOTT:**Okay. Well I am glad you 

### 2 clarified that. I could not remember. I knew we were 

### 3 discussing that in the fish test the last time and I could not 

### 4 remember which was which. 

### 5 I would only comment on the T4. That is a 

### 6 universally measured metric in humans and (inaudible). 

### 7 And if we are going down the adverse outcome 

### 8 pathway, I will just use an example. A panel or two back where 

### 9 we had an example with triclosan and you all showed how we could 

### 10 go down that adverse outcome pathway. You all were using T4, I 

### 11 believe. I am pretty sure it was T4. 

### 12 We measure that in dolphins. And because we 

### 13 had triclosan measurements, we could actually go back and look 

### 14 at that in our dolphins. And I really think, where we have 

### 15 these readily established methods where we can do that, that are 

### 16 good reliable methods, that is going to help us where we do have 

### 17 some benchmarks. And since you are asking the question, at 

### 18 least I just thought I would mention our experience of being 

### 19 able to piggy-back off of where you all were going in other 

### 20 species. 

### 21 The other question I had related to 

### 22 histopathology, and not so much the sectioning and the 

### 23 processing of samples but in the reading of samples. In the 

### 24 current guidelines, are there positive controls sent to folks 

### 25 reading the slides or some set of slides where they should be 

### 26 telling you the same thing?

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1 **DR. SIGMUND DEGITZ:**Not in the current 

### 2 guideline. But as Rodney mentioned in his presentation, we are 

### 3 building a web-based tool, so all this glass feeds that 

### 4 web-based tool at this point. 

### 5 **DR. GEOFFREY SCOTT:**Could we just say it 

### 6 would be good to have those benchmarks. 

### 7 **DR. SIGMUND DEGITZ:**Here is what an estrogen 

### 8 looks like and here is what an androgen looks like, I agree. 

### 9 **DR. GEOFFREY SCOTT:**I just thought I would 

### 10 make that comment. 

### 11 **DR. MARTHA SANDY:**Okay. And now we will hear 

### 12 from Dr. Touart. 

### 13 **DR. LESLIE TOUART:**And this is just kind of a 

### 14 follow-up on the point that Dr. Scott made. 

### 15 We are planning to have a histopathology 

### 16 workshop similar to what we had done for the Tier 1 in frogs to 

### 17 go over some of these aspects to bring on some of the work being 

### 18 done to try to develop these web-based guidances, but also to 

### 19 try to look a little bit more in terms of the histo techniques 

### 20 to be utilized as well as the pathologist themselves to be 

### 21 utilized. And this would include pathologists that had been 

### 22 part of the validation studies in terms of the readings, but 

### 23 others that would have expertise on the taxa that we would be 

### 24 looking at, which would include fish, frogs and birds. And the 

### 25 tentative plans for this would be later this summer or early 

### 26 fall, but actual dates have not been set just yet.

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1 **DR. GEOFFREY SCOTT:**Well very good. You all 

### 2 are thinking in the right way. Thank you. 

### 3 **DR. MARTHA SANDY:**And now Dr. Denver. 

### 4 **DR. ROBERT DENVER:**Say, were you also unable 

### 5 to measure T4 using the ELISA before ethanol extraction in your 

### 6 lab? I am just wondering. 

### 7 **DR. SIGMUND DEGITZ:**Yes, we could measure it, 

### 8 but what was happening to us, it was incredibly inconsistent. 

### 9 So the CVs were huge. And by precipitating out a lot of protein 

### 10 during that process, we were able to get the values to be much 

### 11 more reproducible. 

### 12 So we basically got the same results that the 

### 13 contract labs did when we tried to use the kit. We went through 

### 14 a lot of perturbations to clean that approach up and it turned 

### 15 out the first thing we tried was ethanol; went on to a lot of 

### 16 other things but that ethanol was still the best. 

### 17 **DR. ROBERT DENVER:**And given the difficulties 

### 18 of obtaining blood from tadpoles, and also the variability that 

### 19 you see at stage 62, why don't you just measure it in the 

### 20 juvenile frogs? I mean you can get plenty of serum from them. 

### 21 They have -- xenopus compared to a lot of -- and urines has 

### 22 detectible T4. 

### 23 **DR. SIGMUND DEGITZ:**You know, in our hands -- 

### 24 maybe in your hands it is different -- we do not detect T4 in 

### 25 xenopus blood of juveniles. 

### 26 **DR. ROBERT DENVER:**Really?

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1 **DR. SIGMUND DEGITZ:**No. So we have used the 

### 2 ICP-MS method pretty extensively with that. Yes. 

### 3 **DR. ROBERT DENVER:**I imagine you would in 

### 4 those thyroid hyperplasic animals, possibly. 

### 5 **DR. SIGMUND DEGITZ:**And then the other reason 

### 6 to get the T4 measure at stage 62 is for consistency, in terms 

### 7 of taking all three of those and considering them 

### 8 simultaneously. But you are right. 

### 9 **DR. ROBERT DENVER:**Right. 

### 10 **DR. SIGMUND DEGITZ:**But again, and something 

### 11 very interesting, I mean we have not found in trops or laevis 

### 12 post metamorphosis; it is below our detection limits and we have 

### 13 some fairly low detection limits with the ICP-MS. 

### 14 **DR. ROBERT DENVER:**Yes, we have used 

### 15 radioimmunoassay in adult xenopus but not juvenile. 

### 16 **DR. SIGMUND DEGITZ:**Yes. 

### 17 **DR. ROBERT DENVER:**The other question I have 

### 18 is about replication. And is four replicate tanks sufficient 

### 19 given that you know you can lose tanks pretty easily if it is 

### 20 diseased. 

### 21 **DR. SIGMUND DEGITZ:**Yes. Yes. 

### 22 **DR. ROBERT DENVER:**And a related question is 

### 23 why the dis-balance in the design? Why eight control and why 

### 24 four? 

### 25 **DR. SIGMUND DEGITZ:**Well I will take that 

### 26 question first. And that is, if you lose a control you lose a

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1 study, in terms of everybody, all of your doses. That is why we 

### 2 are balanced on the controls. 

### 3 You know you lose a middle of the dose 

### 4 response, you lose one tank and you are left with three, yeah, 

### 5 your power is reduced but you have not lost the others. So that 

### 6 is why the control is stat. We did some power analysis -- I do 

### 7 not have it with me -- and we looked -- and, you know, you 

### 8 really have to start to get those replicates up pretty high 

### 9 before you are gaining a whole lot. 

### 10 Now I understand your idea about insurance 

### 11 policy, is that can you work with three -- you know, and four is 

### 12 okay. You know, if you cannot work with three -- and we did 

### 13 some looking at that and it is really a compromise between how 

### 14 complex the exposure system gets because these are not the small 

### 15 Medakas, you know, where you have got multiple animals per tank. 

### 16 So that is why we are weighted for the controls. And you could 

### 17 always have more replicates but how doable is it? Does that 

### 18 answer your question? I do not mean to skirt around it. I mean 

### 19 I am trying to answer it. 

### 20 **DR. ROBERT DENVER:**Yes. I mean you said that 

### 21 you have sufficient data to evaluate the power in that four is 

### 22 sufficient. 

### 23 **DR. SIGMUND DEGITZ:**Yes. What we have found 

### 24 is it took quite a bit of increase to get you -- you did not 

### 25 make gains with one or two replicates, which is what we are 

### 26 talking about. So at this point we are talking 28 tanks in

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1 these studies. Then the next jump would be 32, 36. 

### 2 **DR. ROBERT DENVER:**Yes. I just have one last 

### 3 question. 

### 4 **DR. SIGMUND DEGITZ:**Okay. 

### 5 **DR. ROBERT DENVER:**Why can't the EPA hold 

### 6 contract labs accountable for following protocol? I am just 

### 7 wondering who has that authority? 

### 8 **DR. SIGMUND DEGITZ:**These guys will answer 

### 9 that question. 

### 10 **DR. LESLIE TOUART:**I can try to answer that a 

### 11 little bit. And within the contract framework, there is some 

### 12 accountability that is there and there is performance reviews 

### 13 that are done of contractors when things come in. 

### 14 We try to hold them, but with any protocol, as 

### 15 we found, individual labs sometimes like to do things and 

### 16 interpret thing in their own way, in a sense, and it is just the 

### 17 reality of how things are dealt with. What we do not have is as 

### 18 big a hammer to use against them if they are starting to color 

### 19 outside the lines or something like that. It is even more 

### 20 difficult when we deal with some of our cooperators where we 

### 21 have an interagency agreement, another federal partner or other 

### 22 collaborator like the Japanese. 

### 23 We try to work and communicate what is 

### 24 important about assays, but in terms of the ability that we have 

### 25 to actually control a contractor to follow everything is a 

### 26 little difficult. We get what we get. And that is part of the

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1 validation exercise itself, because the contract labs that we 

### 2 are using are the contract labs that are likely to be the ones 

### 3 foremost in conducting these tests in routine basis. 

### 4 So the results that we see are likely the 

### 5 things that they would be doing and they point out where we need 

### 6 to modify our guidance and specifications in terms of the test 

### 7 methodologies that we give them in terms of the test guidelines 

### 8 and specifics; what things we feel are really must, what things 

### 9 are more recommended kind of things. So it is a little 

### 10 difficult and that is the best I can say. 

### 11 **DR. STEVEN BRADBURY:**As Les was saying, and 

### 12 Sig and Rodney, as we are going through the development of the 

### 13 protocols, labs are trying it out. I think some of the 

### 14 reflections are some things that we need to describe perhaps 

### 15 more clearly or directly. This is a really key critical 

### 16 component. 

### 17 But indirectly speaking as we go through the 

### 18 test guidelines generically, sort of the test requirements that 

### 19 come in, the register test that we already got in play, when we 

### 20 lay out the test guidelines, we lay out what we expect to see in 

### 21 terms of certain performance criteria to be met. 

### 22 And if a study comes in and it does not have 

### 23 certain quality characteristics, we will not accept the study 

### 24 and we will require the study to be rerun. And we try to be 

### 25 very clear about what are the decision points and we try to make 

### 26 sure we are looking at all the information to make sure we are

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1 just not doing check box because a lot of animals are being 

### 2 scarified to do these studies and we want to make sure we're 

### 3 getting all the information that we can. 

### 4 I think the incentive, once you start moving 

### 5 into a regulatory program is clarity over what it takes to have 

### 6 an acceptable study. You have to have acceptable data to 

### 7 maintain the registration. If you do not provide any data that 

### 8 meets our quality criteria, you will have to do it again, and 

### 9 there is a cost to doing it again. 

### 10 So I think you are just sort of seeing the 

### 11 evolution of what we are learning about, development of a method 

### 12 versus documenting what it takes to have a method work. And it 

### 13 is reasonable that that method can work if you follow key 

### 14 protocols properly, and then that is the expectations for the 

### 15 data when it comes in to actually make our regulatory 

### 16 determinations. 

### 17 **DR. MARTHA SANDY:**And Dr. Touart? 

### 18 **DR. LESLIE TOUART:**Again, just a quick 

### 19 follow-up. But one of the things with the test guidelines that 

### 20 we have before test methods, you know, we have the proposed test 

### 21 guidelines and there are some performance criteria that are 

### 22 listed within that. 

### 23 So I would really ask the Panel to look at 

### 24 those and determine should we have some more stringent criteria 

### 25 listed or should they be presented in a different way because 

### 26 there are some difficulties as Dr. Bradbury pointed out, in

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1 terms of our ability to accept studies in terms of the quality 

### 2 factors that we think are appropriate. 

### 3 So we would hopefully be able to look at those 

### 4 and provide some comments in terms of, you know, are these too 

### 5 laxed or do we need to specify some more. And it really is part 

### 6 of the laboratory exercises that we do. These are to allow us 

### 7 to test to see what things really matter and what things that 

### 8 seem to be the ability to have a little bit more flexibility. 

### 9 **DR. MARTHA SANDY:**Dr. Perkins? 

### 10 **DR. EDWARD PERKINS:**Sig, can you comment on 

### 11 the reproducibility in these other labs and the other obviously 

### 12 histopathology/pathology problems? It seems like there was a 

### 13 lot of variability in growth endpoints and a lot of other stuff 

### 14 like that. I guess, is this really reproducible? 

### 15 **DR. SIGMUND DEGITZ:**Well I guess that is the 

### 16 question that we are here to talk about at the end, that is the 

### 17 take-home. 

### 18 I think in terms of the contract laboratories 

### 19 electing to sort of go on their own in terms of feeding, like 

### 20 the one laboratory that increased their feeding by 30 percent 

### 21 and did not tell us, or the other laboratory that uses a 

### 22 different diet formulation. 

### 23 If you look at some of those things, you know, 

### 24 when you start to manipulate diet and you start to affect growth 

### 25 to an extent that you see being effected, that is also 

### 26 associated with the devil is in the details; but a fairly

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1 protracted period of time that it takes that group of 

### 2 individuals to reach stage 62. So now we start to see it all 

### 3 wrinkling through all of our endpoints then. You know, 

### 4 something that starts at feed. You know, I guess if you can 

### 5 read and follow directions, I think that this protocol is 

### 6 reproducible. 

### 7 **DR. EDWARD PERKINS:**But you did not have that 

### 8 requirement from the beginning? 

### 9 **DR. SIGMUND DEGITZ:**We forgot to state it, I 

### 10 guess. 

### 11 You know, the problem is that the labs -- one 

### 12 of the labs was very experienced with frogs, and it was probably 

### 13 the worse performance we saw. The other lab was fairly new to 

### 14 it and they did pretty darn good, except for the extra feed by 

### 15 30 percent. Aside from that, their first study was really nice. 

### 16 But what ends up happening is -- so you 

### 17 increase feed relative to what everyone else does, and all of a 

### 18 sudden those animals are on a different growth rate. That 

### 19 changes your sensitivity to be able to pick up the chemicals 

### 20 effect on growth. So if you are producing excessive growth, you 

### 21 are going to be probably way more sensitive than another 

### 22 laboratory that is not producing excessive growth. Does that 

### 23 make sense? 

### 24 **DR. EDWARD PERKINS:**No, it does. So as a 

### 25 result of what you are saying, do you think you need to repeat 

### 26 the whole inter-laboratory studies with more stringent criteria

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1 for actual people that read the protocols to really show this 

### 2 reproducibility? 

### 3 **DR. SIGMUND DEGITZ:**I guess that is what the 

### 4 SAP is here to judge. You know, it is reproducible in our 

### 5 hands, and reproducible in some of the people we work with. The 

### 6 key is that more contact is better. The folks that we had 

### 7 collaborations with were much more successful than the contract 

### 8 laboratories. 

### 9 **DR. MARTHA SANDY:**Okay. I have seen two 

### 10 other hands, Dr. Propper, then Dr. Klaine and then Dr. Scott. 

### 11 **DR. CATHERINE PROPPER:**So along these lines 

### 12 then, because the variability was there -- and I was actually 

### 13 surprised because there were places where there was more 

### 14 consistency than I would have expected, but there is variability 

### 15 both in overall outcome and especially in LOECs. How do you 

### 16 then interpret your data to actually set regulatory LOECs? And 

### 17 maybe this is a question for -- 

### 18 **DR. MARTHA SANDY:**I am sorry. We are 

### 19 supposed to be asking questions of clarification. 

### 20 **DR. CATHERINE PROPPER:**Clarification? Okay. 

### 21 How do you interpret the different LOECs? 

### 22 **DR. SIGMUND DEGITZ:**Okay, start over. I was 

### 23 following you and I was anticipating you and then we got 

### 24 changed. 

### 25 **DR. CATHERINE PROPPER:**Right; and I apologize 

### 26 I think I went beyond the scope of the question period. The

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1 different LOECs that you get, do you find that that makes 

### 2 interpretation also the inter-laboratory variation tricky? 

### 3 **DR. SIGMUND DEGITZ:**Well it depends on to 

### 4 what degree you are making that interpretation. You know, when 

### 5 you have multiple endpoints such as -- okay. So if you have 

### 6 four labs and one is the odd lab out, it is a pretty easy 

### 7 situation to be in, right? You have got two labs and which one 

### 8 is the odd lab out? And in regulatory context, most of the time 

### 9 there is one lab, one study, and we do not know what the 

### 10 variability is. And beyond that, I do not know what else to 

### 11 say. 

### 12 **DR. MARTHA SANDY:**Mary Manibusan? 

### 13 **MS. MARY MANIBUSAN:**So just for clarification 

### 14 because it falls into a regulatory domain, typically as Dr. 

### 15 Degitz had indicated, the Agency received one study, and that 

### 16 one study will be judged based on its performance. 

### 17 So those proficiency criteria, as Dr. Touart 

### 18 had indicated is going to be really critical to nail down and be 

### 19 very specific in order to judge the quality of that study. When 

### 20 that quality of that study meets our acceptance for regulatory 

### 21 determination, that one study will give us, perhaps, the point 

### 22 of departure we need for risk assessment. You will not, 

### 23 usually, typically have multiple labs running the same study for 

### 24 submission to an agency. But again, the quality of that study, 

### 25 as we have learned through our inter-laboratory evaluations, 

### 26 will help us nail down that test guideline so that it speaks to

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1 the conductor of that study so they can demonstrate the 

### 2 performance that we need. And we will not be in a situation 

### 3 where we have such a wide variability in our evaluation in our 

### 4 LOECs. So that is our goal here. 

### 5 And so it is important to split out the 

### 6 difference between laboratory performance that we are seeing in 

### 7 the inter-lab. And quite frankly you are right, we have picked 

### 8 a variety of labs from inexperienced all the way to experienced 

### 9 laboratories, but that information is very important to the 

### 10 Agency to, again, helping us to achieve a test guideline that 

### 11 can speak to a range of laboratories so that they can all begin 

### 12 to submit acceptable test guideline studies. 

### 13 **DR. MARTHA SANDY:**Dr. Klaine? 

### 14 **DR. STEPHEN KLAINE:**So following up on that. 

### 15 So when a laboratory submits a report and you deem it 

### 16 unacceptable; so in an inter-laboratory comparison test, you 

### 17 don't make them repeat it? Because it seems like you accepted 

### 18 the test but just did not count it in the analyses. 

### 19 **DR. SIGMUND DEGITZ:**Are you talking about for 

### 20 the inter-laboratory? 

### 21 **DR. STEPHEN KLAINE:**Yes. 

### 22 **DR. SIGMUND DEGITZ:**So she is talking in 

### 23 terms of -- 

### 24 **DR. STEPHEN KLAINE:**I know what she is 

### 25 talking about. But you were talking about comparison between a 

### 26 regulatory laboratory submission versus the submissions for what

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1 we are dealing with now. 

### 2 **DR. SIGMUND DEGITZ:**Right. As I understand 

### 3 it -- and I will just cut to the chase on this -- is the way 

### 4 these contracts are established. They are paid for the work 

### 5 they do, not for the product they produce. 

### 6 **DR. STEPHEN KLAINE:**Okay. 

### 7 **DR. SIGMUND DEGITZ:**Yes. 

### 8 **DR. STEPHEN KLAINE:**So if you get complete 

### 9 control failure, you just toss it out -- pay them anyway. 

### 10 **DR. SIGMUND DEGITZ:**I was going to say that 

### 11 earlier, but I did not think it was my place, but I do not think 

### 12 you guys got the direct answer that you were looking for so I 

### 13 just did that. 

### 14 **DR. STEPHEN KLAINE:**Thanks. 

### 15 **MS. MARY MANIBUSAN:**But these are 

### 16 subcontractors. 

### 17 **DR. MARTHA SANDY:**Yes, Dr. Touart? 

### 18 **DR. LESLIE TOUART:**Just a clarification as 

### 19 Sig said. I mean the contract vehicles that we utilize with the 

### 20 government are somewhat limited. And in the most part, it is a 

### 21 level of effort type contract. So when the work is performed, 

### 22 it would be concluded as you see time limitations that are 

### 23 involved as well. And so you could run into some situations 

### 24 that where if you needed the study to be done a different way 

### 25 you are going to have to pay for a new study in a sense. Often 

### 26 there is not time to do that.

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1 So the context is that in an inter-laboratory 

### 2 exercise, we have established certain performance that we feel 

### 3 would make the study scientifically readable, in some sense. 

### 4 And so the context is that, if that study fails to meet those 

### 5 quality factors, then we can deem it as unacceptable if there is 

### 6 too much control survival or some other factors that we use, and 

### 7 some of those are performance criteria that we have established 

### 8 within the proposed guidelines that we have. So if it fails 

### 9 those, we cannot compare that. 

### 10 We cannot use a bad study and then compare 

### 11 that in terms of its CVs to good studies. We want to try to 

### 12 identify how -- with what we are perceiving as good studies -- 

### 13 how these compare with each other. And so that is why some 

### 14 laboratories would fall out of an evaluation. 

### 15 **DR. STEPHEN KLAINE:**Just real quick here. I 

### 16 was just expecting to see the test repeated. You are asking him 

### 17 to repeat the test, that is all, but now I know why you do not 

### 18 ask him. 

### 19 **DR. MARTHA SANDY:**So Dr. Scott? 

### 20 **DR. GEOFFREY SCOTT:**Just a couple of quick 

### 21 comments for clarification. In all of the assays you all have 

### 22 presented today, the fish and everything, the frog as well; I am 

### 23 particularly thinking of slide 60 that you showed on the fish, 

### 24 Rodney. You gave what were the control metrics for a successful 

### 25 -- you need to put a lot of emphasis on what that is because in 

### 26 some instances we are doubling the number of controls to compare

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1 with, in some instance. 

### 2 So you really need to focus on are the labs 

### 3 producing that in the absence of those. And then it would seem 

### 4 to me everything else then falls into compliance with the dosing 

### 5 in other aspects of the metrics you are measuring. But if you 

### 6 know your controls are good, that is at least the starting point 

### 7 of quality assurance. So it would seem to me in defining this 

### 8 that the emphasis or the lab should be focused on the control 

### 9 metrics. And we have not seen that clarity on every 

### 10 presentation today, but I just was taken by slide 60. I thought 

### 11 that was really nice, so I would go back -- if you all do this 

### 12 -- focusing on that control metric to me would seem appropriate. 

### 13 **DR. MARTHA SANDY:**So Dr. Scott, thank you. 

### 14 Hold that thought for the charge questions. Okay. Dr. Scanes? 

### 15 **DR. COLIN SCANES:**I want to follow-up Dr. 

### 16 Scott's question, and that is, Dr. Scott talked about the issue 

### 17 of controls. And I wonder if one of the lessons learned might 

### 18 be there needs to be a positive control used, so that if you 

### 19 have got different labs doing tests you want to see if they get 

### 20 a response. If they get no response to the positive control 

### 21 that equally may be a criteria to be considered. 

### 22 **DR. MARTHA SANDY:**And I would like to remind 

### 23 the Panel that those are excellent comments, but those are more 

### 24 along the lines of answering charge questions, I think, than 

### 25 clarifying questions. 

### 26 Okay. Do we have any other clarifying

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1 questions from the Panel? Yes, Dr. Bentley? 

### 2 **DR. GEORGE BENTLEY:**Going back to the thyroid 

### 3 histopathology in your T4 assay -- I should have asked this 

### 4 earlier, but I am curious; do you ever run any correlations 

### 5 between the size of the thyroid and the circulated T4? And then 

### 6 following on from that, do you ever consider looking for changes 

### 7 in expression in the enzymes in the liver? 

### 8 **DR. SIGMUND DEGITZ:**I would answer yes to all 

### 9 of your questions in our research life, okay? Not in our 

### 10 regulatory life. I mean the ORD does that future looking type 

### 11 of research to understand relationships. You know, can volume 

### 12 predict? Could the other things predict? You know, we are 

### 13 always looking to the future that some day we could shelf these 

### 14 archaic approaches we are using today and come up with something 

### 15 better. 

### 16 So yes, we do look at that. It is hard at 

### 17 this point in time to implement that and bring that into the 

### 18 regulatory context. 

### 19 **DR. COLIN SCANES:**The reason I ask is I am 

### 20 thinking about biologically meaningful endpoints of what you are 

### 21 measuring, and these would be quite simple things to measure. 

### 22 It could actually make life a lot easier from a regulatory 

### 23 standpoint. 

### 24 **DR. SIGMUND DEGITZ:**Agreed. The key is going 

### 25 to be for ORD to work through these approaches and work with the 

### 26 program office so they understand how they can assist in

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1 implementation. 

### 2 **DR. COLIN SCANES:**Okay, thank you. 

### 3 **DR. MARTHA SANDY:**At this point, I would like 

### 4 to thank you very much Dr. Degitz for your presentation. We 

### 5 need to take a short five-minute break, just five minutes. 

### 6 Please be back. We have one more presentation to get through 

### 7 today. Thank you. 

### 8 (Whereupon a break was taken) 

### 9 **DR. SHARLENE MATTEN:**Mary Christman and 

### 10 Dallas Johnson have written down in plain English the more 

### 11 specific questions they wanted to ask about clarifications of 

### 12 the reanalysis, if that is the appropriate word, of the 

### 13 statistical StatCHARRMS package, and I am typing those. For 

### 14 those who are listening, I am typing those because it is in 

### 15 handwritten form. 

### 16 I am sending it to Mary Manibusan who is 

### 17 sitting across from me and she is going to get it to the team 

### 18 and they will provide some clarifications to the best of their 

### 19 ability in the morning sometime or sometime in the near future, 

### 20 not at 6 a.m. or -- it does not have to be that. But they are 

### 21 going to consider the questions and try to clarify them. Is 

### 22 that clear? 

### 23 **DR. MARTHA SANDY:**Thank you, Dr. Matten. And 

### 24 now we are ready for our fourth presentation today by Dr. Touart 

### 25 on the Mysid. 

### 26 **DR. LESLIE TOUART:**Okay. Thank you, Chair.

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1 And I know it has been a long day and I know people are starting 

### 2 to get weary, so I will try to walk through this as quickly as I 

### 3 can and try to cover the key points. 

### 4 And what I am going to do is talk about the 

### 5 Mysid two-generation test as well as the copepod development and 

### 6 reproduction test, which was kind of an additional aspect that 

### 7 we evaluated. And I will talk a little bit about why we did 

### 8 that. 

### 9 The basic outline of this presentation is just 

### 10 to go back and cover a little bit of the purpose and overview of 

### 11 an invertebrate test as part of the EDSP Tier 2, you know, why 

### 12 is it fitting in here. A little bit of the history of the 

### 13 validation process for both the mysid copepod and then talk 

### 14 specifically about the mysid in terms of the protocols, some 

### 15 example test results, some generation comparisons and lessons 

### 16 learned. And similarly, with the copepod, to talk about its 

### 17 protocols, some basic results seen and some advantages, 

### 18 limitations. And then talk about a little bit of the 

### 19 recommendation that we have in terms of why we feel we prefer 

### 20 just to pursue the mysid. 

### 21 In terms of overview -- and again we are not 

### 22 talking about the more important taxa to be considered and that 

### 23 is the invertebrate. 

### 24 The estuaries are an important ecosystem and 

### 25 recipient of many EDCs. And I mention estuaries because the 

### 26 species, both the mysid as well as the copepod are estuarine in

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1 a sense. They are crustaceans and these are often among the 

### 2 more sensitive organisms and they are important individuals 

### 3 within the ecosystems themselves. 

### 4 There are many insecticides that are really 

### 5 putative EDCs in some context. There are some that are 

### 6 exploited specifically to interact with the insect hormone 

### 7 systems. And crustaceans are also effected by these, so we 

### 8 understand the context of how endocrine disruption can happen 

### 9 because that is used to try to eradicate some species in a 

### 10 sense. 

### 11 But in the context, the invertebrates have 

### 12 hormones. They have endocrine systems that are very well 

### 13 developed. And ecdysteroids, which is a molting hormone, is 

### 14 also important for reproduction in these individuals, these 

### 15 occur. 

### 16 In context the endocrine systems of 

### 17 invertebrates differ from that of vertebrates in some 

### 18 fundamental ways, but the response of invertebrates to an EDC 

### 19 are kind of expressed differently but they can also be affected 

### 20 by compounds that will affect vertebrate hormones. 

### 21 Crustaceans have true endocrine glands, but we 

### 22 have seen, at least in some cases were are vertebrate active 

### 23 materials -- and I just give a couple of examples here like 

### 24 testosterone interferes with ecdysteroid regulation, at least 

### 25 suggested. And aromatase inhibitor can interfere with 

### 26 ecdysteroid synthesis because it is just, you know, steroid

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1 production kinds of things. 

### 2 So these are just some examples. But the real 

### 3 key here is not so much that would cure their invertebrate 

### 4 active compounds will have similar effects like on estrogens, 

### 5 androgens and thyroids, which are not active in these 

### 6 individuals, but that these activities, if we have identified an 

### 7 invertebrate active compound, that this may have an adverse 

### 8 effect within the invertebrate. 

### 9 And with some of the validation work, we will 

### 10 show that these invertebrates are sensitive to some of these 

### 11 compounds because some of these vertebrate compounds that we 

### 12 have here have been active. 

### 13 So the context is, when we have identified a 

### 14 particular material as having some potential endocrine activity, 

### 15 at least vertebrate endocrine activity, there is a question as 

### 16 to how this is going to adversely effect invertebrates. And to 

### 17 do that we need a test that would comprehensively evaluate some 

### 18 of the adverse effects that are plausible before that, and that 

### 19 is what the purpose of the test really is. 

### 20 And so with both mysids and copepods, these 

### 21 have been used for a number of years now in toxicity tests. 

### 22 Mysids especially have been used in regulatory testing both 

### 23 acute and life-cycle tests for over 30 years now. There are 

### 24 standard test protocols for these mysids, American mysids bahia. 

### 25 And the Amphiascus Tenuiremis as a copepod exist as like ASTM 

### 26 standard methods for conducting tests. But the copepods are not

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1 used as much as mysid, but there are methods that have been 

### 2 established. 

### 3 In terms of a history for the mysids, the 

### 4 initial event was an evaluation of fenoxycarb. You know this is 

### 5 a growth regulating type compound and this was evaluated in a 

### 6 multigeneration mysid test and was a basis for an initial draft 

### 7 test guideline back in 2001. 

### 8 There was an invertebrate detailed review 

### 9 paper that was initiated and completed in 2004 to look at a 

### 10 variety of invertebrate methodologies and recommendations in 

### 11 terms of what may be appropriate to evaluate in terms of an 

### 12 invertebrate test. 

### 13 Through the course of time from 2004 through 

### 14 2009 there were a number of demonstration tests that were done 

### 15 with the particular protocol that we have that we are proposing 

### 16 and these are a variety of compounds that have been used. Some 

### 17 of these are not specifically a vertebrate-active material, at 

### 18 least we know of, in terms of like the dichlorophenol and 

### 19 compounds like fipronil, but clearly some of these are ones that 

### 20 we have already talked about today in the validation of some of 

### 21 our other vertebrate test with things like prochloraz, compounds 

### 22 like flutamide, ketoconazole which is a steroidogenesis. We 

### 23 have atrazine and a perfluorodecanoic acid as another compound. 

### 24 And then in our inter-laboratory evaluation, 

### 25 we had labs look at lindane as a compound as kind of a standard 

### 26 toxicant, but also octylphenol. And we had a laboratory

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1 evaluate vinclozolin. 

### 2 For the copepods, the efforts with copepods 

### 3 initiated more as an international effort through the auspices 

### 4 of the OECD, that is the Organization of Economic Co-operation 

### 5 and Development. There was a proposal by Sweden to development 

### 6 a copepod test methodology, and so they proposed a particular 

### 7 protocol. And the initial protocol really included three 

### 8 different harpacticoid copepod species as well as a calanoid 

### 9 species to investigate. 

### 10 Subsequent, the detailed review paper that was 

### 11 completed that considered mysids as well as copepods as well as 

### 12 some other crustacean, you know, these were considered finalized 

### 13 in this 2004 time period. But within the OECD there were two 

### 14 phases that were completed, one with a DCP and then a second 

### 15 phase with lindane and atrazine that were completed. 

### 16 But the outcomes of these left a couple of 

### 17 questions lingering and it was considered that copepods may 

### 18 offer some advantages over the mysids in context of maybe being 

### 19 a more logistically easy or a shorter study to do. It takes 

### 20 less space, and I will talk about that a little bit later when 

### 21 we talk about copepod methodology itself. 

### 22 But EPA decided that we would include the 

### 23 copepod as part of our inter-laboratory effort where we were 

### 24 doing lindane and vinclozolin for the mysid. So we felt, well 

### 25 we will take a look at this and perhaps copepod might be 

### 26 considered an alternative or even a replacement for the mysids

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1 in context. 

### 2 So just a brief overview in context of what 

### 3 these two methodologies were like. With the mysid, this is a 

### 4 two-generation method. The copepod is just a single life-cycle. 

### 5 With the mysid, we are dealing with Americamysis bahia. We are 

### 6 dealing with the flow-through system. We recommend solvent-free 

### 7 continuous flow. The duration of this test is 60 days or a 

### 8 little bit longer, depending on the development rate of the 

### 9 species. Design is basically 30 animals per tank with 15 per 

### 10 chamber. There is a reproductive chamber and a growth chamber 

### 11 that is utilized, and each tank would be a replicate. There are 

### 12 three of these in each treatment group. There would be five 

### 13 treatments in seawater control, and if one was not using a 

### 14 solvent-free system, one would need to add a solvent control as 

### 15 well. 

### 16 The exposure duration begins with individual 

### 17 offsprings that are less than 24 hours, so that is less than 24 

### 18 hours post-release from the brood pouch of their parent. And 

### 19 this goes through the median second brood release in controls 

### 20 plus seven days, and this is within the second generation. 

### 21 For the copepod, it is semi-static. These are 

### 22 done in really the microwell plates. It is 36-day test, time 

### 23 definite. There are 30 animals used per plate, three rows of 

### 24 10. There are three replicates of the treatments of 90 animals 

### 25 per treatment level. There would be five concentrations solvent 

### 26 control and seawater control. And these are also 24 hours

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1 post-release through day 36, and that should include two brood 

### 2 releases within the time period. 

### 3 So the mysid two-generation test is really 

### 4 just an adaptation and continuation of the existing OCSPP 

### 5 850.1350 Mysid Life-Cycle Test. The mysid life-cycle test is 

### 6 currently a 28-day time definite study where it begins the same 

### 7 with less than 24-hour old individuals and carried for 28 days. 

### 8 Typically there are two brood releases within that time, but 

### 9 depending upon developmental rates, it could be one or more, 

### 10 even as much as three within the context of that test. 

### 11 And what we are doing in the two-generation is 

### 12 you initiate the life-cycle test, but you take the offspring 

### 13 from this second brood of this life-cycle test and initiate the 

### 14 second generation with that and grow those into adulthood and 

### 15 reproduction. 

### 16 So the purpose, again, to evaluate adverse 

### 17 reproductive and developmental effects of vertebrate endocrine 

### 18 active compounds that would effect an invertebrate, and 

### 19 characterize a dose response that would be relevant for 

### 20 ecological risk assessment. 

### 21 There are multiple endpoints that allow for 

### 22 diagnostic and definitive evaluation of these compounds but they 

### 23 are basically evaluating endpoints of survival, growth and 

### 24 reproduction. 

### 25 This is just a schematic that talks a little 

### 26 bit about evaluating potential endocrine effects and

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1 considerations of the activation of reversible type effects. 

### 2 These are considered those that occur post-developmental. These 

### 3 are adults that have been developed. So effects that would 

### 4 occur here are really more of these activational and reversible 

### 5 where you would anticipate that removing exposure would return 

### 6 the individual to a healthy state. 

### 7 On the organizational level, these are 

### 8 generally developmental exposures that would occur at 

### 9 developmental stages like embryo, larval or juvenile. And these 

### 10 may be organizational and considered maybe irreversible if I 

### 11 have affected something within, say, larval development that is 

### 12 latent and manifest later in terms of its reproductive 

### 13 viability. 

### 14 This is a schematic of the two-generation test 

### 15 where the test would initiate with these new released 

### 16 offsprings. These would mature and grow to adulthood. 

### 17 A common name for mysid is opossum shrimp 

### 18 because they carry their brood in a pouch. The embryos will 

### 19 develop until they are really a fully metamorphosed juvenile 

### 20 that are then released at a time. So once it has gone through 

### 21 one life-cycle, which would be consistent to the existing mysid 

### 22 life-cycle test, the offspring from the second brood would be 

### 23 used to initiate the second generation and carry those through 

### 24 adulthood and reproduction. 

### 25 This is a little bit more of a specific 

### 26 overview in terms of species delivery. We talk about the

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1 endpoints that are included or survival. There is dry weight, 

### 2 time to maturation, time to brood release, inter-brood 

### 3 durations, number of young per female, total number, offspring 

### 4 viability, sex ratio, and also percent reproductively active 

### 5 female. 

### 6 And test validity criteria that we utilized in 

### 7 this test really relate to the purity delivery of the chemical 

### 8 ensuring that we are maintaining the expected exposure 

### 9 concentrations to a level that would be appropriate. 

### 10 We are looking for an average production in 

### 11 the controls greater than eight young/control female through the 

### 12 first and second broods to demonstrate enough reproduction so we 

### 13 have enough power to detect reproductive events. Water quality, 

### 14 looking at greater than/equal to 75 percent survival and greater 

### 15 than 75 percent females producing young in controls. 

### 16 So this gets into some of the results that we 

### 17 have seen. In this case, this is the lindane comparison with 

### 18 mysid. Three laboratories participated in this test. Two 

### 19 laboratories achieved the performance criteria but one lab did 

### 20 not and so it was dropped from further inclusion in the 

### 21 analyses. 

### 22 In terms of effects, what we are seeing is 

### 23 effects on length, days to brood and offspring production were 

### 24 affected within one laboratory. In the other lab, the length 

### 25 and days to brood release were affected, but some of the other 

### 26 endpoints like reproduction and survival were not as affected to

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1 the same extent. 

### 2 But in context, the LOEC based upon a more 

### 3 sensitive endpoint like length were somewhat comparable in 

### 4 context. So in this case, the LSE comparisons were somewhat 

### 5 similar. 

### 6 This is, again, inter-lab, and this is the 

### 7 number of offspring in the two broods. And the interesting 

### 8 thing here -- this is the SMV lab and this is the UNT lab. The 

### 9 context is that there is somewhat of a U-shaped response curve 

### 10 that is occurring in the first generation, and this is repeated 

### 11 in the second generation with somewhat lower effects, or at 

### 12 least more significant effects from the control. 

### 13 The interesting thing also is that in the 

### 14 other lab, although we were not seeing significant effects on 

### 15 reproduction, there were similar trends in terms of the pattern, 

### 16 you know, that we see with some declines, but also a kick-up in 

### 17 the number of offspring at the higher concentrations. And so 

### 18 that is repeated. 

### 19 So the context here is that we are seeing 

### 20 something similar. The actual significant concentrations were a 

### 21 little bit different. 

### 22 This is another case for octylphenol where two 

### 23 laboratories participated and the nature of the effects seen. 

### 24 And again, the most sensitive effects seen here were in length 

### 25 and in offspring production. And the LOECs are very close in 

### 26 context between the two laboratories.

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1 In this case for the days to brood, the 

### 2 concentration, which was equivalent to the more sensitive LOEC, 

### 3 this occurred only at the lower concentration so that the higher 

### 4 concentrations did not have the significant effect, except, I 

### 5 think it is one of the highest. 

### 6 This is, again, the table that demonstrates 

### 7 this with the up arrow is significant, the down arrow is the 

### 8 decrease that is occurring. And of course all concentrations 

### 9 for some of these endpoints we were seeing effects. This is 

### 10 within the SMV laboratory which had the, I guess, greater 

### 11 magnitude of effects that were seen. 

### 12 This table which is a little hard to see, but 

### 13 I will try to point out a couple of things. This was done to 

### 14 evaluate significant treatment effects, and so this is looking 

### 15 at is there a difference between treatment and control for the 

### 16 variety of endpoints for the F0 and the F1. 

### 17 And the next slide kind of highlights one of 

### 18 the examples and this is for the 14-day length. And the context 

### 19 here is that, yes, there was a difference seen here. These are 

### 20 the different laboratories. The S and U are different 

### 21 laboratories for the lindane. 

### 22 But the next slide kind of points out, well 

### 23 what are we talking about in terms of a significant trend that 

### 24 we are seeing between control and the treatments. And here you 

### 25 see the, from the lower concentration to the higher, a trend 

### 26 that would have effects. So these are seen across many of the

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1 various endpoints and characterized in contents. 

### 2 And along similar lines, we looked at 

### 3 significant treatment effects comparing generations, the 

### 4 difference between the F0 versus the F1 generation for different 

### 5 endpoints, again between the laboratories that were involved 

### 6 where we had two generations to look at. 

### 7 And again, pulling one of these in terms of 

### 8 the time to first brood where, yes, we see a generational effect 

### 9 in both laboratories but at least more pronounced within this 

### 10 one laboratory. The context here is that, within the F0 

### 11 generation you see the responses across the different 

### 12 concentrations. But in the F1, the duration higher, so there is 

### 13 a significant difference between the F1 generation, brood 

### 14 duration versus the F0. 

### 15 So trying to summarize a little bit more of 

### 16 effect differences between generations, this is another example 

### 17 using ketoconazole with the F0 mysids and looking at the second 

### 18 brood release, where in the F0s, the significant effect was seen 

### 19 at the 33.7 micrograms per liter and in the F1 mysids, much 

### 20 lower in terms of the lowest concentrations. 

### 21 And the context as I get into some of these 

### 22 others, is, for a variety of these chemicals, some of the 

### 23 vertebrate active compounds, the actual effect concentrations in 

### 24 the mysids are very low. Like octylphenol, we are seeing .36 

### 25 micrograms per liter or 3.9. Both of these are lower than the 

### 26 lowest test concentrations say in the frogs or fishes in

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1 context. So the mysids are more sensitive, and so if we are not 

### 2 including these then you may miss information in terms of 

### 3 potential adverse effects in these type of species. 

### 4 What I have done here is highlighted in red 

### 5 the more sensitive endpoint between like F0 and F1, so you can 

### 6 see that in some cases the F0 generation is more sensitive. In 

### 7 other cases the F1 generation is more sensitive. And in others, 

### 8 there really is not much of a difference between the 

### 9 concentrations. 

### 10 Just a point of clarification with atrazine 

### 11 and the mysid, the highest concentration was 193, and there were 

### 12 no significant effects that were seen, so LOEC would be greater 

### 13 than that. But the question mark is that there was a trend that 

### 14 was identified in the F1s. Because of the variabilities, it was 

### 15 not deemed significant, so that is why the question marks. 

### 16 And this is going to be more difficult to see 

### 17 because I cannot really see it myself. The context here was the 

### 18 numbers -- and this was ranges of coefficient of variation in 

### 19 the F0s and the F1s. 

### 20 And the reds are those where the coefficient 

### 21 of variation was greater than 20. All the blacks are those 

### 22 where the coefficient of variations for those endpoints are less 

### 23 than 20. But in many of these, even though it is greater than 

### 24 20, they are not much greater; I mean, you were dealing with 

### 25 things like 24, 26 or 21, they are some of the endpoints that 

### 26 the coefficients of variation did get up to a little bit higher

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1 level. But by and large, these are -- at least in my experience 

### 2 of aquatic test -- CVs at 20 percent or that neighborhood are 

### 3 very respectable in context. 

### 4 So limitations of the mysid studies; again, 

### 5 there are difficulties in evaluating your dose selection 

### 6 methodologies in terms of how you set your concentrations so you 

### 7 can determine actual no effects. In some of the tests that we 

### 8 had, we had effects down at the lowest concentrations tested, so 

### 9 those tests would need to be repeated to identify a real NOEC to 

### 10 be evaluated. 

### 11 There are problems with food limitations in 

### 12 the studies. Food availability for mysids is an important thing 

### 13 to do and one has to ensure that they are getting adequate feed 

### 14 and that feed is made available to them multiple times during 

### 15 the day. 

### 16 Few endpoints were considered responsive based 

### 17 on signal to noise. Also blocking design was often ineffective 

### 18 and may have resulted in some erroneously high errors in ANOVA 

### 19 because of the blocking that was done. Sometimes the F1 

### 20 generation is too small to test endpoints, meaning that the 

### 21 effects in the F0 were so great that we did not have enough 

### 22 individuals to fully populate the F1 to evaluate potential 

### 23 effects. 

### 24 Again, difficult to test substances are 

### 25 difficult to test in any aquatic system. And with some of these 

### 26 difficult materials where we had high CVs of measured

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1 concentrations, these may overlap which confounds ones ability 

### 2 to detect effects later on. 

### 3 In terms of lessons learned with the mysid, 

### 4 first are these test designs. Again we prefer a completely 

### 5 randomized test unless some form of blocking would have been 

### 6 required. And it just cautions in terms of how the blocking 

### 7 would be utilized. 

### 8 In some of the earlier studies, they employed 

### 9 self-starting siphons and it was found that we get a lot of 

### 10 impingement with the mysids. The mysids in their systems, they 

### 11 are usually kept within little screened compartments. And as 

### 12 the water levels in these self-starting siphons would deplete, 

### 13 the mysids would get impinged or attached to the side of these 

### 14 containers and so they would dry out and then be lost. So 

### 15 avoiding the self-starting siphons and just having a direct 

### 16 overflow seem to reduce the numbers of individuals lost through 

### 17 the impingement. 

### 18 Feeding; what we found out was that brine 

### 19 shrimp alone was insufficient nutrition for these individuals. 

### 20 And what we began to use were enriched brine shrimp where these 

### 21 brine shrimp are grown with an additional enriching media that 

### 22 adds more fatty acid content to the brine shrimp themselves. 

### 23 And these are commercially available enrichment materials that 

### 24 can be used. 

### 25 We also found from the initial study where we 

### 26 were using just mysids that we need to increase those to 20 to

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1 have sufficient numbers to carry through the test. 

### 2 In our dry weight measures, initially we had 

### 3 0.1 micrograms which requires a very sensitive balance to 

### 4 detect. But we found that perhaps one microgram would be 

### 5 sufficient to utilize for that measure. 

### 6 Exposure duration we feel should be terminated 

### 7 at second brood release or the seven days median time post the 

### 8 control brood release. And the context here is that once an 

### 9 individual has, like in controls or treatment, has had its 

### 10 second brood release, that individual could be sacrificed and 

### 11 utilized at that particular time. You do not want to 

### 12 necessarily carry those individuals until you have actually 

### 13 achieved the seven-day median. 

### 14 So once that brood release is made it is done. 

### 15 If they have not released brood within seven days of the median 

### 16 control release, then the whole test is terminated and any 

### 17 individuals left over would be captured. And it is felt that if 

### 18 they had not released, that is an endpoint within itself in 

### 19 terms of the duration per release. So it would be a 

### 20 reproductive effect. 

### 21 For survival, we found out that sometimes if 

### 22 we lost one of the individuals in one of the reproducing pairs 

### 23 that could be replaced by a like individual within the growth 

### 24 chambers. So if we lost a male in a reproductive chamber and 

### 25 replaced that with a male from the growth chamber that is in the 

### 26 same replicate that would be satisfactory. It is just we have

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1 to reconcile that in terms of how the survival calculation is 

### 2 made in terms of individuals that are lost and where those go. 

### 3 And for reproduction, we felt that we need the 

### 4 cumulative measure of number of offsprings per female per day 

### 5 would be the best summary statistic to utilize for comparisons. 

### 6 Now switching to the copepods; again, this is 

### 7 a single generation method originally proposed through OECD. 

### 8 The purpose is, again, similar to the mysid to evaluate adverse 

### 9 effects and characterize dose response. 

### 10 The attraction of the copepod was that there 

### 11 were fewer endpoints in the mysid, but it still includes some 

### 12 development reproductive endpoints and has shorter duration and 

### 13 less lab space requirement. And so the thought was maybe this 

### 14 might be more logistically practical. 

### 15 The mysid tests, for those that are familiar 

### 16 with them have a history of concerns that are difficult, 

### 17 rejection rates could be relatively high compared to some of the 

### 18 other standard methodologies. And it is a difficult test 

### 19 because, again, in marine system, trying to maintain sufficient 

### 20 marine water, especially for inland-based laboratories where 

### 21 they have to use artificial seawater and stuff, it can be 

### 22 complicated. But with copepods being a much smaller critter, 

### 23 there was thought that we could have some advantages here. 

### 24 And this is a little bit of a description of 

### 25 what goes on through the development. So there are discrete 

### 26 life stages beginning with embryos; two days to hatch to

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1 nauplii, and then going through six nauplius stages to develop 

### 2 into a copepodite, there are five stages, and then developing 

### 3 into adults and reproducing in context. So there are 

### 4 development that can occur. 

### 5 The copepod has sexual dimorphism in that 

### 6 males have characteristics that separate them from female, for 

### 7 instance antennae for clasping can be seen. 

### 8 And this is another kind of schematic of the 

### 9 life-cycle and the context of using like 96 well plates where 

### 10 you have individuals within each particular plate to evaluate 

### 11 and carry forward. 

### 12 Overview of the method; again, semi-static 

### 13 systems with 250 microliter per well, one animals per well, 

### 14 three rows of 10, three plates and survival going on, number of 

### 15 offsprings and clutches. And then validity criteria, again, 

### 16 similar where you have got delivery of test compound, water 

### 17 quality, maintaining survival and controls and survival within 

### 18 any given plate that is utilized, an average of eight produced 

### 19 within the clutches. 

### 20 In terms of the test that were performed, this 

### 21 is just to identify concentrations, you know, differences that 

### 22 were kind of -- these were the nominal concentrations; for 3, 

### 23 5-DCP, three laboratories; atrazine, three labs; lindane, there 

### 24 were overall seven laboratories and these are representing some 

### 25 of the more sensitive effect concentrations that were seen 

### 26 across these.

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1 And the next slide looks at an inter-lab 

### 2 comparison with the lindane. And this was the last comparison 

### 3 with the lindane that was done with EPA with the three contract 

### 4 laboratories that were utilized. And again, with the 

### 5 concentrations tested, the lowest effect concentration was five 

### 6 micrograms in two of these and 10 micrograms across all three 

### 7 laboratories, and higher concentrations having effect. So that 

### 8 is reasonably fair concordance across the laboratories in terms 

### 9 of the copepod comparison. 

### 10 Advantages of copepod, again, laboratories are 

### 11 generally able to culture the species. They can harvest gravid 

### 12 copepods and hatch healthy nauplii to conduct the test. 

### 13 Microplate testing allows for individual-based 

### 14 tracking. And this species has a rapid life-cycle, which the 

### 15 time is money in the context of these types of tests. 

### 16 There are limitations though, and we ran into 

### 17 substantial technical difficulties in the laboratories in terms 

### 18 of misidentifying copepodites in adults with gender 

### 19 misidentifications and increased handling time, these kinds of 

### 20 problems. And that may obscure some of the treatment-related 

### 21 effects. 

### 22 The misidentification of copepodites as adults 

### 23 decreases reliability. There is some questionable sensitivity 

### 24 but I will talk about that a little bit more. Largely that is 

### 25 due to the number of compounds that we looked at to really 

### 26 understand what is the copepod sensitivity.

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1 Chemical exposures are problematic because 

### 2 these are done in semi-static and in microwell plates so that we 

### 3 are not able to reverify exposure concentrations in the actual 

### 4 test chambers themselves as part of the plate. I only would be 

### 5 able to monitor it in the actual media that would be used to 

### 6 fill those. There might be some solvent-related toxicity that 

### 7 could also obscure effects. 

### 8 So trying to consider the rationale for mysid 

### 9 versus copepod, while both mysids and copepod require 

### 10 significant laboratory experiences, there seems to be a little 

### 11 bit more substantial experience with the mysids and copepods. 

### 12 There are more labs that utilize that primarily because it is a 

### 13 standard species on pesticide testing for estuarine 

### 14 invertebrates. 

### 15 There is a large amount of published toxicity 

### 16 data with the mysid. And the available evidence suggests that 

### 17 mysids are generally more sensitive in general of the test 

### 18 species. 

### 19 We may have a little bit more understanding of 

### 20 the biology of mysids than of the copepods. And mysids have 

### 21 been used for evaluating reproductive toxicity for a long time 

### 22 so we are familiar with that. And it has potential to 

### 23 development some more mechanistic endpoints, although we have 

### 24 not explored those specifically for our method. Similar to 

### 25 copepod, mysids are of ecological importance and would be 

### 26 important there.

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1 The mysid system allows for a continuous 

### 2 intermitted flow exposures so we can maintain the exposure 

### 3 concentrations to a better extent. The greater body size of the 

### 4 mysids makes them more amenable to other measures but also 

### 5 easier to handle than the copepods. The mysid test includes 

### 6 well-established endpoints, and the copepod does not include 

### 7 growth measurements, per se, just the difference between their 

### 8 developmental stages. Although data obtained through both the 

### 9 mysid and copepod can be used for population modeling, the mysid 

### 10 has data through two generations, which might be an advantage. 

### 11 So in considering sensitivity and in those 

### 12 compounds that we had similar studies for the mysid and copepod, 

### 13 it is a little bit mixed where for the DCP, the copepod was a 

### 14 factor to more sensitive. For atrazine, the copepod did have 

### 15 significant effects, whereas the mysid we did not see the 

### 16 significance at the concentrations testing. 

### 17 For lindane, you know there -- or is the 

### 18 magnitude more sensitive for the mysid in this case. And for 

### 19 vinclozolin, an anti-androgen, the copepod mysid are of really 

### 20 equivalent sensitivities. 

### 21 So trying to summarize the results is that 

### 22 laboratories were generally able to complete both the mysid and 

### 23 copepod. The inter-laboratory, lowest observed effect 

### 24 concentrations were comparable for some but not all endpoints in 

### 25 terms of the results individually for mysids and individually 

### 26 for copepods.

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1 The copepod exhibited substantial technical 

### 2 difficulties, which required some additional time and effort to 

### 3 overcome. In conversations with the laboratory personnel, the 

### 4 amount of effort is fairly substantial for those individuals. 

### 5 So the context that we have a logistical improvement is 

### 6 questioned in that sense. 

### 7 So based on limitations with the copepod, that 

### 8 leads us to somewhat prefer the mysids since it is an extension 

### 9 of an existing life-cycle test, and that commercial laboratories 

### 10 are already familiar with the method and so they would be able 

### 11 to apply that. 

### 12 So with that, that is the conclusion of the 

### 13 presentation. So if there are some clarifying questions, I am 

### 14 happy to respond. 

### 15 **DR. MARTHA SANDY:**Thank you very much for 

### 16 that presentation. And we will go with Dr. Christman first. 

### 17 **DR. MARY CHRISTMAN:**Quick question. You 

### 18 mentioned blocking but I never saw a definition of what a block 

### 19 was in the design. And it could be I was just blanking out at 

### 20 the time, I apologize, but I am having a hard time understand 

### 21 what was your block. 

### 22 **DR. LESLIE TOUART:**In the blocking, this was 

### 23 more considered in terms of -- because this was in relation to 

### 24 the mysid test and it is in the context that, in the laboratory 

### 25 setup where the individual controls or experimental units are 

### 26 placed, they may be in different locations, so that your

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1 blocking would be associated with where they occur. 

### 2 There is a context that within each replicate 

### 3 chamber, meaning tank, you have a reproductive chamber where you 

### 4 have got individually paired mysids in context. 

### 5 **DR. MARY CHRISTMAN:**Right, you add those two 

### 6 baskets. 

### 7 **DR. LESLIE TOUART:**And then you have got your 

### 8 growth chambers that are utilized. And so if one is selecting 

### 9 individuals in a purely random form you could have individuals 

### 10 that may be subject to other, I guess you would say experimental 

### 11 influences that would not be seen by others. So if you are 

### 12 blocking you are trying to identify some of those. But I think 

### 13 what we found out that any blocking schemes that we used really 

### 14 did not provide any advantage to what was actually seen. 

### 15 **DR. MARY CHRISTMAN:**So let me repeat back to 

### 16 you if I understand this correctly. What you are saying then is 

### 17 that, within the tank -- because you had two blocks. You had 

### 18 the reproductive and then you had the growth, I guess it was, 

### 19 the other part? 

### 20 **DR. LESLIE TOUART:**Growth, where as the 

### 21 length measure from the individuals would be sacrificed at 

### 22 different times. 

### 23 **DR. MARY CHRISTMAN:**Right. So you had them 

### 24 separated out, although I supposed there was some movement. So 

### 25 what you are saying is that if you identified individuals as 

### 26 belonging to a basket as opposed to a tank, then you found that

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1 that was too noisy. Because it repeatedly says that it just 

### 2 made the variance too high. 

### 3 **DR. LESLIE TOUART:**Correct. In context, as 

### 4 far as I understand, that is correct in terms of how that was 

### 5 employed and how that increased the variance that was seen. So 

### 6 I think I am thinking clearly right now. I think that is 

### 7 correct from your observation. 

### 8 **DR. MARY CHRISTMAN:**So the blocking then 

### 9 refers to baskets within a tank? 

### 10 **DR. LESLIE TOUART:**Correct. I can check that 

### 11 to verify to make sure. 

### 12 **DR. MARY CHRISTMAN:**But like I said, I could 

### 13 not find a good definition that is why I asked. 

### 14 **DR. LESLIE TOUART:**I think it is explained a 

### 15 little bit more clearly within the ISR itself. 

### 16 **DR. MARY CHRISTMAN:**I did a "find" and -- 

### 17 **DR. LESLIE TOUART:**Okay. I will check on 

### 18 that for you and provide further clarification. 

### 19 **DR. MARY CHRISTMAN:**Okay. Thank you. 

### 20 **DR. MARTHA SANDY:**Thank you. Dr. Klaine? 

### 21 **DR. STEPHEN KLAINE:**Could we go back to slide 

### 22 12? It is always good to start at the beginning. 

### 23 And sorry for this, but I am trying to get 

### 24 back at where we started today, which was building this house. 

### 25 And this is Mary's slide actually. And so there is a connecting 

### 26 between Tier 1 and Tier 2 tests. My understanding is, if I

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1 interpreted what you said and read the slide correctly that when 

### 2 you do Tier 1 testing, you are going to get some indication as 

### 3 to prioritizing Tier 2 testing. 

### 4 And I can see a link between Tier 1 testing 

### 5 and Tier 2 testing for the other Tier 2 tests, but I am not 

### 6 finding a link for this one. And I just thought do you have a 

### 7 link or an idea of what data you would get in Tier 1 that would 

### 8 cause you to pull up the mysid test in Tier 2? 

### 9 **DR. LESLIE TOUART:**Let me try to explain. 

### 10 Again, this goes back to the EDSTAC recommendations in terms of 

### 11 consideration that invertebrates make up 98 percent plus of the 

### 12 animal (inaudible). And the consideration that if we have a 

### 13 chemical that is demonstrated to be endocrine active in 

### 14 vertebrate systems because we are focused on estrogens, 

### 15 androgens and thyroids, the concern is that, okay, if it is 

### 16 active there and we want to do a comprehensive risk assessment, 

### 17 well is there a likelihood that that might influence an 

### 18 invertebrate? 

### 19 And there are some suggestions. I mean it 

### 20 extends in context but there are some studies in the literature 

### 21 that demonstrate that there are activities that do cross over, 

### 22 you know, where you have materials that, even though it may be 

### 23 active on, say, estrogen pathway, it may influence something in 

### 24 an ecdysteroid pathway in the compound. How it does that, we do 

### 25 not fully understand in context, but it has that potential. 

### 26 Now when you look at steroidogenesis

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1 inhibitor, then clearly that might inhibit some steroidogenesis 

### 2 process in terms of the ecdysteroid. There are some similar 

### 3 pathways that are involved in those syntheses. But the context 

### 4 is that it potentially could have some effect. 

### 5 So if we see an activity, then the question is 

### 6 well what does it do to an invertebrate that may be sensitive? 

### 7 And so that is the rationale, I guess, in terms of why that 

### 8 might then trigger the Tier 2 tests, since invertebrates would 

### 9 be exposed, and the concern is, well what is that consequence? 

### 10 And if you remember the side for the mysids in 

### 11 terms of evaluating some of these compounds, you see the 

### 12 sensitivity of these as yeah, these concentrations are fairly 

### 13 low and lower than what we might see in fishes or birds or 

### 14 something. And so the invertebrate could be a driver in terms 

### 15 of a risk assessment. 

### 16 Now, trying to understand what is going on, I 

### 17 do not think is the intent because the estrogen/androgen thyroid 

### 18 pathways at least in crustacean are not so prevalent. For other 

### 19 invertebrates there are suggestions that there might actually be 

### 20 some connection, but in crustaceans that is not the case. But 

### 21 there is potential cross-talk that exist, and so that 

### 22 determination would likely be made if we anticipate that there 

### 23 is likely environmental exposure and concern what that material 

### 24 might pose to invertebrates. 

### 25 **DR. STEPHEN KLAINE:**Okay. And I certainly 

### 26 agree with everything you said, in terms of the sensitivity and

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1 the need for some invertebrate testing. I guess in my mind, I 

### 2 was trying to figure out just where this test would fit, Tier 1 

### 3 or Tier 2? So in your mind, if I am interpreting what you have 

### 4 said, is that you expect to call up the invertebrate test every 

### 5 time you see a positive in the Tier 1 test; is that correct? 

### 6 **DR. LESLIE TOUART:**Well I mean that decision 

### 7 has not been made. I mean we are looking at more of other 

### 8 information that draws into those types of conclusions in terms 

### 9 of how one may decide what test within Tier 2 are actually 

### 10 selected. 

### 11 There are some considerations. I mean if you 

### 12 go back to EDSTAC recommendations, I mean they had kind of a 

### 13 default concept that, everything else being equal, you would ask 

### 14 for all of your Tier 2 tests if you have positive in Tier 1. 

### 15 But they also said that you could consider 

### 16 other targeted methodologies based on exposure, based on some 

### 17 other considerations, other information that you might have 

### 18 available to you. And so we were working through exactly what 

### 19 those other criteria might be in terms of how it can make that 

### 20 particular determination. 

### 21 But I think the context is that when you have 

### 22 identified endocrine potential, the next question is, well what 

### 23 is the adverse consequence. And if you have other pathways to 

### 24 try to determine what that is, then you can use those. If you 

### 25 do not, then the question is well how do I evaluate that in 

### 26 terms of the Tier 2 tests which were developed to try to address

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1 the adverse effects that may occur. 

### 2 **DR. STEPHEN KLAINE:**Okay. 

### 3 **MS. MARY MANIBUSAN:**So I just want to maybe 

### 4 shorten Dr. Touart's response into maybe a couple of points. 

### 5 What I am hearing is the assumption that, if I have a Tier 1 rat 

### 6 positive, like a pubertal positive, then that would correlate to 

### 7 a Tier 2 and I would call up the rat 2-gen or the extended 

### 8 1-gen. And similarly, if I see the fish or the frog lighting up 

### 9 in Tier 1, I would ask for the corollary Tier 2. 

### 10 And this slide, although I have probably 

### 11 failed in the attempt, was meant to clarify the distinction 

### 12 between Tier 1 and Tier 2 in that they are answering very 

### 13 different questions, and they are allowing for different levels 

### 14 of uncertainty. 

### 15 So the Tier 1 is meant to only answer the 

### 16 question, again, whether a chemical has a potential to interact 

### 17 with E, A or T pathways. And pathways would start from an AOP 

### 18 concept of having a molecular initiating event or early key 

### 19 event like ER-binding, ERTA, uterotropic lighting up and then 

### 20 you run into the in vivo cells of the pubertals, the fish and 

### 21 the fish study. So that is a pathway analysis. 

### 22 And we are not looking at Tier 1 in terms of 

### 23 species-specific types of interaction, we are looking at them 

### 24 altogether and just answering that blunt question; yes, no, does 

### 25 it have a potential, not whether it is yes only in rat or yes 

### 26 only in fish or whatever. You see what I am saying?

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1 And once you have answered that question that 

### 2 it does have the potential to interact, then you make the 

### 3 decision, "Okay. So now do I need Tier 2 testing? And if I do, 

### 4 which ones would they include?" And as Dr. Touart indicated, 

### 5 there are different sets of conditions or criteria for making 

### 6 that decision. One could be that we ask for all five. That is 

### 7 a default that EDSTAC allow for. 

### 8 The other condition is we ask, well what is a 

### 9 use patterns? What is exposure pattern? Is it 

### 10 residential-only, for example, where the only concern about the 

### 11 human health risk assessment, and therefore we naturally call in 

### 12 the two-generation or extended one-generation, or does it have 

### 13 an environmental exposure pathway? And there, it is split up 

### 14 across the ecological test in answering the question, well which 

### 15 species do I need to call up to again answer the question that 

### 16 we are posing in the risk assessment framework. 

### 17 So again, Tier 1 is a screen. We are allowing 

### 18 for a false positive, so it is not directly that, oh you see 

### 19 something lighting up in Tier 1, therefore I must see something 

### 20 in Tier 2. That is not the question, right? So Tier 1 we're 

### 21 allowing for some chemicals to fall through the sieve -- if you 

### 22 think about this as a filter because we want to be conservative, 

### 23 it is meant to be a screen. It is not meant to have that higher 

### 24 level of statistical power to get at the questions that Tier 2 

### 25 was intended to answer. So I hope that clarifies that. I do 

### 26 not know if I have muddied it a little bit more.

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1 **DR. STEPHEN KLAINE:**That does; thanks. 

### 2 **DR. GEOFFREY SCOTT:**Thanks Les, for that 

### 3 great presentation. 

### 4 On slide 173, which is the one where you are 

### 5 looking at the coefficient of variance in the mysid metrics. 

### 6 And I was struck by your comment where we were looking at over 

### 7 20 percent. In many of those instances, I noticed it may be the 

### 8 duration of exposure endpoints in some instances. Sometimes it 

### 9 is not. 

### 10 But in going through that as you look at those 

### 11 ones that are in red, you know, if you were to go back and look 

### 12 at the variation, say, within 10 to 20 percent of that 

### 13 coefficient of variation, I assume like you said, probably some 

### 14 of those would go away, and that is actually pretty good 

### 15 concordance; just your comment to make sure I am understanding 

### 16 what you are saying. 

### 17 **DR. LESLIE TOUART:**I think that is correct. 

### 18 And the other point I was trying to make -- and again, this 

### 19 slide is not easily read. 

### 20 In context, for many of the endpoints, also, 

### 21 even though they are greater in 20 percent, they are not that 

### 22 much greater so they are still kind of in the same ballpark. 

### 23 And how much that differs, and given the small number of tests 

### 24 that have been utilized, I am sure those can change. 

### 25 But the main context here was just to indicate 

### 26 that, yes, we have evaluated sort of the variety of endpoints

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1 that we have coefficient of variations, and they are not so bad 

### 2 in terms of being able to determine what may be going on. 

### 3 For a couple of these, those CVs might be 

### 4 fairly large that they would cause some difficulty in trying to 

### 5 interpret, but some of that might be cleared up with more 

### 6 experience or understanding. But for the key endpoints that we 

### 7 are concerned about with reproduction and growth measures, I 

### 8 believe that they are satisfactory. 

### 9 I would say though that something like dry 

### 10 weight -- and we have some very high CVs for dry weight -- that 

### 11 is a difficult thing to measure in these individuals because 

### 12 they are so small. And usually, especially for the day 0s and 

### 13 day 7s, you are pooling new individuals. 

### 14 The context of whether dry weight gives you 

### 15 anymore information, because length is an easier measure for 

### 16 these critters, so the context is that perhaps length might be 

### 17 more appropriate to use then you could drop the dry weight 

### 18 measure, in context. 

### 19 **DR. GEOFFREY SCOTT:**Yes. That is kind of 

### 20 interesting because you would think weight is a more -- you 

### 21 know. If you take a pair of calipers out or however you are 

### 22 doing it, you would think it is a more consistent measurement 

### 23 with a metric, a balance, to have a balance or something as you 

### 24 do this. But that is what the data says. That surprised me. 

### 25 In slide 163 you mentioned in the mysid assay 

### 26 that one lab was excluded, and I was just curious as to which of

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1 those metrics on 173 were not met. I was just curious as to 

### 2 what led to that exclusion. 

### 3 **DR. LESLIE TOUART:**I am trying to recall. I 

### 4 think it primarily was control reproduction that was affected 

### 5 because the laboratory was having some difficulty in being able 

### 6 to get sufficient numbers produced. And this was one of the 

### 7 lessons learned in terms of being able to modify, because they 

### 8 were using just a straight brine shrimp diet and they were not 

### 9 fortifying that with the additional enrichment, you know, to add 

### 10 the fatty acid, what the other labs had keyed onto to improve 

### 11 their production of numbers. 

### 12 And so that laboratory was dropped here for 

### 13 the lindane, but we utilized them again in the octylphenol and 

### 14 they had sufficient performance there so that the changes that 

### 15 they made were sufficient so that they could actually complete 

### 16 the test correctly as well. 

### 17 **DR. GEOFFREY SCOTT:**Slide 174, you mentioned 

### 18 high coefficient of variance in dose levels for the mysid assay 

### 19 as limitation. You also mentioned that in the copepod assay as 

### 20 a rationale for problems with it. So I was trying to understand 

### 21 the magnitude of those two statements, given that it peers in 

### 22 some instances, both assays has problems delivering dose, so if 

### 23 you could just clarify that. 

### 24 **DR. LESLIE TOUART:**Yes. In this particular 

### 25 side, the high CVs is in the actual maintaining concentrations 

### 26 to ensure that they were consistent. Typically, we would like

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1 to see exposure concentration with CVs of less than 20 percent 

### 2 to be able to have high confidence in the effect. 

### 3 On the copepods, I am not sure if I 

### 4 understood. 

### 5 **DR. GEOFFREY SCOTT:**Well in your slide, I 

### 6 forget which one it was. 

### 7 **DR. LESLIE TOUART:**Because I do not think for 

### 8 the copepod it was CVs in exposure. It was more CVs in terms of 

### 9 -- 

### 10 **DR. GEOFFREY SCOTT:**No, it was not CVs, it 

### 11 was chemical exposure less consistent from the static; just 

### 12 trying to clarify that. 

### 13 **DR. LESLIE TOUART:**Again, I think for the 

### 14 copepod, remember these are done in these microwell plates, and 

### 15 so you are adding material in those and you really do not get 

### 16 good verification of what the actual exposure concentration was 

### 17 because the volumes are too small. You are usually doing it by 

### 18 a relative sense, in terms of the media that you have prepared 

### 19 to add to those, but it declines over time. I mean it is being 

### 20 replaced every three days or so, but the context is that the 

### 21 actual exposure is a little bit more different. 

### 22 Typically it is just going to be expressed in 

### 23 terms of a nominal exposure and that may be difficult for a 

### 24 compound. And the difficulty that we saw with the copepod was 

### 25 for the vinclozolin as an example that has a fairly short 

### 26 hydrolytic half-life understanding, well what did they actually

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1 see in terms of exposure was question. 

### 2 But in the bottom line when you look at the 

### 3 actual results that were seen in terms of comparing the LOEC of 

### 4 the copepod to the mysid, they were close. It may not have 

### 5 mattered that much in context, but in our ability to project 

### 6 effects and things, it is difficult from a risk assessment 

### 7 standpoint if you have uncertainty in your exposure metric. 

### 8 **DR. MARTHA SANDY:**And Dr. Scott, I am sorry. 

### 9 Given the late hour, I would like to suggest that we stop here 

### 10 today and we pick up again the morning, all right? So we will 

### 11 be starting at 9 a.m. tomorrow morning. 

### 12 I would really like to thank all the 

### 13 presenters from EPA for hanging in there, and all the co-authors 

### 14 that are in the audience. I would also like to thank all the 

### 15 Panel members for sticking with these presentations and being so 

### 16 diligent in their questions. Dr. Matten has something to say 

### 17 before we adjourn. 

### 18 **DR. SHARLENE MATTEN:**I just want to meet with 

### 19 the Panel members in the back room for about five minutes just 

### 20 to get a game plan for everyone. 

### 21 And also, I sent Mary Manibusan the questions 

### 22 from the statistical duo/trio via email so they can -- well Ken 

### 23 Portier says he is still going to check his email. 

### 24 So the expectation is you will write 

### 25 something, say something. And I will put those questions, since 

### 26 it is formally written, in the public docket for the public and

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1 today's presentations. 

### 2 And with that, if anyone who is still here has 

### 3 not signed up for public comments, just think about it and you 

### 4 can make more public comments tomorrow or tell me tomorrow. And 

### 5 with that, we will be adjourned for today and we will start 

### 6 again at 9. Thank you very, very much. 

### 7 (Whereupon the meeting was adjourned for the day) 

### 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. SHARLENE MATTEN:**Good morning. My name 

### 2 is Sharlene Matten. I'm the designated federal official for 

### 3 this FIFRA Scientific Advisory Panel meeting. This is the 

### 4 second day of the Scientific Advisory Panel meeting to review 

### 5 and consider the scientific issues associated with the 

### 6 environmental Endocrine Disruptor Screening Program's proposed 

### 7 Tier 2 ecotoxicity tests. It's my job to maintain the 

### 8 regulatory part of the process here under the Federal Advisory 

### 9 Committee Act, to make sure all the ethics requirements are met. 

### 10 I have already loaded into our Docket 

### 11 Management System for quality assurance, quality control the EPA 

### 12 presentations made yesterday, the panel's question that was 

### 13 given to EPA, the public comment that's going to be read into 

### 14 the record that we have. And after that, we will have the 

### 15 public comments from the group that's representing the Endocrine 

### 16 Policy Forum. 

### 17 If there's anyone here who hasn't registered 

### 18 with myself or the EPA Scientific Advisory Panel staff for 

### 19 public comments, please see Laura Bailey and the FIFRA 

### 20 Scientific Advisory Panel staff at this table, over to my right. 

### 21 With that, I'm going to turn the microphone 

### 22 over to Dr. Martha Sandy, and the panel will go around and 

### 23 introduce themselves. Thank you. 

### 24 **DR. MARTHA SANDY:**Good morning. I am Martha 

### 25 Sandy, and I'm chairing this session. I'd like to welcome 

### 26 everyone back to this meeting. And we're just going to go

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1 around quickly and introduce ourselves. I'm Martha Sandy. I'm 

### 2 a toxicologist, and I'm with the Office of Environmental Health 

### 3 Hazard Assessment of California EPA. 

### 4 **DR. BARRY DELCLOS:**Barry Delclos from the 

### 5 FDA's National Center for Toxicological Research. 

### 6 **DR. JAMES McMANAMAN:**Jim McManaman, 

### 7 University of Colorado. 

### 8 **DR. DANIEL SCHLENK:**Dan Schlenk, University 

### 9 of California, Riverside. 

### 10 **DR. STEPHEN KLAINE:**Steve Klaine, Clemson 

### 11 University. 

### 12 **DR. MARION EHRICH:**Marion Ehrich, Virginia 

### 13 Tech. 

### 14 **DR. GEOFFREY SCOTT:**Geoff Scott, NOAA. 

### 15 **DR. VICKI BLAZER:**Vicki Blazer, U.S. 

### 16 Geological Survey. 

### 17 **DR. REYNALDO PATINO:**Reynaldo Patino, U.S. 

### 18 Geological Survey, Texas Cooperative Fish & Wildlife Research 

### 19 Unit. 

### 20 **DR. CATHERINE PROPPER:**Cathy Propper, 

### 21 Northern Arizona University. 

### 22 **DR. EDWARD PERKINS:**Ed Perkins, U.S. Army 

### 23 Corps of Engineers. 

### 24 **DR. ROBERT DENVER:**Bob Denver, University of 

### 25 Michigan. 

### 26 **DR. JOHN McCARTY:**John McCarty, University of

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1 Nebraska, Omaha. 

### 2 **DR. DALLAS JOHNSON:**Dallas Johnson, Professor 

### 3 Emeritus, Kansas State University. 

### 4 **DR. COLIN SCANES:**Colin Scanes, University of 

### 5 Wisconsin, Milwaukee. 

### 6 **DR. SETH KULLMAN:**Seth Kullman, North 

### 7 Carolina State University. 

### 8 **DR. SHEA TUBERTY:**Shea Tuberty, Appalachian 

### 9 State University. 

### 10 **DR. JENNIFER FREEMAN:**Jennifer Freeman, 

### 11 Purdue University. 

### 12 **DR. MARY CHRISTMAN:**Mary Christman, MCC 

### 13 Statistical Consulting. 

### 14 **DR. GEORGE BENTLEY:**George Bentley, U of C, 

### 15 Berkeley. 

### 16 **DR. MARTHA SANDY:**Okay. I would like to 

### 17 remind all panel members who are -- we need to speak directly 

### 18 into the microphone so that the webcast and audio recording is 

### 19 clear. 

### 20 So as we left off yesterday evening, we had 

### 21 just heard the last presentation from EPA staff on the mysid and 

### 22 copepod assays. And we hadn't quite finished with some 

### 23 questions. And I think we were in the middle of Dr. Scott's 

### 24 question. So I'll turn to him. 

### 25 **DR. GEOFFREY SCOTT:**Thank you, Dr. Sandy. 

### 26 Good morning. Geoff Scott, NOAA. Good morning, Les. Just a

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1 couple more questions, and again, thank all of you all for the 

### 2 great presentations yesterday. 

### 3 I just had a couple of last final questions, 

### 4 and that really gets to the addition of the additional lifecycle 

### 5 to the conventional mysid assay. And I say this not lightly 

### 6 because I do think if you look at the use of the conventional 

### 7 mysid assay, it has already been used to regulate known 

### 8 endocrine disruptors, IGRs, things such as that. 

### 9 So one of the questions that I have is in 

### 10 Rodney's presentation he had a slide, I think it was like 74 -- 

### 11 you don't need to put it up. All it was doing was comparing his 

### 12 F0, F1, which is more sensitive, the number of tests and the 

### 13 compounds. 

### 14 And I guess as we were looking at your data, 

### 15 it seems to me a summary like that would be helpful because it 

### 16 appeared to us that only in two or three instances did we 

### 17 actually see the F -- I'm particularly thinking of Slide 172. 

### 18 There only seemed to be a couple instances where that additional 

### 19 lifecycle provided additional protection. 

### 20 And I'm going to kind of go back and draw an 

### 21 analogy, Les, to lifecycle testing. We can't predict with -- 

### 22 whether it's grass shrimp or mysids -- whether larvae or 

### 23 post-larvae stages are most sensitive. Some compounds it's one, 

### 24 some it's the other. 

### 25 And I kind of used that analogy of not being 

### 26 able to predict that. It's sort of like looking at this, an

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1 analogous situation here to the multigen testing because it 

### 2 appears just like we can't predict whether larvae or post-larvae 

### 3 are most sensitive, I mean the good thing about the assays is 

### 4 you have it. You're embracing all those life stages and in some 

### 5 instances multiple times. 

### 6 So I guess just make sure we understand the 

### 7 data and get your comments on that for clarification. 

### 8 **DR. LESLIE TOUART:**Yeah. Thanks, yeah, for 

### 9 the observation. I think the context with the two-generation, 

### 10 the concern was as presented, you know, there was concern how 

### 11 may this play out into another generation, and how valuable 

### 12 would that be in terms of population modeling or subsequent 

### 13 analysis of that sort. And it's all well and good, but the 

### 14 question still comes down is well, what is that value of the 

### 15 additional time and effort into the next generation? And so 

### 16 that was one of the efforts to try to evaluate, you know, the 

### 17 performance of this test. 

### 18 And in the mysid case, it's I think a good 

### 19 case in a sense that the first generation, you know, the F0, is 

### 20 really identical to the existing lifecycle test. So you 

### 21 contained that or incorporated that in the context of the 

### 22 two-generation to say okay, well what other information do I 

### 23 glean from that next generation? And as you point out, and we 

### 24 did in this Slide 172, there were a couple instances where, you 

### 25 know, there was a little bit more sensitivity, I guess, in that 

### 26 next generation.

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1 Whether that's a statistical anomaly, you 

### 2 know, based on the results in terms of okay, this one was 

### 3 significant, that maybe the other one was close but not quite, 

### 4 if you had redone it. But the context is that this is, you 

### 5 know, likely to happen, as you've indicated, in terms of 

### 6 particular life stages might be sensitive sometimes, maybe not 

### 7 the other time. 

### 8 So the question comes in terms of when is it 

### 9 really a question? I mean we could live with the existing mysid 

### 10 lifecycle test because that's already on the books, I mean it's 

### 11 standard. But the question comes is well, would this carry 

### 12 forth into another generation? And in that context, you know, 

### 13 there may be occasions when it would be lower. 

### 14 Now what are the kinds of compounds that would 

### 15 likely do that? Well, potentially they could be a material that 

### 16 is endocrine active. And if it's a vertebrate active would that 

### 17 do the same? The one thing we did notice for the mysids were 

### 18 that the sensitivity of mysids for these vertebrate active 

### 19 compounds was generally, you know, more sensitive than the 

### 20 vertebrates themselves, maybe not for the same reason, but at 

### 21 least there's indication. 

### 22 So I think there's utility in that sense, but 

### 23 in terms of ultimate value I think that's where we would seek 

### 24 additional guidance from the panel, you know, given that if 

### 25 you've only got a few instances where you've got some 

### 26 improvement, you know, where your NOEC or your level of concern

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1 might change slightly; is that sufficient to, you know, for 

### 2 everybody to go into one of these tests? 

### 3 When you test just a small number of 

### 4 compounds, I mean with mysids we have, I don't know, maybe a 

### 5 dozen, but the, you know, a few of them were more sensitive than 

### 6 the others in that next generation. So that's a question as to, 

### 7 you know, whether we would want to go forward. 

### 8 One thing I would add, though, is that in the 

### 9 development and optimization for the two-gen, we were able to 

### 10 refine certain aspects that would improve the existing lifecycle 

### 11 test as it goes in terms of being more consistent in proving the 

### 12 production of offspring so that you have a little bit more 

### 13 ability to detect where we are, you know, setting performance 

### 14 criteria of at least eight mysids through the two broods. 

### 15 The other context is that one thing we notice 

### 16 is that we really want to focus the test more on the actual 

### 17 biological performance, not on a time dependent like the 

### 18 existing lifecycle test is 28 days. And it's stopped whether 

### 19 you had two broods or not. But if you key it into a brood 

### 20 production and brood duration as endpoints, then that gives you 

### 21 another indication of all the effects that may be occurring. So 

### 22 there would be improvements in that context. 

### 23 But I think in our view, there would be some 

### 24 value in that next generation. The question is, is that value 

### 25 sufficient to warrant this as an additional, you know, guideline 

### 26 that could be utilized in terms of Tier 2 for endocrine-active

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1 materials. 

### 2 **DR. GEOFFREY SCOTT:**Thank you for that. And 

### 3 I did not realize the addition -- I'm glad you clarified that 

### 4 improvement in -- that can be also put forward to the, you know, 

### 5 the conventional test through this process. And that's really 

### 6 important. 

### 7 I have just two more questions. When you made 

### 8 the slide at the end with the comparison between the copepod 

### 9 assay and the mysid assay at the end, were you surprised at how 

### 10 similar some of the results were, and in some instances, there 

### 11 were differences, but were you surprised that both assays for 

### 12 those compounds you had listed were as close as they were, I 

### 13 guess is what I'm saying, going in? Just your perspective on 

### 14 that. 

### 15 **DR. LESLIE TOUART:**I think I was a little 

### 16 surprised only in the sense that we had done that little bit of 

### 17 literature review because there are a lot of published studies 

### 18 done on the copepod, the Amphiascus. Tom Chandler at South 

### 19 Carolina has done a lot of work for a lot of different 

### 20 compounds. And for many of those we have mysid data as well 

### 21 that have been done based on just mysid lifecycle type 

### 22 information. And from those comparisons we've seen that mysids 

### 23 tend to be more sensitive. 

### 24 But as with any species comparison, sometimes 

### 25 one's more than the other. But for the few compounds that we 

### 26 had matched with the mysid and copepod in our exercise, the

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1 results were as we saw. Really only I think lindane you had a 

### 2 dramatic difference. The others, you know, were in the same 

### 3 ballpark. 

### 4 And that was a little surprising in one 

### 5 context. The lindane wasn't so surprising because mysids 

### 6 typically have been a lot more on the more sensitive side. 

### 7 The other thing that I think, for me, that was 

### 8 surprising about the mysids was through, well, two generations 

### 9 where the copepod was still within its lifecycle component and 

### 10 to be in the same ballpark for the compounds that we could match 

### 11 up, you know, said a lot about the copepod itself. 

### 12 But again, our rationale for saying, well, 

### 13 let's just stick with the mysid was that the thought that the 

### 14 copepod could be an easier, quicker, you know, cheaper type test 

### 15 to proceed with kind of fell apart when we went into inter labs 

### 16 and found the difficulties that laboratories had trying to apply 

### 17 the method. Even though it would conceptually seem simpler, it 

### 18 had its own difficulties and problems that would make it a 

### 19 little bit more cumbersome to try to deal with in terms of 

### 20 routine type testing. 

### 21 **DR. GEOFFREY SCOTT:**Slide 171, if you could 

### 22 turn to that just for clarification, Les; the asterisk there 

### 23 referred to differences with what? 

### 24 **DR. LESLIE TOUART:**In this case, these are 

### 25 significant difference, but in the statistics that were done on 

### 26 this test, this was to the vehicle control. So they would

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1 compare it -- 

### 2 **DR. GEOFFREY SCOTT:**To the vehicle -- within 

### 3 each generation? 

### 4 **DR. LESLIE TOUART:**Right. 

### 5 **DR. GEOFFREY SCOTT:**Okay. 

### 6 **DR. LESLIE TOUART:**The treatment effect, you 

### 7 know, so that in the F0s it would be the seven offspring, you 

### 8 know, per second brood compared to the vehicle control of 9.87. 

### 9 **DR. GEOFFREY SCOTT:**So it's not comparing 

### 10 among generations? 

### 11 **DR. LESLIE TOUART:**In this slide, no. This 

### 12 is just looking for where we had significant difference, and in 

### 13 this case, the 33.7 was significant for the F0s and for the 

### 14 others, the concentrations down to 2.15 with the 4.19, not 

### 15 statistically different. 

### 16 **DR. GEOFFREY SCOTT:**Okay. One last question 

### 17 and it will be the last one and same question I asked Rodney. 

### 18 Since we're trying to look at international harmonization on 

### 19 these criteria and some of the information you provided with 

### 20 some of the OECD parallel trials, what's the current status in 

### 21 terms of OECD in terms of these assays? Could you kind of give 

### 22 us an update on where they are so that -- and have you informed 

### 23 them of your, you know, just like Rodney I asked are you getting 

### 24 comments back on moving forward with just the mysid? 

### 25 **DR. LESLIE TOUART:**And thanks for that 

### 26 question. As it happens, the OECD invertebrate expert group did

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1 have a meeting this past week, last Monday, and I 

### 2 teleconferenced in to give them a little bit of a briefing where 

### 3 I showed them basically the same slides that I've shown the 

### 4 panel here today to give them some indication or update in terms 

### 5 of how the mysid and copepod have been developed. 

### 6 And I informed them that this panel will be 

### 7 providing comments, and so they're interested in knowing the 

### 8 outcomes of that within the OECD process where expert groups 

### 9 provide advice and information in terms of test guideline 

### 10 development. 

### 11 It then goes to their -- what they have now 

### 12 with validation management groups, and there's one for 

### 13 ecotoxicity testing, and the validation management group kind of 

### 14 reviews the data that supports the test guidelines that have 

### 15 been, you know, I guess consulted through the expert groups. 

### 16 And, you know, then they would make a recommendation as to 

### 17 whether the guidelines should go forward to international 

### 18 acceptance where the national coordinators within the test 

### 19 guideline program across multiple nations would consider. 

### 20 And to be adopted by the OECD test guideline 

### 21 has to be unanimously approved. If there's one member country 

### 22 that's not accepting of a particular guideline then it doesn't 

### 23 move forward. 

### 24 In the development of the invertebrate assays 

### 25 for the copepod and the mysid, the copepod was one that was 

### 26 initially proposed by Sweden and has been going through the

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1 process as I kind of outlined in the historical perspective, and 

### 2 so that one has some interest. 

### 3 From the European standpoint, they are also 

### 4 interested in having an estuary test guideline. They currently 

### 5 don't have a method similar to the mysid to evaluate, so they 

### 6 would like to have that even, you know, regardless of endocrine 

### 7 activities or not. And so that was part of the original 

### 8 interest that the copepod had. But they're interested in seeing 

### 9 endocrine methodology as well. 

### 10 In their discussions, they're also concerned 

### 11 about invertebrate relevant endocrine disruption, not just 

### 12 vertebrate, but the context as they can understand and there is 

### 13 some interest. 

### 14 On the mysid side, I would say that there is 

### 15 not as much international interest on the mysid as has been 

### 16 shown on the copepod, but they're looking to see what are 

### 17 recommendations. If the U.S. ultimately adopts a particular 

### 18 guideline they would likely want to pursue it. They haven't 

### 19 pursued the mysid lifecycle to date, but that doesn't mean that 

### 20 it may be something that they would like to see. 

### 21 But the outcome of this meeting will have 

### 22 influence because that would get discussed at their validation 

### 23 management group meeting which is tentatively scheduled for this 

### 24 fall in the October timeframe. And at that meeting, they would 

### 25 then decide whether to advance the method into the test 

### 26 guideline, you know, to be adopted by OECD.

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1 **DR. GEOFFREY SCOTT:**Just a quick follow-up. 

### 2 In their validation, are they looking at those assays as a Tier 

### 3 1 or Tier 2, I mean relative to your schema? 

### 4 **DR. LESLIE TOUART:**With the OECD, they have a 

### 5 different framework. They have five levels within their 

### 6 endocrine testing scheme where a first level is really more 

### 7 computational in-vitro moving on through a level five which 

### 8 would be considered a definitive test. So the mysid tests as 

### 9 proposed and the copepod tests would fall within their level 

### 10 five, so this would be their definitive test. 

### 11 They don't consider Tiers in a concept, they 

### 12 feel you can enter and exit in any different point, so they have 

### 13 a little bit different context than the U.S. program, but we've 

### 14 got kind of a two-tiered approach. So for them, they built a 

### 15 toolbox of assays so one can pick and choose where within the 

### 16 framework one might want to apply a particular tool to make the 

### 17 determination. 

### 18 **DR. GEOFFREY SCOTT:**Well, thank you for that 

### 19 clarification because that wasn't clear in the OEC -- I'm not 

### 20 familiar with that whole process and that was really helpful to 

### 21 understanding. Thank you, Les. 

### 22 **DR. MARTHA SANDY:**I saw Dallas Johnson first. 

### 23 **DR. DALLAS JOHNSON:**Thank you. While we're 

### 24 on this particular slide, some of the slides that we've been 

### 25 presented have the standard error. This one looks like it's 

### 26 standard deviation, and I guess I'm curious as to which it

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1 really is in this case because I think if that were standard 

### 2 deviations there would be a lot more significant differences 

### 3 there than what are shown. So I wonder if this isn't standard 

### 4 errors that are on this slide as well. 

### 5 **DR. LESLIE TOUART:**It's reported as standard 

### 6 deviation, so my assumption is that's what it is. But I didn't 

### 7 do the stats for this. I would have to go back and review that 

### 8 particular study to see if perhaps it was just transcribed 

### 9 differently in terms of what came up here. 

### 10 **DR. DALLAS JOHNSON:**I guess the point is that 

### 11 if you were comparing two means controlled to one other mean, 

### 12 then the standard error of the difference would sort of be 

### 13 involved with sample size. So if the sample size were to go in 

### 14 as a denominator, which would make those standard errors, what 

### 15 are reported as standard deviations, they'd be a whole lot 

### 16 smaller because you're going to divide them by the number of 

### 17 samples or N, number of replicates, I guess, depending on what 

### 18 the sampling unit is. And so I'm just -- it might be worth 

### 19 checking on that. I just noted that when I saw that particular 

### 20 slide a little bit ago. 

### 21 **DR. MARTHA SANDY:**Dr. Tuberty. 

### 22 **DR. SHEA TUBERTY:**Les, in Slide 172, the next 

### 23 slide, if you come to that; looking through this list of which 

### 24 of the generations were more sensitive, 3 of the 11 times there, 

### 25 the F1 was more sensitive or protective than the F0. Are these 

### 26 based on the brood or any of the outcomes that were measured at

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1 about 20 different endpoints that were measured, the mysids? 

### 2 Was this just looking at one, any one of them that might have 

### 3 been more protective or was it the outcomes of the F1 brood 

### 4 reproduction? 

### 5 **DR. LESLIE TOUART:**For all these except the 

### 6 ones noted, it was on the most sensitive endpoint identified. 

### 7 And that could have been growth, it could have been first brood 

### 8 or inter-brood duration. 

### 9 For the ketoconazole and then I think for 

### 10 vinclozolin, we separate out those that were on the 

### 11 reproductive, so these would be offspring production, and so 

### 12 those were based on that particular endpoint. 

### 13 For the vinclozolin, what we did was looked at 

### 14 the reproduction endpoint but also considered the others because 

### 15 there was a big difference between the sensitivity of some of 

### 16 the other endpoints that were growth related or in that context. 

### 17 18 So in this sense, I'd have to go back to look 

### 19 at the specific endpoint that was responsive. But my guess is 

### 20 it would probably be like a 7-day length or a 14-day length on 

### 21 say the vinclozolin and for ketoconazole this would have been 

### 22 the second brood production because the slide previous to that 

### 23 showed the 2.15 as the lowest effect concentration. 

### 24 **DR. SHEA TUBERTY:**All right. So for 

### 25 clarity's sake, yesterday when we were looking through some of 

### 26 the other assays and the results and the decision-making

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1 process, there were more or less equal numbers of more 

### 2 protective in second generation than first. And in this case, 

### 3 like I said, 3 of the 11 were actually more protective going 

### 4 through the second brood all the way to the second brood of the 

### 5 F1 generation. 

### 6 Since two of them show that the F0 generation 

### 7 was actually more protective and all the rest of the seven or 

### 8 eight there were actually equal in both, it really seems like 

### 9 the -- going all the way to the second brood of the F1 

### 10 generation is, you know, using the thinking from yesterday it 

### 11 wouldn't have been the decision-making process to go forward 

### 12 with that. Does the EPA feel that in the big scheme of things 

### 13 that going all the way through the F1 generation is going to be 

### 14 more protective? 

### 15 **DR. LESLIE TOUART:**I think that's somewhat of 

### 16 a big question. But in terms of would that next generation be 

### 17 more protective, potentially in some cases that would be the 

### 18 case. 

### 19 The actual risk assessments and decisions that 

### 20 are made carry other, you know, considerations and things as 

### 21 well. We want to look at magnitude of a particular fact and 

### 22 numbers of endpoints and stuff that it would look like. 

### 23 But I think the context is that one could make 

### 24 an argument that it is more protective, but how much more 

### 25 protective, you know, might it be? 

### 26 Are you sufficiently protective if you could

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1 use just F0 information, you know, kinds of things, I think that 

### 2 is the question. We see that with these numbers how much of a 

### 3 difference in a risk assessment would the difference between say 

### 4 13 part per billion versus 32 part per billion. That would come 

### 5 into play depending up on where your exposures might be in terms 

### 6 of whether you would exceed a level of concern or not. So in 

### 7 some cases there could be a difference that could occur. 

### 8 In the case of like the octylphenol where we 

### 9 had a much more sensitive endpoint in the F0 where your F1 was 

### 10 higher is an indication that there had been some type of maybe 

### 11 compensation. Those individuals that survived to the F1 were 

### 12 more, you know, less -- or at least less susceptible in a 

### 13 context. So in that context, the next generation isn't giving 

### 14 you anything other than maybe confounding some information if 

### 15 you're adding that into a population model because it might give 

### 16 you a different sense of security. 

### 17 It's somewhat I think a value judgment in 

### 18 terms of what one would see. We are interested in the Panel's 

### 19 thinking. You know, my thinking and speaking personally is that 

### 20 yeah, there is some value. How much of a value, it's debatable. 

### 21 But I would be concerned that that question would still exist 

### 22 if I only have the one generation. What would happen if I can 

### 23 carry it on into the next generation, would that have lowered 

### 24 effects or not? Not knowing that, how much that weighs into 

### 25 decisions is a different consideration 

### 26 I wish there was an easier way to say or be

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1 able to predict when that next generation would be more 

### 2 sensitive. If that were a case, then you may be -- given the 

### 3 nature of the kinds of compounds that we were exploring, which 

### 4 were primarily vertebrate-active not invertebrate-active, the 

### 5 consideration that we actually did see some instances that were 

### 6 more sensitive in the next generation makes me think, well, how 

### 7 often does that need to take place before I would want to see 

### 8 that more universally? You know, 10 percent of the time, 20 

### 9 percent of the time, you know, 50 percent? How many times is 

### 10 that appropriate to try to look at? So that's the 

### 11 consideration. 

### 12 I think one could evaluate it in a couple of 

### 13 different ways, but I think that we have demonstrated there is 

### 14 some value. It is just does that value offset the additional 

### 15 time and cost and potential complications of maybe having to 

### 16 repeat the test if you fail in that next generation because of 

### 17 some happenstance in the laboratory? 

### 18 **DR. SHEA TUBERTY:**I appreciate you sharing 

### 19 your personal views on that. I feel the same. I think there 

### 20 seems to be some complication there from the loss of sensitivity 

### 21 or increased tolerance in the second generation which we 

### 22 wouldn't expect unless there was some culling out of the first 

### 23 generation. 

### 24 Another question I have is in looking at 

### 25 copepods versus mysids and the types of endpoints that were 

### 26 chosen to look for endocrine disruption specifically -- and this

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1 goes back to some of the comments that were being made yesterday 

### 2 that when endpoints seem to be more growth related, they were 

### 3 weighed less important for endocrine disruption. And certainly 

### 4 you've commented several times and many of the endpoints that 

### 5 were listed as the most sensitive for mysids was, in fact, 

### 6 growth. 

### 7 Now as an invertebrate zoologist and 

### 8 physiologist, I'll give you that molting processes controlled by 

### 9 ecdysteroids ultimately control growth. But the amount of 

### 10 growth between the molting steps, the ecdysis events, could, in 

### 11 fact, be controlled by many other things including just 

### 12 nutritive state and the amount of resources available. And 

### 13 we've talked in all the assays about the importance of good 

### 14 nutrition and food stocks during the assays. 

### 15 So back to my point, given that the copepod 

### 16 tests are actually looking at timing between molting events, 

### 17 which is a direct measure of the active effects of ecdysteroids, 

### 18 rather than in just growth per se; in mysids you're just looking 

### 19 at the final size of your animals or lengths of your animals, 

### 20 not the number of ecdysal events that have happened during that 

### 21 period. Would you say that the copepod assays are actually 

### 22 better at measuring specific ecdysteroid events or 

### 23 endocrine-mediated processes? 

### 24 **DR. LESLIE TOUART:**And that's an interesting 

### 25 question in context. I think the ecdysteroid effects would show 

### 26 up apically in the mysid. But the copepod would have a little

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1 bit more diagnostic capability of being able to link that. 

### 2 Just to go back in terms of the purpose for 

### 3 the mysid and/or copepod is to evaluate adverse consequence of 

### 4 vertebrate-active material. So even though it's an endocrine 

### 5 test, we are not so focused on the endocrine activities to be 

### 6 able to measure because the method does not have direct measures 

### 7 of those. These could be added in with extra complications, but 

### 8 they could be in terms of being more specific to molting stages 

### 9 and things like that which can be measured. 

### 10 But the context is that we're concerned about 

### 11 what are the adverse effects to the endpoints like growth or 

### 12 reproduction. 

### 13 For growth with mysids as other crustacea, a 

### 14 measure like length is going to go up more stepwise because with 

### 15 each molt the length if pretty well set until the next molt kind 

### 16 of thing. 

### 17 So that's an indication of, you know, how that 

### 18 individual has fared in that sense, but recognizing there can be 

### 19 other factors in strictly endocrine. When we look at a compound 

### 20 like DCP, you know, this inhibits oxidative phosphorylation in 

### 21 mitochondria. So the context is that yeah, this is likely 

### 22 something that would reduce energy, metabolism and uptake. 

### 23 So it's not likely directly an 

### 24 endocrine-active material, but we do see effects carry into 

### 25 another generation that are resulting from that. So the adverse 

### 26 effect is of importance even though there's no direct indication

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1 that that was disrupting the endocrine process of the 

### 2 individual. 

### 3 But I think the context again is that if one 

### 4 were interested in the actual endocrine mechanisms and being 

### 5 able to diagnose the disturbances of those endocrine processes, 

### 6 whether the ecdysteroids or molt-inhibiting hormones or others 

### 7 that would be present, one would have to add some additional 

### 8 endpoints to observe those and be confident of that. But if 

### 9 it's strictly apical concern, then the reproductive growth 

### 10 endpoints are appropriate. 

### 11 **DR. SHEA TUBERTY:**Thank you. Another point I 

### 12 wanted to make related to growth was on 168, Slide 168. You 

### 13 highlighted this data here and showed that in the inset dataset 

### 14 there, and I'm not sure if this was from lindane or octylphenol 

### 15 exposure. 

### 16 **DR. LESLIE TOUART:**It's lindane. 

### 17 **DR. SHEA TUBERTY:**Okay. So this is looking 

### 18 at dry weights of I guess adults in response to lindane exposure 

### 19 in the F0 generation. And with increased exposure you show 

### 20 significantly increased growth of the animals. How would EPA 

### 21 judge this as positive or negative in this adverse effects 

### 22 assessments? 

### 23 **DR. LESLIE TOUART:**And just for 

### 24 clarification, this is the Day 14, so these would be individuals 

### 25 that would be approaching their first brood release in a 

### 26 context. Mysids will typically begin releasing brood from about

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1 Day 14 through maybe 18 depending upon the particular strength. 

### 2 But in this case, as you see, there is a significant size 

### 3 increase. 

### 4 In combination since there were some other 

### 5 effects seen with inter-brood duration being increased and 

### 6 things like that that, these are linked in a context that the 

### 7 longer the individuals have not released brood, you know, you do 

### 8 have an effect on dry weight and things like that. So there are 

### 9 some indications. 

### 10 In terms of whether just by size alone, like 

### 11 weight alone, if that's increasing with concentration, that may 

### 12 not be perceived as adverse unless it starts to be reflected in 

### 13 other endpoints. A decrease in growth would be considered a 

### 14 little bit more adverse. But biological changes, as I'm sure 

### 15 you're aware, are changes if it's a statistically significant 

### 16 change there may be a biological significance to that change if 

### 17 it's different in terms there is energy input to grow and what 

### 18 is at the expense of that, so one would have to judge that. 

### 19 If it's an endpoint like this that stands 

### 20 alone, it would be difficult to make a regulatory judgment on 

### 21 that by itself and one would have to evaluate whether that's 

### 22 really a true response or a statistical anomaly. 

### 23 So when we see a dataset like that, that tends 

### 24 to indicate that that's more of a real response. And it would 

### 25 garner concern, and likely we would set a NOEC/LOEC around that 

### 26 particular endpoint, but it would be referenced as such so when

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1 it carries into a risk assessment one would be considering that 

### 2 aspect, that it would be a growth stimulant in a sense and 

### 3 whether that plays out or makes any other determinations would 

### 4 depend on other factors within risk assessment. 

### 5 **DR. SHEA TUBERTY:**Thanks, Les. And was 

### 6 there, in fact, concomitant decrease in reproduction in this 

### 7 group, or was there increased reproduction in this group with 

### 8 the increased growth? I only ask because in many -- there was a 

### 9 comment yesterday, somebody was saying that they didn't know of 

### 10 a clear link between size and fecundity. But in my experience, 

### 11 it's always been the largest females by far the largest 

### 12 fecundity, and I thought there was a direct relationship there. 

### 13 So is that true in the mysids a well? 

### 14 **DR. LESLIE TOUART:**I'm trying to move the 

### 15 slide back. But if you go back on Slide 163, there's a lindane 

### 16 comparison that was done where survival, weight, length, days to 

### 17 brood release and offspring production are included for the two 

### 18 laboratories, and you can see from that that the weight wasn't 

### 19 the more sensitive endpoint. Length was in this context. But 

### 20 there is connection to offspring production and others in that 

### 21 particular laboratory. 

### 22 **DR. MARTHA SANDY:**Dr. Scanes. 

### 23 **DR. COLIN SCANES:**Can we flip to Slide 171 

### 24 for a second? You said that the data with the stars and the 

### 25 highlight are significantly different from control. But you've 

### 26 got two controls, vehicle control and control. Which are they

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1 different from? And then I'll come back. 

### 2 **DR. LESLIE TOUART:**As I mentioned before, the 

### 3 comparisons for the treatments in this particular case was the 

### 4 treatment groups compared to the vehicle control. 

### 5 **DR. COLIN SCANES:**In which case, you seem to 

### 6 have quite a marked effect to the vehicle in the F1 generation. 

### 7 How do you explain that? 

### 8 **DR. LESLIE TOUART:**I think the context is 

### 9 there was not a significant difference between the dilution 

### 10 control and the solvent control, but in terms of differences, 

### 11 I'm not exactly, you know, clear in the sense that the vehicle 

### 12 seems to, you know, just numerically have greater root in the 

### 13 next generation. Why that is occurring would be speculative on 

### 14 my part. I don't know that that's always the case in terms of 

### 15 these particular studies. 

### 16 **DR. COLIN SCANES:**It does make you start 

### 17 wondering whether one shouldn't be doing the statistical 

### 18 comparison against both controls because if you've got a 30 

### 19 percent increase in the vehicle control compared to the control, 

### 20 that's not seen in the F0 generation. And that would, if you 

### 21 did it against both controls, my betting is the 2.15 would drop 

### 22 out. 

### 23 **DR. LESLIE TOUART:**That's possible. I think 

### 24 the context, and there are differing considerations in terms of 

### 25 how one is more appropriately to analyze, you know, studies in 

### 26 terms of comparing to vehicle control, if a vehicle's used,

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1 comparing to dilution water control, cooling controls in a 

### 2 context. 

### 3 You know, typically what we want to be able to 

### 4 do is to evaluate to ensure that there is no difference between 

### 5 the controls in terms of the vehicle versus control. If there's 

### 6 not a statistical difference, then we would consider that the 

### 7 vehicle likely didn't have an effect. 

### 8 In cases where the vehicle control is 

### 9 different from your dilution water, that raises the question of 

### 10 the vehicle then imposing some effects that you are aware of, 

### 11 and so how you deal with that, whether you want to invalidate a 

### 12 study and repeat it or whether you would accept it somehow by 

### 13 pooling. Pooling control does add some additional power to your 

### 14 test. But there are questions as to how you would deal with 

### 15 that. 

### 16 There is a guidance document that was produced 

### 17 by the OECD, Statistical Approaches to Analyzing Ecotoxicity 

### 18 Tests. And it discussed some of these kinds of concepts and 

### 19 recommendations in terms of how one would utilize it. The 

### 20 current practice within the Office of Pesticide Programs is to 

### 21 basically compare to dilution water control and to keep that 

### 22 somewhat consistent. But that's, you know, depending upon what 

### 23 would be, you know, most appropriate in the context of a 

### 24 particular test. 

### 25 The best approach is to avoid solvents. And I 

### 26 know that the EPA Duluth laboratory has gone to great extent to

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1 development methodologies to solvent-free systems. And they're 

### 2 pretty much solvent free now, even for very difficult test 

### 3 materials like the vinclozolin material able to do in 

### 4 solvent-free type systems. 

### 5 So these are what's recommended, but many 

### 6 contract laboratories have not developed their techniques or 

### 7 skills to the point of being completely solvent free, though 

### 8 there has been movement in that direction. And I would 

### 9 anticipate in the future that we see more solvent-free type 

### 10 systems where this becomes more of a historical consideration 

### 11 than something in the future. 

### 12 **DR. MARTHA SANDY:**Dr. Scott. 

### 13 **DR. GEOFFREY SCOTT:**Just a follow-up on that 

### 14 comment, Les. I think that is one of the advantages of the 

### 15 mysid system is those solvent-free systems have been developed 

### 16 with those flow-through diluters, right? I mean I think that's 

### 17 one of the advantages, right? 

### 18 **DR. LESLIE TOUART:**Yeah. That's correct in a 

### 19 sense that one would be able to adapt that. 

### 20 There are some added difficulties with, you 

### 21 know, mysids because it's seawater system, there at 20 parts per 

### 22 thousand. So that makes some materials less soluble and maybe a 

### 23 little bit more difficult to deal with in the solvent-free 

### 24 system. But it is adapted to that, and that could be developed 

### 25 on the maybe extreme cases what solvents you could pursue for 

### 26 that.

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1 But again, contract labs currently would still 

### 2 opt for a solvent-based system because that's how they're set 

### 3 up, and their diluter systems are designed with syringe 

### 4 injectors and things to be able to do that. But going forward 

### 5 they recognize that there are advantages in going solvent free 

### 6 and we're seeing those kinds of adaptations being made by some 

### 7 contractors. And we would anticipate, more or less, that that 

### 8 will become more commonplace. 

### 9 **DR. MARTHA SANDY:**Any other questions of 

### 10 clarification from the panel? 

### 11 Seeing none, I would like to give the panel a 

### 12 chance, with Dr. Touart here, are there any questions that came 

### 13 to mind overnight on the quail assay? 

### 14 Yes, Dr. Scanes. 

### 15 **DR. COLIN SCANES:**A couple of questions, and 

### 16 I'll go through one by one. First of all is the issue of the 

### 17 end, the number of replicates. For a number of the endpoints 

### 18 the number of end -- the end was either five or, in some cases, 

### 19 even three. What was the rationale behind that? 

### 20 **DR. LESLIE TOUART:**Can you be more specific 

### 21 upon which endpoints you would be referring to in terms of the 

### 22 ends? I mean primarily with the avian study, we're dealing 

### 23 with, you know, 12 pairs for collection and the ends are going 

### 24 to be fairly large in a lot of cases. But in some of the 

### 25 sub-sampling primarily when we get into some of the biochemical 

### 26 or histopathology we are sub-sampling and it may be male only or

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1 female only so there would be just a few in some of those 

### 2 instances. 

### 3 **DR. COLIN SCANES:**Being more specific then, 

### 4 for the hormonal and the histopath data, the end is down to five 

### 5 and in some cases we've lost samples and it gets much lower than 

### 6 that. 

### 7 **DR. LESLIE TOUART:**Yeah. Again, for the 

### 8 samples, what we were trying to do is, you know, select and 

### 9 we're basically sampling from the individuals that have survived 

### 10 to the end. So for some of the treatments there may not be, you 

### 11 know, sufficient pairs of individual. In controls, what we 

### 12 would be trying to do would be collect from the particular sex 

### 13 and one's making tradeoffs between what goes to histopath, what 

### 14 may be going to hormone analyses. 

### 15 **DR. COLIN SCANES:**Related question. The 

### 16 report makes a lot of the histopath data and yet no statistics 

### 17 was done. What's the rationale for the lack of statistical 

### 18 analysis? 

### 19 **DR. LESLIE TOUART:**I think the context is we 

### 20 didn't do any statistics on the pathology on the bird side. On 

### 21 StatCHARRMS statistics program that had been identified and had 

### 22 been utilized for the fish and frogs has the capability of doing 

### 23 some statistical analyses of that. We didn't apply that on the 

### 24 bird side because we're still developing and for the datasets 

### 25 that we had they just were not conducive to the adaptations and 

### 26 StatCHARRMS was developed more for the fish test paradigm.

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1 With the pathologists, they're generally used 

### 2 to evaluating their information and making their judgments 

### 3 through nonstatistical methodologies because of the nature of 

### 4 the kinds of responses that are seen. So we didn't do the 

### 5 analyses because it was not deemed to be necessary for the 

### 6 results that we were observing. 

### 7 **DR. COLIN SCANES:**Looking at your data, if 

### 8 you're putting histopath data into a scoring system of either 1, 

### 9 2, 3, 4 and 5 or 0, 1, 2, 3 and 4, that is then amenable to 

### 10 statistical analysis. 

### 11 **DR. LESLIE TOUART:**Yeah. I mean I would 

### 12 agree that one could be able to evaluate, you know, some of 

### 13 that. But in the context and judgment of the pathologists, 

### 14 especially when we went through the vinclozolin study, there 

### 15 weren't very many remarkable responses to try to evaluate in 

### 16 such a manner. And the nature of the responses that were seen 

### 17 for the epididymis, you know, these were observed and concluded 

### 18 to be legitimate effects. 

### 19 Now the context in terms of whether that might 

### 20 be appropriate in retrospective or moving forward to apply some 

### 21 statistical techniques to evaluate that, I think that the 

### 22 further advancement and development of the StatCHARRMS program 

### 23 and as that is converted to the "R" statistic base, then I think 

### 24 it would be useful to apply such a technique, you know, to the 

### 25 evaluations of birds or really any other datasets that we would 

### 26 have.

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1 **DR. MARTHA SANDY:**Could I remind panel 

### 2 members to keep their questions to clarifying questions and not 

### 3 a back and forth discussion. I don't mean to cut people off, so 

### 4 any other clarifying questions on the avian assay? 

### 5 No. Thank you very much, Dr. Touart. I'd 

### 6 like to now give the panel a chance, if you've thought of any 

### 7 questions of clarification you'd like to ask Dr. Rodney Johnson 

### 8 about the Medaka assay. 

### 9 Dallas Johnson. 

### 10 **DR. DALLAS JOHNSON:**Yes. I'm still confused 

### 11 with blocks and stuff and hopefully -- let me see if I can pull 

### 12 the slides up. Got too many files open here. 

### 13 I guess the reason I'm confused is we talked 

### 14 yesterday what a block was, and the block was one of those nice 

### 15 pictures that you showed. Yeah, if you know where it is. 

### 16 **DR. RODNEY JOHNSON:**I think I can find it. 

### 17 **DR. DALLAS JOHNSON:**Slide 85 or something 

### 18 around in there, I think. 

### 19 **DR. RODNEY JOHNSON:**Am I going in the right 

### 20 direction? 

### 21 **DR. DALLAS JOHNSON:**No. You're going the 

### 22 wrong direction. 

### 23 **MALE SPEAKER:**Wrong direction. 

### 24 **DR. LESLIE TOUART:**It's Slide 77. 

### 25 **DR. DALLAS JOHNSON:**You can do a search on 

### 26 blocks, the way I usually find it.

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1 **DR. RODNEY JOHNSON:**Okay. Yeah. 

### 2 **DR. DALLAS JOHNSON:**Yeah. Now I guess the 

### 3 reason I'm getting confused again is back on the previous slide 

### 4 you talked about the experimental design consisting of seven 

### 5 blocks, five for chemical and two for control. And then the 

### 6 next slide you indicated that this represented a block which had 

### 7 -- and I asked you about the fact that there were 6 pairs there, 

### 8 and I thought there was going to be 12. And the reason I 

### 9 thought that there was 12 is on the next slide, you talked about 

### 10 using 12 treatment pairs rather than 6 treatment pairs. So I'm 

### 11 all mixed up with respect to the number of treatment pairs and 

### 12 anything that you can say that would help clarify that in my 

### 13 mind would be appreciated. 

### 14 **DR. RODNEY JOHNSON:**Okay. So the first thing 

### 15 I think I would say is that I'm concerned about using the word 

### 16 "block" but let's say that this is a representation of the 

### 17 replication structure of any particular treatment, let's say, 

### 18 and let's not even talk about it in the context of the controls 

### 19 right now. 

### 20 All right. As we go from left to right, in F0 

### 21 we start with six replicate breeding pairs. And the embryos 

### 22 produced by those six pairs are all pooled. 

### 23 **DR. DALLAS JOHNSON:**Yeah. I'm okay with that 

### 24 part. 

### 25 **DR. RODNEY JOHNSON:**Okay. Then we go to what 

### 26 would be incubation in F1, which has six replicates.

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1 **DR. DALLAS JOHNSON:**My question is mostly 

### 2 about the F0, the first column. 

### 3 **DR. RODNEY JOHNSON:**Okay. 

### 4 **DR. DALLAS JOHNSON:**You have six pairs there. 

### 5 Excuse me. Turn my microphone. 

### 6 You have six pairs there, and this six pairs 

### 7 are going to be -- let's say they're treated with the 100-level 

### 8 dose. 

### 9 **DR. RODNEY JOHNSON:**Correct. 

### 10 **DR. DALLAS JOHNSON:**Okay. Then are those the 

### 11 only six pairs that are in the 100-level dose, or is there 

### 12 another six pairs somewhere else that's in the 100-level dose, 

### 13 or are there six of these blocks that are within the 100-level 

### 14 dose? 

### 15 **DR. RODNEY JOHNSON:**Okay. This would be on 

### 16 the left-hand side where there are six breeding pairs that would 

### 17 be six breeding pairs per treatment. So in let's say 100 -- a 

### 18 dose of a 100, level of 100, there are going to be six breeding 

### 19 pairs at dose level 100. 

### 20 There will be six breeding pairs at the next 

### 21 lower dose, which let's say is 50, et cetera, until we get to 

### 22 the controls. And at the controls, there would be two times 

### 23 that or 12. 

### 24 **DR. DALLAS JOHNSON:**And then how does that 

### 25 relate to the next slide where you did the power analysis and I 

### 26 thought I interpreted from that that you were going to use 12

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1 treatment pairs or recommending use of 12 treatment pairs rather 

### 2 than 6? 

### 3 **DR. RODNEY JOHNSON:**Okay. So in F1, first of 

### 4 all, you should recognize or I want to state that there are no 

### 5 data collected here. They simply are the brood stock, if you 

### 6 will, for starting the exposure within the next generation which 

### 7 then, of course begins at this line or in here where we have 

### 8 embryos being incubated within the F1 generation, or they are 

### 9 the beginning of the F1 generation. 

### 10 Now at Week 9, where we measure the effects, 

### 11 the diagnostic or the AOP effects, we measure that at Week 9, 

### 12 but we also at Week 9 take those six replicate tanks, and we 

### 13 take two males and two females from each tank, and we produce a 

### 14 common tank of males and a common tank of females which are then 

### 15 distributed into 12 reproductive pairs, and those 12 

### 16 reproductive pairs are the data by which reproduction is 

### 17 assessed. 

### 18 **DR. DALLAS JOHNSON:**Yeah. I think that 

### 19 clarifies it for me. 

### 20 **DR. MARTHA SANDY:**Dr. Blazer. 

### 21 **DR. VICKI BLAZER:**I was curious, with the MTT 

### 22 and the fact that you're doing the whole fish histopathology, 

### 23 why there was nothing with thyroid? Do you actually look at the 

### 24 thyroid at all? 

### 25 **DR. RODNEY JOHNSON:**The protocol specifies to 

### 26 explicitly look at the gonads, explicitly look at the kidneys,

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1 explicitly look at the liver and to evaluate other organs for 

### 2 effects. But it does not specify which organs and what to 

### 3 evaluate them for. So the thyroid is implicitly in the 

### 4 guideline, but it is not explicitly called out to be evaluated. 

### 5 The principal rationale for that is that the 

### 6 -- as of course you know, the thyroid of fishes is not an 

### 7 encapsulated organ. It tends to be rather diffuse and it is not 

### 8 an easy organ to evaluate in sagittal section in particular 

### 9 because it is not real predictable where you're going to get 

### 10 good sections of the thyroid. 

### 11 You will almost always get sections of 

### 12 follicles in parasagittal sections, almost always. The most 

### 13 important caveat would be that it's difficult to establish a 

### 14 subjective, sort of qualitative evaluation of extent of changes. 

### 15 Now it is clear that one can evaluate things like follicular 

### 16 cell hypertrophy and some things like that, but it's quite 

### 17 difficult to definitively and subjectively provide a good 

### 18 assessment of extent. So it would be more, I think, a hallmark 

### 19 of a remark rather than something that would be defined as a 

### 20 definitive endpoint in the fish. 

### 21 **DR. MARTHA SANDY:**And Dr. Jennifer Freeman. 

### 22 **DR. JENNIFER FREEMAN:**Hi. Thank you. I just 

### 23 had a clarifying question on whether or not dose was analyzed if 

### 24 there's any tissue analysis that was done in the assay? 

### 25 **DR. RODNEY JOHNSON:**I'm sorry. I didn't hear 

### 26 the last phrase of the question clear.

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1 **DR. JENNIFER FREEMAN:**Yeah. Was tissue 

### 2 analysis completed at all during the assay to determine dose? 

### 3 **DR. RODNEY JOHNSON:**No tissue analyses are 

### 4 done to determine dose. Only the water concentration is 

### 5 measured for dose. 

### 6 **DR. JENNIFER FREEMAN:**All right. In light of 

### 7 that, I have a follow-up quick question about your thoughts on 

### 8 how the revised MRT without assessing the F2 reproductive end 

### 9 points, whether or not the assay would be able to assess if the 

### 10 chemical is bio-accumulating in the F1? 

### 11 **DR. RODNEY JOHNSON:**There's quite a bit of 

### 12 literature describing determinates of bioaccumulation. So I 

### 13 think one may be able to ascertain whether or not 

### 14 bioaccumulation is something to be considered based on the 

### 15 physical chemical properties of the chemical in question. 

### 16 But there is sort of one sort of check, if you 

### 17 will, and it's sort of an indirect check, and that would be 

### 18 looking at the toxicity with respect to hatching in F1 compared 

### 19 to F2. And with those chemicals that would not bio-accumulate, 

### 20 you would expect those values to be similar. 

### 21 On the other hand, if there was a major 

### 22 difference in the hatching success rate in F1 compared to F2, 

### 23 especially if F2 was quite a bit lower or was lower, I think one 

### 24 would be very concerned that it perhaps was due to 

### 25 bioaccumulation and would make you consider your adequacy of the 

### 26 protocol on that basis.

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1 **DR. MARTHA SANDY:**Any other questions of 

### 2 clarification? 

### 3 Seeing none, thank you very much, Dr. Johnson. 

### 4 I'd now like to have Dr. Degitz come here, and 

### 5 anyone have any questions for him on the amphibian assay? Looks 

### 6 like we have all our questions answered. 

### 7 I think we should take a break. We're 

### 8 scheduled for a break now. Ten minutes or fifteen? I'm not 

### 9 sure. Let's go with a ten-minute break. Let's give ourselves 

### 10 12 minutes. Let's come back at 10:15. Thank you. 

### 11 (Whereupon a break was taken) 

### 12 **DR. MARTHA SANDY:**Mary is seated. For those 

### 13 who are listening. Okay. 

### 14 **MS. MARY MANIBUSAN:**Thank you. I want to 

### 15 thank Dr. Christman and Dr. Johnson for their clarification 

### 16 questions on the statistical analyses that were conducted in 

### 17 part of the reanalysis that was issued from the errata. 

### 18 Because of time differences and receiving 

### 19 those questions late yesterday, we're having a slight challenge 

### 20 with time zone differences. But we anticipate bringing before 

### 21 you the final responses sometime today. We'll be checking in 

### 22 around the noon hour to help solidify when that will be done. 

### 23 We want to make sure that we are firm in our response and clear 

### 24 and accurate. Thank you. 

### 25 **DR. MARY CHRISTMAN:**Thank you, Mary. 

### 26 Appreciate that a lot. Fortunately, since it relates to

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1 StatCHARRMS, and they don't come up until probably tomorrow and 

### 2 the following day, we're good. 

### 3 **MS. MARY MANIBUSAN:**Thank you. But we'd like 

### 4 to make sure we provide those responses as swiftly as possible 

### 5 for your consideration. So thank you again. 

### 6 **DR. MARTHA SANDY:**Thank you very much, Mary. 

### 7 All right. I think now we're ready to turn to 

### 8 the public comments. And we have received comments from two 

### 9 groups of individuals. One set of comments the person that 

### 10 submitted them has asked that that be read into the record. So 

### 11 I've been asked to read this. So I think we'll start with this 

### 12 public comment and then we'll move to comments that have been 

### 13 submitted by the Endocrine Policy Forum, and there we have 

### 14 presenters that are here physically in the room. 

### 15 So if you'll bear with me, I've been asked to 

### 16 read these comments. They're submitted by Patricia Bishop, and 

### 17 she's providing these comments on behalf of the People for the 

### 18 Ethical Treatment of Animals, the Physicians Committee for 

### 19 Responsible Medicine and the Humane Society of the United 

### 20 States. 

### 21 These comments are a summary of written 

### 22 comments that these groups have submitted for the panel's 

### 23 consideration. They're in the Docket, and we've all been sent 

### 24 them and read them. Okay. So here are the comments. 

### 25 The three vertebrate ecotoxicity assays, 

### 26 the Japanese quail two generation, the Medaka

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1 Multigeneration and the Larval Amphibian Growth and 

### 2 Development Assay are complex tests of long duration. 

### 3 They use large numbers of animals. The fish study uses 

### 4 about 3,000 animals, the bird study uses about 1,500 

### 5 animals, and the amphibian test uses at least 750 animals. 

### 6 These numbers do not include animals used in optimization 

### 7 and range-finding studies or cold animals. Thus, we must 

### 8 have assurance that these tests are fit for purpose and 

### 9 will definitively characterize a chemical's action as an 

### 10 endocrine disruptor in a clear dose response fashion. 

### 11 The results of the validation studies 

### 12 indicate that the reliability as definitive tests is 

### 13 suspect because numerous areas exist for introduction of 

### 14 error and variability that could confound interpretation 

### 15 of findings. Thus, we believe that there is a high 

### 16 potential for inexperienced laboratories to generate poor 

### 17 quality data which will result in the repeating of tests 

### 18 that will kill even more animals. 

### 19 While it is important to show the 

### 20 consistency and use of a protocol as achievable, this 

### 21 should follow careful development of the test. The main 

### 22 focus during development and evaluation of the Tier 2 

### 23 assays must be the identification of endocrine-sensitive 

### 24 endpoints and characterization of sensitivity as well as 

### 25 specificity of these endpoints. It is not clear in the 

### 26 case of the three tests discussed here that there has been

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1 sufficient, or any, characterization of a sensitivity 

### 2 and/or specificity of these endpoints. It is critical 

### 3 that careful attention be paid to the endocrine 

### 4 sensitivity of endpoints included in any new reproductive 

### 5 tests that are not measured in the standard suite of 

### 6 toxicity tests used in pesticide registration. 

### 7 With respect to the inter-laboratory 

### 8 comparisons, one of the most obvious issues noted in all 

### 9 of the inter-laboratory comparisons was a lack of 

### 10 consistency across labs in identifying effects. The JQTT 

### 11 inter-laboratory study using a single reference chemical 

### 12 test substance, the androgen receptor antagonist 

### 13 vinclozolin, showed little in the way of treatment-related 

### 14 effects. However, there were several sources of variation 

### 15 such as the strain of the bird that could lead to serious 

### 16 problems with interpretation of results. 

### 17 The MMT inter-laboratory comparison also 

### 18 used vinclozolin as the single reference substance for the 

### 19 inter-laboratory study and the only significant conclusion 

### 20 regarding effects across labs seems to be that all three 

### 21 labs did not see decreased reproduction. In the case of 

### 22 the LAGDA inter-lab comparison, where two different 

### 23 chemicals were used, there also were few instances where 

### 24 all labs consistently reported the same significant 

### 25 finding. 

### 26 Use of vinclozolin as a reference

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1 chemical in two of the three studies and the ambiguous 

### 2 responses reported seems to have raised more questions 

### 3 than it answered, since even EPA noted that it posed a 

### 4 challenge as a test substance in a validation exercise, 

### 5 leading to uncertainty in the expected response. It 

### 6 clearly did not result in a well defined 

### 7 concentration-response relationship, the development of 

### 8 which is one of the primary aims of the Tier 2 tests and 

### 9 essential to the assessment of inter-laboratory validity. 

### 10 One of the stated objectives of the JQTT 

### 11 study was to show that the endpoints added to this assay 

### 12 beyond the ones in the standard avian reproduction 

### 13 toxicity assay were, in fact, sensitive to endocrine 

### 14 disruptors. It seems counterintuitive to select a 

### 15 reference chemical such as vinclozolin to test this 

### 16 hypothesis when there were questions about whether effects 

### 17 would even be seen. Use of vinclozolin as a reference 

### 18 chemical did not appear to adequately demonstrate the 

### 19 usefulness of the assay or the appropriateness of the 

### 20 selective endpoints for detecting endocrine disruptors. 

### 21 During the fish studies, there were 

### 22 issues of labs being unable to demonstrate consistent 

### 23 control responses. This does not lead to confidence in 

### 24 the ability of commercial labs to successfully conduct 

### 25 this test or confidence in the results produced. Cases of 

### 26 erroneous sexing of fish occurred as well which obviously

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1 introduces substantial error into the results. 

### 2 Genotypic, phenotypic sex mismatching 

### 3 was noted by EPA to be a problem by labs in performance of 

### 4 the Tier 1 fish short-term reproduction assay as well. 

### 5 Very few significant effects were noted in the MMT 

### 6 inter-lab comparison. One of the three labs showed no 

### 7 significant effects at all, and there was little 

### 8 consistency among labs in the effects that were observed. 

### 9 10 The failure of the three labs to discern 

### 11 effects of vinclozolin in a consistent manner raises a 

### 12 question as to whether this is an artifact of the method's 

### 13 inability to detect weak endocrine disruptors or if too 

### 14 many sources of variability introduced into the 

### 15 experiments masked any significant results. 

### 16 With respect to the LAGDA validation, 

### 17 several significant sources of variability introduced by 

### 18 the labs were indentified in the study. Inter-lab 

### 19 comparisons demonstrated how slight differences in feeding 

### 20 regimens alone can alter the study results. Also, it was 

### 21 noted that interpretation of pathology results by 

### 22 different pathologists could significantly alter study 

### 23 findings. During the prochloraz study, one lab reported 

### 24 treatment-related liver pathologies that were vastly 

### 25 different from what was reported by the other three labs. 

### 26

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1 EPA speculated that this may be 

### 2 explained by disease in the fish, but the cause was never 

### 3 determined. The fact that one lab could have such 

### 4 dramatically different results from the other three 

### 5 suggests that this assay may also not be ready for use in 

### 6 the Tier 2 Endocrine Disruptor Screening Program. 

### 7 We conclude from the studies provided 

### 8 that none of the three vertebrate assays appears ready for 

### 9 use as a valid Tier 2 test. More critically, the tests 

### 10 have not been shown to include endpoints that are 

### 11 sufficiently sensitive to endocrine-active substances to 

### 12 definitively characterize the suspected endocrine 

### 13 disruptors. 

### 14 In addition, inconsistencies among labs 

### 15 in reporting significant findings, variability introduced 

### 16 into the tests by strains of animals used, feeding 

### 17 regimens, pathology interpretation and other unknown 

### 18 factors indicate that more evaluation and refinement of 

### 19 the methods are necessary. 

### 20 Therefore, neither the relevance nor the 

### 21 reliability of these tests has been sufficiently shown. 

### 22 These tests are lengthy and complex new protocols 

### 23 requiring a high level of expertise to perform properly 

### 24 and due to lack of reproducible significant effects 

### 25 amongst labs participating in the inter-laboratory 

### 26 validation studies, validation has not been adequately

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1 demonstrated. 

### 2 That said, we are extremely concerned by 

### 3 the announcement yesterday that EPA has initiated the 

### 4 process for collecting Tier 2 data from some of the List 1 

### 5 chemicals by issuing an information collection request for 

### 6 Tier 2 testing. This action appears to be premature for 

### 7 the reasons I have already described. Most importantly, 

### 8 it is not assured at this time that the Tier 2 ecotoxicity 

### 9 tests will be positively reviewed and supported by the 

### 10 SAP. 

### 11 So again, I have just read into the record, by 

### 12 request, the comments submitted by Patricia Bishop on behalf of 

### 13 the People for the Ethical Treatment of Animals, the Physicians 

### 14 Committee for Responsible Medicine and the Humane Society for 

### 15 the United States. 

### 16 **DR. SHARLENE MATTEN:**So the sound system for 

### 17 the webcast works, but some folks, it's individual specific, so 

### 18 I'm demonstrating how you need to speak into the microphone. 

### 19 Martha is heard. Mary Manibusan is heard. I'm heard. Dallas 

### 20 was heard. You basically have to go like this. And I don't 

### 21 have a singing career or acting career, but in radio land, my 

### 22 husband works for radio, you do hold the microphone really close 

### 23 to your mouth. And if you have to move, move the microphone 

### 24 with you. All right. That applies to the panel, too. So I 

### 25 don't have as booming as Mary Manibusan, but I do my best. 

### 26 That's a little laughter here. That's always

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1 good. And now Martha will state where we are going next. 

### 2 **DR. MARTHA SANDY:**Thank you, Dr. Matten. 

### 3 So now we'll turn to the next group of public 

### 4 commenters and these are comments made on behalf of the 

### 5 Endocrine Policy Forum. I'll let you sit down and introduce 

### 6 yourself, please. 

### 7 **DR. ELLEN MIHAICH:**Good morning. Thank you 

### 8 for letting me speak. My name's Ellen Mihaich and I am the 

### 9 scientific advisor for the Endocrine Policy Forum. I know I did 

### 10 this initial slide last month, since many of us were together. 

### 11 But since there's some new faces, I just wanted to be able to do 

### 12 this again to set the stage for what you're going to be hearing 

### 13 next. I also want to thank you for being here and being willing 

### 14 to participate in this review. It is a huge amount of 

### 15 information and we feel for you for having to try to get through 

### 16 it. 

### 17 The next four presenters will actually take 

### 18 each one of the tests separately and go through some of the high 

### 19 points that we saw in going through the data and then I will be 

### 20 back up again to do a final sort of wrap up. 

### 21 Just a brief introduction to who the Endocrine 

### 22 Policy Forum is. We're a consortium of about 95 percent of the 

### 23 List 1 test order holders so the recipients, the people that 

### 24 have been doing this testing. So for the SAP last month, we 

### 25 were here with all of the experience of actually having to do 

### 26 these in a regulatory forum of the 11 screens. So clearly we

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1 have a pretty significant interest in how the Tier 2 tests are 

### 2 going to move forward because we're going to be the ones that 

### 3 have to do it in a regulatory setting. 

### 4 So the other thing that I want to point out to 

### 5 you is we're a group that the List 1 is predominantly, not all, 

### 6 but predominantly from the crop protection industry. And if you 

### 7 know anything about what it takes to get a pesticide registered, 

### 8 it's millions and millions of dollars of testing, regulatory 

### 9 testing, and we do a lot of these chronic studies. 

### 10 So we have a lot of expertise in running these 

### 11 in contract labs and being able to evaluate them. So we came at 

### 12 this from looking at that, trying to look at the data that's 

### 13 here and thinking about having to do this in a regulatory 

### 14 setting. But I think, again, as we go through this, you know, 

### 15 you're being charged with deciding whether or not these tests 

### 16 are valid for their purpose, and that's where we were looking 

### 17 at, too. 

### 18 So with that, I was just setting that up, and 

### 19 then the next four, as I said, will take each of the four tests 

### 20 separately, and then I'll come back to fill in a few overarching 

### 21 comments. Thank you. 

### 22 **DR. MARTHA SANDY:**Thank you. And I gather 

### 23 the first presenter will be Tim Fredericks, and there will be 

### 24 four presentations of about 15 minutes each. Right? 

### 25 **DR. TIM FREDERICKS:**Yes. This is Tim 

### 26 Fredericks. Okay. I'll be commenting on the Japanese Quail

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1 Two-generation Test. 

### 2 Can I advance the slides? 

### 3 So we're going to start with some conclusions, 

### 4 just to give everything a background of what I'm going to 

### 5 explain and we'll come back to why these are important 

### 6 throughout the next about 15 slides. So as was mentioned 

### 7 yesterday, there's no guidance on when this test design would be 

### 8 triggered from Tier 1 testing to Tier 2 testing. This is fairly 

### 9 important. This test system has not been validated and thus is 

### 10 not appropriate for regulatory studies. We would consider this, 

### 11 what's happened, a pre-validation. 

### 12 So poor performance in ring testing, too many 

### 13 measurement endpoints, limited test sensitivity to 

### 14 endocrine-mediated effects. So it includes both population 

### 15 level, relevant endpoints for risk assessment, but it's limited 

### 16 in its ability to definitively link these to endocrine mechanism 

### 17 of action. So the big question is how does the Japanese Quail 

### 18 Two-generation Test enhance our understanding of population 

### 19 level effects in birds versus a one-generation test that's 

### 20 available for a lot of these compounds? 

### 21 So the background, quail and duck have been 

### 22 used in regulatory testing for greater than 20 years following 

### 23 the one-generation test guideline. So routinely conducted for 

### 24 pesticides, the F generation, most F0 and F1 generation are 

### 25 pretty much the basis for the endpoints that are in the 

### 26 two-generation test. All of these are in the two-generation

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1 test, and then two-generation test builds on these. 

### 2 So the specific rationale for selecting the 

### 3 Japanese quail is well presented for the -- in the ISR. Again, 

### 4 there needs to be specific guidance on when this would be 

### 5 required based on the Tier 1 testing. And although 

### 6 theoretically there are reasons presented in the ISR as to why 

### 7 an avian species should be tested in the Tier 2 test, these are 

### 8 mostly theoretical and have not been proven based on the 

### 9 validation testing and the pre-validation work that's been done. 

### 10 So specifically, Charge Question 2, 

### 11 toxicological relevance of this test design; so there were 106 

### 12 endpoints analyzed statistically, and a lot of these were 

### 13 analyzed over multiple time points during the test. So you 

### 14 ended up at almost 155 statistical comparisons in this one test 

### 15 design. And on top of that, there was endpoints that were 

### 16 collected and actually not analyzed statistically or not 

### 17 analyzed at all. They were just archived, these samples. So 

### 18 there were more endpoints then were actually presented. 

### 19 The diversity of endpoints selected for this 

### 20 test monitor wide variety of adverse effects to birds and the 

### 21 connection to endocrine-related effects has not been made. 

### 22 Specific endpoints have not been determined to give definitive 

### 23 answer to endocrine activity versus overt toxicity. And this is 

### 24 particularly important in regards to how this will be used in 

### 25 the risk assessment which is the goal of Tier 2 testing. 

### 26 So the assay endpoints. I've already listed

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1 that there's probably too many endpoints included that have not 

### 2 been validated in responses for endocrine-active pathways. So 

### 3 pretty much all of these endpoints can be influenced by overt 

### 4 toxicity and the difference between overt toxicity and 

### 5 endocrine-mediated effects has not been clearly made. 

### 6 There's a large degree of interrelatedness 

### 7 between a lot of these endpoints. You may measure eggs 

### 8 collected, but then you're going to measure the egg set of eggs 

### 9 collected. You're going to measure the egg set of the eggs laid 

### 10 and eggs laid per hen, per day. So although there may be 106 

### 11 endpoints, a lot of those rely on other endpoints to make those 

### 12 endpoints. And again, a lot of these endpoints are included in 

### 13 the current guideline for one-gen reproduction study. 

### 14 So one of the endpoints that was collected and 

### 15 not analyzed were fecal steroids. And to quote the ISR on page 

### 16 54, "Fecal samples reflect more consistent effects than 

### 17 biochemical analyses." So biochemical analyses were analyzed 

### 18 but fecal steroids weren't. So this is probably a major limiter 

### 19 for this. 

### 20 And then it's difficult to collect blood, so 

### 21 fecal steroids could be collected from the 15-day-old chicks 

### 22 before they hatched, so they'd be actually 15-day-old embryos. 

### 23 So those embryos will have a fecal sack that will be easily 

### 24 collected. What was currently done is blood was taken from that 

### 25 15-day-old embryo, and a lot of the samples did not have the 

### 26 quantity to be analyzed for the biochemical analyses, and you

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1 could do a correlation between the fecal sack and blood 

### 2 chemistry and determine if that relationship exists and then 

### 3 have a more simple sampling technique. 

### 4 And then fecal steroids for the adults, 

### 5 there's a lot of sample. These birds are in cages for 10, 12, 

### 6 14 weeks. They're defecating every day, and you could separate 

### 7 it by males and females, but it has not been correlated between 

### 8 blood steroid levels and fecal steroid levels, but this could 

### 9 easily be done. 

### 10 Histopathological endpoints have not been well 

### 11 validated for avian species, as some of the commenters mentioned 

### 12 today. Sample size was an issue here, and the statistical and 

### 13 the interpretation of these endpoints was limited by the number 

### 14 of samples that were collected and analyzed. And from the 

### 15 histological report, there were a lot of cases of missing, 

### 16 insufficient or not submitted tissues, especially for the 

### 17 embryos. They didn't have the tissue available for a lot of 

### 18 these to be able to do a valid histological comparison. 

### 19 And then biochemical analyses, as was pointed 

### 20 out yesterday, these are highly variable. It was one of the 

### 21 most variable endpoints measured across the adults, the chicks 

### 22 or the embryos. And again, there's a couple of issues with the 

### 23 sampling that happened for both the histopath and the 

### 24 biochemical. These samples were selected, not selected 

### 25 randomly; they were selected as the best samples that were 

### 26 submitted. So there seems to be a sampling disconnect there and

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1 a statistical design disconnect there as well. 

### 2 And specifically for the histopath, we 

### 3 mentioned yesterday the epididymis as the one endpoint that was 

### 4 noted as statistically different across the study designs 

### 5 through the labs. And so I looked last night and that 

### 6 epididymis was statistically dose response at the one lab, and 

### 7 when you looked at those individual pens for those individual 

### 8 males that had valcuolization or dilation in the epididymis, 

### 9 they all produced chicks at 14 days in the second generation or 

### 10 the first generation if they were the F0s or F1s. 

### 11 So the reproductive endpoints were all okay. 

### 12 They were able to produce chicks, which is the main goal of this 

### 13 test is to see if we're going to have population level effects. 

### 14 So despite having a potential histopathological effect, it was 

### 15 not realizing a population level effect. 

### 16 So again, the background studies, a lot of 

### 17 these are one generation and this would be what we would 

### 18 consider pre-validation testing. Most of these were developing 

### 19 the endpoints that would have gone into this ring test. But in 

### 20 reality, we were still pre-validating in the ring test because 

### 21 we don't have validated methods for these endpoints or 

### 22 responses. 

### 23 And just to point out the trenbolone that was 

### 24 talked about yesterday that was used in the two-generation 

### 25 design, and there were effects in both the F1 and the F2 or 

### 26 there was just effects in the F1 in each of those tests that

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1 were run. So the second generation didn't add anything to the 

### 2 interpretation of these results. The results were the same 

### 3 based on the F1 or the F2. So you would expect a similar if you 

### 4 ran one-generation test instead of a two-generation test. 

### 5 So statistical methods in the ISR were not 

### 6 well defined but were generally appropriate. Again, you run -- 

### 7 oh so there are two things -- you're running multiple 

### 8 statistical tests on a single endpoint. So you're running a 

### 9 Dunnett test and JT test for trend. And really, you shouldn't 

### 10 analyze the same endpoint with two statistical tests. 

### 11 So there needs to be some correction for false 

### 12 discovery rate. It was discussed in the ISR, and just based on 

### 13 the sheer number of variables analyzed when you take into 

### 14 account the different days that food consumption, body weights 

### 15 and the different generations that each of these were measured 

### 16 for, you end up with over 155 endpoints instead of the 106 that 

### 17 you started with. And so just sheer five percent alpha value, 

### 18 you're looking at quite a few false detections with 155 

### 19 variables. 

### 20 So the standard one-generation avian repro 

### 21 data is very robust and reliable. These studies have been done 

### 22 for over 20 years. The additional endocrine-specific endpoints 

### 23 have not been rigorously evaluated. So a; the endpoint list 

### 24 needs to be refined, and b; these specific endpoints haven't 

### 25 been linked to endocrine-mediated effects. 

### 26 No endpoints were consistently statistically

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1 significant across all three laboratories following vinclozolin 

### 2 exposure. And when you compare across the labs for some of the 

### 3 endpoints that were flagged, you see at some labs that endpoint 

### 4 may have been up regulated, and some labs that may have been 

### 5 down regulated. 

### 6 So when you're running this test as part of a 

### 7 Tier 2 test design, how are you to interpret your results if it 

### 8 changes between the three labs that were running the validation? 

### 9 So could that result that was maybe tempered by the fact that 

### 10 you had two other studies to compare it to result in a 

### 11 regulatory decision if there was a trend at one lab but not the 

### 12 other two or the other two trended the opposite way? 

### 13 So transferability between labs; so strain 

### 14 selection needs to be addressed and this is probably a positive 

### 15 and the negative for the assay because yes, you could choose a 

### 16 specific strain to temper this variability, but is that really 

### 17 appropriate at the larger population level? 

### 18 So refinement to the protocol; the protocol 

### 19 hasn't been well refined, and that's why this should be 

### 20 considered pre-validation work. There's large inner and 

### 21 intra-laboratory variability across many of the variables, 

### 22 especially the new variables that aren't in the standard 

### 23 one-generation test design. 

### 24 So dose response relationships were not 

### 25 present, especially for vinclozolin. And then a lot of the 

### 26 other compounds that were run through the one-gen and two-gen

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1 study designs didn't have multiple comparisons of the same 

### 2 chemicals. So how valid are those responses at a single lab? 

### 3 Endpoint responses were generally not present 

### 4 or consistent when present. 

### 5 Strengths of this study design; species 

### 6 selection is well justified in the ISR. It uses a precocial 

### 7 species that matures rapidly, and this is important for a test 

### 8 that runs as long as this test does. So includes both apical 

### 9 endpoints for overt toxicity, and these are the endpoints that 

### 10 are really useful for risk assessment at the population level. 

### 11 So there's the potential to detect endocrine-mediated effects 

### 12 fairly well, but this hasn't been well validated. 

### 13 So the limitations to the Japanese Quail 

### 14 Two-generation Test. There's large variability in endpoint 

### 15 response, and a lot of this could be based on the sample size 

### 16 and not having adequate sample size. And actually, it's 

### 17 probably not the sample size it's the analysis of the samples. 

### 18 So the samples could have been there, and they may have decided 

### 19 to not analyze these for cost reasons. 

### 20 So there's limited data to justify the 

### 21 necessity of a second generation. Poor reproducibility between 

### 22 labs and non-endocrine-mediated toxicity may complicate test 

### 23 interpretation for determining endocrine-specific mechanisms of 

### 24 action, but this would not affect risk assessment. 

### 25 So histopathological expertise and experience 

### 26 in avian tissues are limited. This is a fairly new analysis, it

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1 hasn't been traditionally included in the test designs as it has 

### 2 for the mammalian systems. 

### 3 And one thing to consider is when the 

### 4 mammalian two-gen versus one-gen test design was discussed there 

### 5 wasn't an overwhelming response that the two-generation was 

### 6 necessary even in a well-validated system like the mammalian 

### 7 system. So limited value is added by that second generation 

### 8 which is why that's staggered as a potential one-gen/two-gen 

### 9 study for the Tier 2 test. 

### 10 So one-gen avian data required for pesticide 

### 11 registrations. So there's extensive experience with the 

### 12 one-generation guideline. So the variability tended to increase 

### 13 from the F0 to the F1 to the F2. It was a slight increase, but 

### 14 it was there and really if you saw something at F1, you might 

### 15 not see it at F2 and the same thing is if you saw it at F1 in 

### 16 the studies we've seen, you saw it at F2. So necessity for the 

### 17 second generation is still questionable. 

### 18 So added value for risk assessment from 

### 19 additional generations has not been well established. 

### 20 This has been highlighted before, but it feels 

### 21 like there was just a major opportunity missed with test 

### 22 substance selection. There were other compounds that were run 

### 23 through one-generation test designs and it would have been nice 

### 24 to see a ring test on a compound that was run through a 

### 25 one-generation test so you can make a comparison from 

### 26 one-generation to two-generation and if the endpoints were

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1 helpful in the second generation. 

### 2 So histopathology was not analyzed in previous 

### 3 validation work for a lot of the tests. So how are you to 

### 4 compare and build a dataset of control values that you're going 

### 5 to look for in these two-generation tests? 

### 6 So the low number of samples in the analysis 

### 7 of the ring test data for vinclozolin, and again, no compound 

### 8 was run through the entire battery for the Tier 1 and Tier 2 

### 9 assays. 

### 10 So back to our conclusions and why all of that 

### 11 leads to these. Again, there's no guidance on when we're going 

### 12 to use this assay. So it has not been validated, adequately 

### 13 validated. This would be considered more pre-validation work. 

### 14 And again, it's not appropriate for regulatory studies as it 

### 15 currently sits. 

### 16 Poor performance, too many measurement 

### 17 endpoints, limited assay sensitivity to endocrine-mediated 

### 18 effects. So the Japanese quail includes population relevant 

### 19 endpoints for risk assessment but is limited in its ability to 

### 20 definitively link these to endocrine-mediated mechanism of 

### 21 actions. 

### 22 **DR. MARTHA SANDY:**Thank you, Dr. Fredericks. 

### 23 I think I'd like to have all the commenters 

### 24 present first, and then we'll open it up to panel questions. So 

### 25 Katie Coady. 

### 26 **DR. KATIE COADY:**I'll just take a moment to

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1 introduce myself. I'm an ecotoxicologist at the Dow Chemical 

### 2 Company. And I'll be providing some comments today on both the 

### 3 Medaka Multigeneration Test and the Medaka Reproduction Test on 

### 4 behalf of the Endocrine Policy Forum. 

### 5 I'm starting also with a summary slide just to 

### 6 show everybody the major points I'm going to be making. One is 

### 7 that it really does seem that the revised protocol, the Medaka 

### 8 Reproduction Test, seems to be an improvement over the multigen 

### 9 medaka test that the Agency started with the validation effort. 

### 10 The second point, however, is that this Medaka 

### 11 Reproduction Test is this new optimized protocol that we're 

### 12 coming out with at the end of the validation effort, and it 

### 13 really has not been adequately ring tested, hasn't been ring 

### 14 tested. So we need to take a step back and look at doing this. 

### 15 Also, both assays, the 2-1/2-generation Medaka 

### 16 Multigen and the Medaka Reproduction Test do include endpoints 

### 17 that are population-level relevant and useful for risk 

### 18 assessment. But as some of the speakers before me, I'll just be 

### 19 making the point that they are limited in their ability to 

### 20 definitively link to an endocrine mode of action. And I'll 

### 21 discuss that a little bit more as I go along here. 

### 22 I think actually this first bullet point was 

### 23 elucidated a little bit better yesterday by Dr. Johnson. There 

### 24 was some more rationale provided, I think, for why the medaka is 

### 25 used as the test species. I just want to point out that it is a 

### 26 different test species that was used in Tier 1. So if there's

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1 going to be a need to incorporate data from Tier 1 and Tier 2 

### 2 together and look at both of those analyses from both of those 

### 3 studies, there's an extra variable now that we have a different 

### 4 species in Tier 2. 

### 5 I think we saw yesterday that the 

### 6 2-1/2-generation Medaka Multigen Test, the MMT, that test design 

### 7 did not contain sufficient replication to adequately assess 

### 8 reproduction. 

### 9 However, the MRT does seem to be designed 

### 10 appropriately to really look at the biological significance of 

### 11 reproduction. That seems to be an enhanced design. It also 

### 12 includes looking at growth, survival and development, all of 

### 13 these things that are important for performing a risk 

### 14 assessment. But again, there has been no demonstration of this 

### 15 particular study design, the Medaka Reproduction Test, for 

### 16 repeatability, reliability or transferability. 

### 17 Okay. This is really specific to Charge 

### 18 Question 2 where it's asking what do the endpoints tell us 

### 19 specifically about endocrine disruption? And the point I want 

### 20 to make with this slide is that some of the endpoint responses 

### 21 that you can see in these assays are not specific to an 

### 22 endocrine mode of action. 

### 23 If a fish is stressed out, there's a lot in 

### 24 the literature that shows us that a fish that's stressed is just 

### 25 not going to reproduce as well. A fish that's stressed will 

### 26 have lower circulating levels of sex steroid hormones, will have

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1 lower secondary sex characteristics and have lower vitellogenin 

### 2 and this is really an ecological principle that goes back to 

### 3 having a finite energy reserve. 

### 4 So if something's stressed, it just doesn't 

### 5 have the energy to put into things associated with reproduction 

### 6 that are energetically costly. I think this was mentioned 

### 7 yesterday, and I'm just reiterating it as well, that decreases 

### 8 in these parameters are not necessarily endocrine specific. 

### 9 For Charge Question 3, the question that's 

### 10 around just the assay endpoints, there's selection, optimization 

### 11 and demonstration; we've heard multiple comments just about the 

### 12 genotyping of the fish. And what we see in the inter-laboratory 

### 13 studies is there's some sort of problem going on here with this 

### 14 and really we just need better guidance to make sure that this 

### 15 important endpoint is done correctly. 

### 16 Again, not all the data may be present in the 

### 17 Tier 2 assays to definitively determine if the adverse effect on 

### 18 population-level endpoints, this is the data you'd use in a risk 

### 19 assessment like reproduction, growth, development, these are 

### 20 apical endpoints meaning that they can be influenced by a lot of 

### 21 different things. 

### 22 So you might see something like a change in 

### 23 vitellogenin at a lower concentration, let's say, but you cannot 

### 24 be absolutely certain that biochemical change is also the same 

### 25 mode of action that's causing your effect on your apical 

### 26 endpoint. You can't make that definitive link. So that's a

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1 limitation that we have in these Tier 2 assays. 

### 2 With this slide, I just do want to point out 

### 3 that there were several different kinds of reference chemicals 

### 4 evaluated in both the full MMT and the abbreviated MMT. 

### 5 The point I want to make here is that, as we 

### 6 heard yesterday, this protocol changed throughout the validation 

### 7 effort. It was being optimized, as it should be, and at the end 

### 8 we have a better, I think, test design, the Medaka Reproduction 

### 9 Test proposed. 

### 10 But I just want to point out that this study 

### 11 design, the MRT, has not been ring tested, hasn't been tested in 

### 12 various laboratories. As you see here on the screen, the 

### 13 increased replication design was tested in one lab with a weak 

### 14 estrogen agonist at one time. So we just have to keep that in 

### 15 mind that that has not been ring tested. 

### 16 The other point I want to make is that it 

### 17 would actually be interesting to run a non-endocrine-active test 

### 18 material through the assay in the validation effort. And this 

### 19 would be potentially something that has another known mode of 

### 20 action, and the reason for doing this would be able to get an 

### 21 evaluation of what these supposedly diagnostic endpoints in the 

### 22 assay, how they would respond to something that has a mode of 

### 23 action that is not primarily endocrine but has some other known 

### 24 mode of action, for instance, a hepatotoxin, what does that do 

### 25 to vitellogenin? Let's put that out there so we can understand 

### 26 the endpoints and how specific they really are for endocrine.

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1 I also again want to point out that there is 

### 2 only one assay with the increased replication as proposed in the 

### 3 MRT that's been tested with only one mode of action. 

### 4 The statistical methods seemed appropriate, 

### 5 but there was some confusion about is it okay to run a Dunnett 

### 6 test and a Jonckheere-Terpstra to determine treatment-related 

### 7 differences from controls, to run both of those tests. 

### 8 So just to solidify that that decision should 

### 9 be made a priori looking at your dataset, if it's monotonic or 

### 10 not, which is the appropriate statistic to employ, and then just 

### 11 one statistical test should be employed to determine 

### 12 significance of your treatment versus your control. I also want 

### 13 to point out that Appendix E in the materials provided which was 

### 14 the MRT protocol, the statistical section didn't match that in 

### 15 the Integrated Summary Report for the MMT, so that just needs 

### 16 some clarification. 

### 17 As brought up before, the solvent control 

### 18 issue needs also clarification. When a solvent control is used, 

### 19 and you do a test to see is your solvent control different from 

### 20 your negative control, if the answer is yes, there's some 

### 21 confusion about what you do there. 

### 22 In the Integrated Summary Report, it was 

### 23 indicated that treatments that should then be compared to your 

### 24 dilution control, your negative control. However, in the OECD 

### 25 guidance document for Statistical Approaches For Ecotoxicity 

### 26 Tests, the guidance is actually to compare to your solvent

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1 control. And that guidance document is also cited in the 

### 2 Integrated Summary Report as something to follow. So again, 

### 3 this is an issue that needs some clarification so that labs are 

### 4 performing the statistics appropriately. 

### 5 I think this has been brought up before, so 

### 6 I'll just go over this very quickly, that the control data and 

### 7 the treatment-related data with vinclozolin, these were all 

### 8 different between labs, and it really seemed that this was due 

### 9 to the differences in the feeding regimen and the technical 

### 10 problems of the genotypic sexing. 

### 11 Also in addition with vinclozolin, there were 

### 12 some issues with test material delivery and just the fact that 

### 13 it didn't provide a really strong response in the assay and 

### 14 therefore it made it hard to compare assay results. So those 

### 15 are some of the issues with repeatability and reproducibility. 

### 16 And as has been acknowledged, a quote from the 

### 17 Integrated Summary Report was that the expectation for any 

### 18 specific response to the AR antagonist vinclozolin is uncertain. 

### 19 20 So unfortunately, the ring test that was done, 

### 21 not on the protocol that's being proposed but on the 

### 22 2-1/2-generation MMT, that abbreviated MMT, really didn't give 

### 23 us a lot of information. So the MMT is not transferrable. The 

### 24 newly proposed MRT, which is shorter in duration and has 

### 25 increased replication and also includes specific guidance when 

### 26 the sub-adults are to be examined at week nine, this has not

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1 been ring tested. This specific design doesn't seem to have 

### 2 even been conducted. So this is a shortcoming in the validation 

### 3 effort. 

### 4 Also, testing with this proposed design should 

### 5 include more than one mode of action. It should be evaluated 

### 6 with various other modes of action. We need to work out the 

### 7 genotypic-phenotypic match problem. There's specific 

### 8 refinements in the proposed MRT protocol needed that have been 

### 9 provided in the written comments. And again, a correlation 

### 10 between the fathead minnow and medaka would be useful if we're 

### 11 going to at any time be thinking about looking at Tier 1 and 

### 12 Tier 2 together with the fish. 

### 13 The strengths of the Medaka Reproduction Test, 

### 14 this is the protocol outlined to move forward. It has a lot of 

### 15 strengths. It's a chronic test. It includes sensitive life 

### 16 stages. It includes apical endpoints that are useful in risk 

### 17 assessment. It does include enough concentrations that you 

### 18 could theoretically do a dose response. It does have additional 

### 19 replication that seems to be much better for assessing 

### 20 biologically significant effects on reproduction. It uses a 

### 21 fairly well-studied fish species, the medaka. It does actually 

### 22 have the potential to detect estrogen agonists and androgen 

### 23 agonists fairly well. 

### 24 And the point I want to make here is that the 

### 25 diagnostic endpoints here are a little bit more specific for 

### 26 these modes of action. For instance, if there's an increased

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1 vitellogenin in males, well, this is likely more specific to an 

### 2 estrogen receptor mode of action. Also, if you do see the 

### 3 presence of male secondary sex characteristics in females, that 

### 4 is likely pretty specific to an androgen receptor agonism. So 

### 5 there's several modes of action that could potentially be picked 

### 6 up fairly well in these assays, we just need to test further to 

### 7 see this. 

### 8 The limitations, these other modes of action, 

### 9 endocrine activity, the anti-androgen, the anti-estrogen and the 

### 10 steroidogenesis inhibitors, because the endpoint responses here 

### 11 are very similar to what you'd see with a generalized stressed 

### 12 response, these are more nonspecific. So it's a little bit 

### 13 harder to nail down these modes of action with this assay. 

### 14 We have some limitations in correlating Tier 1 

### 15 to Tier 2 if we're going to do that at any time because we have 

### 16 a different test species. Just coming from the lab in my own 

### 17 personal experience, any time you run a long assay you're afraid 

### 18 because there's just going to be a chance with a longer assay 

### 19 that random events will occur, especially in your mechanical 

### 20 stuff, to deliver a waterborne exposure. So that can be a 

### 21 limitation. 

### 22 The route of exposure is potentially 

### 23 difficult, and I think Dr. Johnson addressed this yesterday, 

### 24 indicating, you know, there's some compounds that have low water 

### 25 solubility that are very hard to deliver, also compounds that 

### 26 can biodegrade over time, and this makes it very tricky to keep

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1 those concentrations at the appropriate level over a chronic 

### 2 test. 

### 3 Again, non-endocrine mediated toxicity, that 

### 4 overt toxicity that could be occurring, that could cloud the 

### 5 interpretation and make it more difficult for determining 

### 6 endocrine-specific mode of action. However, overall, this 

### 7 doesn't affect the endgame which is to get an endpoint that can 

### 8 be used in an environmental risk assessment. 

### 9 In closing, there's not good reproducibility 

### 10 or transferability right now of the MMT design. And another 

### 11 limitation is that laboratories are going to need not only 

### 12 expertise in environmental toxicology but analytical, genomic, 

### 13 histopathological expertise as well. And the laboratories that 

### 14 could do this under one roof are few and far between, so that 

### 15 can be a limitation for getting these conducted in a reasonable 

### 16 timeframe. 

### 17 This is particularly to Charge Question 8, 

### 18 what's the concern potentially with the loss of the F2, and in 

### 19 really looking at the LOEC values for the F1 and the F2 on the 

### 20 whole. In the MMTs that have been conducted to date, there 

### 21 doesn't seem to be a great loss with that not including the F2 

### 22 up through reproduction. The data indicate that lower NOECs and 

### 23 LOECs values would not routinely be expected in the F2. So 

### 24 terminating the assay after F2 hatch appears to be supported in 

### 25 the current dataset. 

### 26 So just in conclusion, I think the Medaka

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1 Reproduction Test holds promise as an improvement over the 

### 2 Medaka Multigeneration Test for all of the reasons listed there. 

### 3 However, we're sort of at the pre-validation stage. We now 

### 4 have a study protocol that's been optimized but not ring tested, 

### 5 so we need to ring test it before it's ready for use. We need 

### 6 to ring test it with a compound that's easier to work with, has 

### 7 a stronger response in the assay that's to be expected and we 

### 8 need to ring test including this revised protocol. 

### 9 So the MMT and the MRT, in closing, they have 

### 10 population-level relevant endpoints useful for risk assessment, 

### 11 but they can be limited in their ability to definitively link to 

### 12 endocrine mode of action. This is just something to keep in 

### 13 mind as interpretation goes forward. Thank you. 

### 14 **DR. MARTHA SANDY:**Thank you very much. I'd 

### 15 like to ask Katie Holmes come up for the next presentation. 

### 16 **MS. KATIE HOLMES:**Hello. My name is Katie 

### 17 Holmes, and I work for BASF, and I also serve as the Endocrine 

### 18 Policy Forum regulatory affairs chairperson. I'm commenting 

### 19 today on the Larval Amphibian Growth and Development Assay, and 

### 20 that's the last time you'll hear me call it that. From here on 

### 21 it will be the LAGDA because it's just a lot faster, and I will 

### 22 certainly run out of time if I say Larval Amphibian Growth and 

### 23 Development Assay every time. 

### 24 Thought I'd start with just a little bit of 

### 25 background. The amphibians are slightly different than some of 

### 26 the organisms. They're still a fairly new organism to our

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1 little world of environmental toxicology testing in the 

### 2 regulatory arena. There is no existing standard guideline EPA 

### 3 study right now. Under the current risk assessment scenario, 

### 4 the fish data are usually used in lieu of the amphibian data. 

### 5 There's a critical review paper that's just getting ready to 

### 6 come out from Velcha, (phonetic) et al., and it looks 

### 7 specifically at the sensitivity between fish and frogs and has 

### 8 concluded that amphibians are equally or less sensitive than 

### 9 fish in most cases. So I would -- it's in press right now in 

### 10 ET&C. 

### 11 I want to make clear during these comments, we 

### 12 are not questioning the scientific validity of this amphibian 

### 13 assay. What we are questioning a little bit is if it is fit for 

### 14 purpose for what we're looking for in the EDSP program which is 

### 15 a really different question. I think the assay itself has a lot 

### 16 of scientific validity. There's a lot of good work that's been 

### 17 put into it. It's just how does it fit into the EDSP program 

### 18 which is the overall question. 

### 19 Let's see. According to the ISR, the LAGDA is 

### 20 intended to serve as a higher-tier test with amphibian for 

### 21 collecting definitive concentration response information on 

### 22 adverse effects suitable for use in the ecological risk 

### 23 assessment. 

### 24 And the key there is ecological risk 

### 25 assessment. Ecological risk assessment has traditionally been 

### 26 done on population-level effects, and that's an important thing

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1 to think about when we're looking at these biomarkers and things 

### 2 like that. We're really looking for endpoints that we can use 

### 3 in ecological risk assessment. 

### 4 If we are going to spend the financial and 

### 5 animal resources necessary for this chronic test, the data 

### 6 generated really must add value to the EDSP assessment and 

### 7 inform the risk assessment. 

### 8 I'm going to go now through the different test 

### 9 endpoints. Mortality, growth and development delay are apical 

### 10 endpoints that have clear relevance to the environmental risk 

### 11 assessment. As was mentioned yesterday and has come up several 

### 12 times, these endpoints may or may not be related to endocrine 

### 13 modulation. Hopefully, by the time we're at Tier 2, we've 

### 14 determined the mode of action in Tier 1 or the proposed mode of 

### 15 action and we can see if they're related. 

### 16 The other thing I want to make a point of here 

### 17 is that these studies are being run in a way that will be 

### 18 protective without the knowledge of mode of action. You all 

### 19 were talking a little bit a while ago about the protective 

### 20 nature of these studies. These are studies that are conducted 

### 21 over the entire lifecycle or more to the lifecycles of an 

### 22 organism where the organism is exposed to concentrations 100 

### 23 percent of the time, 24 hours a day, all day, every day over the 

### 24 life cycle and often multiple lifecycles. And they're often 

### 25 exposed at concentrations that are above anything that's 

### 26 environmentally relevant. So it's hard to imagine that that

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1 won't be protective when we get the responses to those studies. 

### 2 I'm trying to be respectful of the time. 

### 3 Due to the linkages with adverse outcomes at 

### 4 the population level, we're questioning the use of the 

### 5 histopathology in the liver somatic indices. They're not 

### 6 traditionally been used in environmental risk assessment. We're 

### 7 anxious to see what the proposal is. Will those be endpoints in 

### 8 the risk assessment, or are they just information as to mode of 

### 9 action in the end? So that's a question that's sort of still 

### 10 lagging out there. I think that's enough for that one. 

### 11 The vitellogenin and T4 are used as biomarkers 

### 12 in this assay. The endpoints are least likely to inform a 

### 13 traditional ecological risk assessment. The vitellogenin 

### 14 induction is already well measured in the Tier 1 FSTRA, so we're 

### 15 wondering if it might be redundant here and if it's something 

### 16 that really is going to inform the long-term goals of this 

### 17 study. T4 measurements seem to maybe be more appropriate as a 

### 18 determinative mode of action in Tier 1. And so if we're going 

### 19 to be measuring T4, perhaps should we consider moving that back 

### 20 to the frog study in Tier 1 where we're specifically tasked to 

### 21 look at mode of action. 

### 22 The HPG axis, we're looking at -- well, what 

### 23 we're thinking, sorry about that, is that the medaka test looks 

### 24 -- does a much better job at looking at HPG axis issues than 

### 25 does the frog study. Medaka includes measures of fecundity, 

### 26 while the LAGDA endpoints are not directly related to

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1 reproduction. 

### 2 We would like to request or suggest that 

### 3 because of this that the EPA consider doing a comparison between 

### 4 the amphibian and fish sensitivity to alterations and gonad 

### 5 differentiation and development and then decide, based on that 

### 6 assessment, if both assays are justified. If they're redundant, 

### 7 they're both taking a lot of time, energy, money and animal 

### 8 resources, do we really need them both? 

### 9 In terms of the HPT axis, there are three 

### 10 endpoints in the LAGDA that are looked at. The first two, T4 

### 11 and thyroid histopathology, don't usually inform risk 

### 12 assessment. They don't quantitatively inform for sure. The 

### 13 delay in development is the only HPT axis ecological risk 

### 14 assessment relevant endpoint. 

### 15 I thought the presentation yesterday did a 

### 16 great job at looking at some of the vulnerability of the study 

### 17 and some of the issues with the transferability of the 

### 18 endpoints. Obviously, some of these things still need work. 

### 19 Hopefully, you've all had a chance to look at our paper. We go 

### 20 into a little bit more detail in the written comments that we've 

### 21 provided. 

### 22 There was a wide variability in the growth and 

### 23 mortality of the control individuals. The vitellogenin ELISAs 

### 24 that were run, EPA had ended up needing to supply the antibody 

### 25 which is something need to be -- it's not something that can't 

### 26 be cleared up, but it's something that needs to be finalized

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1 before industry is asked to run many of these studies. 

### 2 Based on the information that came out just 

### 3 Friday in the ICR, looking at Tier 2; there are 26 of our 

### 4 compounds maybe will be moving forward to Tier 2 testing. And 

### 5 if that's true, and the frog study is one of those that's based 

### 6 on exposure, the study itself, I'll mention later, is going to 

### 7 be somewhere around $500,000 and PETA said 750 organisms. 

### 8 So we better be justified in running the study 

### 9 by the time we get there. Even if only half of those, that's 

### 10 almost $10 million' worth of studies, and I can't do the math 

### 11 fast enough on the organisms. But we need to make sure that we 

### 12 really know what we want and that the data are going to be used 

### 13 before we get to the end of the process. 

### 14 T4 was only measured adequately in two out of 

### 15 the five labs. None of this is unsolvable, but these things 

### 16 should be addressed prior to asking people to run these studies, 

### 17 and that's really our point, not that they're a bad idea or they 

### 18 can't be done, but it's not ready if we can't do that yet. 

### 19 And then finally, just a reality check. There 

### 20 are exactly four laboratories in the U.S. right now that can do 

### 21 the frog histopath. That's it, that industry knows of. Now 

### 22 there may be some academics, but they may not be working under 

### 23 GOP. We won't be able to use that data. There are four. 

### 24 That's not a lot of labs. We have Tier 1 studies that have both 

### 25 fish and frog, and now we have the Tier 2 tests that will also 

### 26 have fish and frog histopath, four labs.

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1 There's not a lot of experience in this, and 

### 2 that's one of the reasons I think we saw so much variation. So 

### 3 we need time to come up to speed and practice. 

### 4 I think you've heard enough about the 

### 5 statistical message. You can take what you heard for the fish, 

### 6 restate it, and we'll move on. 

### 7 In terms of test reproducibility and 

### 8 repeatability, the way that the ISR was written, it was very 

### 9 difficult for us to look at individual studies themselves. When 

### 10 we're working in a regulated arena, we only get one study. We 

### 11 don't get three or four or two and a half to look at. And so we 

### 12 need to be able to rely on the results of that one study to 

### 13 inform our decision making. 

### 14 And things being generally consistent across 

### 15 studies aren't good enough. We need a study that can stand 

### 16 alone, and right now, there are enough endpoints within the 

### 17 context of this study that aren't quite defined that that would 

### 18 be difficult to trust that you have a good study at the end. 

### 19 This particular table was really interesting 

### 20 to me. I pulled it out and put it in here. First, I want you 

### 21 to notice this is an NOEC table. We're looking at the growth of 

### 22 the amphibians in the prochloraz study. Because it's an NOEC 

### 23 table, what that really means is where we see the 170 and the 

### 24 164 in Lab A and Lab C, the LOEC which is what most people have 

### 25 been talking about through this meeting are actually above those 

### 26 levels. So your LOEC is really greater than 170.

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1 If you look at Lab D and Lab B, those are both 

### 2 effects studies. Lab A and C are no effects studies. The 

### 3 difference between greater than 170 and 20 or 59 is very 

### 4 different than looking at this data. 

### 5 Having been in the lab for a long time, can 

### 6 say that the dose setting on these -- and this is an issue that 

### 7 goes across all four, and I think it's really important -- dose 

### 8 setting is going to be very hard. We are looking at bracketing 

### 9 both biomarkers and the lifecycle development of an organism. 

### 10 In this study, the concentrations were set in 

### 11 a way that would be acceptable to OECD but generally not 

### 12 acceptable to EPA unless you had some extenuating circumstance. 

### 13 The dilution factor here is based on .3 which means it goes 180 

### 14 to 60 to 20, the numbers. We are for our chronic studies, 

### 15 unless it's going to be changed, really obligated to use .5 

### 16 which would mean the only way that we could cover this same 

### 17 range would be to add additional concentrations which would be 

### 18 additional frogs, additional cost, additional time. 

### 19 So I understand this is a validation and when 

### 20 the rubber hits the road, and we're running these studies not 

### 21 very long from now, the ICR is out. We're going to need to set 

### 22 the doses for these and anything you all can have to say about 

### 23 that would be really useful for us. It's going to be difficult 

### 24 to cover both ends of the spectrum unless we can do a wider 

### 25 concentration range, but when we do that we lose some of the 

### 26 ability to come up with reasonable NOECs and LOECs in our risk

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1 assessment because our range gets too broad. 

### 2 The strengths of the LAGDA are that it 

### 3 provides chronic toxicity in a class of organisms that are not 

### 4 typically tested in environmental toxicology. The measures in 

### 5 survival and growth are particularly relevant for environmental 

### 6 risk assessment. The mechanistic data that's generated, while 

### 7 potentially of limited use for the ecological risk assessment, 

### 8 may help resolve issues with specific chemicals with concerns in 

### 9 amphibians. 

### 10 The limitations, we don't believe that 

### 11 transferability between labs was demonstrated with respect to 

### 12 husbandry or some of the endpoints. Many of the endpoints 

### 13 required are not necessarily relevant to the ecological risk 

### 14 assessment. 

### 15 This is likely going to be the most expensive 

### 16 single ecological test that we're required to perform under the 

### 17 standard test guidelines right now. There is no final protocol. 

### 18 But what there is right now suggests that the cost of the 

### 19 biology without any of the histopath and things is around 300- 

### 20 to $350,000. When you add to that the histopath and the 

### 21 biomarker measurements, it could easily get to $500,000. And 

### 22 that's for one of the studies. So it needs to be defined, and 

### 23 we need to have a good study before we start running it. 

### 24 It's unclear if the labs can adequately 

### 25 perform some of the more technically challenging measurements. 

### 26 It's not that they can't we just need practice and to make sure

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1 that those guidelines are out there and then it's been tried 

### 2 before we start. And then the lack of experience with histopath 

### 3 with amphibians will happen as we get experience with histopath 

### 4 and amphibians. 

### 5 Finally, our conclusions, we don't believe the 

### 6 LAGDA has been adequately validated. Inconsistent husbandry 

### 7 across the labs needs to be resolved. While the T4 and 

### 8 vitellogenin measurements have potential to inform mode of 

### 9 action, if they can't be inform reliably their measurement 

### 10 provides little value and should be reconsidered as required 

### 11 endpoint. 

### 12 Medaka is likely protective of HPG effects, 

### 13 eliminating the need to run LAGDA past metamorphosis. Just 

### 14 again, those two tests need to be put together and see which 

### 15 provides value where. 

### 16 And finally, there's limited value in the 

### 17 ecological risk assessment of relevant thyroid-related 

### 18 endpoints. 

### 19 And that is all I've got. Thank you very 

### 20 much. 

### 21 **DR. MARTHA SANDY:**Thank you very much. 

### 22 And I'd like to call up John Brausch, please. 

### 23 **DR. JOHN BRAUSCH:**Good morning. Thank you. 

### 24 My name is John Brausch, and I'll be presenting the public 

### 25 comments regarding the Tier 2 invertebrate studies once I get 

### 26 slides.

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1 Thank you. 

### 2 So as we've seen before, I'm going to start 

### 3 with the conclusions. And this will really allow us to focus on 

### 4 some of the important points with an understanding of where it's 

### 5 all going to lead in the end. 

### 6 So Dr. Klaine brought up yesterday that 

### 7 there's really very little guidance on when this invertebrate 

### 8 study would be needed, as there's no E, A or T pathways in 

### 9 invertebrates. We've got some feedback on how this study is 

### 10 going to be implemented in the Tier 2, but we need a lot more 

### 11 information, and then these decisions are going to need to be 

### 12 thoroughly vetted, so we all have confidence that doing this 

### 13 study is actually going to enhance the EDSP program or 

### 14 protecting aquatic invertebrates. 

### 15 I'm going to abbreviate the studies as MTTT, 

### 16 which is the Mysid Two-gen, or the HCDRT, which is the copepod. 

### 17 And we feel that neither has been appropriately validated, and 

### 18 thus with the current status they're likely not suitable for 

### 19 regulatory studies. 

### 20 In the ISR, it pretty clearly states that the 

### 21 mysid shrimp are the species that we want to move forward with, 

### 22 and we would agree with this. So as we move forward, I'm going 

### 23 to focus primarily on the mysid but just point out little things 

### 24 with the copepod when appropriate. And also, I hope we get to 

### 25 the point where we realize that the mysid chronic studies that 

### 26 are currently conducted for pesticide registration and

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1 re-registration might be a suitable replacement for the MTTT if 

### 2 we're looking to replace or just to further enhance our 

### 3 understanding. 

### 4 The one question I kind of want to remind 

### 5 everybody to think about the entire presentation, and as you're 

### 6 going through the ISRs, really how does this study enhance our 

### 7 understanding of population-level effects? And even more so, 

### 8 how does it enhance the EDSP program as stated? 

### 9 So as we've heard, mysid have been used for a 

### 10 long time in regulatory testing. One of the common studies is 

### 11 the mysid chronic study which is 28 days. What's important 

### 12 about this is the endpoints that are looked at. We look at 

### 13 survival, growth and reproduction already for the first 

### 14 generation, and then there's also the possibility to look at 

### 15 second generation survival and length, and if you take it a 

### 16 couple extra days even the sex ratio. 

### 17 A lot of these endpoints are the exact same 

### 18 endpoints that we're looking at in the MTTT, but rather than 

### 19 being a 60-day assay we're looking at a closer to 28-day assay. 

### 20 So this could be beneficial for a lot of the problems that have 

### 21 been observed thus far. 

### 22 I want to follow kind of the charge questions, 

### 23 and I'll kind of run through them quickly. But before we do, I 

### 24 want to remind everyone what EDSTAC's envision was for the Tier 

### 25 2 studies. The Tier 2 studies are to characterize the dose 

### 26 response relationship of endocrine disruption of the E, A and T

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1 pathways in humans and wildlife. So as we think about that, how 

### 2 does this study actually fit into that initial definition? 

### 3 Well, there are some positives of using an 

### 4 invertebrate. Invertebrates are very common throughout the 

### 5 world, and 95 percent of the species worldwide. An aquatic 

### 6 invertebrate even furthermore, as aquatic systems tend to be 

### 7 syncs for anthropogenic compounds. 

### 8 But there's also a number of negatives using 

### 9 an invertebrate and an aquatic invertebrate. As a whole, 

### 10 invertebrates' endocrine systems are really poorly understood, 

### 11 and this could lead to many difficulties in trying to determine 

### 12 the potential for endocrine disruption. Additionally, 

### 13 invertebrates have no estrogen, androgen or thyroid system. So 

### 14 this is almost contrary to what the Tier 2 studies were 

### 15 initially to look even though we've got some further information 

### 16 yesterday on how the invertebrate could potentially add to the 

### 17 system. 

### 18 I think what this shows is that invertebrates 

### 19 and aquatic invertebrates are definitely justified for chemical 

### 20 regulation. We know they're sensitive. We know they're common. 

### 21 But this doesn't actually justify its use in the EDSP studies 

### 22 specifically. 

### 23 Real quickly, hormone systems in invertebrates 

### 24 are generally very different than vertebrates. There's two main 

### 25 types or sets of hormones in invertebrates, and those would be 

### 26 similar to vertebrates such as serotonin, for example, or the

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1 invertebrate specific such as ecdysone and the ecdysteroids. 

### 2 Ecdysone and similar compounds are responsible 

### 3 for all growth, reproduction and embryogenesis in invertebrates, 

### 4 very important class of steroids. In the detailed review paper 

### 5 from 2004, they mention this, and they talk about it, and they 

### 6 say that they're even analogous to a number of vertebrate 

### 7 hormones. But it's very important to remember they're 

### 8 structurally, functionally and metabolically different from the 

### 9 vertebrate hormones themselves, and thus, they very likely have 

### 10 very different responses than what would be seen in invertebrate 

### 11 study. 

### 12 I think it's clear that mysids are 

### 13 ecologically relevant and sensitive. But this alone does not 

### 14 actually justify a multigeneration exposure for them. We'd have 

### 15 to take into account what that multiple-generation exposure 

### 16 actually enhances in our understanding. 

### 17 One example I'd like to give was from the ISR, 

### 18 and that's for methoprene. Methoprene would be a characteristic 

### 19 endocrine modulator in invertebrates. It is an analog to 

### 20 juvenile hormone. 

### 21 The results from this study demonstrate that 

### 22 numerous endpoints were affected, but all of these endpoints are 

### 23 observed in the chronic mysid study, once again, further 

### 24 illustrating how this mysid chronic study could be beneficial. 

### 25 There are greater than 20 endpoints analyzed 

### 26 in the mysid two-gen for both generations. None of the

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1 endpoints are specifically endocrine specific, and all endpoints 

### 2 are influenced by overt toxicity. 

### 3 One of the problems with these endpoints is 

### 4 there's a large degree of interrelatedness. Things such as 

### 5 female body length and offspring produced per female are 

### 6 interrelated. Therefore, if you see an effect on offspring per 

### 7 female, you'd have to almost do your own weight of evidence 

### 8 approach to try to determine where that effect is coming from. 

### 9 And once again, virtually all these endpoints 

### 10 can be assessed in the mysid chronic study, and the ISR even 

### 11 hints at that when it says that additional information obtained 

### 12 in this second generation was not obvious. 

### 13 Here's a table similar to what we've seen, but 

### 14 I want to point something out. We're going to be looking at 

### 15 NOECs here versus LOECs. So that's why you're going to see 

### 16 differences in numbers. The reason why I'm presenting the NOEC 

### 17 is because that's actually the regulatory value of concern. 

### 18 That's the number that the chemical industry gets analyzed on, 

### 19 so I want to present those because they give a much better 

### 20 understanding of what the impacts of these studies could 

### 21 potentially be. 

### 22 In general, the first-generation NOEC was 

### 23 lower than the two-generation NOEC, indicating that a 

### 24 shorter-term study would be appropriate to determine effects and 

### 25 to promote safety of aquatic invertebrates. 

### 26 For the inter-laboratory validation, only two

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1 of the three labs appropriately conducted the different studies 

### 2 for the different compounds. And I would suggest only one 

### 3 compound was actually used for validation, and that would be 

### 4 lindane. 

### 5 We saw octylphenol results from yesterday. 

### 6 The one issue with the octylphenol results is that although it 

### 7 met the performance criteria in that the controls in the second 

### 8 generation produced sufficiently, none of the exposed 

### 9 individuals reproduced, and therefore, no results from that 

### 10 second generation can be used. So basically, for one lab we're 

### 11 comparing a two-generation study, and in the other lab we're 

### 12 comparing a two-generation for the controls and no -- a 

### 13 one-generation for the exposed. So it's not quite an 

### 14 apples-to-apples comparison. 

### 15 The one compound that was adequately validated 

### 16 was lindane. When you look at the actual results, only one of 

### 17 the 25 endpoints that showed an effect -- so there were a lot of 

### 18 endpoints that did not show any effect. But for the response 

### 19 endpoints, only one of the 25 endpoints showed definitive 

### 20 concurrence. 

### 21 There were trends that lined up. There was a 

### 22 trend in one and a definitive statistical difference in one. 

### 23 But for ones that actually had statistical significance, there 

### 24 was only a single endpoint. And to kind of show what that 

### 25 means, it was actually for the second generation, the Day 14 

### 26 length but only at the second lowest concentration. I would

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1 suggest that's not very good concurrence between the labs. 

### 2 Once again, there's transferability issues 

### 3 between the labs that we saw major differences in the control 

### 4 groups specifically. And when you take it even past the control 

### 5 groups, if you look at this regulatory value of concern, the 

### 6 NOECs actually differed by almost twentyfold for lindane. That 

### 7 would be a very large difference if you're doing a risk 

### 8 assessment. 

### 9 For the test substances, we feel that the test 

### 10 substance selection was not optimal. Only one compound that was 

### 11 tested had known endocrine effects in invertebrates, and that 

### 12 was fenoxycarb. That was done very, very early on in the 

### 13 process. 

### 14 But more so, we feel a major opportunity was 

### 15 missed. I mentioned this mysid chronic study that is routinely 

### 16 done for registration and re-registration. However, only two of 

### 17 the compounds that they looked at actually had this mysid 

### 18 chronic data. 

### 19 There's a whole list of compounds that could 

### 20 be investigated, and then that way you could compare between the 

### 21 mysid chronic study and the two-gen to really see what value was 

### 22 added. However, the only two compounds, atrazine and fipronil, 

### 23 unfortunately had non-definitive endpoints in the mysid two-gen, 

### 24 so no real comparisons could be made. 

### 25 For invertebrates it's also really important 

### 26 that compounds are run through the entire battery. We don't

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1 know how mysids are going to respond to vertebrate hormones or 

### 2 vertebrate endocrine-active compounds. The only way to 

### 3 determine that is additional research. This would be a great 

### 4 opportunity to kind of retroactively go back and run compounds 

### 5 through the Tier 1 battery and then the additional Tier 2 test, 

### 6 and then you could really get a better feel of how the mysid 

### 7 comes into the EDSP and really what it adds to it. 

### 8 The number of strengths and limitations, some 

### 9 of the strengths of the mysid specifically is they've got a long 

### 10 history regulatory testing. Analytical verification is possible 

### 11 with the mysid two-gen. That's one thing we didn't hear. The 

### 12 copepod, currently there's no way to do analytical verification 

### 13 of the test compounds due to the minimal amount of water used. 

### 14 So that's something to keep in mind. 

### 15 Also, the mysid also provides the needed 

### 16 information for population modeling. But this is also true for 

### 17 the mysid chronic study. The same authors that really came out 

### 18 with ways to use the two-generation for population modeling, if 

### 19 you'd look at their other papers that they have come up with, 

### 20 they've also used it for the mysid chronic study. So these are 

### 21 basically similar between the two. 

### 22 There are a number of limitations with the 

### 23 mysid and the copepod assays, and that would be just the limited 

### 24 understanding of endocrine function and really the negligible 

### 25 value of the additional generations. The large variability in 

### 26 endpoint responses between the labs is definitely concerning,

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1 especially at the large degree we saw. 

### 2 And that kind of takes us back to where we all 

### 3 started 15 minutes or so ago, I hope. Mysid shrimp are probably 

### 4 the most appropriate species, but really, are they appropriate 

### 5 for the EDSP? We would suggest currently probably not. 

### 6 However, at some point they might be, but additional research 

### 7 would need to be done, and really this system would need to be 

### 8 understood. We also feel there's really no need for a second 

### 9 generation, as the first generation would be sufficient for 

### 10 protection goals and to protect aquatic invertebrates. 

### 11 So I'd like to just leave with a couple last 

### 12 words, just some quotes from the ISR directly. But I asked one 

### 13 thing in the beginning was to remember the question what does 

### 14 the MTT add to both aquatic risk assessment for invertebrates as 

### 15 well as the EDSP? From my point of view, I don't think it 

### 16 really adds a whole lot, as the mysid chronic study likely 

### 17 incorporates all the necessary endpoints as well as provides the 

### 18 necessary safety for aquatic invertebrates. Thank you. 

### 19 **DR. MARTHA SANDY:**Thank you very much. 

### 20 The last speaker is Dr. Mihaich. Welcome. 

### 21 **DR. ELLEN MIHAICH:**Thanks. Slides? Sorry. 

### 22 So what I want to do is just talk a little bit 

### 23 about some of the overarching themes I think that we've been 

### 24 hearing over the last four talks. And again, I'm Ellen Mihaich, 

### 25 and I am the scientific coordinator for the Endocrine Policy 

### 26 Forum.

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1 So I wanted to start with sort of questions to 

### 2 be asked. You know, you're here to determine the validity of 

### 3 these four ecotoxicity tests, and then after that, we're the 

### 4 ones that are going to have to use them. So we have come up 

### 5 with, in these last four presentations, and in our fairly 

### 6 extensive 60 pages of written comments, which I'm sure you've 

### 7 read cover to cover since you've only had a couple days to do it 

### 8 in; I'm sorry about that. 

### 9 But we really feel like there's insufficient 

### 10 validation to determine if the studies are reliable and relevant 

### 11 which means they're fit for purpose. 

### 12 In Appendix A of each of the studies, you 

### 13 know, you've got a description of what validity means, and this 

### 14 is a regulatory program that there is a mandate for valid tests. 

### 15 So in asking the questions then, will sufficient 

### 16 inter-laboratory testing with more modes of action be allowed to 

### 17 assess the reliability, sensitivity and specificity of these 

### 18 studies? 

### 19 Is one of these species or two of these 

### 20 species more sensitive than others? That's going to be the most 

### 21 relevant for risk assessment, you know, in that can we look at 

### 22 the endpoints that there, you know, I know the EPA has talked 

### 23 about which -- this is not a battery. They'll be picking and 

### 24 choosing. 

### 25 Well, is there something that we can look at 

### 26 to decide which ones would be, if we had an idea of a particular

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1 mode of action, which ones would be the most relevant to be 

### 2 choosing to help us? Could some endpoints be removed? I mean 

### 3 unfortunately, I don't think that there's enough information to 

### 4 date yet to look at somewhat of a sensitivity analysis, look at 

### 5 the CVs around some of these endpoints to know whether or not 

### 6 they're useful. I mean because every endpoint you choose to do 

### 7 is just one more test you have to evaluate and one more way to 

### 8 sort of muddy the waters because you might have inconsistent 

### 9 responses. 

### 10 And does testing in the second generation 

### 11 really provide any additional information that would impact the 

### 12 risk assessment? Remembering that Tier 2 data is for risk 

### 13 assessment, and given that ecological risk assessment, we look 

### 14 at population-level effects of survival, growth and 

### 15 reproduction. Is it really critical to define a particular or 

### 16 specific mode of action? I mean I think that's a good question 

### 17 to ask. 

### 18 And these are, again, these are going to be 

### 19 guideline studies in the end, so 870 or 890 series guidelines. 

### 20 And for those of you that were here last month for the last SAP, 

### 21 I had brought up some of the challenges we had in the Tier 1 

### 22 guidelines which were that they weren't -- we wanted to have 

### 23 some public comment, and so I'm just asking again will public 

### 24 comment be sought on the draft test guidelines. I mean you have 

### 25 protocols in the ISR, not necessarily guidelines. 

### 26 So I'm just going to go separately through

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1 some of these if I can. 

### 2 Okay. So looking at the question of 

### 3 validation; really you're being asked a very important point 

### 4 here, and that is because since Tier 2 results are for risk 

### 5 assessment, we need to make sure that we understand scientific 

### 6 validity of these studies. I mean this is important. They're 

### 7 going to be used to determine whether or not a chemical is going 

### 8 to be a problem in the environment or not. And so we need to 

### 9 have the confidence, we all need to have the confidence of the 

### 10 scientific validity of these studies. 

### 11 So that means we have to understand fully the 

### 12 sensitivity, specificity, reliability and relevance. This needs 

### 13 to be determined before test orders are sent out to be able to 

### 14 do it. I think there's been a lot of question about why 

### 15 couldn't labs do things. Well, I think we've been working as -- 

### 16 and this is not a problem, this is just the way the process 

### 17 goes. We're still learning a lot, but it shouldn't happen after 

### 18 it's for a regulatory program. It should happen before. 

### 19 And I think that we've all seen that there's 

### 20 been some inconsistent and sometimes contradictory results in 

### 21 the studies that we've been presented with. There's no common 

### 22 test substance that's gone through Tier 2 or across both tiers 

### 23 to really determine sensitivity and specificity. 

### 24 And as I think Katie brought up, Katie Coady, 

### 25 no negative or non-endocrine toxic chemical has been run in 

### 26 these to try to establish the actual specificity of the

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1 endpoints. So you know, it's not going to be telling us very 

### 2 much if you're trying to look at these biomarker endpoints but 

### 3 they're still impacted. 

### 4 And we know that many of them are impacted by 

### 5 overt, systemic type of toxicity and that's okay, we just need 

### 6 to understand. And it's also important for -- we talk a lot 

### 7 about adverse outcome pathways. Well, if that language is going 

### 8 to keep coming up, we really need to understand what that 

### 9 biomarker means to an adverse effect so that we can get there. 

### 10 We're not there yet. 

### 11 And I have to say that, you know, 

### 12 unfortunately, and I know we're all under time pressure and all 

### 13 of that. But in this case, a data evaluation appeared to be 

### 14 somewhat rushed. I know the whole discussion about the 

### 15 StatCHARRM challenge, and luckily it was caught. It was 

### 16 actually one of our scientists that caught it and then discussed 

### 17 it with the agency, but these things happen. It's not a 

### 18 problem. You go, you find it and you fix it. 

### 19 But if you're trying to rush through an 

### 20 evaluation to meet some timeline, then the scientists involved 

### 21 don't get the opportunity to really sit back and make sure it 

### 22 makes sense. You know, and that's all we're saying is we need 

### 23 to make sure that if this is going to be something for risk 

### 24 assessment, and we're going to rely on it, you need to have 

### 25 enough time to really look at the information. 

### 26 And of course, then there's this whole issue

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1 of, you know, this requirement for Tier 2 and remembering, we've 

### 2 heard and it's true, unlike Tier 1 the Agency has been very 

### 3 clear that Tier 2 is not a battery, and we appreciate that. But 

### 4 we don't really understand yet what that's going to mean. What 

### 5 are the criteria? What's the rationale for the requirement of 

### 6 these specific tests. 

### 7 And you have an opportunity here to help weigh 

### 8 in on what seems to make sense, what might be the endpoints that 

### 9 in a particular mode of action and what studies might be the 

### 10 ones that would be the most relevant to look at. 

### 11 And as been said by the previous speakers, we 

### 12 need to know that there is value added for running these in the 

### 13 assessment. So you know, having again an understanding of the 

### 14 sensitivity of endpoints to meet their relevance and the 

### 15 sensitivity of the species is, I think, important. 

### 16 Again, we've said it over and over, Tier 2 

### 17 data is for risk assessment. But these studies have both 

### 18 mechanistic and apical endpoints. And the apical endpoints are 

### 19 good for the risk assessment. The mechanistic endpoints can 

### 20 help us understand what's going on, to some extent. It's not 

### 21 necessarily -- I mean there's -- we just don't have the length 

### 22 that the mechanistic endpoints are the cause of the adverse 

### 23 effect, and we have to always keep that in mind, but that's 

### 24 okay. I mean that helps us in this whole weight of evidence to 

### 25 try to understand this. 

### 26 There is a little bit of a concern to me,

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1 though, and that's what was in the EDSTAC report and was also 

### 2 said in the Federal Register Notice, and that's that quote 

### 3 there. "Tier 2 has the purpose to characterize the nature and 

### 4 likelihood in dose response relationship with endocrine 

### 5 disruption of estrogen, androgen and thyroid in humans and 

### 6 wildlife." 

### 7 Now I'm not a mammalian toxicologist. I'm not 

### 8 going to speak to the rat study. Maybe there you feel a little 

### 9 more comfortable with what those endpoints mean. But again, if 

### 10 you look at these four ecotox studies, we don't have that link 

### 11 to say it's endocrine disruption, and we need to be very careful 

### 12 about what any biomarker endpoint means to an adverse effect. 

### 13 So identifying that lead mode of action is 

### 14 going to be quite challenging in these. Again, that's okay, in 

### 15 my opinion as an environmental toxicologist, because those 

### 16 population-level endpoints of survival, growth and reproduction 

### 17 integrate toxic responses from all modes of action that are 

### 18 going on. And those are the things that we care about because 

### 19 that's what we're going to be, you know, we want to know that 

### 20 when we use this chemical in the environment, it's going to be 

### 21 safe. 

### 22 So just please keep in mind that most of these 

### 23 biomarker endpoints are correlative, not causative, and that 

### 24 hopefully, as we move forward, and this is the plan for the 

### 25 Agency, and I think we'll, you know, I'm hoping someday we will 

### 26 get there, that we will determine some causal links; we'll get

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1 there to the adverse outcome pathways. It's still a work in 

### 2 progress. Okay. 

### 3 And I was disappointed by seeing adverse 

### 4 outcome pathway listed all through the ISRs because I think 

### 5 unfortunately sometimes things can take on a life of their own 

### 6 if you keep reading about it; oh, it must be so. We're not 

### 7 there yet, and I think we just have to be careful about that. 

### 8 The causal links are not necessarily established, not in every 

### 9 case. I think they can be. We're just not there yet. 

### 10 And in the end, what we're going to be doing 

### 11 is looking at a weight of evidence using Tier 1 data, Tier 2 

### 12 data and other scientifically relevant information so that we 

### 13 can make strong, robust conclusions on what this chemical is 

### 14 doing. It may include the ability to have some understanding of 

### 15 the endocrine activity, but I think it's going to be very 

### 16 difficult to necessarily pin it down. So again, just trying to 

### 17 keep that in mind. 

### 18 It's a bit of a soapbox for me, this guideline 

### 19 development thing. Again, you have protocols, there are 

### 20 protocols and there are guidelines. Okay. So you saw protocols 

### 21 for the most part, even though they had kind of the word 

### 22 "guideline" on some of them in the ISRs. 

### 23 In Tier 1, the 11 assays, there was no public 

### 24 review of the guidelines before they were put out. And again, 

### 25 thinking about this whole question of well, why can't a lab do 

### 26 what you're telling them to do? Well, if it's not real clear in

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1 the guideline, or if there are some things in there that really 

### 2 aren't quite worked out, that's why a lab can't necessarily do 

### 3 it because they go about doing what they think is the best thing 

### 4 forward. So we need to be very careful about what guideline 

### 5 goes out. 

### 6 In the case of the Tier 1's, many were overly 

### 7 prescriptive. There were errors in the Tier 1 guidelines that 

### 8 needed to be corrected, and the Agency has done that on the 

### 9 website, not necessarily in the guideline, but they have really 

### 10 worked that way. However, if we again had had the opportunity 

### 11 to look at that ahead of time, we might have been able to fix it 

### 12 before they were finalized. 

### 13 And we did notice some similar problems in 

### 14 those draft two protocols, some errors in things that were in 

### 15 there. And again, let's just fix it up front, and then we can 

### 16 be done with it. Some of the specific things are this issue of 

### 17 the solvent pooled the negative controls. I think you've seen 

### 18 that there was some inconsistency there where it said we're 

### 19 following the OECD 2006 guidelines. Those guidelines say 

### 20 compared to the solvent control if you can't pool, but yet the 

### 21 policy typically is to look at the negative controls. So we 

### 22 just need some clarity there. 

### 23 Requirements for multiple statistical tests. 

### 24 It's not good statistical -- I'm not a statistician, but I hear 

### 25 from my friends that it's not good statistical practice to 

### 26 evaluate the same response two different ways because you can

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1 come up with inconsistencies. We need to answer the question on 

### 2 the need for multiple generations, and I think it's a good 

### 3 opportunity to do some sensitivity analysis on some of the 

### 4 endpoints. 

### 5 So in conclusion, again, all stakeholders need 

### 6 confidence in these Tier 2 tests, and we need to know that 

### 7 they're validated for purpose, and that purpose is for use in 

### 8 risk assessment. 

### 9 The risk relevant apical endpoints; really 

### 10 we've got to be very careful not to just link them to an 

### 11 endocrine pathway by default just because they're in there. We 

### 12 need to be very careful not to do that. We need to have some 

### 13 good understanding there. 

### 14 We need to make sure that the Tier 2 tests and 

### 15 the guidelines are well vetted, and they need to be finalized. 

### 16 We need to have an opportunity for stakeholder input before 

### 17 they're finalized and before being required in Tier 2 testing. 

### 18 And we really do need to take the time to thoroughly evaluate 

### 19 the data and heed the advice of the stakeholders as well as the 

### 20 panel. 

### 21 And I was, just as the earlier comments from 

### 22 PETA and the Humane Society, we were pretty surprised to see 

### 23 that release of the information collection request the day 

### 24 before the Science Advisory Panel. And in that information 

### 25 collection request, that we now have 60 days to comment on, it 

### 26 has all five of the studies just like you're looking at today,

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1 including the MMT, doesn't even talk about the MRT. 

### 2 Now I know we can, you know, we'll have time 

### 3 to comment on them, but this will be, for the most part, the 

### 4 last time you get to really comment on them. And given the fact 

### 5 that you haven't even had a chance to give your input, that's a 

### 6 little disappointing. 

### 7 So with that, I am finished. 

### 8 **DR. MARTHA SANDY:**Thank you very much -- 

### 9 **DR. ELLEN MIHAICH:**Sure. 

### 10 **DR. MARTHA SANDY:**-- for your comments. 

### 11 If I could ask all your fellow presenters to 

### 12 come up as well, and we'll give the panel a chance, if they have 

### 13 any clarifying questions, to ask them now. If you have any, 

### 14 this is an opportunity. Do I see any? 

### 15 Yes, Dr. Scott. 

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

### 17 really excellent presentation. And I just want to go to a 

### 18 couple of points that I think were very interesting in your 

### 19 comments. 

### 20 And Ellen, I'll start with yours since it was 

### 21 the last. If we're going to go to a weight of evidence 

### 22 approach, h we have to have, in my opinion, some mode of action 

### 23 assessment. I don't know how else we distinguish between overt 

### 24 toxicity and endocrine disruption. It seems like you all are 

### 25 wrestling with that and we are, too. And I know we'll have a 

### 26 panel in a few weeks. We'll get into that, but in the absence

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1 of that right now, I just wanted to have any more clarification 

### 2 on what you think we need to do to remedy that dilemma or 

### 3 address that dilemma. 

### 4 **DR. ELLEN MIHAICH:**Geoff, I have to say we 

### 5 are wrestling with that. I mean this is a really tough thing to 

### 6 talk about because from the standpoint we see this as important 

### 7 for risk assessment. I mean what we're doing here is we want to 

### 8 know that if we get through, you know, I don't have a problem 

### 9 with doing the testing, okay. But you need to know what the 

### 10 endgame is and is it meaningful. Otherwise, it's a lot of 

### 11 wasted resources on everybody's part. So it becomes really hard 

### 12 when you see the purpose being to identify endocrine activity, 

### 13 but yet you know full well that what we're looking, and 

### 14 especially from ecotox, is population-relevant endpoints, 

### 15 survival, growth and reproduction. 

### 16 My opinion? I don't think you need to know 

### 17 exactly the specific mode of action. I think you need to know 

### 18 what that number is that when you look at the risk assessment 

### 19 you're being protective. 

### 20 Now I think that, you know, you -- I look at 

### 21 Tier 1 as saying okay, well, maybe we were going to work on what 

### 22 could be a mode of action. And then I think we just need to 

### 23 make sure that the studies in Tier 2 are making sure that you're 

### 24 going to pick up anything that might be impacted by that mode of 

### 25 action. So I think we've seen some good -- I mean we've seen 

### 26 some -- definitely some good work out of EPA Duluth in possibly

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1 moving forward with some changes or some amendments, some 

### 2 improvements to current protocols that maybe do a better job of 

### 3 picking some of these things up. 

### 4 The same thing happened in the rat study, the 

### 5 rat two-gen. Now interestingly, in there you see the two-gen is 

### 6 now being a one, you know, a one-gen, extended one-gen. Being a 

### 7 little more focused on some of the reproductive, anything that 

### 8 could be more endocrine. I think we can do that on some of 

### 9 these, too. But I think that to me it's more important to make 

### 10 sure that we know that we're being protected. 

### 11 Does anybody else need to -- want to weigh in? 

### 12 Okay. 

### 13 **DR. GEOFFREY SCOTT:**Well, I think that's 

### 14 really good because EPA has successfully regulated, at least on 

### 15 the invertebrate side of known endocrine disruptors safely. I 

### 16 think John pointed out methoprene, for example, in his slides. 

### 17 If I could ask a second question, and John, I 

### 18 really enjoyed your presentation. One of the things that I 

### 19 found most interesting was your NOEC comparison and I don't -- 

### 20 I'm not saying this is what it implies, but when you see higher 

### 21 NOECs in second gen, that may suggest resistance, the 

### 22 development, and we know particularly with insecticides the 

### 23 development of resistance is a common phenomena. 

### 24 And I guess one of the questions I see that 

### 25 may be helpful, and I know this gets into proprietary 

### 26 considerations, but I'd certainly like to know industry's view

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1 on this, you all gather data for the target test on some of that 

### 2 type of information and we're not -- right now, that's 

### 3 proprietary, and we're not using it. But it seems like for 

### 4 weight of evidence that could or could possibly be considered. 

### 5 I don't know whether industry would be willing to share any of 

### 6 that kind of data, but it certainly seems like if we see that 

### 7 evidence in the screening, it would be worth looking at that 

### 8 kind of information and I don't -- you all may not have an 

### 9 answer for that today, but I just simply throw that out as an 

### 10 observation and something to think about. 

### 11 **MS. KATIE HOLMES:**Mode of action of 

### 12 registered compounds is not a secret. So I don't guess that 

### 13 there would be -- I mean I can't speak for everybody. I think 

### 14 that that would not necessarily be a big concern. But again, I 

### 15 can't speak for the industry overall. 

### 16 **DR. GEOFFREY SCOTT:**Well, I'm not going to 

### 17 put you on the spot and ask you to say -- I'm only asking to see 

### 18 if you think that's within the realm of consideration in getting 

### 19 all the information out without having to spend more money is 

### 20 all I'm thinking. 

### 21 Yeah. There was one other question, John, I 

### 22 had. Let me just real quickly go through your slides because 

### 23 you had some really good points on the invertebrate test. Just 

### 24 real quickly. I have them written down. 

### 25 Okay. Yeah. The present mysid test you guys 

### 26 mentioned, and John in particularly paying attention to that, I

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1 happen to agree with that because it has served us well. And I 

### 2 guess I just wanted to agree and clarify the process as we go 

### 3 forward in looking at those EDSTAC charges of looking at A, E 

### 4 and T pathways for the invertebrates, do you think consulting 

### 5 that information where we have available one-gen mysid would be 

### 6 a first step in that weight of evidence analysis? I'm just 

### 7 trying to make sure I follow in your logic. 

### 8 **DR. JOHN BRAUSCH:**Yeah. Yes. I completely 

### 9 agree. I think we have sufficient information on a wide range 

### 10 of pesticides specifically, and if we can look at that data with 

### 11 what we've got for vertebrate systems, I think that would be a 

### 12 good first step to try to elucidate which compounds may or may 

### 13 not function similarly between inverts and verts. 

### 14 **DR. GEOFFREY SCOTT:**Thank you. 

### 15 **DR. MARTHA SANDY:**Dr. Ehrich. 

### 16 **DR. MARION EHRICH:**It seemed there was an 

### 17 underlying theme that went through all these talks that I need 

### 18 some clarification on is that you're looking for 

### 19 population-definitive endpoints, and there was discussion of 

### 20 fecal steroids, blood things and histopathology as individual 

### 21 points. Am I hearing you right? Is that the overall theme that 

### 22 all of you seem to go for? You need the population things for 

### 23 Tier 2? 

### 24 **DR. ELLEN MIHAICH:**Typically from a 

### 25 regulatory perspective on the eco side of things, we care more 

### 26 about the populations than an individual. Now that's not saying

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1 that we don't still, even in the current guideline studies have 

### 2 things that we would consider to be biomarkers or something like 

### 3 a biomarker that might help tell us something. 

### 4 But we typically do not regulate, we're not 

### 5 regulated on those. For example, we're not regulated on the 

### 6 vitellogenin concentration. Vitellogenin may be helpful in 

### 7 understanding what else might be going on, but no, you get 

### 8 regulated on survival, growth, reproduction kinds of things 

### 9 because that's the most important for the survival of the 

### 10 population. 

### 11 **DR. MARTHA SANDY:**Dr. Scanes. 

### 12 **DR. COLIN SCANES:**I was very much struck by 

### 13 John Brausch's comments about the need for value added for 

### 14 multigeneration tests. And I have a question for Tim 

### 15 Fredericks. On one of your slides, you talked about the use of 

### 16 quail and ducks being used for 20 years. We talked about there 

### 17 are measures that include the F0 reproduction. Now how would a 

### 18 compound that had an endocrine disruptor effect be detected with 

### 19 the one-generation test? 

### 20 **DR. TIM FREDERICKS:**I think the point, and it 

### 21 was made by Marion's comment recently, is we're looking for 

### 22 population-level effects. And so whether it is endocrine 

### 23 mediated or not, if you see a decrease in chicks that are 

### 24 produced, whether it's at the F1 or the F2, if you do to the 

### 25 two-gen test, that's where you'll pick it up. 

### 26 So you may have an endocrine-mediated effect

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1 in the epididymis in the two-gen test. And that wasn't picked 

### 2 up in chicks produced at the F1 or in chicks produced at the F2 

### 3 from those specific males that had the potential 

### 4 histopathological effect in the adults. 

### 5 **DR. COLIN SCANES:**However, if one looks at 

### 6 the effect of trenbolone on the F2 generation when picked up an 

### 7 effect on reproduction in terms of a marked decrease in 

### 8 reproduction, wouldn't that test have been missed in the present 

### 9 one-tier test? 

### 10 **DR. TIM FREDERICKS:**So you're saying there 

### 11 was an endpoint picked up from trenbolone in the F2 that was 

### 12 presented yesterday by Les, in that study, but it was on a chick 

### 13 production, right? It was number of chicks at the F2? 

### 14 **DR. COLIN SCANES:**Number of eggs. 

### 15 **DR. TIM FREDERICKS:**So number of eggs at the 

### 16 F2. That endpoint was also statistically significant at the F1. 

### 17 So that's an endpoint that's measured in a one-generation test. 

### 18 **DR. COLIN SCANES:**But it wasn't significant 

### 19 at F0. 

### 20 **DR. TIM FREDERICKS:**Okay. So when you say F0 

### 21

-- 

### 22 **DR. COLIN SCANES:**F0 is the -- 

### 23 **DR. TIM FREDERICKS:**Eggs produced to make the 

### 24 F1. 

### 25 **DR. COLIN SCANES:**Correct. 

### 26 **DR. TIM FREDERICKS:**So that would be

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1 considered the F1 endpoint. So eggs produced by the F0 is an F1 

### 2 endpoint. So that if you look at -- 

### 3 **DR. COLIN SCANES:**No -- 

### 4 **DR. TIM FREDERICKS:**-- eggs produced by the 

### 5 F1, that happens in the F2. That's considered the second 

### 6 generation, the F1 eggs. The F0 eggs are the F1. 

### 7 **DR. COLIN SCANES:**I don't think so but -- 

### 8 **DR. ELLEN MIHAICH:**I think it's just an 

### 9 understanding of how the study goes, and I have to admit, I 

### 10 don't know the two-gen as well. But you start with adults. 

### 11 Okay. 

### 12 So you're exposing adults, and in the current 

### 13 for Bobwhite quail and mallard duck, you expose them for ten 

### 14 weeks, and then you start egg collection. Okay. And so then 

### 15 that egg collection is then what makes the next generation. And 

### 16 what I think Tim is saying is that the eggs were reduced in the 

### 17 way we would currently do the study, and it would have been 

### 18 picked up at the same level as in the new study that goes on, 

### 19 you know, raises those chicks up and does it again. Okay. So 

### 20 the F0 are the adults. 

### 21 **DR. COLIN SCANES:**I think what we're 

### 22 concerned about is the possibility of either sensitization or an 

### 23 effect that is only seen on the embryo in terms of development 

### 24 of the reproductive system. 

### 25 **DR. TIM FREDERICKS:**And I would say that 

### 26 there isn't a study that's presented that shows that.

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1 **MS. KATIE HOLMES:**The F0 generation is 

### 2 adults. They have eggs that are then exposed through their 

### 3 whole -- the adults have been exposed the entire time those eggs 

### 4 are being made. Those eggs are laid. That's your F1 

### 5 generation, and what Tim is saying is that we saw an effect in 

### 6 that F1 generation that's the same as what you saw. That F1 

### 7 generation went on to have an effect in the eggs it laid also. 

### 8 But F0 starts as adults, so there are no eggs exposed in F0. Is 

### 9 that -- no? Okay. 

### 10 **DR. MARTHA SANDY:**Yes. Dr. Tuberty. 

### 11 **DR. SHEA TUBERTY:**John, back to you again. 

### 12 What tier -- well, now, let's get away from the EDC testing. 

### 13 What in the regulatory realm is a requirement of you for 

### 14 invertebrate testing that would be related to some of the mysid 

### 15 endpoints? 

### 16 **DR. JOHN BRAUSCH:**Okay. So for invertebrate 

### 17 testing, we are required to conduct two different species, 

### 18 daphnid and mysid. 

### 19 For the daphnid we've got acute as well as 

### 20 chronic lifecycle study. For mysids we've got an acute study. 

### 21 We also have the chronic study and how the requirements are 

### 22 currently written, it's a conditional study. So certain 

### 23 toxicological or (inaudible) parameters have to be met, and if 

### 24 they're met then you have to continue on with the mysid chronic 

### 25 study. 

### 26 However, recently, over the last couple years,

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1 the mysid chronic studies have been conducted whether or not 

### 2 they meet those guidelines. And as we move forward, those are 

### 3 becoming almost a nonofficial required study, I guess is the 

### 4 best way to put it. 

### 5 **DR. SHEA TUBERTY:**Okay. Thank you. And I 

### 6 guess what I'm thinking about is would those suffice to serve as 

### 7 a Tier 1 in your opinion? 

### 8 **DR. ELLEN MIHAICH:**Sorry. Let me field that 

### 9 one, if you don't mind. So you're asking if it goes into what 

### 10 is already the Tier 1 battery? 

### 11 **DR. SHEA TUBERTY:**No. I know there's no 

### 12 invertebrate Tier 1 requirement. 

### 13 **DR. ELLEN MIHAICH:**Right. Okay. 

### 14 **DR. SHEA TUBERTY:**What I'm saying is are the 

### 15 current requirements for registration, in your opinion, 

### 16 fulfilling the requirements of a Tier 1 for EDCs? Are you going 

### 17 to find the sensitive measures of reproduction, development and 

### 18 growth in those assays? 

### 19 **DR. ELLEN MIHAICH:**Okay. If that study was 

### 20 -- and we need to be a little careful here that these are 

### 21 requirements -- 

### 22 **DR. SHEA TUBERTY:**I understand. I 

### 23 understand. 

### 24 **DR. ELLEN MIHAICH:**-- for pesticides, not 

### 25 safe drinking -- 

### 26 **DR. SHARLENE MATTEN:**Ellen, excuse me.

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1 **DR. ELLEN MIHAICH:**Sorry. Yeah. 

### 2 **DR. SHARLENE MATTEN:**Before we go down 

### 3 further in this road, just let me remind the panel that we're 

### 4 looking at scientific issues. You're putting our colleagues in 

### 5 the public in a bit of a predicament because they're speaking to 

### 6 policy matters and regulatory matters that for the point of the 

### 7 panel are not relevant and would potentially be very confusing. 

### 8 9 So if you could talk amongst yourself over 

### 10 lunch or a beer, just to speculate, but just -- it would be 

### 11 prudent just to stick to the slides that they mentioned or any 

### 12 of the background documents if there's any understanding 

### 13 questions, and the same goes for colleagues over in the corner 

### 14 because it's great to have a little chitchat, but really we're 

### 15 looking for clarifying questions. 

### 16 And any comments that you make for charge 

### 17 questions, great, and so I just -- I know this is very exciting. 

### 18 I really do. And I appreciate the excitement, how hard you've 

### 19 worked to get here. 

### 20 **DR. SHEA TUBERTY:**All right. I apologize. 

### 21 So you mentioned that you feel that the mysid assay was more 

### 22 appropriate than the copepod assay. Can you give me some 

### 23 specific reasons for your opinions on that? 

### 24 **DR. JOHN BRAUSCH:**Yeah. Probably the major 

### 25 one is the mysids have been used in the lab for so long that we 

### 26 have a very good idea of husbandry and everything that needs to

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1 be done. None of the current CROs currently have the copepod in 

### 2 house. So all new husbandry methods would need to be developed, 

### 3 and it's just a lot of back work that would need to be conducted 

### 4 before we could ever even get to the point of being able to use 

### 5 a copepod in an assay. 

### 6 Also, the problem with analytical verification 

### 7 or the lack of analytical verification in the copepod assay 

### 8 would be a major concern moving forward, especially if you were 

### 9 to see an effect, and you wouldn't actually be able to relate it 

### 10 to an actual concentration other than what you'd expect to be 

### 11 there. 

### 12 There's also some concern -- the copepod that 

### 13 was chosen is typically a benthic copepod. So we are now 

### 14 exposing a copepod that is usually associated with sediment in a 

### 15 96-well plate, water only. So this could also add some 

### 16 uncertainty to the results you'd observe. I believe 

### 17 reproduction is one of those endpoints that when looking at the 

### 18 copepod in a water sediment situation, there's very good 

### 19 reproduction. As soon as you take it to a water-only situation, 

### 20 the reproduction drops off dramatically. 

### 21 **DR. MARTHA SANDY:**Dr. Perkins. 

### 22 **DR. EDWARD PERKINS:**I have a question for 

### 23 John. On your chart on comparing the one-gen NOEC to the 

### 24 two-gen NOEC, can you clarify how you derive this and how it 

### 25 relates to the comparison by generation in mysids that Leslie 

### 26 presented yesterday?

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1 **DR. JOHN BRAUSCH:**I can't really compare to 

### 2 what Les presented. I never looked at the NOEC values, so I 

### 3 actually have no relation -- I have no way to speak on the 

### 4 difference between the two. 

### 5 One thing I will point out for the NOEC, there 

### 6 was a bit of -- looking at the endpoints, there were a lot of 

### 7 endpoints that had effects at one concentration, then no effects 

### 8 for a couple concentration, and then an effect for another 

### 9 concentration. There was no typical dose response observed. 

### 10 In situations like that, it's usually looked 

### 11 at as you try to find the actual dose response and then take 

### 12 those endpoints. So some of these endpoints, they might be a 

### 13 bit different because the LOEC could be established at one point 

### 14 where there was no dose response and simply just a response at a 

### 15 single endpoint. These NOECs were derived from where a dose 

### 16 response trend was observed. 

### 17 **DR. EDWARD PERKINS:**Okay. Thank you. 

### 18 **DR. MARTHA SANDY:**Okay. I think we've 

### 19 exhausted our questions. Thank you very much to the presenters, 

### 20 public commenters. I think this closes our public comment 

### 21 period. And we will now go to lunch. I'd like people to return 

### 22 in an hour, that would be 1:15, and we'll start on the first set 

### 23 of charge questions. Thank you. 

### 24 (Whereupon a lunch break was taken) 

### 25 **DR. MARTHA SANDY:**3:00:42 Good afternoon. 

### 26 I'd like all the panel members to take their seats please. So

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1 thank you all. I think we'll get started now. Thank you for 

### 2 getting back on time. Dr. Matten has a few things to say. 

### 3 **DR. SHARLENE MATTEN:**I just wanted to let 

### 4 everyone know that Mary Manibusan, Les Touart and Patience Brown 

### 5 provided a description from a summary study in the invertebrate 

### 6 area to address the consideration of block effects or blocking 

### 7 effects. And I have then two things. 

### 8 I extracted out the highlighted pages that Les 

### 9 Touart pointed out to me and distributed that to the panel. And 

### 10 then I also provided the full summary study if you want to see 

### 11 the whole details. And this is in response to a question that 

### 12 Mary Christman asked after Les Touart's talk on the invertebrate 

### 13 assays. 

### 14 Oh and it will also be put into the public 

### 15 Docket. I probably will put that in later this evening for QA 

### 16 and QC. 

### 17 **DR. MARTHA SANDY:**Thank you for providing 

### 18 that. Now we're ready, I believe, to start the series of charge 

### 19 questions on the quail assay. I don't know who is going to be 

### 20 reading them. Dr. Touart. 

### 21 **DR. LESLIE TOUART:**Okay. I can go through 

### 22 these. And there is some preamble information that just talks 

### 23 about, you know, general purpose of what to see the Tier 2s go 

### 24 and talk about the integrated summary reports. 

### 25 **DR. MARTHA SANDY:**And could I ask you to 

### 26 speak clearly into the microphone. Close to the microphone.

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1 **DR. LESLIE TOUART:**Okay. Sorry about that. 

### 2 I'll try to keep the microphone in front of me. Moving, 

### 3 basically to the first charge question and reading through; a 

### 4 rationale for the test method should be available including a 

### 5 clear statement of scientific basis in the regulatory purpose in 

### 6 need of the test method. The EDSTAC described the Tier 2 test 

### 7 as having the purpose to characterize the nature likelihood and 

### 8 dose response relationship of endocrine disruption of estrogen, 

### 9 androgen and thyroid in humans and wildlife. 

### 10 Tier 2 tests were designed to be definitive 

### 11 tests that generates sufficient data to characterize a specific 

### 12 hazard of the substance and provide sufficient information on 

### 13 dose response and adverse effects to permit risk decisions. So 

### 14 given that please comment on the rationale and purpose of the 

### 15 assay as part of the Tier 2 testing in the EDSP as described in 

### 16 Sections 2.5 and 2.6 of the Japanese Quail Two-generation Test 

### 17 ISR. 

### 18 **DR. MARTHA SANDY:**And Dr. McCarty is the lead 

### 19 discussant on this? 

### 20 **DR. JOHN MCCARTY:**Okay so the group of us 

### 21 assigned to the avian protocol included myself, George Bentley, 

### 22 Colin Scanes and Dallas Johnson, Ed Perkins and then Ken Portier 

### 23 also provided some input to this. And so we'll be the lead and 

### 24 associate discussants on these eight questions. 

### 25 After the presentations yesterday and then 

### 26 this morning Drs. Bentley, Scanes, Johnson and I met to compare

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1 our reviews of the protocol documents and discuss issues raised 

### 2 in the presentations and so we've attempted to produce some 

### 3 synthesis of what we thought were the most important points that 

### 4 each lead person will read and then after that associates will 

### 5 provide additions, corrections, disagreements, et cetera. 

### 6 So first we are asked is there a clear 

### 7 statement of the scientific basis in regulatory purpose and in 

### 8 general we found the rationale and purpose behind the principle 

### 9 of an avian two-generation test to be well supported. It's well 

### 10 supported in Section 2 of the report as well as elsewhere in the 

### 11 meeting documents. 

### 12 The science is clear that birds may react in a 

### 13 fundamentally different way than mammals and the endocrine 

### 14 disrupting chemicals both because of the mechanism of sex 

### 15 determination as we've heard with males being homogametic sex, 

### 16 females heterogametic as well as because of other differences in 

### 17 hormone pathways among distantly related Taxa. 

### 18 In terms of the goal of using the results of 

### 19 the Tier 2 tests as a component of risk assessments, it makes 

### 20 sense to have Taxa on specific information for birds which will 

### 21 be an important focus of possible ecological risk assessments. 

### 22 Next we were asked about the need for the test 

### 23 method and we also thought the need for an avian specific test 

### 24 method was well explained in the documents. The OECD document 

### 25 in Appendix D goes into great detail on this and the arguments 

### 26 outlined convinced us of the need for additional endocrine

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1 relevant endpoints and for protocols that expose a greater range 

### 2 of life stages than our founding current approved test 

### 3 protocols. 

### 4 I'd like to emphasize in our discussions that 

### 5 we overall agreed that the modifications might be called for. A 

### 6 strong case has been made for an Avian specific test protocol as 

### 7 part of the Tier 2 testing program. So we don't want that 

### 8 message to be lost in our recommendations for improvement that 

### 9 follow. 

### 10 That said the group did have some concerns as 

### 11 we moved from the principle behind the test approach to some of 

### 12 the specifics of the currently proposed test. 

### 13 In particular, if we look at the wording on 

### 14 the screen there that the Tier 2 tests were designed to be 

### 15 definitive tests that generates sufficient data to characterize 

### 16 the specific hazard of the substances and provide sufficient 

### 17 information on dose response and adverse effects for risk 

### 18 assessments sets a pretty high bar for this test. 

### 19 And members of the avian group thought some of 

### 20 the decisions behind the details of the current protocol needed 

### 21 additional justification if this goal was to be met. 

### 22 In particular, a general theme that will run 

### 23 through many of our comments has to do with the choice of the 

### 24 Japanese quail as the sole subject of these tests. The detailed 

### 25 OECD report in Appendix B does a good job of summarizing the 

### 26 rationale for choosing Japanese quail over Bobwhite for this

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1 test. That review clearly lays out the advantages of Japanese 

### 2 quail and that they're strictly practical in nature. 

### 3 They're a domesticated species, easy to raise 

### 4 in captivity, they have an usually short generation time, they 

### 5 have precocial development that allows for production of large 

### 6 numbers of eggs that can be artificially incubated. 

### 7 Now the group is well aware of the importance 

### 8 of ensuring that any test protocol is logistically practical to 

### 9 implement if these tests are to be successfully used on a large 

### 10 scale. So we don't want to discount the importance of the 

### 11 practical factors and the attractiveness of using Japanese 

### 12 quail. 

### 13 However, the reliance on Japanese quail needs 

### 14 more justification if these tests are to be the definitive tests 

### 15 providing sufficient information to do an ecological risk 

### 16 assessment. Virtually everything that makes Japanese quail 

### 17 uniquely easy to work with in the lab also makes it less easy to 

### 18 extrapolate to wildlife populations that may be the subject of 

### 19 risk assessments. 

### 20 This issue will probably come up several times 

### 21 in our comments and we want to emphasize we're not suggesting 

### 22 that these concerns mean we're recommending dropping the 

### 23 Japanese quail. Instead it's a concern that the tests relying 

### 24 solely on this for input are unlikely to fully characterize 

### 25 adverse effects in birds. 

### 26 We'd recommend that as part of the protocol

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1 the agency carefully outline the areas where additional 

### 2 information about other groups of birds may be needed in order 

### 3 to apply results of the Tier 2 tests to risk decisions. 

### 4 As an example, one of the practical advantages 

### 5 of Japanese quail is that they're young and precocial and can be 

### 6 raised with little intervention from parents or lab managers. 

### 7 However, the majority of bird species and all passerines are 

### 8 altricial. They hatch earlier in development and are dependent 

### 9 on extensive parental care. 

### 10 Now there is no evidence to support the idea 

### 11 that either precocial or altricial species will be more 

### 12 sensitive to the effects of endocrine disrupting chemicals but 

### 13 they are likely to respond differently to endocrine disrupting 

### 14 chemicals. 

### 15 And obvious an example of the differences 

### 16 would be in the importance of parental care in altricial 

### 17 species. A set of behaviors crucial to successful reproduction 

### 18 therefore relevant in ecological risk assessment and known to be 

### 19 vulnerable to endocrine disruption and so that makes a direct 

### 20 link between EPAs focus on population level responses and 

### 21 wildlife for risk assessment. 

### 22 This difference between Japanese quail and a 

### 23 majority of birds runs more deeply. Recent phylogenies of birds 

### 24 place quail and their relatives in a lineage distinct from the 

### 25 vast majority of birds. Likewise there is concern about 

### 26 evolutionary and ecological differences between Japanese quail

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1 and bird species of greatest concern for risk assessment. 

### 2 The gap between the proposed tests that rely 

### 3 on only Japanese quail and the goal of being a definitive test 

### 4 and fully characterizing adverse effects could be partly 

### 5 addressed by incorporating supplemental data from an altricial 

### 6 species such as Zebra finches, at least in some cases. 

### 7 The zebra finch is a passerine, it's got 

### 8 altricial development, it's also a model lab organism with many 

### 9 of the same practical advantages that Japanese quail have, 

### 10 including a very short generation time. 

### 11 Now overall while the basis for the choice of 

### 12 Japanese quail over Bobwhite is clearly articulated, the zebra 

### 13 finch has advantages of its own, including the recent 

### 14 publication of the species genome, which may facilitate future 

### 15 advances in testing. 

### 16 It will be helpful in future application of 

### 17 Tier 2 tests if the protocol included explicit guidance on when 

### 18 additional information from other groups of birds will be needed 

### 19 before going forward for ecological risk assessments. 

### 20 **DR. MARTHA SANDY:**And then I'll turn to Dr. 

### 21 Bentley. Any additional comments? 

### 22 **DR. GEORGE BENTLEY:**No. Dr. McCarty has 

### 23 summarized the discussion quite well as far as I'm concerned. 

### 24 **DR. MARTHA SANDY:**Thank you and Dr. Scanes? 

### 25 **DR. COLIN SCANES:**I fully agree with all 

### 26 points John raised.

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1 **DR. MARTHA SANDY:**Dr. Johnson. 

### 2 **DR. DALLAS JOHNSON:**This item is probably 

### 3 outside my area of expertise and so I have no comments to add. 

### 4 **DR. MARTHA SANDY:**Dr. Perkins. 

### 5 Dr. Edward Perkins: I have no further 

### 6 comments to add. 

### 7 **DR. MARTHA SANDY:**Any comments from other 

### 8 members of the panel? 

### 9 **DR. DALLAS JOHNSON:**I might just add that 

### 10 there were some comments from Ken Portier but I think they've 

### 11 been incorporated in the discussion as been given so far. 

### 12 **DR. MARTHA SANDY:**Thank you. No other 

### 13 comments? I think we're ready for the next charge question. Oh 

### 14 I'm sorry. So EPA do you have any questions of clarification? 

### 15 **DR. LESLIE TOUART:**Yes. Just a point of 

### 16 clarification because I understand, I appreciate your comments 

### 17 on the Japanese quail being relevant but -- with a big but in 

### 18 terms of other species to be considered -- now typically within 

### 19 a test guideline we focus on the actual test methodology that 

### 20 would be utilized in that context. And given the suggestion 

### 21 that now we consider some criteria of sorts as to when to go to 

### 22 maybe another species for additional information, I think that's 

### 23 relevant. I'm not sure how that fits within a test guideline 

### 24 per se. 

### 25 So, you know, two things, one do you have 

### 26 suggestions of how that might be suggested in terms of the say

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1 data interpretation and additional information to be sought and 

### 2 second in considering a recommendation such as you have with the 

### 3 zebra finch and we have looked into zebra finch as well as some 

### 4 other species like canary altricials to evaluate, but these pose 

### 5 big difficulties for long term tests especially carry on through 

### 6 reproduction. 

### 7 So do you have suggestions that potentially 

### 8 could be made in terms of whether there is available methods 

### 9 that we could adapt or whether this would be more of a research 

### 10 developmental program that considered zebra finch as an 

### 11 auxiliary species in future testing? 

### 12 **DR. MARTHA SANDY:**Dr. Scanes? 

### 13 **DR. COLIN SCANES:**I was actually signaling 

### 14 for John to take the question. 

### 15 **DR. JOHN MCCARTY:**Yes, thanks. Thanks for 

### 16 that. I'm going to ask for input from the other members. So 

### 17 one thing to clarify the wording in the charge that this is 

### 18 definitive and that this will be sufficient is what motivated us 

### 19 to emphasize this. I realize you're designing a protocol, but 

### 20 you're setting a high bar if you say this is the definitive and 

### 21 sufficient test. 

### 22 That said, I'd say the first step one of our 

### 23 recommendations is there is a lot known about the endocrinology 

### 24 of zebra finches and to outline the pathways that are likely to 

### 25 be most vulnerable. 

### 26 So for example, parental care is an obvious

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1 one. Song is another obvious one where these are different and 

### 2 if given the other information that is going through the whole 

### 3 screening program here perhaps that would provide the evidence 

### 4 of when there is going to be need for additional information. 

### 5 And then outside the realm of this particular 

### 6 screening program, if the focus of an ecological risk assessment 

### 7 is likely to target species that are passerines that are 

### 8 altricial that would be another motivation. But I defer to the 

### 9 endocrinologists as well. 

### 10 **DR. MARTHA SANDY:**Dr. Bentley. 

### 11 **DR. GEORGE BENTLEY:**If I remember what you've 

### 12 asked correctly you asked for recommendations on how a passerine 

### 13 species could be included within this test? Could I ask you a 

### 14 question back, you said you had considered the uses of zebra 

### 15 finches and canaries, has that actually been attempted and if so 

### 16 can you tell me what happened? 

### 17 **DR. MARTHA SANDY:**I think I need to interrupt 

### 18 a little bit. We don't want -- this is not a back and forth. 

### 19 It's really you've answered the charge question and then we're 

### 20 asking EPA if they understood the answer and if they got -- if 

### 21 you've fully answered the question. We don't want to have a 

### 22 long back and forth. 

### 23 **DR. GEORGE BENTLEY:**Okay let me answer in a 

### 24 different way. Zebra finches, as I'm sure you are aware, are 

### 25 quite easy to breed in captivity, pretty much as easy as 

### 26 Japanese quail housed in the correct conditions. So it is my

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1 opinion that if it were deemed suitable to test one of these 

### 2 chemicals in a passerine species to help with this question of 

### 3 whether this test is definitive in terms of risk assessment, 

### 4 then it should be fairly straight forward with slight 

### 5 modification of the protocol described in here. 

### 6 **DR. MARTHA SANDY:**Dr. Scanes. 

### 7 **DR. COLIN SCANES:**I want to come back to your 

### 8 question and I'm agreeing with my colleagues but suggesting or 

### 9 encouraging EPA to consider some research using zebra finch and 

### 10 the same compounds that have been applied in other situations. 

### 11 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 12 **DR. EDWARD PERKINS:**Yes, I second Colin's 

### 13 comments and I would add that one other way to extend this test 

### 14 is potentially include genomics where you can look at 

### 15 fundamental pathways in the brain that might be related to the 

### 16 song and learning across species. 

### 17 I know there is -- genomics are available in 

### 18 Japanese quail also. We've done studies comparing quail and 

### 19 zebra finch correctly. 

### 20 So looking at -- that can establish a 

### 21 commonality of impacts on function that would provide some 

### 22 weight-of-evidence to pursue a targeted -- is there an impact on 

### 23 song development or is there developmental impact in passerine 

### 24 birds. So that would be more of an extension in of the test 

### 25 rather than a whole new prescribed series. And do it more in a 

### 26 hypothesis testing manner consistent with the general approach

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1 of risk assessment. 

### 2 **DR. MARTHA SANDY:**Okay. For the discussants 

### 3 on Charge Question 1 if you can make sure you include some of 

### 4 the suggestions you just discussed in your written comments so 

### 5 that EPA has them in front of them and I'm asking you Dr. Touart 

### 6 is that enough clarification there? 

### 7 **DR. LESLIE TOUART:**Yes I understand the 

### 8 responses and it's helpful thank you. 

### 9 **DR. MARTHA SANDY:**I think we're ready for the 

### 10 next question. 

### 11 **DR. LESLIE TOUART:**Going to Charge Question 

### 12 2. Test methods and their associated endpoints should be 

### 13 scientifically relevant to the biological processes of interest 

### 14 and should be demonstrated to be responsive to the specific type 

### 15 of effect/toxicity of interest. Each species presents unique 

### 16 characteristics from a biological perspective and allows for 

### 17 specialized endpoints to address a specific toxicological mode 

### 18 of action. 

### 19 Please comment on the biological and 

### 20 toxicological relevance of the assay in regards to the stated 

### 21 purpose of characterizing endocrine disruptors, as described in 

### 22 Sections 3 and 4 of the ISR. 

### 23 **DR. MARTHA SANDY:**Dr. Scanes. 

### 24 **DR. COLIN SCANES:**I'm going to start with the 

### 25 overview. The test methods and their associated endpoints are 

### 26 clearly scientifically relevant to the biological processes of

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1 interest. The available evidence on the effects on 

### 2 environmental toxicants in birds is highly supportive of 

### 3 including eggshell thickness as an endpoint. Indeed its absence 

### 4 would impact the credibility of the Agency. There is a clear 

### 5 consensus that the behavioral indices should be maintained and 

### 6 were justified. 

### 7 Then we'll come on to some less major points 

### 8 but nonetheless important in terms of areas where the protocol 

### 9 could be improved. And it was often areas where a little bit of 

### 10 extra clarification could be helpful. 

### 11 An example would be to ensure that there were 

### 12 at least 12 pairs remaining in the study. Another example would 

### 13 be the information on water quality for the birds should be 

### 14 included. Another example would be to ensure that the dose 

### 15 response curve is done in the same way between different service 

### 16 labs, contract labs. 

### 17 We've seen in some of the materials that we've 

### 18 had use of Log to the base 2, Log to the base 2.5, log to the 

### 19 base 3 and sometimes with jumps. I think the protocol needs to 

### 20 be clear whether it's Log to the base 2, 2.5, 3. Whatever 

### 21 figure the top dose there needs to be a clear consensus as to 

### 22 how does one determine what is the top dose in the dose response 

### 23 curve. 

### 24 The use of vehicle to dissolve the test agent. 

### 25 At least one of us was concerned that use of acetone and then 

### 26 not blowing off the acetone could be a confounding factor.

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1 The issue of number of replicates we will be 

### 2 coming back to on a number of occasions. But I think that it's 

### 3 clear that we're viewing the number of replicates for the 

### 4 hormonal assay, for the histopath should be much greater. Maybe 

### 5 a minimum of 8. We're very concerned on the loss of sample. 

### 6 One study they only found one pineal out of eight birds. Now I 

### 7 just collect a lot of pineals in my life, but I've never found a 

### 8 bird without a pineal. 

### 9 The review which was very well done, talked 

### 10 about some of the quail models that the Japanese are using. 

### 11 There was some concern that without full genome data it's not 

### 12 possible to know if this is really a very unusual model that a 

### 13 Japanese worker is using, Oshima and colleagues 2012. I'll come 

### 14 back to the histopathology when on Question 3. 

### 15 And the other areas in terms of looking at 

### 16 this are concerns whether we should be also looking at any other 

### 17 hormones. And I know my colleague George Bishop will be coming 

### 18 back to that. 

### 19 **DR. MARTHA SANDY:**So George Bentley. 

### 20 **DR. COLIN SCANES:**George Bentley, I'm sorry. 

### 21 **DR. GEORGE BENTLEY:**That's okay. I know you 

### 22 got me muddled up with another person from the same lab years 

### 23 ago. You wanted me to come back to that now or later? 

### 24 **DR. COLIN SCANES:**I thought later. 

### 25 **DR. GEORGE BENTLEY:**Okay. 

### 26 **DR. MARTHA SANDY:**Dr. Scanes are you

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1 finished? 

### 2 **DR. COLIN SCANES:**Yes. 

### 3 **DR. MARTHA SANDY:**So now I am turning to the 

### 4 other people on this question and the first one is Dr. Bentley 

### 5 if he has anything to add to Question 2 response. 

### 6 **DR. GEORGE BENTLEY:**Nothing else to add apart 

### 7 from this is going to be a recurrent theme throughout the 

### 8 questions is that the behavioral analyses we feel are very 

### 9 important and we need to make sure that they're carried out 

### 10 adequately. But we will get back to that too. 

### 11 **DR. MARTHA SANDY:**Dr. McCarty. 

### 12 **DR. JOHN MCCARTY:**Yes. I just would agree 

### 13 with both what Colin said and George's addition about the 

### 14 importance of parental care and behavior as endpoints. Sorry. 

### 15 **DR. MARTHA SANDY:**Dr. Johnson and if you want 

### 16 to speak for Dr. Portier as well. 

### 17 **DR. DALLAS JOHNSON:**Okay. I have nothing to 

### 18 add and I'm currently looking at what Dr. Portier -- reviewing 

### 19 what he had said again, and why don't we pass by and I'll see if 

### 20 I want to add anything. I don't want to waste your time. 

### 21 **DR. MARTHA SANDY:**That's fine. Dr. Perkins. 

### 22 **DR. EDWARD PERKINS:**The only thing I would 

### 23 add is that it would be nice on the behavioral to have more -- 

### 24 to be able to examine more impact on learning behavior 

### 25 especially on song, which isn't too easy to do in quail. I'm 

### 26 referring back to the first one, maybe if some effects point

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1 towards some potential effects you might also investigate in an 

### 2 alternate species. 

### 3 **DR. MARTHA SANDY:**Dr. Bentley? 

### 4 **DR. GEORGE BENTLEY:**I thoroughly agree with 

### 5 that. I was going to comment on that more later but learning 

### 6 and cognition are aspects of behavior that are probably more 

### 7 easily tested in the passerine species than in Japanese quail. 

### 8 **DR. MARTHA SANDY:**Dr. Dallas Johnson. 

### 9 **DR. DALLAS JOHNSON:**Yes, Dr. Portier had put 

### 10 some indications in this section dealing with power of some of 

### 11 these tests and there is discussion in the reports about certain 

### 12 types of 80 percent power for certain types of percentage 

### 13 increases and he brought up the idea, I guess, as to whether 

### 14 these percentage increases in the end variables are biologically 

### 15 relevant or not and that there need to be some indication of 

### 16 what the biological relevance is of some of the endpoints that 

### 17 they might be looking for changes in. 

### 18 In other words is a 20 percent decrease in 

### 19 body weight, is that biologically relevant or statistical 

### 20 significance doesn't imply biological significance and vice 

### 21 versa. I think the point he was trying to get at. 

### 22 **DR. MARTHA SANDY:**Does anyone else have 

### 23 anything to add on this Question 2? No? Okay I'll turn over to 

### 24 EPA. Do you understand the answers to this charge question? 

### 25 **DR. LESLIE TOUART:**Yes, I understand. I 

### 26 appreciate, but just a couple of clarifications in the

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1 conversation in terms of the behavior and its importance in 

### 2 trying to add that. I gather that's going to be something else 

### 3 discussed in terms of some more specific recommendations later. 

### 4 Is that correct or? 

### 5 **DR. GEORGE BENTLEY:**Yes, I am going to bring 

### 6 it up later, but we can also discuss it now if you'd like. 

### 7 **DR. LESLIE TOUART:**I think the main thing is 

### 8 just to whatever specific recommendations or suggestions in 

### 9 terms of how to add that in terms of the Japanese quail but also 

### 10 if there are alternative, you know, suggestions to make those 

### 11 prevalent as well. 

### 12 But the other clarification point that I had 

### 13 was the question about dose spacing that Dr. Scanes brought up. 

### 14 And I don't know if he recommends a specific dose separation 

### 15 that we should use. Because right now within the guideline it's 

### 16 left somewhat open and flexible, but I gather that the 

### 17 suggestion is to have that a little bit more stringent to use a 

### 18 specific geometric progression based upon whatever a high dose 

### 19 might be identified as. 

### 20 **DR. COLIN SCANES:**The view was that it should 

### 21 be consistent. Because then if you're using four doses and 

### 22 you're using log to the base 2 you got one spectrum and if 

### 23 you're using log to the base 3 you got a whole different 

### 24 spectrum. If you're using log to the base 5, different 

### 25 spectrum. 

### 26 And so without -- we were in many ways looking

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1 for consistency, such that there could be ready comparison 

### 2 between compounds of potential toxicants. 

### 3 **DR. LESLIE TOUART:**I guess my question then 

### 4 is what would you recommend? Is there a specific dose spacing 

### 5 that you think is best to maybe span two hours of magnitude. 

### 6 **DR. COLIN SCANES:**We did not discuss this. 

### 7 For myself I tend to like a log to the base 3 because you spend 

### 8 more concentrations. But that's a personal view. We have not 

### 9 discussed this. 

### 10 **DR. MARTHA SANDY:**Mary Manibusan. 

### 11 **MS. MARY MANIBUSAN:**Just as an over arching 

### 12 request from the Agency. Where you are seeking to provide the 

### 13 Agency recommendations, if you can be as specific as possible, 

### 14 so an example would be dose setting. An example also would be 

### 15 how to establish that top dose so that we're not tripping into 

### 16 overt toxicity for example. That is a resonating issue across 

### 17 all these assays as well as the Tier 1 assays. 

### 18 So if you can be please specific in your 

### 19 recommendations in the final report and provide any references 

### 20 that you believe the Agency would benefit from having that would 

### 21 be greatly appreciated. Thank you very much. 

### 22 **DR. MARTHA SANDY:**So I'll suggest to this 

### 23 group addressing this question you can think about it and talk 

### 24 about it later as the day goes on and tomorrow and I'll give you 

### 25 a chance if you come to any decision and recommendation to make 

### 26 it at a later point. Okay. Are we ready to move on to Question

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1 3? Let's do that. 

### 2 **DR. LESLIE TOUART:**Reading the third question 

### 3 and it's a two part question but we'll take the first part first 

### 4 and the test protocol should be sufficiently detailed and should 

### 5 include a description of what is measured and how it is 

### 6 measured. 

### 7 The selection of endpoints within the assay 

### 8 should be reflective of the biological processes of interest and 

### 9 the endpoints should be intrinsically relevant and have 

### 10 established sensitivity. The test protocol should demonstrate 

### 11 the ability to measure the endpoints and provide adequate 

### 12 performance criteria for evaluation. 

### 13 Please comment on the selection, optimization 

### 14 and demonstration of the assay endpoints as outlined in Sections 

### 15 4 and 5 of the ISR. 

### 16 **DR. COLIN SCANES:**We will go through the 

### 17 endpoints first then come to some suggestions for improvements. 

### 18 The first endpoints I'd like to pull together are egg 

### 19 production, growth, embryonic development, sex reversal and 

### 20 mortalities. 

### 21 These endpoints meet the criteria for 

### 22 reliability, for relevance. They are well demonstrated in the 

### 23 protocol. One issue that we raise on these and that is the 

### 24 issue of adding a positive control. And certainly the more 

### 25 we've seen of inter-lab differences, the issue of a positive 

### 26 control could make a lot of sense.

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1 Egg shell thickness is an obvious measure. It 

### 2 meets the criteria of reliability and relevance but -- the but 

### 3 is the lack of standardization. If one looks at the OECD report 

### 4 it provides clearly a range, and to the best of my memory it's 

### 5 from 0.18 to 0.21. We saw some reports in the appendices where, 

### 6 when one was seeing values of over .4 and in one case over .9. 

### 7 There needs to be both training and standards 

### 8 that could be used for inter-lab comparison and actually 

### 9 intra-lab comparisons. 

### 10 Consideration should be given to the addition 

### 11 of epigenetic endpoints. Now this work was designed in the late 

### 12 1990s to early 2000s and that is not a criticism, it's the 

### 13 endpoints included were state of the art when they were first 

### 14 envisioned, but now there is a case to look at for future 

### 15 research. 

### 16 The behavioral indices are labor intensive but 

### 17 they add a novel and very important biological parameter. The 

### 18 time to first mount, the number of successful copulations are 

### 19 good measures, but they are restricted to male behavior; some 

### 20 indices of female behavior would be nice to see. 

### 21 Our overall recommendation on the behavioral 

### 22 indices is that they should continue, they are important 

### 23 endpoints, they meet the criteria of relevance. We do have a 

### 24 recommendation and that is for the behavioral indices, there 

### 25 needs to be an appropriate training program. Now that may be a 

### 26 web based training program, but nonetheless it could reduce the

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1 coefficient of variation and make this an even more powerful 

### 2 test. 

### 3 Another set of endpoints that make a lot of 

### 4 sense are spermatozoa characteristics. However, spermatozoa 

### 5 characteristics were only looked at in epididymal samples. And 

### 6 even there there was tremendous variation. In one case the 

### 7 number between the F0 generation and the F1 generation, the 

### 8 number of spermatozoa declined by 94 percent. And that's in the 

### 9 controls. 

### 10 Our recommendation is for spermatozoa to 

### 11 continue to be an important endpoint, but consideration be given 

### 12 to having a semen sample collected. Now semen samples are 

### 13 collected in turkeys, they're collected in chickens, I'm told 

### 14 they are collected in starlings and so that a method to collect 

### 15 semen from quail is an area where it is not outside the bounds 

### 16 of science to develop such a method. It would require some 

### 17 research but I think it would provide a greater reliability were 

### 18 there to be major changes. 

### 19 Again, because of the variation we would 

### 20 encourage the development of web based training for spermatozoa 

### 21 characteristics. Or web based or other way of doing training, 

### 22 we're not prescribing that. 

### 23 Cloacal area should in theory be a very good 

### 24 index of androgenic or antiandrogenic activity. However, there 

### 25 was little evidence provided in any of the studies suggesting 

### 26 that cloacal area was a useful measure. To the best of my

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1 knowledge it was not looked at prior to photostimulation and so 

### 2 the potential androgenic effects may well be missed. 

### 3 So our recommendation is without supporting 

### 4 any supporting data or without investigating the situation in a 

### 5 short day length, non-reproductively active quail, this 

### 6 parameter may or may not be useful. 

### 7 Hormone assays. Hormone assays are 

### 8 appropriate, useful and clearly meet the criteria for 

### 9 reliability and relevance. 

### 10 That said; we consider the number of 

### 11 replicates was very low and for the protocol if it's in revision 

### 12 or further clarified to up the end -- and we would say probably 

### 13 to an end of eight rather than an end of five. And particularly 

### 14 where samples get lost where you're then getting down to ends of 

### 15 three and four. And you could be pretty well guaranteed not to 

### 16 get anything significant. 

### 17 An area that requires further research is the 

### 18 area of vitellogenin. The information provided was such that it 

### 19 really was not done in a consistent way. It was not clear if it 

### 20 was being measured by ELISA -- if it can be measured by ELISA. 

### 21 Certainly the ability to use real time BCR would make a lot of 

### 22 sense. 

### 23 Recommendation; there is a need for further 

### 24 research to assess the validity of assays for plasma 

### 25 vitellogenin in the Japanese quail and whether this is a good 

### 26 endpoint for endocrine disruptors.

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1 A further suggestion or recommendation for 

### 2 research was the potential to develop in ovo or in vitro 

### 3 vitellogenin production as almost like a screen one, so looking 

### 4 at whether chick embryo hepatocytes respond to estrogen or to 

### 5 endocrine disruptors; something that might be worth considering 

### 6 by the Agency. 

### 7 This concludes the summary report. 

### 8 **DR. MARTHA SANDY:**So Dr. Bentley. 

### 9 **DR. GEORGE BENTLEY:**Well I agree with 

### 10 everything that Dr. Scanes has said. Let me just move this 

### 11 over. I would just like to add that there are a couple of good 

### 12 vitellogenin assays available to measure plasma vitellogenin in 

### 13 birds and qPCR to measure messenger RNA for vitellogenin in the 

### 14 liver. It works very well in several labs. So that should be 

### 15 possible. 

### 16 **DR. MARTHA SANDY:**Dr. McCarty. 

### 17 **DR. JOHN MCCARTY:**Two points about selection 

### 18 of endpoints. 

### 19 One is fairly straightforward and that has to 

### 20 do with increased aggression, which in the current protocol is 

### 21 treated as a husbandry issue. It's mentioned in Section 4.1.6 

### 22 as a possible problem. But I guess I'm wondering why it can't be 

### 23 an endpoint especially given that if you look in your summary of 

### 24 other studies. For example, 5.3.1 it's found that one of the 

### 25 effects of the treatment chemical was increased aggression. So 

### 26 that seems fairly straight forward to keep track of that as an

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1 endpoint through the process. 

### 2 The other more vague and harder to address 

### 3 addition would be given its -- I'm a little concerned that 

### 4 immunofunction is not given the attention it deserves given both 

### 5 its links to endocrine disruption and then given the attention 

### 6 we've heard about wanting to have these endpoints be relevant to 

### 7 an ecological risk assessment. Immunofunction would certainly 

### 8 be relevant in that context as well. And in my written comments 

### 9 I'll try to be more specific about how you might do that, but 

### 10 there are certainly lots of methods that are well established 

### 11 for testing the immunofunction in birds. 

### 12 **DR. MARTHA SANDY:**Dr. Johnson. 

### 13 **DR. DALLAS JOHNSON:**I had no comments but I 

### 14 would like read into the record what Dr. Portier had to say and 

### 15 in case he wants to expand upon it in the written report. 

### 16 He indicated in Section 4 it provides detailed 

### 17 discussion on 15 different classes of assay endpoints. This 

### 18 discussion should be followed up with a section on how an 

### 19 evaluator might choose one or more of these endpoints to be used 

### 20 in combination with the information in Tables 11 to 13 in the -- 

### 21 I think it's Appendix C is it. He gives a reference to it but I 

### 22 believe it's one that is known as Appendix C. 

### 23 To guide and justify the highest or second 

### 24 highest test concentration the information to create this 

### 25 guidance is available in the two documents that just need to be 

### 26 put together. The discussion in Section 5 provides another set

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1 of information to support guidance that matches detection 

### 2 distances that give power to key assay endpoints in Section 5. 

### 3 Each chemical has a discussion of the levels used in the 

### 4 endpoint the researchers found most important on which to 

### 5 comment. 

### 6 For example, the estradiol studies Section 

### 7 523.2 neural systems and bursitides as key assay endpoints. If 

### 8 one were to design a new JQTT for estradiol would we again use 

### 9 0.550 over 500 milligrams per egg or would a sample size 

### 10 analysis point to a very different sequence. 

### 11 And then he also said I recommend Section 5.5 

### 12 be expanded and integrated with the information from the power 

### 13 analysis. 

### 14 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 15 **DR. EDWARD PERKINS:**I have a couple of 

### 16 points. On the protocol in general I think it might -- and I 

### 17 noticed this with most of the models that there seems to be a 

### 18 lot more technique and execution problems with these life stages 

### 19 between multiple laboratories, and it may do to have a library 

### 20 of YouTube videos based to actually show people how to hold 

### 21 things, how to do things, troubleshoot things. Sort of an 

### 22 idiot's guide to toxicology, so that they have something to look 

### 23 back to that they can easily look at. 

### 24 In regards to positive controls and standards 

### 25 and probably the supply may also apply the Question 3.2 actual 

### 26 standard slides, histo slides that you can show that you know

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1 the results on that you can give people to see how their ability 

### 2 to actually interpret what your interest in, the endpoints are 

### 3 interest in. Some sample studies might help out quite a bit to 

### 4 get a standardized analysis results and get people to practice 

### 5 basically. 

### 6 As far as endpoints I think one thing and I 

### 7 hate to add things to it, but I guess that is sort of our job. 

### 8 In a system sort of context, one of the things I sort of view 

### 9 the Tier 2 as and it's actually stated in the EDSTAC, 

### 10 characterizing the nature likelihood and dose response 

### 11 relationship of types of endocrine disruption. One of the 

### 12 things I don't really see is the animals being look at as a 

### 13 system very well. 

### 14 We're looking at multiple endpoints. We're 

### 15 not really relating one to another really tightly, I don't 

### 16 really see analysis for that. 

### 17 So one of the things I wanted to recommend was 

### 18 the possible inclusion of transcriptomics or metabolomics. One 

### 19 advantage is transcriptomics may provide some underlying causes 

### 20 that relate different signaling pathways together and can also 

### 21 help provide weight-of-evidence for androgen and antiandrogen 

### 22 effect. 

### 23 That in combination with the apical effects 

### 24 and the secondary sexual characteristics and so forth, these can 

### 25 make really nice evidence that support each other towards a more 

### 26 defined conclusion than independent things themselves. And

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1 through that you can also address some of the epigenetic effects 

### 2 with methylation status and things like that. Oh and 

### 3 metabolomics. 

### 4 **DR. MARTHA SANDY:**Dr. Bentley. 

### 5 **DR. GEORGE BENTLEY:**Just to follow up from 

### 6 Dr. Perkins' comment about the YouTube idiot's guide, I think 

### 7 that would be extremely helpful but I also firmly believe that 

### 8 there is nothing as powerful as hands on experience. So perhaps 

### 9 before different labs embark on this test, maybe there can be a 

### 10 workshop to cover some of these more problematic techniques. 

### 11 We see this theme coming up of inter-lab 

### 12 variation throughout these tests so why not have a workshop and 

### 13 then make videos during that time and then the people can refer 

### 14 back to them. 

### 15 **DR. MARTHA SANDY:**This is Dr. Martha Sandy. 

### 16 I have a couple of questions of my own. I wondered, Dr. Scanes, 

### 17 in your reading of the group's response you mentioned that they 

### 18 should consider measuring female behavior endpoints and I 

### 19 wondered if you had any example endpoints to suggest or if you'd 

### 20 like to put them in the written comments. 

### 21 **DR. COLIN SCANES:**I think the female endpoint 

### 22 would be probably much better done in zebra finch. Apparently 

### 23 males do all the decision making in terms of sexual selection. 

### 24 And females do not have that same role. Where as in zebra finch 

### 25 the females have a much stronger role, and this is providing 

### 26 some support for the use of zebra finch because of the ability

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1 to measure some female behavioral parameters. 

### 2 **DR. MARTHA SANDY:**Dr. Bentley. 

### 3 **DR. GEORGE BENTLEY:**Again, I agree with that 

### 4 and I would just like to add that, well reaffirm that female 

### 5 quail don't seem to have much of a choice when it comes to 

### 6 mating. Female zebra finches, their mate choice behavior has 

### 7 been quite well documented. They seem to make decisions based 

### 8 on symmetry of males. The perfection of the male song and this 

### 9 gets back to the question about learning vocalizations in zebra 

### 10 finches. 

### 11 Quail do not learn the vocalizations. They 

### 12 don't have a neuro song control system. So I think if we're 

### 13 going to look at aspects of female behavior, which are, in my 

### 14 opinion, critical at a population level for most bird species 

### 15 then you need to test it in the species where it is easily 

### 16 measurable. 

### 17 **DR. MARTHA SANDY:**And I also wondered, would 

### 18 you want to expand on -- oh Dr. McCarty. 

### 19 **DR. JOHN MCCARTY:**Just very quickly, one 

### 20 female behavior that could be done in Japanese quail that's 

### 21 ecologically relevant would be incubation behavior. 

### 22 **DR. MARTHA SANDY:**Thank you. The other 

### 23 question I had was in suggesting a positive control be included 

### 24 in the experiment. Can you say a little more about how you 

### 25 would chose a positive control? 

### 26 **DR. COLIN SCANES:**Without having talked to

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1 the group first, I'm a little loathe to do this but I'm going to 

### 2 do it anyway; and that would be if one is looking at an agent 

### 3 that has androgenic activity to include something like 

### 4 trenbolone. If something is a putative estrogen, to include 

### 5 estradiol and if one gets no effect let's say with estradiol 

### 6 when you're expecting one that tends to mean that you're kind of 

### 7 surprised with that. So it's a similar agent to the agent that 

### 8 you're testing. 

### 9 **DR. MARTHA SANDY:**Dr. Delclos. 

### 10 **DR. BARRY DELCLOS:**Would you then - I mean 

### 11 this -- this is complications when you're doing a Tier 2 type of 

### 12 test. You use one dose of the positive control or two doses. 

### 13 You're also going to have analytical chemistry costs involved 

### 14 and so forth. 

### 15 **DR. COLIN SCANES:**If there is substantial 

### 16 data in the literature showing an effect, and certainly with the 

### 17 trenbolone there is and at 20 parts per million the data that 

### 18 Les so well showed yesterday, that would provide confidence that 

### 19 the contract lab was performing the test in an adequate and 

### 20 satisfactory manner. 

### 21 **DR. MARTHA SANDY:**Dr. McCarty. 

### 22 **DR. JOHN MCCARTY:**I was going to say this 

### 23 later but I'll address it now. I am sympathetic with yes it is 

### 24 going to add complications and it's going to add cost. On the 

### 25 other hand I'm concerned that some combination of poor 

### 26 contractor lab performance or low statistical power is going to

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1 result in production of nothing but negative results that we 

### 2 don't know if they are false negatives or real negatives. 

### 3 And if those are accepted for regulatory 

### 4 purposes and they're false negatives, then we've wasted all the 

### 5 money. Yes, it's an added expense, it's an added effort, but 

### 6 given the results we see here, I think it's a necessary addition 

### 7 in my opinion. 

### 8 **DR. MARTHA SANDY:**Dr. Bentley. 

### 9 **DR. GEORGE BENTLEY:**I entirely agree. I 

### 10 think it would be a false economy not to include a positive 

### 11 control of that sort. And I think a single dose at a level that 

### 12 has been proven to work before in the literature would have the 

### 13 predicted effect before in the literature would be entirely 

### 14 appropriate. 

### 15 **DR. MARTHA SANDY:**Other comments from the 

### 16 panel on Question 3.1? And EPA any questions? 

### 17 **DR. LESLIE TOUART:**Just for clarification on 

### 18 the positive control. Do I understand correctly that you're 

### 19 suggesting that a positive control be included with every test 

### 20 or are you suggesting that it might be appropriate as maybe a 

### 21 kind of a one time or periodic inclusion to demonstrate 

### 22 proficiency at that particular laboratory? 

### 23 **DR. COLIN SCANES:**We were suggesting that it 

### 24 would be included in every test which would give, I think, a 

### 25 degree of confidence in the results of that individual test with 

### 26 those same personnel, that same lab.

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1 **DR. MARTHA SANDY:**I believe we're ready to 

### 2 move on to Question 3.2. 

### 3 **DR. LESLIE TOUART:**Okay. Reading Question 

### 4 3.2. Pathological evaluation of histological tissue 

### 5 preparations is an established, sensitive and integral endpoint 

### 6 in the assessment of effects in long-term in vivo assays. The 

### 7 tissues targeted for histopathological evaluation should be 

### 8 shown to be sensitive to exposure and relevant to a mode of 

### 9 action or pathway determination. Tissue samples from several 

### 10 organs or glands for example kidney, liver, thyroid, gonads, 

### 11 underwent histopathological examination in the Japanese Quail 

### 12 Two-Generation Test. Inter-laboratory validation and a 

### 13 discussion of histopathology as an endpoint is provided in 

### 14 Section 4.1.13 of the ISR. 

### 15 Please comment on the value of 

### 16 histopathological analyses in the Japanese quail test assay for 

### 17 each of the tissue types examined and what, if any, critical 

### 18 information is gained from their inclusion or would be lost if 

### 19 histology were not examined. 

### 20 **DR. MARTHA SANDY:**Dr. Scanes. 

### 21 **DR. COLIN SCANES:**I'm going to answer this, 

### 22 first of all, in the abstract. In principle histopathology 

### 23 needs to be included. Abnormal histopathology provides very 

### 24 strong evidence for endocrine disruption. However, the data 

### 25 that is presented is insufficient to make definitive 

### 26 prescriptive conclusions. There will be a series of tentative

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1 recommendations proposed. 

### 2 Before I get to that I'd like go through -- 

### 3 I'll go through these recommendations and then I'll go through a 

### 4 detailed analysis of what we did. For the multiple endpoints 

### 5 employed in the Japanese quail Two-generation Test there was 

### 6 predominantly a lack of consistent responses to the putative 

### 7 endocrine disrupter. 

### 8 Histological data albeit limited was 

### 9 consistent. So the first thing is we've got a consistency in 

### 10 the data. There is a very strong case for epididymal and 

### 11 gonadal histology. 

### 12 The gonadal histology providing evidence of 

### 13 sex reversal, the epididymal histology was consistent with a 

### 14 lesion. There is a reasonable case for liver histology. In the 

### 15 absence of testing a known goitrogen in any of the tests that we 

### 16 saw, it was not possible to draw conclusions on whether or not 

### 17 thyroid histology should be part of the test. 

### 18 So it's an area that needs to be researched on 

### 19 whether there are changes in the thyroid and certainly 

### 20 consistently you would expect them with a goitrogen, but we saw 

### 21 no data on that area. 

### 22 There is an overwhelming case for research to 

### 23 develop quantitative morphometric indices for epididymal liver 

### 24 and possibly thyroidal histology and one should add in gonadal. 

### 25 This would greatly facilitate statistical analysis. The number 

### 26 of replicates must be -- should be sufficient to draw

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1 conclusions. In some of the cases we were down to ends of 

### 2 three, four and at most five. Consideration should be given to 

### 3 in situ or immunocytic chemistry. 

### 4 I want to go through some of the data and 

### 5 first of all complement the contract labs for the quality of the 

### 6 histology. The histology was very, very nice. 

### 7 Five replicates should have been used for each 

### 8 treatment. We'll come and see that this wasn't always the case. 

### 9 The pathologists used a grading or a severity score. And it 

### 10 states in one of the references "pathologists for each study 

### 11 laboratory conferred to harmonize diagnostic terms and severity 

### 12 grading." 

### 13 The pathologist used a severity grading. They 

### 14 talked about Grade 1 for the testing for the gonads. Morphology 

### 15 consistent with the male. Grade 2 was 80 percent containing 

### 16 spermatogonia and less than 20 percent containing oogonia. 

### 17 Grade 3 gonad contained distinct foci of ovarian and testicular 

### 18 tissue that encompassed about 50/50. 

### 19 We go onto Grade 4 and Grade 5 

### 20 proportionately. When looking at the liver a system was used 

### 21 for non response -- the liver and other tissues I should 

### 22 clarify. A non response of less than 5 percent of the tissue 

### 23 affected; minimal 5 to 30 percent; mild 30 to 60 percent; 

### 24 moderate 60 to 80 percent; severe over 80 percent of the cells 

### 25 affected. 

### 26 Now if you've got -- first of all if you've

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1 got a score, one could do statistical analysis on those scores. 

### 2 That was not done. But if you're talking about percentages, why 

### 3 not actually go to those percentages and say that 5.7 percent of 

### 4 the slide was showing abnormality or 5 percent of the cells. 

### 5 If one has a continuous measure, one can use 

### 6 regular statistical analyses. It also means one can use images 

### 7 and use automated systems. Dr. Proper yesterday mentioned some 

### 8 of those techniques that can be used. So I actually went back 

### 9 and just on the scores and I looked at the scores and will 

### 10 include this in the appendix in the report, I converted the 

### 11 scores into just initially doing a mean and standard on the 

### 12 scores and then comparing them between labs. 

### 13 In comparing them between labs there was 

### 14 remarkable consistency. It was not picked up in the summary 

### 15 report or in the reports on the histology. It gave a lot of 

### 16 reason to have confidence in the data. An example was looking 

### 17 at the testes. Zero testes showed abnormalities. Now that's a 

### 18 pretty powerful piece of evidence that you're getting no change. 

### 19 On the other hand with the epididymal data, we 

### 20 were seeing on average in the toxicant treated group scores of 

### 21 twos and uniformly twos. Now we're dealing with here sometimes 

### 22 ends get down to three, four, samples get lost. That's why we 

### 23 are saying we want to have an end of at least eight. 

### 24 Now we also have a concern on the histology 

### 25 and the histopathology; that the pathologists used a 

### 26 representative section. Now we would much prefer either to have

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1 a randomized situation or to systematically go through the organ 

### 2 and take every fifth slide or fifth section. The issue of 

### 3 saying well this is the most representative brings in very much 

### 4 a subjective viewpoint as opposed to using an objective 

### 5 viewpoint, and we're trying throughout this discussion on the 

### 6 histology to look for this to be as objective as possible and 

### 7 amenable to statistics. 

### 8 So in summary in principle we view 

### 9 histopathology as very important. The data was not strong 

### 10 enough to make definitive conclusions but strong enough for us 

### 11 to make recommendations that were tentative to say that we 

### 12 should at least continue with testes, epididymis and liver. And 

### 13 look to the development with research of morphometric analyses, 

### 14 quantitative estimates. 

### 15 **DR. MARTHA SANDY:**Thank you. Any additional 

### 16 comments from Dr. Bentley? 

### 17 **DR. GEORGE BENTLEY:**Thank you. Well I agree 

### 18 with what Dr. Scanes had said. I would just like to add that it 

### 19 wasn't really clear which individuals were actually selected and 

### 20 sampled for analysis, because not all individuals in a group 

### 21 were selected. So I would like information or I'd like to see 

### 22 some standardization as to how individuals were selected, 

### 23 whether they're preferably randomized. 

### 24 And also as described in Docket Article No. 69 

### 25 describes the evaluation of the sections; and it says "after the 

### 26 initial evaluation the study pathologist performed a

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1 reevaluation of all specimens." The single most representative 

### 2 section was histologically evaluated from each tissue. 

### 3 So I would like to see in the absence of any 

### 4 further systematic analysis, that Dr. Scanes described, how is 

### 5 that most representative section decided upon and what are the 

### 6 criteria for that. This might go away if you do a systematic 

### 7 analysis that Dr. Scanes described. 

### 8 **DR. MARTHA SANDY:**Dr. McCarty. 

### 9 **DR. JOHN MCCARTY:**My only addition would be 

### 10 that to the extent the goal of this test is to identify the 

### 11 mechanism then I see this as an extremely important set of 

### 12 information to both try to establish endocrine disruption or to 

### 13 evaluate the presence of toxicity in the birds. 

### 14 **DR. MARTHA SANDY:**Dr. Johnson. 

### 15 **DR. DALLAS JOHNSON:**Yes, I don't have 

### 16 anything to personally add. I did want to read what Dr. Portier 

### 17 had written with response to this item. 

### 18 He indicated that the national toxicology 

### 19 program has extensive experience with histopathological analysis 

### 20 of tissue from toxicology studies. That experience is primarily 

### 21 with rats and mice, but the lesson learned over 30 years of this 

### 22 kind of analysis are sure to be applicable for these analyses 

### 23 performed on Japanese quail tissue. 

### 24 My recollection -- well then this is the 

### 25 second paragraph -- my recollection of the take home message for 

### 26 statistician from these discussions is that if data from

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1 histopathological analysis is to be useful, pathologists have to 

### 2 be extensively trained, continually tested and challenged with 

### 3 samples from known conditions or possible multiple pathologists 

### 4 should be used for key studies. 

### 5 **DR. MARTHA SANDY:**Thank you and Dr. Perkins. 

### 6 **DR. EDWARD PERKINS:**I have no further 

### 7 comments to add. 

### 8 **DR. MARTHA SANDY:**Do any other members of the 

### 9 panel want to add anything to Question 3.2? No. All right I 

### 10 think we're ready. EPA do you have questions of clarification? 

### 11 **MS. MARY MANIBUSAN:**Just another general 

### 12 request from the panel as we move forward and I request this as 

### 13 part of your verbal recommendations, but certainly in the 

### 14 written final report speaking to the specificity. 

### 15 As the Agency has been hearing some of the 

### 16 verbal recommendations so far, we really appreciate that 

### 17 distinction that you're making in terms of areas and additional 

### 18 measurements or methods where we must have for the assays to be 

### 19 reliable and relevant. And where there are areas as we 

### 20 understand there are advancements of science and technology and 

### 21 methods that are always coming on line, areas where we might 

### 22 want to investigate further and those areas are more research 

### 23 focused. If you can please make that distinction to the Agency 

### 24 that would be greatly helpful. 

### 25 And on top of that if you can provide just 

### 26 justifications or rationales for those additional measurements

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1 and by not having them what are we missing. And then in the 

### 2 research areas by having them what would we gained. That would 

### 3 be again just very, very helpful to the Agency. Thank you. 

### 4 **DR. MARTHA SANDY:**Okay. Thank you. Sorry. 

### 5 We will go on to Question 4 for now please. 

### 6 **DR. LESLIE TOUART:**Reading Question 4. 

### 7 Demonstration of the test method performance should be based on 

### 8 the testing of reference chemicals representative of the types 

### 9 of substances for which the test method will be used. 

### 10 Test substances should adequately represent an 

### 11 appropriate range of responses and physical/chemical properties 

### 12 for which the test method is proposed to be appropriate. The 

### 13 selection of the most appropriate statistical approaches depends 

### 14 in part on the nature of the data and also on the design of the 

### 15 validation study. Statistical and non-statistical methods used 

### 16 to analyze should be described. 

### 17 Please comment on the selection of test 

### 18 substances and methods, (analytical and statistical where 

### 19 appropriate) chosen for the demonstration and validation of the 

### 20 Japanese quail assay. 

### 21 **DR. MARTHA SANDY:**Dr. Johnson is the lead 

### 22 discussant on this. 

### 23 **DR. DALLAS JOHNSON:**Are we ready to go for 

### 24 the rest of the day. Hopefully that's a joke, but I'm not sure 

### 25 whether it will be by the time I get finished or not. 

### 26 I'd like to break the charge down into three

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1 questions I guess. Asked to please comment on the selection of 

### 2 test substances and I'm going to let my associate discussants 

### 3 address that because I don't feel like I have any expertise to 

### 4 address that particular issue and I didn't feel comfortable with 

### 5 taking whatever they might provide to me and being sure that I 

### 6 could get it out in a manner that would make sense. And so I'm 

### 7 going to rely upon the associate discussants to address the 

### 8 selection of the test substances. 

### 9 A second part of the charge has to do with the 

### 10 analytical methods that are used and whether they're appropriate 

### 11 for demonstration and validation of the quail assay. And again 

### 12 that's an area where I don't think I have the proper expertise 

### 13 to judge and I'm going to again rely upon the associate 

### 14 discussants to address that part of the charge. 

### 15 The part of the charge which I feel completely 

### 16 comfortable with is the part that has to do with statistical 

### 17 methods. And I do have -- part of this -- maybe I'm going to 

### 18 add something into the charge that is not there because 

### 19 somewhere along the line I guess I was looking for something 

### 20 that might be related to the protocol design and whether the 

### 21 protocol design is appropriate or not and there really hasn't 

### 22 been in any of the charges throughout the four studies that 

### 23 hasn't been addressed. But I feel it's an important issue and I 

### 24 want to take some time to discuss it. 

### 25 I had mentioned yesterday, and the order that 

### 26 I have these is probably not the best order for our report and

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1 hopefully when we do the report it will be in a better order. 

### 2 The order that I have them in is sort of the order that they 

### 3 arose to me as I was reading the ISR. And so if you feel like 

### 4 I'm jumping around a little bit it's because the order is fixed 

### 5 by the ISR itself and I apologize for that. 

### 6 One of the things that I did ask a question 

### 7 about yesterday and I just say it again today just to make sure 

### 8 that we can talk about it in the report is there is a statement 

### 9 made in the report that said most of the variability in the 

### 10 controls is due to strained differences. And I asked a question 

### 11 about whether the different laboratories used different strains 

### 12 or whether the different strains were interspersed across the 

### 13 laboratories and I believe the answer to that was that the 

### 14 strains used within the laboratory were pretty consistent but 

### 15 they were different across laboratories. 

### 16 And so with that in mind then laboratory 

### 17 effects and strain effects are completely confounded with one 

### 18 another and there is no statistical way that we can separate 

### 19 those. So any separation that you make with respect to those is 

### 20 based on expert opinion and not on the statistical analysis. So 

### 21 if you believe the differences are due to strains and not 

### 22 differences due to laboratories then have at it. I don't have 

### 23 any problem with that it's just not a statistical test that we 

### 24 can make. 

### 25 The study designs and a lot of what I'm going 

### 26 to say here today I think probably can relate to maybe each of

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1 the studies that we're evaluating. And I'm not sure that all 

### 2 the points will need to be made every day. So hopefully the 

### 3 next day and the next day and the next day when I get a chance 

### 4 to talk I won't talk so long and just say refer back to what I 

### 5 said about the Japanese quail study. Because a lot of the 

### 6 issues I think are consistent across all of those studies. 

### 7 And this study design as well as the others do 

### 8 a good job I think of discussing factors that can be controlled 

### 9 like the amount of chemical to use, the dose levels, the birds 

### 10 used, the cages that they're done in. But they say very little 

### 11 about how randomization is used and how it should be used. 

### 12 I did find in a statement somewhere that they 

### 13 were supposed to do something impartially and I didn't know 

### 14 whether impartially was another acronym for randomization or 

### 15 whether it meant something different. So I have a feeling that 

### 16 it's not quite as much as randomization but you're not supposed 

### 17 to bias your results. You're supposed to look at them 

### 18 impartially and so I think that is important to replace that, I 

### 19 think, with some mechanism to ensure that randomization is used. 

### 20 Now just some questions about the quail study 

### 21 that come to mind and I don't know the answers and I didn't try 

### 22 to clarify these with the discussion yesterday, but they are 

### 23 questions that arise and I think that if you're going to propose 

### 24 a protocol for other studies to be done and laboratories to do 

### 25 these things, I think it is important to get things a bit more 

### 26 standardized and a bit more under control in terms of -- so the

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1 results are less dependent on the laboratory and more dependent 

### 2 on the chemicals that are being studied. 

### 3 So some of these things that came to mind with 

### 4 respect to the pairs of birds that are placed in the same cage. 

### 5 We have in this study I think there is 12 caged pairs and one 

### 6 male and one female, but then it doesn't say anything about what 

### 7 are done with the cages. So the questions I have are the cages 

### 8 in the same room, are they in different rooms? If they are all 

### 9 in the same room where are they located in that room? Were 

### 10 these locations selected randomly? How is randomization used to 

### 11 select the pairs to be mated? And then eggs are taken from the 

### 12 pairs that are put together and how are these eggs selected. 

### 13 Are they selected randomly? 

### 14 In some sense you select all the eggs because 

### 15 you count the eggs so you have them all there but then some 

### 16 tests are done on some of those and other tests are done on 

### 17 other ones. So which eggs are used for which tests and how are 

### 18 they selected. I think just to make some comments I guess on 

### 19 the process that should be done in some random manner. 

### 20 The feed and water -- how is feed and water 

### 21 placed in to the cages, are they going in the same order every 

### 22 day, was there a different order used every day. If the order 

### 23 randomized in the order in which the feed and water was placed 

### 24 in the cage. And then are all the birds in a given treatment 

### 25 group housed together. 

### 26 In other words if the control group is located

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1 in one portion of the room and the -- one of the treating groups 

### 2 is in another portion of the room and another treated groups in 

### 3 another potion of the room, then you got differences in 

### 4 treatments that are confounded with the location in the room. 

### 5 And so those locations in the room that I think in which -- or 

### 6 wherever they're at some thought and care should be taken to 

### 7 make sure that those sorts of ideas are getting randomized and 

### 8 so that the idea of randomization is to guarantee -- there are 

### 9 certain things you know might bias the data and so you want to 

### 10 randomize to avoid those things but there are other things that 

### 11 are unknown that might also bias the data that you don't know 

### 12 about but you want to guard against them. And so the idea of 

### 13 randomization is to guard against all those things that might 

### 14 bias the data whether they are known or unknown and to try to do 

### 15 things in as random a process as is possible. 

### 16 What wasn't clear, I guess I wasn't able to 

### 17 tell whether in this study involving the Japanese quail so we 

### 18 have 12 pairs under each treatment. Say there is maybe 24 cages 

### 19 for the controls and 12 cages under each of the dose levels and 

### 20 then it's not -- I couldn't really tell in the report whether it 

### 21 was all done at the same time, did they all start at day one and 

### 22 they all finished on the day the thing ended or was this 

### 23 staggered out throughout the study period and again something 

### 24 needs to be -- I think it's important that something needs to be 

### 25 said about the order in which these things are done and how they 

### 26 are done and where randomization is just taking place and where

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1 not. 

### 2 And I guess in this I also threw in a question 

### 3 about how are the initial birds chosen for each of the 

### 4 laboratories. Later on in the report it stated that dose level 

### 5 during the sixth and seventh post treatment weeks, if that dose 

### 6 level does not produce at least 40 eggs then that level is 

### 7 dropped from further analysis to the test. 

### 8 And it wasn't clear to me whether that meant 

### 9 40 eggs from a single pair, or 40 eggs from all the pairs that 

### 10 are in a single dose level. This wasn't clear to me what that 

### 11 was referring to. And so I think that can be clarified a bit. 

### 12 A statement was made at four weeks of age the 

### 13 F1 control treatment group birds are weighed and then randomly 

### 14 allocated to pens by pairs with male offspring from odd numbered 

### 15 parental pairs being paired with females from even numbered 

### 16 parental pairs. And even numbered pen males being compared with 

### 17 odd numbered females; I guess that is randomization coming into 

### 18 play there. 

### 19 But the question that arose for me is does 

### 20 that guarantee that paired birds do not come from the same 

### 21 lineage or does that matter? 

### 22 Earlier it was important there somewhere that 

### 23 the birds come from different lineages and then I think in a 

### 24 follow up statement it said that the process should be a 

### 25 stratified random process to assure to the extent possible that 

### 26 each of the F0 pairs are contributing genetically to the F1

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1 breeding population in order to represent individuals that may 

### 2 vary in sensitivity to chemical exposure. 

### 3 And again it's just a little bit confusing to 

### 4 me about what all that means. Maybe to the scientists in the 

### 5 room that all makes sense, but I was not really able to follow 

### 6 it through myself. 

### 7 Another statement that says the frequency of 

### 8 diet renewal should not be more than once a day and not less 

### 9 than once a week. That's a pretty big range. And so there is 

### 10 another area where I think things could be tightened up a bit to 

### 11 where you do it every other day or you do it every day. So at 

### 12 least that one lab is not doing it once a day and another lab is 

### 13 doing it once a week, I think that is an issue than can be 

### 14 straightened up a bit or tightened up a bit and made less 

### 15 variable. 

### 16 While I'm thinking about it I don't have this 

### 17 written down here but I hope I don't forget about it, is I know 

### 18 when we do studies with animals back at Kansas State University 

### 19 and anytime we're involving weight, it sort of became a standard 

### 20 practice that you kept them from drinking water over night and 

### 21 then you weighed them the first thing in the morning before they 

### 22 had any chance to eat or drink or do anything so that you sort 

### 23 of took away some of the variability that might occur in weight 

### 24 due to the amount of water that they've ingested. 

### 25 So I'm wondering if things like that should be 

### 26 incorporated into the weight of Japanese quail. If you're going

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1 to weigh these things, should you weigh them at some certain 

### 2 time of the day when after they've been -- after they've been 

### 3 removed from food and water for a few hours or so or overnight 

### 4 so that maybe that can take some of the variability out of that 

### 5 response and might make the assays and other kinds of analyses 

### 6 more powerful in that way. 

### 7 There is a question I had about incubators and 

### 8 hatchers that said incubators and hatchers preferably with 

### 9 automatic temperature and humidity controls and an egg turning 

### 10 device are necessary. In addition suitable equipment is 

### 11 required to maintain stored eggs within the temperature and 

### 12 humidity ranges specified. And so I guess my question there is 

### 13 there an incubator for each pair or are all the eggs placed in 

### 14 the same incubator at the same time and if they're all placed in 

### 15 the same incubator at the same time are their locations in the 

### 16 incubator randomized? 

### 17 I don't know how big these incubators are, are 

### 18 they really huge and do you get a whole bunch of eggs in it or 

### 19 are they real small so I can only put one egg in one at a time. 

### 20 And again a place where -- again the idea is to try to guard 

### 21 against any affects that might bias the results whether you 

### 22 think they might bias or whether they might not bias. 

### 23 I guess my concern, I usually have a -- put it 

### 24 in here that some of the things when I teach classes I try to 

### 25 tell the students that when I - my concern is that when people 

### 26 do things in a certain way because it's convenient to do it that

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1 way, certainly a lot of ways that are more convenient to do 

### 2 things than it is randomization. And randomization is not easy 

### 3 and it's not very convenient to do it. 

### 4 But I try to tell my students that convenience 

### 5 is an enemy of good experimental design. If you do something 

### 6 because it is convenient to do it that way there is probably a 

### 7 better way. And so that's sort of my driving philosophy I guess 

### 8 behind some of the comments and things that I'm making here. 

### 9 There is a statement in the reports that says 

### 10 chicks should be identified individually or by pen of origin. 

### 11 Maybe be housed in groups of approximately equal number by 

### 12 treatment group. And I guess that sent up a red flag. 

### 13 Are we taking chicks that came from one pair 

### 14 of adults and mixing them up with chicks that came from another 

### 15 pair of adults so that we can get them in groups of 

### 16 approximately equal numbers. So it's a little bit like Medaka 

### 17 thing there yesterday where we're putting all the fish in a 

### 18 basket and stirring up the basket and then selecting out to go 

### 19 on to the next generation. 

### 20 And so it's not clear to me in my mind as to 

### 21 how that is being done here and but I am a bit concerned about 

### 22 what effects it might have on the statistical analysis and about 

### 23 mixing chicks from different pairs. And I think its -- maybe 

### 24 it's not an issue if you're not trying to follow the adult pair 

### 25 that you started with all the way through the process. If you 

### 26 want to do that and you wanted to compare F2 to F1 then you

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1 probably need to follow that thing through. 

### 2 If you just want to talk about F2 by itself 

### 3 and F1 by itself, then perhaps this idea of mixing might be 

### 4 okay. I haven't really made up my own mind on that yet. But 

### 5 there again I realize this is a convenient way to do things but 

### 6 again I wrote down that convenience is an enemy of good 

### 7 experimental design. 

### 8 There is a statement that made all F0 birds 

### 9 used in a test should be from the same hatch. And I don't know 

### 10 enough about Japanese quail to know what that means. I'm sure 

### 11 it doesn't mean that they should all come from the same parents 

### 12 so just for us non scientists, I guess, that can be clarified a 

### 13 bit. 

### 14 It's indicated that the eggs were stored in a 

### 15 cold storage facility for a maximum of two weeks prior to 

### 16 setting them in the incubator and again were the eggs placed in 

### 17 that cold storage facility. I'm sure that some parts of the 

### 18 facility are colder than other parts. And the temperature is 

### 19 probably not constant in there. Are the eggs placed at random 

### 20 locations in the cold storage facility or are eggs from the same 

### 21 parents kept together. 

### 22 A statement was made that during the study one 

### 23 egg per pen is collected from odd numbered pens -- in odd 

### 24 numbered weeks and even numbered pens and even numbered weeks to 

### 25 evaluate egg shell strength. I guess in some sense, going odd 

### 26 and even is part of a random process and I can live with that,

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1 but is the egg selected for looking at egg shell thickness is 

### 2 that done at random. And if so how is the randomization 

### 3 assured. Here I can see that somebody trying to do something 

### 4 impartially might not be the same as being randomized. 

### 5 Aren't you glad you invited a statistician to 

### 6 be a part of this panel? 

### 7 Fertility and embryo viability in all 

### 8 incubated eggs are determined by cattling the eggs after eight 

### 9 days of incubation, EDA and two days before hatching at 

### 10 embryonic day 15. Eggs will be transferred to a hatcher. And 

### 11 then is randomization involved in this process and if so how. A 

### 12 statement was made that some sample sizes were too small for 

### 13 statistical analyses and I don't know how small is too small? 

### 14 Well there is again some statements that came 

### 15 about comparing the data from the three labs that involved 

### 16 strains again, but I think we've already determined that. 

### 17 Power determinations were carried out for each 

### 18 endpoint based on the control group within run variance. I'm 

### 19 not sure that I understand exactly what is meant by "within run 

### 20 variance." I think I know what is meant but it would be helpful 

### 21 to have that clarified. 

### 22 In these power analyses and I think this also 

### 23 relates to the co-efficience of variation that are talked about 

### 24 several times throughout all of these reports is that we have 

### 25 these different sources of variability. There is sometimes pen 

### 26 to pen variability and sometimes there is variability within

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1 groups and variability between groups and when you look at a 

### 2 co-efficient of variation you would look at the variance divided 

### 3 by the mean. Standard deviation divided by the mean. 

### 4 Well then the question arises which standard 

### 5 deviation are we using? And it's not always clear what standard 

### 6 deviation is used in calculating that ratio. So is it the bird 

### 7 to bird variation or fish to fish variation or in the Medaka 

### 8 study is it the tank to tank variation. Just what type of 

### 9 standard deviation is being used when the co-efficience of 

### 10 variation are calculated. 

### 11 In Table 14, this is I guess in Appendix C. 

### 12 It was also, I think, a table that was shown, it has the means 

### 13 and standard errors in comparisons of avian vinclozolin 

### 14 concentrations to control across laboratories and they're 

### 15 reported as the mean in the standard error and in Lab 1 I'm just 

### 16 looking at the adult body weight. 

### 17 This sort of issue occurs in all the variables 

### 18 that you're looking at in Table 14 but the one I copied in and 

### 19 put in here is adult body weight. 

### 20 And in Lab 1 you have -- and this is in the 

### 21 control group -- 190.6 standard error of 3.36. Lab 2 is 202.7, 

### 22 standard error 4.357. Lab 3 is 113.4, standard error 1.8. But 

### 23 then when you go to the average the average is 170.4 which is 

### 24 kind of between -- somewhere around the 113, the 190 and the 202 

### 25 so it looks like a reasonable average, but then instead of 

### 26 standard error like 3.36 and 4.35 and 1.82 you've got a standard

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1 error of 29.5. 

### 2 And so there again it comes in as to what 

### 3 variance is being used and what quantity is being incorporated. 

### 4 Now that could be that that is the right standard error report 

### 5 because now you have to have lab to lab variation that goes into 

### 6 that variance. But it looks a bit -- if you're comparing the 

### 7 control group to the other groups, then you get a lot different 

### 8 comparisons within labs than you might get across labs. And if 

### 9 you're doing it across labs you ought to be able to compare 

### 10 controls to treated groups within a lab, get that difference in 

### 11 standard error and then take a weighted average of those short 

### 12 of a metaanalysis to do a comparison across labs. 

### 13 So just taking the average and the standard 

### 14 errors associated with that average and doing a comparison 

### 15 between control and treating groups, certainly wouldn't be the 

### 16 most powerful way to do it. And I'm not even sure it would be 

### 17 an unbiased way to do it. 

### 18 I struggled a lot with Table 11. I think now 

### 19 I'm getting to where I feel a little bit better about it. But 

### 20 I'll probably have some comments about it in the report. 

### 21 This is a table where you give the control and 

### 22 then you give the standard error the control, you give the 

### 23 sample size per group and then you indicate the detection that 

### 24 can be detected at a certain distance with 80 percent power. 

### 25 And then you have a lower limit and an upper limit, which appear 

### 26 to be confidence limits on the control mean.

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1 Then if you look at the detection distance and 

### 2 the standard error, take the ratio of those two then that's 

### 3 constant across all variables. So there is evidently some sort 

### 4 of critical point in there. I'm trying to decide in my own mind 

### 5 whether that critical point makes sense or not. It's about 4 

### 6 point something and originally I thought it was way too big but 

### 7 then I forgot about the fact that you might need to incorporate 

### 8 a fact -- a standard -- a square root of two in that to account 

### 9 for the fact that you're comparing a control group to a treated 

### 10 group and then if you put that square root of two in there then 

### 11 it seems to bring it back into line. 

### 12 So I'm mentioning it here just so if I want to 

### 13 write about it in the report, I can. I at least brought it up 

### 14 but I'm not sure that I'm going to have to write about it in the 

### 15 report. I'll talk to Mary about that. 

### 16 There is a section that deals with statistical 

### 17 analyses in -- there is some sort of bolded remarks there that I 

### 18 don't have much of a problem with. Need to consider and allow 

### 19 for nonlinear data analysis. Analysis with many endpoints may 

### 20 dilute the detection of effects, primary responsive endpoints 

### 21 must be identified for assessment, parametric tests preferred 

### 22 but many of the data do not meet statistical assumptions. 

### 23 Depending on the measurement endpoint the statistical analysis 

### 24 must be preplanned to accommodate a range of issues that are 

### 25 inherent in the types of data to be collected. 

### 26 Assessment should include some acknowledgement

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1 of variability as an indicator of effect. And then it goes into 

### 2 nonparametric tests may be appropriate for behavioral tests and 

### 3 ordinal data and standardized methods should be prescribed so 

### 4 that there is consistency in the analyses. 

### 5 I want to address this, these non parametric 

### 6 tests a bit. But before I do that I want to talk a little bit 

### 7 about the transformations of data because transformations of 

### 8 data for analysis have been suggested. They have been suggested 

### 9 when the data are not normal and they have been suggested when 

### 10 the data had unequal variances. 

### 11 I guess I agree that transformations are 

### 12 useful when nonnormal data can be transferred in a way that 

### 13 makes the data more closely normally distributed, but 

### 14 transformations that deal with unequal variances, it seems to me 

### 15 at this day and age are no longer necessary. We have 

### 16 statistical software that can deal with analyses of data with 

### 17 heterogeneous variances. 

### 18 And so I feel pretty good with transformations 

### 19 to try to make the data more like the analysis. In other words, 

### 20 a lot of our analysis methods are based on normally distributed 

### 21 data and the good thing about those analyses is they're fairly 

### 22 robust than non-normal data, more robust than what some might 

### 23 think. 

### 24 So even though the data are not entirely 

### 25 normally distributed they probably still do a pretty good job in 

### 26 my opinion. It's the report and not just this report but some

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1 of the other reports suggest that you do a Shapiro-Wilk test and 

### 2 test for normality and if it is rejected you try a 

### 3 transformation, log, square root, arcsine whatever 

### 4 transformation comes to mind and try to find a transformation 

### 5 that is more normally distributed. 

### 6 My experience has been with these normality 

### 7 tests is that they are highly dependent on sample size. That if 

### 8 you have a large sample size you'll reject any distribution you 

### 9 try. If you have a small sample size you won't reject any. And 

### 10 so I'm not sure these tests on normality are all that useful. 

### 11 I think that whether the data are normal or 

### 12 not probably is better based on your experience. These data 

### 13 endpoints that you're looking at are endpoints that have been 

### 14 used over and over and over again in many, many studies about 

### 15 which people have lots and lots of experience, or at least I 

### 16 would think they would, and the appropriate units for analysis 

### 17 ought to be known. And that seems to me that those are the 

### 18 appropriate units that should be used whether the data are 

### 19 normal or not. 

### 20 For example in the classes I teach I think I 

### 21 used -- I'm not a chemist so I never get it right. Anyway so pH 

### 22 is the transformation of the negative ion concentration or 

### 23 something like that. Is that right? Hydrogenised ion 

### 24 concentration? Hydrogenised Ion concentration? Okay. We'll 

### 25 get that right in the report. 

### 26 But the idea is that there is a variable that

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1 was known to be non-normal and so it got transferred to pH and 

### 2 so everybody does the analysis in terms of pH because that 

### 3 variable tends to have a more normal distribution. And it seems 

### 4 to me that many of these data that we have for endpoints here, 

### 5 there ought to be able to make a good scientific guess on what 

### 6 the appropriate distribution might be and if you know what the 

### 7 appropriate distribution is then you perhaps know what the 

### 8 appropriate transformation might be. Or there are 

### 9 transformations that are made for lots of kinds of data. Like 

### 10 if you're looking at proportions, proportions is often 

### 11 transformed using an arcsine transformation or something similar 

### 12 to that. 

### 13 And so it's sort of the way that the variables 

### 14 come about and the way they're used it seems to me that you 

### 15 ought to be able to decide what the appropriate unit of analysis 

### 16 is or what the appropriate distribution is and then base 

### 17 transformations on that. 

### 18 I guess I made a statement here that I have 

### 19 some issues with letting the data drive the analysis. I think 

### 20 in some sense the science should drive the analysis and there 

### 21 maybe -- if you're looking at something completely new and you 

### 22 never looked at it before and you don't know anything about it 

### 23 then yes, maybe you should try to decide what the appropriate 

### 24 unit for analysis is, but on a lot of these things that you're 

### 25 looking at, time to first mount, time measurements often has an 

### 26 exponential distribution or a log normal distribution or some

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1 Weibull distribution so there ought to be a distribution that 

### 2 might be able to be used for that. Okay. Enough about 

### 3 transformation. 

### 4 The next thing I wanted to address is the 

### 5 nonparametric stuff. It seems like this report in other words 

### 6 talks about doing the analysis of variance on ranks as sort of a 

### 7 way to treat the data nonparametrically and that was a process 

### 8 that was introduced a fairly long time ago by a very good friend 

### 9 of mine and is a major professor. 

### 10 And turns out it works pretty good under 

### 11 certain circumstances but studies have been shown on later that 

### 12 nonparametric analyses and these ANOVA on ranks don't work very 

### 13 well under certain types of conditions. 

### 14 I sort of knew in my mind what these 

### 15 conditions were, but I was too lazy to go find the books and get 

### 16 them in so I Googled it; got Wikipedia and I'll just give you 

### 17 some of this information that is there and I tend to believe 

### 18 these things and I come across it several times in the past and 

### 19 so on. 

### 20 One of the places that these rank 

### 21 transformations don't work at all is if you have interaction. 

### 22 So if you have a one-way treatment structure and you're just 

### 23 doing analysis of a one-way experiment then the rank transforms 

### 24 seems to be working pretty good and they give you almost as good 

### 25 a result as you might get with normal, even if the data are 

### 26 normal.

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1 But if you have interaction they don't work at 

### 2 all. There is an increase in type one error resulting in a 

### 3 complete failure of the statistics with as high as 100 percent 

### 4 probability of making a false decision or type one error. 

### 5 And they also found -- I don't know that it's 

### 6 appropriate or worried about it here -- but it's found that rank 

### 7 transformation increasingly fails in the two dependent samples 

### 8 as the correlation between the pre-test and post test scores 

### 9 increase. 

### 10 Transforming ranks and I think at least one of 

### 11 the reports talks about transforming ranks by sort of looking at 

### 12 the ranks and then transforming them into the quantiles of the 

### 13 normal distribution or some other distribution so they are 

### 14 sometimes called a quantile normalized data. The statement in 

### 15 Wikipedia is that random normal scores and expected normal 

### 16 scores transformation have been shown to greatly inflate type 

### 17 one errors and severely reduced statistical power. And so that 

### 18 sort of technique should not be done. 

### 19 The reports seem to indicate that the ANOVA on 

### 20 ranks might be appropriate when you have unequal variances. 

### 21 Again quoting from Wikipedia and this is, I think, what I've 

### 22 known to be true in the past so I'm not just completely relying 

### 23 on Wikipedia, but the ANOVA on ranks has never been recommended 

### 24 when the underlying assumption that homogenous variances have 

### 25 been violated either by itself or in conjunction with a 

### 26 violation of the assumption of population normality.

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1 In general rank based statistics become 

### 2 nonrobust with respect to type one errors for departures from 

### 3 homoscedasticity even more quickly than their parametric 

### 4 counterparts that share the same assumption. 

### 5 But there again the good news is that we now 

### 6 have software that we can analyze with unequal variances very 

### 7 easily and we can do it accurately and control type one/type two 

### 8 error rates and so on. So we really don't need nonparametric 

### 9 methods in the case when we have unequal variances. And as this 

### 10 states here they should not be used. 

### 11 And then there is a series of references that 

### 12 are given in that and their references that I sort of relied on 

### 13 when I started doing my look into the Wikipedia stuff. I think 

### 14 that's all I have for now. 

### 15 **DR. MARTHA SANDY:**Thank you. So I think it's 

### 16 a good time to take a break. It's 3:10. Why don't we come back 

### 17 at 3:25. 

### 18 19 (Whereupon a break was taken) 

### 20 21 **DR. MARTHA SANDY:**As we start up again we're 

### 22 in the middle of the response to Question 1 and Dr. Johnson who 

### 23 is the lead has suggested that we break this response up -- 

### 24 sorry four. I'm looking at four but yes. Thank you. 

### 25 He's broken up the questions so he's addressed 

### 26 the statistical methods and design study protocol issues that he

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1 had. I'd like to ask the other panel members that were listed 

### 2 as discussants on Question 4 if they have anything to add on the 

### 3 statistical issues and if not we'll turn to the whole panel. Is 

### 4 that alright? Okay. I will let you yes, Mary Christman please 

### 5 go ahead. 

### 6 **DR. MARY CHRISTMAN:**Hi, I just wanted to 

### 7 comment on Dallas' discussion. Very quickly I promise. 

### 8 Basically I wanted to reiterate what he said 

### 9 earlier about trying to understand what the true underlying 

### 10 probability distribution of your endpoint is. Use the 

### 11 literature, use prior knowledge, use similar experiments just 

### 12 like when you do power analyses. You use this prior knowledge. 

### 13 Take advantage of it. And in this day and age there is a lot of 

### 14 statistical methodology out there that would allow you to use 

### 15 the true distribution and there is methods for checking the 

### 16 diagnostics of the true distribution. So you don't need to 

### 17 always do a transformation. There are times when you want to 

### 18 actually model it as a count variable, like poisson or something 

### 19 or as my advisors used to call it a poisson. That would just 

### 20 confuse the heck out of me. 

### 21 The second thing I wanted to mention was 

### 22 everybody seems to think that nonparametrics means no 

### 23 assumptions. The reality is nonparametrics have assumptions on 

### 24 them as well. They just don't tell you what the shape is, but 

### 25 they demand that the shape be the same in every treatment and 

### 26 two, that the variances be equal. So you weren't getting away

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1 from this requirement to meet assumptions, it just doesn't tell 

### 2 you what that shape is. So keep that in mind when you're 

### 3 thinking about doing nonparametrics. 

### 4 And last but not least one more thing that 

### 5 causes nonparametrics to breakdown are when you have a lot of 

### 6 ties. So if you do have things like counts or small sample 

### 7 sizes where you have the same value, that results in the ranking 

### 8 order being ties and you can't use nonparametrics for when you 

### 9 have many ties. Thank you. 

### 10 **DR. MARTHA SANDY:**Okay. And a tie is 

### 11 multiple -- two. 

### 12 **DR. MARY CHRISTMAN:**Ties, they're ties. 

### 13 **DR. MARTHA SANDY:**Dr. Bentley. 

### 14 **DR. DALLAS JOHNSON:**Just to add to that if I 

### 15 might. In some of the cases where you're looking at these type 

### 16 scales, you know one, two three four five and so you're ordering 

### 17 the data according to some numerical value dealing with severity 

### 18 and if you're using one, two, three, four, five or one, two, 

### 19 three or one through seven or whatever you're using, you're 

### 20 going to end up with a lot of ties just because there is not 

### 21 enough information in those variables to distinguish between a 

### 22 five and a five or a four and a four and so on. 

### 23 **DR. GEORGE BENTLEY:**I don't have anything 

### 24 further to add on the statistical analysis. 

### 25 **DR. MARTHA SANDY:**And Dr. Scanes. 

### 26 **DR. COLIN SCANES:**Ditto.

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1 **DR. MARTHA SANDY:**Dr. McCarty. 

### 2 **DR. JOHN MCCARTY:**And nothing to add on the 

### 3 stats. 

### 4 **DR. MARTHA SANDY:**Dr. Perkins. 

### 5 **DR. EDWARD PERKINS:**Nothing more to add. 

### 6 **DR. MARTHA SANDY:**And Dr. Johnson did Dr. 

### 7 Portier send in anything on this one? 

### 8 **DR. DALLAS JOHNSON:**Surprising he said less 

### 9 about the statistics than anything else. So not on the 

### 10 statistics for the quail he didn't seem to add anything in that 

### 11 we needed to worry about. 

### 12 **DR. MARTHA SANDY:**Okay so any other panel 

### 13 members want to comment on the statistical issues here. No. 

### 14 Okay. So among the other associate discussants on Question 4 

### 15 shall we see who has any comments on the selection of test 

### 16 substances, those issues related to that? And I'll start with 

### 17 Dr. Bentley. 

### 18 **DR. GEORGE BENTLEY:**Well first I should 

### 19 mention that we really haven't discussed these subcomponents of 

### 20 this question as a group so these are my own opinions and we 

### 21 have briefly discussed them. 

### 22 My main question about the selection of test 

### 23 substances is something that came up yesterday I believe or the 

### 24 day before. I don't know how long we've been here now. 

### 25 And that is whether the Tier 1 testing is 

### 26 wholly appropriate for birds given that the Tier 1 tests appear

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1 to be geared more towards mammalian systems and looking for 

### 2 effects of endocrine disruptions via mammalian or potential 

### 3 effects via mammalian receptors. There may well be subtle 

### 4 differences in avian hormone receptors that would provide a 

### 5 false positive in the Tier 1 testing. Now I had stressed -- 

### 6 **DR. MARTHA SANDY:**So excuse me are there any 

### 7 avian tests in Tier 1? I don't think there are. No, so I'm not 

### 8 clear what your commenting on when you're saying Tier 1 tests 

### 9 and avian assays. 

### 10 **DR. GEORGE BENTLEY:**Oh with the initial 

### 11 screening of chemicals to go to a Tier 2 test. There are no -- 

### 12 right so. 

### 13 **DR. MARTHA SANDY:**Okay so you're saying -- 

### 14 **DR. GEORGE BENTLEY:**Chemicals are selected 

### 15 for Tier 2 tests as a result of Tier 1 test. Right? 

### 16 **DR. MARTHA SANDY:**That is correct. 

### 17 **DR. GEORGE BENTLEY:**And there are no avian 

### 18 specific Tier 1 test? 

### 19 **DR. MARTHA SANDY:**That is correct. 

### 20 **DR. GEORGE BENTLEY:**So if a chemical is not 

### 21 selected for a Tier 2 test that might not -- because it doesn't 

### 22 have an effect in a mammalian system that doesn't mean it's not 

### 23 going to have an effect in an avian system. 

### 24 **DR. MARTHA SANDY:**Okay so that's your point. 

### 25 Okay. Thank you. Dr. Scanes. 

### 26 **DR. COLIN SCANES:**I've got a couple of

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1 points. One I do think that George Bentley's comment about the 

### 2 absence of any avian Tier 1 test is very appropriate. A small 

### 3 change in the receptor could change its affinity, could change 

### 4 whether a compound binds and so that one could be either having 

### 5 false positives in terms of using a mammalian test or a false 

### 6 negative. 

### 7 And so a compound that doesn't work in Tier 1 

### 8 could still have, let's say, an estrogenic effect in Tier 2 and 

### 9 so I hope I'm saying that correctly George. 

### 10 **DR. GEORGE BENTLEY:**You're paraphrasing me 

### 11 quite well. You're explaining me much better than I can myself. 

### 12 **DR. COLIN SCANES:**So that's a point of 

### 13 agreement. Two points within the test though I think need to be 

### 14 addressed in the protocol. 

### 15 One is the issue, and it's a very simple 

### 16 thing, of measurement of spillage of food. It says in the 

### 17 protocol spillage is noted. Well, you know, it could be 

### 18 weighed. And I think that would be preferable. 

### 19 Further issue, which could confound things, 

### 20 particularly if cages are randomized, is the possibility of dust 

### 21 or spilled food moving into a different bird's feeder; and so 

### 22 that issue of either dust contamination or spilled food 

### 23 contamination should be addressed because that could easily get 

### 24 confounding effects particularly when we've got marked 

### 25 differences in concentrations. 

### 26 And the final point that I'd like to make was

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1 in terms of the test material that was used I thought trenbolone 

### 2 was a very good test subject. The other material was less 

### 3 compelling. 

### 4 **DR. MARTHA SANDY:**Okay. Dr. McCarty. 

### 5 **DR. JOHN MCCARTY:**Yes I think a lot of our 

### 6 comments -- so regarding the choice of the test substance for 

### 7 the inter-lab comparison and that's the most complete comparison 

### 8 -- you know I think the comments yesterday the EPAs response to 

### 9 the question of why vinclozolin was chosen that something along 

### 10 the lines of it looks harder to defend in hindsight. You know, 

### 11 I think a lot of our uncertainty about the tests goes from that. 

### 12 And it certainly -- several of our questions would be clarified 

### 13 if inter-lab tests had been run with something with more clear 

### 14 cut expectations of what the results should be. 

### 15 Right now, you know if you look at the 

### 16 inter-lab comparisons we don't know how much of it is birds 

### 17 really don't respond to the chemical or the protocol wasn't good 

### 18 or the labs weren't any good or the statistical power isn't 

### 19 strong and because, you know, if a different substance had been 

### 20 chosen it would be easier to evaluate the outcome of those 

### 21 inter-lab comparisons. 

### 22 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 23 **DR. EDWARD PERKINS:**I agree with what John 

### 24 said. I think for the purposes of validating, doing round robin 

### 25 inter-lab comparison a chemical with a more defined 

### 26 characteristics or toxicological affects probably should have

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1 been used. 

### 2 **DR. MARTHA SANDY:**Do other panel members have 

### 3 any comments on selection of test substance? Then we'll go to 

### 4 the third and final part of this question. Comments on the 

### 5 analytical methods that were used. 

### 6 **DR. GEORGE BENTLEY:**I thought that in 

### 7 addition to histology on the gonads for example and the brain, 

### 8 methods such as qPCR could have been used and this could be done 

### 9 without increasing the number of animals used. You could do a 

### 10 qPCR on one testes for example and histology on the other 

### 11 testes. And that way you could target a particular MRNA you 

### 12 want to look at such as star protein, LH receptor, FSH receptor, 

### 13 17-beta, et cetra. 

### 14 This could also be done in the brain. You 

### 15 could do histology and qPCR on the same brain using alternative 

### 16 brain section and therefore look at changes in the expression of 

### 17 GnRH-1 in addition to doing immunocytic chemistry for the GnRH-1 

### 18 peptide. 

### 19 And I think that can give you a lot better 

### 20 picture of what's going on in terms of these endpoints in 

### 21 females; qPCR could be done on individual follicles, for 

### 22 example, as long as they were standardized as to which follicle 

### 23 you were performing it on. 

### 24 And again this could be done without 

### 25 increasing the number of animals used and it could be done quite 

### 26 easily.

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1 **DR. MARTHA SANDY:**Dr. Scanes. 

### 2 **DR. COLIN SCANES:**No further comment. 

### 3 **DR. MARTHA SANDY:**Dr. McCarty. 

### 4 **DR. JOHN MCCARTY:**No. I think any of the 

### 5 other comments on analytical methods that I have are going to be 

### 6 captured in Question 5 and 6 and no need to cover them here. 

### 7 **DR. MARTHA SANDY:**Dr. Perkins. 

### 8 **DR. EDWARD PERKINS:**I would just support what 

### 9 George is saying that there is potential for supporting evidence 

### 10 in terms of weight-of-evidence for looking at expression. 

### 11 **DR. MARTHA SANDY:**Other members of the panel. 

### 12 No questions or comments. And EPA staff do you have any 

### 13 clarifying questions? 

### 14 **DR. LESLIE TOUART:**Just a clarification in 

### 15 terms of test substance and remarks made about vinclozolin and 

### 16 that we needed to maybe have looked at a more defined compound 

### 17 when -- vinclozolin we pretty much understand an androgen 

### 18 antagonist. So we knew that -- that context we didn't know how 

### 19 it was going to respond in bird specifically. 

### 20 I'm not sure what more we would have gained, 

### 21 again, given that this is a more kind of definitive, you know, 

### 22 test so there is nothing to compare it to. I mean there is 

### 23 nothing to say what we could have expected per se. 

### 24 So the context in terms of what more might we 

### 25 have determined given that we did have good correspondence in 

### 26 terms of the endpoints that were looked at and measured. It was

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1 just those didn't have a particular response. 

### 2 So is the concern that, you know, compounds 

### 3 should have had more responses across different endpoints to be 

### 4 able to evaluate kind of reproducibility or would it be that the 

### 5 question would be, you know, that we're trying to have more 

### 6 definitive information on say vinclozolin per se as opposed to 

### 7 the method as it is. 

### 8 **DR. JOHN MCCARTY:**I guess what I'm referring 

### 9 to are those cases where we don't have a response and so take 

### 10 behavior for example, and given the chemical we don't have that 

### 11 information on -- we don't have detailed information on the 

### 12 transfer of this to different labs. Because the three labs used 

### 13 a chemical that we're not really sure to expect and they found 

### 14 negative results. Well is that a reflection on vinclozolin or 

### 15 is that a reflection on the protocol. And that's all I was 

### 16 referring to, that some of our other questions would be answered 

### 17 if we had say inter-lab comparisons running this protocol with 

### 18 something we know has strong effects on birds in the endpoints 

### 19 of interest. 

### 20 **DR. MARTHA SANDY:**Les Touart. 

### 21 **DR. LESLIE TOUART:**And I guess the question 

### 22 then with Trenbolone that was used, and again, only two 

### 23 laboratories in that context but there were measures comparable 

### 24 in terms of the effects that we're seeing. I don't know if 

### 25 that's considered. 

### 26 But I guess I kind of understand what you're

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1 saying, I'm just not clear as to if additional tests are done 

### 2 exactly what they might be able to inform us beyond what we've 

### 3 kind of seen and expect in terms of what we can actually 

### 4 measure. 

### 5 **DR. JOHN MCCARTY:**First I'd reiterate what 

### 6 Colin said. I think the Trenbolone tests were useful. On the 

### 7 other hand we've been -- either Colin or George can correct me 

### 8 on this but we've emphasized that we think behavior are 

### 9 important set of characters and my understanding the sensitivity 

### 10 of male behavior is very sensitive for example to estradiol. 

### 11 And so right now we don't know is the protocol at fault for not 

### 12 finding behavioral responses in the males or is it the fact that 

### 13 the chemical just doesn't affect behavioral development in 

### 14 males. And that would be the added benefit there in my opinion. 

### 15 **DR. MARTHA SANDY:**Dr. Scanes. 

### 16 **DR. COLIN SCANES:**I think my reaction was 

### 17 based upon all of the models. Not only the quail model that the 

### 18 need for consistency across the contract labs. And that's why 

### 19 our recommendation is to include a positive control. Because by 

### 20 having the positive control where you will hopefully see an 

### 21 effect of the positive control adds confidence to the data of 

### 22 the negative control. Particularly if the test agent has no 

### 23 effects, that is exactly what one wants to see from a regulatory 

### 24 point of view. That is an agent that has no effects, the 

### 25 positive control works and the negative control is the same as 

### 26 the test agent.

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1 **DR. MARTHA SANDY:**All right are we ready to 

### 2 move on to Question 5? 

### 3 DR. LESLIE TOUART. Reading the Charge 

### 4 Question No. 5. Considering that variability inherent in 

### 5 biological and chemical test methods, a test method needs to be 

### 6 repeatable and reproducible. A test is robust and reliable if 

### 7 the results are repeatable and reproducible within a laboratory 

### 8 and between different laboratories, respectively. A test 

### 9 protocol should provide sufficient guidance to ensure proper and 

### 10 consistent performance across labs and chemicals. 

### 11 Please comment on the test method robustness 

### 12 and reliability and the repeatability and reproducibility of the 

### 13 results obtained with the Japanese quail assay. 

### 14 **DR. MARTHA SANDY:**Dr. Bentley you're the lead 

### 15 discussant on this. 

### 16 **DR. GEORGE BENTLEY:**Okay thank you. Well a 

### 17 few of these things have been touched upon by Dallas already and 

### 18 his comments on statistical analysis, I'm not going to comment 

### 19 on them from a statistical viewpoint but I will also try to keep 

### 20 repetition to a minimum. 

### 21 As you've heard a recurrent theme is that we 

### 22 feel the assay would benefit from the use for positive control 

### 23 where possible. This would allow us to evaluate how 

### 24 reproducible and repeatable the test is. And we've talked about 

### 25 the use of vinclozolin showing not much in the way of treatment 

### 26 effects in any lab and as is already mentioned we can't tell if

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1 it's a result of any issue of the test reliability, lab rat 

### 2 reliability or if it's a real result. And the use of a positive 

### 3 control would greatly help with our interpretation of the data. 

### 4 There were other potentially identifiable 

### 5 sources of variation between labs and one of those has been 

### 6 commented on, is a strain differences in quail and we have a 

### 7 recommendation about that at the end of my spiel. 

### 8 It could also be differences resulting from 

### 9 subtle differences in incubation parameters of the eggs. For 

### 10 example, humidity, egg storage temperature. These can all 

### 11 influence the hatchability of the eggs. 

### 12 One issue that doesn't appear to have been 

### 13 accounted for is the light intensity the birds were exposed to. 

### 14 And this is not only a potential issue between labs but within a 

### 15 lab and within a room. If quail are housed in racks then the 

### 16 birds at the top of the rack will receive exposure to much 

### 17 greater light intensity than birds who are on subsequently lower 

### 18 racks. 

### 19 And this can really greatly influence that 

### 20 photoperiodic response when you transfer from short to long 

### 21 days. And they'll interpret lower light intensities as shorter 

### 22 days than they are actually housed upon once they're below a 

### 23 certain threshold of light intensity. 

### 24 The issue of the strained differences is 

### 25 potentially a big one but we think this could be somewhat 

### 26 addressed using a single supplier to standardize the quail

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1 source and then we have a recommendation on that that I'll get 

### 2 to. And the other parameters that we'll discuss really could be 

### 3 accounted for using a more standardized SOP and increasing 

### 4 training. Overall it appears the SOP needs to be tightened and 

### 5 adhered to to decrease variability in data. 

### 6 Another issue that wasn't apparently counted 

### 7 for is that within a strain of quail there are so called 

### 8 personality differences. There are often high stress and low 

### 9 stress lines. And these different lines have very different 

### 10 behavioral and physiological responses to a stressor. 

### 11 And as a result circulating glucocorticoids 

### 12 might well change differently between these different lines and 

### 13 that is going to influence the amount of circulating gonadal 

### 14 steroids and that interaction between corticosterone binding 

### 15 protein and the gonadal steroids. So that might well influence 

### 16 the response of these quail to endocrine disrupting chemicals. 

### 17 So we feel that circulating corticosterone 

### 18 needs to be measured to ascertain the responsiveness of the HPA 

### 19 axis to handling many of these animals used. So this is not 

### 20 just measuring the baseline corticosterone which would be 

### 21 measured by measuring fecal corticosterone metabolites, but we 

### 22 would like to see how these animals respond to a stressor and 

### 23 whether that is uniform across groups. More so we would like to 

### 24 see the parameters of if CORT clearance could differ as a result 

### 25 of treatment. 

### 26 We talked quite a bit about histology. We

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1 will refer back to our earlier comments on histology there. And 

### 2 as we have already mentioned the data from behavioral assays are 

### 3 quite highly variable and again just solidifying the SOPs, 

### 4 increasing training, identifying where the variance is and why 

### 5 it is there could help interpret the data. 

### 6 Another comment is that several of the tests 

### 7 have very high CVs and apparently low power to detect changes 

### 8 and again we're not given enough detail to distinguish between 

### 9 variability and introduced by the test protocol itself and/or 

### 10 inherent biological variability. So if the variabilities are 

### 11 function of different labs using slightly different procedures 

### 12 then that could be corrected by correcting the SOPs or being 

### 13 more specific in those SOPs. But if it reflects biological 

### 14 variation then in certain circumstances the sample size may need 

### 15 to be increased. 

### 16 So overall recommendations here are one; 

### 17 consistent use of a positive control; the second one is to 

### 18 standardize the use of the quail strain. Perhaps have a single 

### 19 supplier. And one suggestion we had was maybe to build a 

### 20 foundation of a single genetic stock that is then housed in more 

### 21 than one location in case of disease or natural disaster wiping 

### 22 out that stock. 

### 23 And then have appropriate genetic husbandry 

### 24 performed between these locations periodically. And perhaps 

### 25 they should be made internationally available. 

### 26 The next recommendation is to have stricter

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1 more standardized SOPs. And housing light intensity should be 

### 2 stated in the protocol and standardized across labs, across 

### 3 rooms. And as Dallas mentioned earlier, the time of day of 

### 4 sampling can be an important source of variabilities not just 

### 5 for body mass but also for hormone measurements. So that should 

### 6 be standardized. 

### 7 Histological assays we've talked about 

### 8 standardizing those and Colin provided some details as to how 

### 9 that can be standardized. This should be at least one measure 

### 10 of the CORT response to stress to ensure it is equivalent across 

### 11 groups. And then behavioral assays which I think should be 

### 12 expanded and standardized and adequate training should be 

### 13 provided for the different labs to perform those assays. So for 

### 14 example, appetitive male behavior could be measured in addition 

### 15 to consummatory behavior. 

### 16 Ideally we would like to see less of a focus 

### 17 on male specific behavior and incorporation of measurements of 

### 18 female behavior and make choice and we've already talked a 

### 19 little bit about this and how that might be difficult to do in 

### 20 quail given that the females don't appear to have much of a 

### 21 choice. And so this is where a passerine model such as zebra 

### 22 finch might be useful. 

### 23 And then finally possibly increase the sample 

### 24 sizes if the use of those stricter SOPs don't reduce the 

### 25 observed variability. 

### 26 **DR. MARTHA SANDY:**And Dr. McCarty any

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1 additional thoughts? 

### 2 **DR. JOHN MCCARTY:**No I think he's covered my 

### 3 comments. 

### 4 **DR. MARTHA SANDY:**Dr. Scanes. 

### 5 **DR. COLIN SCANES:**He's covered my comments 

### 6 but I want to point to the research in the appendices supports 

### 7 the robustness of the Japanese quail three generation test. 

### 8 **DR. MARTHA SANDY:**Dr. Johnson. 

### 9 **DR. DALLAS JOHNSON:**I don't think I have 

### 10 anything to add except that in the preamble to the charge it 

### 11 indicates a test protocol should provide sufficient guidance to 

### 12 ensure proper and consistent performance across labs and 

### 13 chemicals. And I guess my feeling it from what I've seen so far 

### 14 is that we're not at that stage yet. 

### 15 **DR. MARTHA SANDY:**Did you have anything from 

### 16 Dr. Portier? We could take that now. While you're looking, 

### 17 I'll turn to Dr. Perkins. 

### 18 **DR. EDWARD PERKINS:**I have nothing more to 

### 19 add. 

### 20 **DR. DALLAS JOHNSON:**Dr. Portier mentions an 

### 21 issue that the document doesn't provide a conclusion regarding 

### 22 test reliability. To the certain extent EPA is asking SAP to 

### 23 make this assessment. The panel can do this if the biologically 

### 24 important detection differences are first identified. 

### 25 Unfortunately EPA has not identified the key endpoints or 

### 26 assigned biological important detection differences. Is this

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1 also something the panel must decide. 

### 2 He adds on EPA should consider creating a 

### 3 database of test endpoint values from all control animals from 

### 4 all test sites to continue to add to this database and continue 

### 5 to add to this database as the test gains popularity in use. 

### 6 This will allow us to establish historical controls. We should 

### 7 allow external assessment as to whether a specific study 

### 8 implemented sufficient test condition controls at critical 

### 9 endpoints from the control animals that are reasonable compared 

### 10 to a historical control. Only in this way can we begin to 

### 11 ensure reproducibility of results and comparability among 

### 12 studies. 

### 13 He also says the test protocol described makes 

### 14 a valued attempt to identify many of the factors that impact 

### 15 outcome variability and to standardize them. It remains to be 

### 16 seen whether all of these factors can/will be controlled by 

### 17 laboratories performing the test. There are a lot of 

### 18 conditions. The test sites implement all controls or only some. 

### 19 If some are the ones that they do chose to control sufficient 

### 20 and that the resulting variability is controlled. 

### 21 Bullet 2 in Section 5.5 gives good advice. 

### 22 Need consistency in test design, photo period, timing of 

### 23 initiation of both photo period and dosing, dosing period, 

### 24 timing of collections, measures and genetic strength. 

### 25 As mentioned previously I feel that dosing 

### 26 amounts need to be tied to some definition of what represents a

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1 biologically significant detectable difference taking into 

### 2 account the power of comparisons at specified sample size. 

### 3 A discussion of reliability in draft versions 

### 4 5.4 it's one of the -0008 Page 11 Section 3B provides a very 

### 5 good summary of what EPA is attempting to examine when they talk 

### 6 about test method reliability. Their concept of reliability 

### 7 ties in exactly with what I have been saying about matching the 

### 8 test sample size to power and the biologically important 

### 9 detection difference. 

### 10 The three types of variability identified 

### 11 within tests, between tests in the same laboratory and between 

### 12 laboratory variability are all important in assessing test 

### 13 reliability. The issue I have with JQTT ISR is that it never 

### 14 provides a conclusion regarding test reliability. To a certain 

### 15 extent EPA is asking the SAP to make this assessment. The panel 

### 16 can do this if biologically important detection differences are 

### 17 first identified. Unfortunately -- maybe I read that already. 

### 18 I think I did. Sorry about that. That's all. 

### 19 **DR. MARTHA SANDY:**Thank you. Other comments 

### 20 from other panel members on Question 5? I see none. I'll turn 

### 21 to the EPA do you have -- was that clear or any questions? 

### 22 **DR. LESLIE TOUART:**I think I understand the 

### 23 comments made, I welcome that. I look forward to any of the 

### 24 additional suggestions the panel will make on that point. 

### 25 **DR. MARTHA SANDY:**So maybe we'll turn to 

### 26 Question 6. Would you like to read that for us please.

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1 **DR. LESLIE TOUART:**Reading Charge Question 6. 

### 2 The test protocol should be descriptive enough to be fully 

### 3 transferable to a competent laboratory. The protocol should 

### 4 describe the methodology of the assay in a clear and concise 

### 5 manner so that a laboratory could comprehend the objective, 

### 6 conduct the assay, observe the measure prescribed endpoints, 

### 7 compile and prepare data for statistical analyses, and report 

### 8 the results. Section 6 of the ISR outlines the process of 

### 9 challenges experienced in the inter-laboratory studies. 

### 10 Please comment on the transferability across 

### 11 labs and provide any suggestions or recommendations for 

### 12 improvement of the Japanese quail assay. 

### 13 **DR. MARTHA SANDY:**And Dr. Bentley is the 

### 14 lead. 

### 15 **DR. GEORGE BENTLEY:**Okay. Well there is 

### 16 going to be a little bit of repetition from the last question 

### 17 but not too much I hope. Again the high variability in some 

### 18 aspects of this assay makes it difficult to interpret 

### 19 transferability across labs. For example, because of the strain 

### 20 difference issue and because of the lack of use of positive 

### 21 controls. So we have some suggestions and recommendations over 

### 22 all and I'll just go through the list. 

### 23 First off, there needs to be clear guidance on 

### 24 which animals will be initially selected for histological and 

### 25 subsequent and statistical analysis. And which tissue from 

### 26 those animals and then within the tissue, which sections are

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1 present. This is vague and we have discussed that. 

### 2 The second one is to measure CORT 

### 3 responsiveness within a strain or the strain to be used to 

### 4 ensure normal distribution to the stress response. Keep the 

### 5 strain as genetically uniform as possible, again using the 

### 6 single supplier of single source. The egg storage and 

### 7 incubation metrics should be standardized. Humidity and as I 

### 8 said slight temperature effects can influence hatchability. Is 

### 9 there a positive control for each endpoint that we think there 

### 10 should be. There were some endpoints not measured that we 

### 11 thought would be valuable to include. This is in addition to 

### 12 the endpoints I mentioned in terms of running qPCR on the gonads 

### 13 and brain tissue. 

### 14 For example GnRH-2, Gonadotropin-releasing 

### 15 Hormone 2 in the mid brain has quite a strong influence on 

### 16 sexual behavior. And that could certainly be measured. It can 

### 17 also influence the HPG access directly as evidence for that. 

### 18 LH and FSH were not measured. It's a critical 

### 19 component of the HPG axis that is impacted by steroid feedback. 

### 20 So they should be measured. Progesterone was not measured. 

### 21 Progesterone is critical for ovulation and for oviduct function 

### 22 in birds. 

### 23 So we feel it is highly appropriate to measure 

### 24 all of these to investigate the potential for population level 

### 25 effects of the chemicals tested. 

### 26 I talked about qPCR on gonadal and brain

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1 tissue. I talked about standardizing the time of day of 

### 2 sampling, just reiterating it here. 

### 3 Just a comment really and I want to emphasize 

### 4 that we think this test seems to be highly appropriate to test 

### 5 potential endocrine disrupting chemicals in galliform species 

### 6 with precocial chicks. Again, I want to mention it is not clear 

### 7 how applicable it will be to avian species in general including 

### 8 the passerine species. 

### 9 Something else to consider is that 

### 10 longed-lived species such as albatross and gulls takes several 

### 11 years to enter into puberty, not just a few weeks like a quail. 

### 12 And as a result they potentially experience a much longer 

### 13 exposure to endocrine disrupting chemicals than quail. Not just 

### 14 as a result at the time of exposure, but also because of 

### 15 bioaccumulation, often higher up in the food chain. 

### 16 And this delayed entry into puberty might make 

### 17 them either more or less sensitive to the chemicals tested. 

### 18 We're not making a recommendation on that, but we wanted to make 

### 19 sure everyone is aware of that. 

### 20 And we've talked about how passerine species 

### 21 with altricial young might be more or less sensitive to 

### 22 endocribe disrupting chemicals than quail. The altricial young 

### 23 hatch in a highly undeveloped state in a way their external 

### 24 embryos and they're often fed insects by the parents. 

### 25 So again bioaccumulation of endocrine 

### 26 disrupting chemicals combined with exposure during critical

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1 developmental stages might make them more sensible to endocrine 

### 2 disrupting chemicals with precocial young. 

### 3 There was a mention of overall fitness 

### 4 measures in the report and we felt that overall fitness might be 

### 5 more easily measured in a song bird model with more pronounced 

### 6 secondary sexual characteristics. Or other parameters that 

### 7 indicate fitness might be more easily measured should I say. 

### 8 And these are infiltrated into sexual 

### 9 selection such as symmetry of the males -- the chest bars on the 

### 10 male zebra finch or accuracy of song, size of song, control 

### 11 nuclei. 

### 12 And again parental care not just incubation of 

### 13 eggs but feeding of hatchlings could also be measured more 

### 14 easily. And as has already been mentioned, we felt that 

### 15 behavioral measures in the adults could be standardized more. 

### 16 We could measure female behavior in a 

### 17 passerine species, we could actually measure female behavior in 

### 18 quail and put the females in a choice chamber and see if there 

### 19 is any preference for males that either have or have not been 

### 20 treated with endocrine disrupting chemicals. And that way there 

### 21 is no confound of the male trying to attack the female. 

### 22 You might want to emphasize that often in labs 

### 23 studied behavior is often considered an easy option versus 

### 24 biochemical assay. So the SOPs are not always as detailed as 

### 25 they might be for an assay and RAA. 

### 26 But we feel that differences in behavior can

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1 be quite subtle and hard to measure to the inexperienced eye so 

### 2 as detailed protocols as for RAA, for example, need to be 

### 3 developed. 

### 4 And we want to emphasize that behavior is an 

### 5 important endpoint in birds because there is a clear link 

### 6 between endocrine disruption as a mechanism and its affects on 

### 7 behavior which in turn provides a good link to the fitness 

### 8 endpoints that are incorporated into an ecological risk 

### 9 assessment. 

### 10 And I mentioned potential measures of 

### 11 appestive behavior as well as consummatory sexual behavior could 

### 12 be used. And as John McCarty mentioned earlier a positive 

### 13 control for behavior could be the use of estrogen as it is known 

### 14 to influence male sexual behavior. 

### 15 So to summarize we think this test clearly has 

### 16 great potential as a standardized assay and it is justified and 

### 17 we feel it will be well suited for its purpose. But at present 

### 18 they need some refinement, particularly in terms of endpoints 

### 19 measured, guidance for transferability across labs and data 

### 20 analysis. We think the test has a great deal of merit in terms 

### 21 of measurement, the effects of endocrine disrupting chemicals in 

### 22 galliform birds. 

### 23 But it is not clear how broadly applicable the 

### 24 findings will be to other orders of birds with very different 

### 25 reproductive life histories. For example phallus size in ducks, 

### 26 song control systems size in birds, behavioral differences,

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1 courtship behavior differences such as directed and undirected 

### 2 singing in zebra finches, effects on female sexual behavior 

### 3 could all be affected by endocrine disrupting chemicals and 

### 4 would not be detected in this assay. 

### 5 So again, we recommend consideration of adding 

### 6 the use of a passerine species such as zebra finches as a 

### 7 component to this test where necessary. These birds breed very 

### 8 well in captivity. They develop very quickly, their behavior is 

### 9 very stereotypical, very easy to quantify or relatively easy to 

### 10 quantify and is well characterized. And as we mentioned the 

### 11 zebra finch genome is available and we know a lot about their 

### 12 physiology, endocrinology, female choice, parental care and 

### 13 sexual differentiation. 

### 14 And the vocal learning in passerine species as 

### 15 we've mentioned is a unique aspect of these birds that may well 

### 16 be affected by endocrine disrupting chemicals. And it's not 

### 17 possible to test this within the quail test per say. And the 

### 18 development of the song control system and the short time window 

### 19 in which zebra finches are able to learn this song, makes this 

### 20 aspect of their neurobiology and behavior potentially, 

### 21 particularly prone to effects of endocrine disrupting chemicals. 

### 22 Okay that's all I have to say. 

### 23 Oh sorry. I do have one more thing to say on 

### 24 the recommendations. In terms of the decreasing of variability 

### 25 across labs and allowing this assay to be transferred across 

### 26 labs easily I thought it might be an idea to have pre-approval

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1 of contract labs based on their proven ability to measure the 

### 2 endpoints decided upon. And we thought there might be a 

### 3 possible involvement of a consortia of public and private 

### 4 partnerships. Okay that is all I have to say. 

### 5 **MARTHA SANDY:**Thank you and now we'll turn to 

### 6 the associates. Dr. McCarty. 

### 7 **DR. JOHN MCCARTY:**The only thing I'd add is 

### 8 that in order to be successfully transferred as we have 

### 9 reiterated it's not just that the protocols be descriptive 

### 10 enough but training. You know, like I think the fish 

### 11 presentation yesterday made a really good job of emphasizing 

### 12 that, you know, hands on training, web training. We've 

### 13 mentioned it before would be a really good addition to ensuring 

### 14 that a good protocol gets adopted correctly. 

### 15 **MARTHA SANDY:**Dr. Scanes. 

### 16 **DR. COLIN SCANES:**Not wishing to reiterate 

### 17 what other members of the team have said but I want to go back 

### 18 to the issue of training and even suggest the consideration of a 

### 19 simple certification program. We as members of this panel have 

### 20 to go through an ethics certification. Does it seem 

### 21 unreasonable for people who are completing the studies to have 

### 22 some form of certification program? 

### 23 The second point to add in is the issue of 

### 24 having standards, certainly for the biochemical measures, 

### 25 certainly for shell, which can be distributed between the 

### 26 different labs doing the testing to ensure that there is good

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1 inter- and intra-lab standardization. 

### 2 And third thing is to reiterate a comment that 

### 3 George made. The need for a random bred genetic stock that is 

### 4 available throughout the U.S. and internationally. If one is 

### 5 thinking about that having it at three locations -- at least 

### 6 three locations and checking the genetics. I think there is 

### 7 really a concern that you could so easily get genetic drift in 

### 8 one supplier and so there does need to be an oversight mechanism 

### 9 to ensure that truly this is a random bred population. 

### 10 **MARTHA SANDY:**Dr. Johnson. 

### 11 **DR. DALLAS JOHNSON:**Perhaps I should 

### 12 apologize I missed the first line of the thing here I guess as I 

### 13 was reading through this and many of my comments that I put in 

### 14 for Question 4 should probably more appropriately be placed here 

### 15 and I'll try to make sure that we do that in the final report. 

### 16 **MARTHA SANDY:**Dr. Perkins. 

### 17 **DR. EDWARD PERKINS:**I have no further 

### 18 comments to add. 

### 19 **MARTHA SANDY:**And Dr. Johnson I'll ask you 

### 20 did Dr. Portier submit anything on this one? 

### 21 **DR. DALLAS JOHNSON:**He did not. 

### 22 **MARTHA SANDY:**Other members of the panel any 

### 23 comments on Question 6? No. Okay and EPA staff any 

### 24 clarification questions? 

### 25 **DR. LESLIE TOUART:**No I think I understood 

### 26 fairly succinctly what the panel suggested and some of the

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1 recommendations so we can move to Question 7. 

### 2 **MARTHA SANDY:**Thank you. 

### 3 **DR. LESLIE TOUART:**Charge Question 7. The 

### 4 purpose of the validation process is to determine the readiness 

### 5 of a test for inclusion in a testing program. A component of 

### 6 readiness of a test is the evaluation of the usefulness and 

### 7 limitations of the test, including the classes and types of test 

### 8 substances that can and cannot be tested. 

### 9 Please comment on the strengths and/or 

### 10 limitations of the Japanese quail assay, as described in Section 

### 11 7 of the ISR. 

### 12 **DR. MARTHA SANDY:**And the lead is Dr. Scanes. 

### 13 **DR. COLIN SCANES:**Starting with an overview 

### 14 that the Japanese quail multigenerational test assay exhibits 

### 15 readiness. It's justified as ready with the caveats that have 

### 16 been mentioned and will be mentioned. Looking at the strengths 

### 17 and weaknesses the first is looking at the strengths of the 

### 18 system and the OECD report of 2002 stated that there are 

### 19 sufficient differences within many avian systems to warrant the 

### 20 development of an avian specific test. We fully concur with 

### 21 that statement. 

### 22 There is a strong background research in 

### 23 endocrine physiology of the Japanese quail, particularly on its 

### 24 reproduction of thyroid function. We fully concur with that. 

### 25 Next is the OECD concerning two species 

### 26 Japanese quail and Bobwhite quail and opted very reasonably in

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1 our opinion for the Japanese quail. Although it is not 

### 2 indigenous to the U.S., it may have undergone extensive 

### 3 domestication and therefore may be less representative of wild 

### 4 species. The OECD recommended it here as the preferred test 

### 5 species because of its small size, high fecundity, well 

### 6 characterized reproductive biology and particularly its very 

### 7 rapid incubation maturation. 

### 8 While we concur with that, we also mention 

### 9 that the zebra finch also exhibits many of these same 

### 10 characteristics. The next in terms of strengths, the 

### 11 multigeneration studies measured egg production growth, embryo 

### 12 development, phenotypic sex, appear to be very strong 

### 13 parameters. I think in particular something that Les showed 

### 14 yesterday, which was the F2 data for trenbolone, that was a very 

### 15 nice demonstration of an endocrine disruptor effect where one 

### 16 was only treating the F0 generation, the F1 generation and in 

### 17 the F2 one was seeing a holdover of effects. 

### 18 It does suggest, by the way, that it might be 

### 19 worth holding in the terms of the test that you keep the F2 

### 20 birds and measure reproduction because that's the only time 

### 21 you'll clearly get demonstration of an endocrine disruptor 

### 22 effect versus a straight toxicological effect. 

### 23 Now I'll put in a caveat on that is it would 

### 24 be nice to see the trenbolone study in a published paper form 

### 25 rather than as an EPA report. 

### 26 Next the effects of the putative endocrine

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1 disruptors on eggshell thickness and on behavior appear to be 

### 2 critical endpoints. So this is among the strengths of the 

### 3 system. Weaknesses or limitations are the strain differences 

### 4 that we talked about before. And this is exemplified by the 

### 5 large differences in body weight between the different 

### 6 laboratories. 

### 7 Second weakness is it's not clear how 

### 8 representative Japanese quail are of birds in general. Quail 

### 9 are in the order Galliformes, the super order Galloanserae and 

### 10 this is considered -- it is generally considered now as a side 

### 11 group within the birds on an evolutionary basis. The precocial 

### 12 development is interesting and very, very important. But it 

### 13 ignores the possibility of an altricial development which is 

### 14 seen in throughout for instance the passerine forms. 

### 15 Weakness again, lack of epigenetic endpoints, 

### 16 but one could equally argue metabolomic or transcriptomic 

### 17 responses. Again we mentioned here the issue of a positive 

### 18 control, lack of consistency of the egg shell thickness needing 

### 19 standardization. 

### 20 Need to clarify terminology and that is the 

### 21 issue that in some parts of the documentation it talks about the 

### 22 P generation as opposed to the F0. And I think it's very 

### 23 important that we have a consistent nomenclature and that 

### 24 nomenclature fits with the one generation Bobwhite or mallard 

### 25 duck nomenclature. Because there is this danger that one is 

### 26 talking across systems and if you use different words for the

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1 same thing or the same word for different things it leads to 

### 2 considerable conclusion. 

### 3 The conclusion of the OECD that the Japanese 

### 4 quail has one of the most sensitive measures of impaired 

### 5 reproduction is the age of sexual maturation didn't seem to be 

### 6 borne out by the data presented. The utility of thyroid weight 

### 7 was questioned. Whether that is a strong parameter except if 

### 8 one is dealing with something which is truly a goitrogen. 

### 9 Although limitations -- and this is one for 

### 10 what it's worth -- and that is in the OECD draft guideline it 

### 11 talks about having 20 replicate pens to increase the likelihood 

### 12 that 16 remain in each group before test termination and yet the 

### 13 number used in the protocol was 12. OECD recommended 

### 14 determination of sperm motility, morphology, fertilization 

### 15 success. It also included circulating corticosterone, it also 

### 16 included vitellogenin. Now these are either not done in the 

### 17 protocols that we saw or done sparingly and no reason was given 

### 18 for their not being done. 

### 19 And I think that is something which EPA may 

### 20 wish to consider. Particularly as OECD mentioned in their 

### 21 report that there is an OECD validation management group, 

### 22 ecotoxicology testing, which is responsible for supervising the 

### 23 planning of and conduct of experimental work in fish, birds, 

### 24 amphibians and invertebrates and that's quoting the OECD report. 

### 25 So that's a limitation for what it's worth. 

### 26 Overall the readiness is justified with the caveats mentioned.

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1 **DR. MARTHA SANDY:**Thank you. Associates 

### 2 we'll start with Dr. Bentley. 

### 3 **DR. GEORGE BENTLEY:**I have nothing further to 

### 4 add. 

### 5 **DR. MARTHA SANDY:**Dr. McCarty. 

### 6 **DR. JOHN MCCARTY:**Nothing to add. 

### 7 **DR. MARTHA SANDY:**Dr. Johnson. 

### 8 **DR. DALLAS JOHNSON:**I don't have anything and 

### 9 Dr. Portier had nothing on this question. 

### 10 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 11 **DR. EDWARD PERKINS:**I just wanted to add that 

### 12 the addition of transcriptomics might be a benefit in the sense 

### 13 it can help extrapolate. One of the advantages of the species 

### 14 is that they both have genomes characterized and you might be 

### 15 able to extrapolate fundamental pathways across species to guide 

### 16 more directed research or provide some weight-of-evidence for 

### 17 impacts on passerine birds. 

### 18 **DR. MARTHA SANDY:**Any comments from other 

### 19 members of the panel? None. And any questions of clarification 

### 20 from the EPA staff. 

### 21 **DR. LESLIE TOUART:**Not questions but just a 

### 22 kind of a plea in terms of some of the recommendations in terms 

### 23 of like for some of the methods mentioned, anything that could 

### 24 be provided in terms of the actual written comments in terms of 

### 25 references or suggestions would be welcome, you know, just to 

### 26 help us in terms of trying to look at these and figure out how

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1 best to try to incorporate some of those into the methodology. 

### 2 **DR. MARTHA SANDY:**We'll ask the panel members 

### 3 to include those references. Shall we move on to the last 

### 4 question for this group? 

### 5 **DR. LESLIE TOUART:**And Charge Question No. 8. 

### 6 There is sufficient evidence to indicate that endocrine 

### 7 disrupting chemicals can disrupt normal development and 

### 8 reproductive success, however the sensitivity of the F2 

### 9 generation compared to the parental or F1 generations is less 

### 10 clearly defined. 

### 11 In the Japanese quail test, there is an 

### 12 increase in endpoint CVs with each subsequent generation. An 

### 13 example given is Table 6-4, which indicates decreased power of 

### 14 discrimination. 

### 15 Please comment from a scientific and risk 

### 16 assessment perspective on the value added of multiple 

### 17 generations in the Japanese quail assay. 

### 18 **DR. MARTHA SANDY:**Dr. McCarty. 

### 19 **DR. JOHN MCCARTY:**Well as a group I think our 

### 20 first message is over all we didn't think there was definitive 

### 21 support for or against the value of the F2 generation based on 

### 22 the documents we reviewed. In principle the information of the 

### 23 F2 generation where exposure is occurring only during that 

### 24 embryonic development does play an important role. Certainly in 

### 25 establishing endocrine disruption as a mechanism for any 

### 26 potential adverse effects seen in the Tier 2 tests. The

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1 inter-lab tests with vinclozolin showed little or no evidence of 

### 2 effects on those F2 birds. If this is a general outcome of 

### 3 those tests, results demonstrating a lack of effects in F2 that 

### 4 are seen in F1 are still potentially important for an ecological 

### 5 risk assessment given work emerging from research lab suggesting 

### 6 that these cross-generation effects are to be expected. So if 

### 7 they're not demonstrated that is still informative. 

### 8 That said members of the group express concern 

### 9 that the inter-lab tests presented may not characterize the 

### 10 potential for adverse effects to be exhibited in the F2. If the 

### 11 lack of a difference are driven by the low statistical power 

### 12 that we've discussed, or protocol issues then the lack of 

### 13 effects in the F2 may prove to be misleading for risk assessment 

### 14 purposes. 

### 15 The framing of the question implies that the 

### 16 answer to this question about the value of the F2 is found 

### 17 simply by looking at the patterns of CVs across generation where 

### 18 it states up there about the sensitivity of the F2s compared to 

### 19 the other generations is suspect because of the greater CVs. 

### 20 And it refers us to Table 6.4 to support that assertion. 

### 21 And in our discussions we didn't agree with 

### 22 that premise. If the pattern of increased variation is actually 

### 23 biological and not an artifact of the way the tests were 

### 24 conducted, then this is a biological result of interest. 

### 25 If within a test variability actually 

### 26 increases in offspring relative to their parents that seems to

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1 actually confirm the need for a multiple generation test, that's 

### 2 a biological change. In that case the limited statistical power 

### 3 that's being referred to associated with those greater CVs 

### 4 becomes a sample size issue and the protocol -- the response 

### 5 should be to revise the protocol adding breeding pairs to ensure 

### 6 that these potentially relevant differences can be detected 

### 7 given the protocol. To us it doesn't automatically say well the 

### 8 F2 isn't worthwhile because that sample size of 12 is not 

### 9 written in stone. 

### 10 Even if we do accept the premise though that 

### 11 an increase in CV across generation indicates something the 

### 12 information provided isn't even convincing us that the results 

### 13 in, for example, that table support the conclusion, the pattern 

### 14 of variation and the CVs doesn't seem straightforward. As 

### 15 Dallas mentioned the methods of determining the CV were a little 

### 16 unclear. We're unable to draw conclusions about the sensitivity 

### 17 of the F2 generations from those results. And if you look at 

### 18 Table 6.4 as the question direct us, the CVs are very test 

### 19 specific. There is a calculation of total CV which the F2 is 

### 20 indeed higher. But at least some of this is because the testing 

### 21 on the F2s includes two variables with very high variation. 

### 22 The biochemistry and behavior are included in 

### 23 that total. But it doesn't include several variables with very 

### 24 low CVs in the other generations, the reproductive effect. So 

### 25 we're not even convinced that there is a very strong pattern of 

### 26 an increase in CVs across generations.

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1 There may be valuable insights to be obtained 

### 2 from analyses of variation but the summary report and our time 

### 3 with it isn't sufficient to lead us to any conclusion on that. 

### 4 Results from the full test using a chemical 

### 5 with a stronger endocrine disrupting effect would be very 

### 6 informative for this. Again, going back to that positive 

### 7 control, several members noted that the inability to detect the 

### 8 effects in the F2 from the inter-lab tests with the vinclozolin 

### 9 is more than offset by the results we saw from Trenbolone where 

### 10 the effects on egg production were clearly demonstrated in the 

### 11 F2. These are in the report in the appendices and were also 

### 12 highlighted in slide 37 of EPAs presentation yesterday. 

### 13 In addition, other results from that study 

### 14 shown in slide 38 of the presentation yesterday indicate that 

### 15 exposure of the F0 generation seemed to increase the sensitivity 

### 16 of F1 ends to endocrine disrupting effects of this compound. 

### 17 This finding argues for the importance of performing dose 

### 18 studies with F2 animals to determine if lower doses might impact 

### 19 the reproductive function with longer intergenerational 

### 20 exposure. 

### 21 It seems prudent at this stage to conduct 

### 22 additional studies of trenbolone and possibly other androgenic 

### 23 compounds on reproductive functions of the F2 generation. The 

### 24 endpoint of F2 generation reproduction is not in the current 

### 25 protocol, however if effects on reproduction of F2s is a 

### 26 standard response of exposure to endocrine disrupting chemicals

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1 or if exposure increases sensitivity of across generations then 

### 2 inclusion of F2 endpoints and possibly expansion of those 

### 3 endpoints seems to be critically important for feeding into 

### 4 population models that the EPA has indicated will play an 

### 5 important role in ecological risk assessment. 

### 6 That said, if the trade off is to eliminate 

### 7 the F2 part of the testing and increase sample sizes in the 

### 8 earlier generations, as was done with some of the other models, 

### 9 perhaps that tradeoff should be considered. We have a general 

### 10 concern in this protocol that sample sizes are small making it 

### 11 difficult to detect biologically and ecologically important 

### 12 differences if they exist. 

### 13 The tradeoff between the F2 and greater sample 

### 14 size is beyond the scope of what we were able to consider. If 

### 15 such a decision were being considered, however, to eliminate F2s 

### 16 and increase sample sizes in the F0 generation, it would be 

### 17 necessary to revisit earlier discussions that the agency had 

### 18 about appropriate dose strategies and the choice to continue 

### 19 treatment through adulthood in the F1. 

### 20 It would also be important to evaluate the 

### 21 impacts on this choice on the goal of using these tests for 

### 22 establishing any adverse effects were associated with the 

### 23 mechanism of endocrine disruption. 

### 24 **DR. MARTHA SANDY:**Thank you. Turning to the 

### 25 discussants. Dr. Bentley. 

### 26 **DR. GEORGE BENTLEY:**I have nothing further to

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1 add. 

### 2 **DR. MARTHA SANDY:**Dr. Scanes. 

### 3 **DR. COLIN SCANES:**The more I can sit through 

### 4 this perhaps my position shifts a little bit. And sometimes 

### 5 kind of listening -- and one of the things that I noticed and 

### 6 that is the only test that definitively showed an endocrine 

### 7 disruptor effect was the F2 reproduction effects of trenbolone. 

### 8 Otherwise the effects could all be viewed as direct toxic 

### 9 effects. 

### 10 But the results that you showed, Les, 

### 11 yesterday, were very clear of an effect which could be mediated 

### 12 through an embryonic development. And so I think there is a lot 

### 13 of justification not only for F2 but to extend F2 to egg laying. 

### 14 And that's a parameter which has a very tight co-efficient of 

### 15 variation. And I think there are ways actually to make it even 

### 16 tighter co-efficient of variation. 

### 17 Where if there is going to be sensitization 

### 18 across generations, or a direct endocrine disruptor effect, and 

### 19 then prove it's an endocrine disruptor effect, the only way to 

### 20 do it is with the F2. 

### 21 **DR. MARTHA SANDY:**Dr. Johnson. 

### 22 **DR. DALLAS JOHNSON:**I have no comments, and 

### 23 Dr. Portier had no comments. 

### 24 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 25 **DR. EDWARD PERKINS:**I have nothing further to 

### 26 add.

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1 **DR. MARTHA SANDY:**Other members of the panel 

### 2 any additional comments on Question 8? I see none. Dr. Touart 

### 3 any clarifications? 

### 4 **DR. LESLIE TOUART:**I think I understood what 

### 5 was being suggested and I don't think we have any further 

### 6 clarifications in terms of suggestions or comments made. 

### 7 **DR. MARTHA SANDY:**Mary Manibusan. 

### 8 **MS. MARY MANIBUSAN:**I just want to add on top 

### 9 of my previous general comments for distinction between 

### 10 additional endpoints or measurements considered as must haves 

### 11 within the assay compared to future research in an area that 

### 12 might be helpful to answering some subsequent questions. If I 

### 13 can add to that list is for those additional measurements or 

### 14 endpoints or methods, if you can please comment within the must 

### 15 have column on its readiness and transferability across contract 

### 16 laboratories for regulatory use that would be very, very helpful 

### 17 to the Agency. Thank you. 

### 18 **DR. MARTHA SANDY:**So I'll ask the members 

### 19 that commented on this question to do that. Thank you. 

### 20 So I think we'd like to get started with the 

### 21 Medaka questions but it's been requested that we take a five 

### 22 minute stretch break and then we'll come back and tackle the 

### 23 fish. 

### 24 (Whereupon a break was taken) 

### 25 **DR. SHARLENE MATTEN:**With just an 

### 26 announcement so everyone can return to their seats or standup

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1 but we're going to start back here. Yesterday Mary Christman on 

### 2 the panel had asked for some clarification of some statistical 

### 3 analysis related to the one way and two way ANOVA for the Medaka 

### 4 multigeneration test and EPA has sent -- it's about a one page 

### 5 or so email that has the answers to those questions. I have 

### 6 made just about eight hard copies of the -- it's a two page Q&A. 

### 7 8 And also Mary Christman had asked another 

### 9 question related to block effects with references to statistical 

### 10 analysis with the Mysids and EPA also provided a study that 

### 11 wasn't included in the 20 that were given to the panel as 

### 12 appendices but this one has a specific details on the testing 

### 13 regime and explanation of block effects which I also sent to the 

### 14 panel by email for their leisure. 

### 15 But I also made eight copies in case somebody 

### 16 would really like to read it now which is fine. And the rest of 

### 17 it I am going to put those into the docket. It's a PDF of the 

### 18 email for the public way, way in the back and also the 

### 19 additional study that has the explanation about the block 

### 20 effects for the Mysid shrimp assay. 

### 21 **DR. MARTHA SANDY:**Thank you Dr. Matten. So 

### 22 now we are ready to turn to the set of questions related to the 

### 23 Medaka Multigeneration test. So is it Dr. Johnson that will be 

### 24 reading the charge questions? 

### 25 **DR. LESLIE TOUART:**I'll read the charge 

### 26 questions.

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1 **DR. MARTHA SANDY:**Oh Les. 

### 2 **DR. LESLIE TOUART:**And for the Charge 

### 3 Question 1 for the Medaka Multigeneration test. A rationale for 

### 4 the test method should be available, including a clear statement 

### 5 of scientific basis and the regulatory purpose and need for the 

### 6 test method. 

### 7 The EDSTAC introduced the Tier 2 tests as 

### 8 having the purpose to "characterize the nature, likelihood, and 

### 9 dose-response relationship of endocrine disruption of estrogen, 

### 10 androgen, and thyroid in humans and wildlife." Tier 2 tests 

### 11 were designed to be definitive tests which generate sufficient 

### 12 data to characterize the specific hazard of the substance and 

### 13 provide sufficient information on dose-response and adverse 

### 14 effects to permit risk decisions. 

### 15 Please comment on the rationale and purpose of 

### 16 the assay as part of the Tier 2 testing in the EDSP, as 

### 17 described in Sections 2.1 and 2.2 of the ISR. 

### 18 **DR. MARTHA SANDY:**Dr. Schlenk is the lead 

### 19 discussant. 

### 20 **DR. DANIEL SCHLENK:**Thank you. I'd like to 

### 21 preference our comments just to say that myself and the 

### 22 associate discussants for this question and the following 

### 23 questions met last night, discussed many of these issues ad 

### 24 nauseam and so my comments, at least in this initial question 

### 25 represent that subset of the panel including Dr. Portier's 

### 26 questions that he had emailed in to me personally.

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1 And one thing I would note in terms of these 

### 2 comments and the comments that will be coming after this, you'll 

### 3 note that there will be fairly several redundancies and I think 

### 4 that is sort of important for at least the Agency to sort of 

### 5 hear that these several issues keep coming up over and over 

### 6 again. So I just want to preface that before we go forward. 

### 7 So with regard to rationale and purpose of the 

### 8 MMT assay as per Item 1 of the ICCVAM validation criteria 

### 9 scientific and regulatory rationale for the test method 

### 10 including a clear statement of its proposed use should be 

### 11 available. 

### 12 Those of us that met largely agree that the 

### 13 MMT and MRT assays provide a clear statement of rationale and 

### 14 purpose. The one exception would probably be Dr. Portier who 

### 15 actually indicated some other issues which we'll get to in a 

### 16 minute. 

### 17 We felt the Section 2.1 of the document meets 

### 18 criteria for justification MMT assay including stated necessity 

### 19 for multigenerational testing, full life cycle assessment and 

### 20 appropriate dosing regime. Dr. Portier thought that Section 2 

### 21 could be written in a more positive tone. He was looking for a 

### 22 more clear bulleted list of advantages for the Medaka for the 

### 23 benefits of using the Medaka. 

### 24 In fact he felt it was too long -- 20 pages 

### 25 was too long before you could actually start seeing those 

### 26 particular benefits such as ease of reproduction, relatively

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1 small genome, growth, et cetera, anal fin papillae, the 

### 2 different sort of things that were listed in the document in 

### 3 Section 2. 

### 4 In contrast a majority of the subpanel that 

### 5 met felt that clear and concise statements are given regarding 

### 6 the advantages of the Medaka model. Although more attention 

### 7 should be given in fact that there is a well-defined mechanism 

### 8 to determine both phenotypic gender and genetic gender in this 

### 9 model. 

### 10 This is an advantage for utilization of the 

### 11 Medaka model that cannot be recapitulated in other small 

### 12 aquarium fish models at least that we know of. Fathead minnow 

### 13 there is some evidence that you can use some things there but it 

### 14 seems to be a little bit more robustness with Medaka. 

### 15 For this reason greater advantage should be 

### 16 utilized in either the MMT and/or the MRT assay for establishing 

### 17 ratios based on phenotypic gender and genetic gender. In fact 

### 18 one of the slides actually indicated this was not an endpoint 

### 19 and we really feel this actually should be an endpoint and it is 

### 20 actually one of the benefits of that particular model. 

### 21 As is the current MMT and MMRT doesn't exploit 

### 22 this aspect, it seems that its sole purpose is to be on a 

### 23 genetic gender for establishing breeding pairs and not really an 

### 24 assessment of genotype or phenotype ratios within either F1 or 

### 25 F2 populations. 

### 26 Section 222 provides a comprehensive

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1 assessment of the conceptual basis for multigenerational test 

### 2 paradigm. Again one panel member thought that more detail 

### 3 should be provided for the less informed reader regarding 

### 4 differences between activational and organizational effects 

### 5 following exposure. In this regard this panel member felt lines 

### 6 463 to 485 were somewhat convoluted, do not clearly state the 

### 7 rationale for timing of adult embryonic exposure. In fact some 

### 8 statements appear somewhat speculative, such as those addressing 

### 9 chemical permeability across the egg corian. 

### 10 There was not consensus regarding the benefit 

### 11 of model evaluating maternal phenyl transfer of EDCs. One panel 

### 12 member indicated little information is available on most EDCs as 

### 13 to maternal transfer from the F0 to F1 populations while 

### 14 experimental design of the assay has been established as 

### 15 safeguard for this event, this aspect of the assay remains 

### 16 equivocal. 

### 17 In contrast another panel member felt that 

### 18 maternal transfer of many chemicals is a significant concern as 

### 19 is exposure of various life stages that may vary in sensitivity. 

### 20 The panel understands the rationale for using Medaka. Again a 

### 21 long history of experimental use, et cetera, easy to maintain in 

### 22 a lab, tolerant to range of environmental conditions and it can 

### 23 be induced to spawn throughout the year. 

### 24 However one panel member felt these very 

### 25 attributes most likely make Medaka less representative of native 

### 26 species of concern than the majority of important and

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1 ecologically and economically native fishes. 

### 2 Many native fishes will accumulate chemicals 

### 3 in the eggs or ovary for months starting in the fall when they 

### 4 begin to incorporate yolk until they spawn in the spring. The 

### 5 young maybe exposed to chemicals in the sediment from maternal 

### 6 sources and through the water. In addition mature fishes will 

### 7 have been exposed for years, not a few months and these issues 

### 8 in relevance need to be noted. 

### 9 The summary report states that the recommended 

### 10 fish Tier 2 test will characterize the nature likelihood and 

### 11 dose response relationship of any adverse outcomes from 

### 12 potential endocrine disruption via E, AEA and/or TT pathways. 

### 13 Again, since the fish used is not native nor 

### 14 representative of many ecologically important species, one panel 

### 15 member indicated the statement may overstate the utility of the 

### 16 model. For instance there have been many studies showing 

### 17 endocrine disrupting chemicals adversely affect disease 

### 18 resistance and the immune response. There is no attempt to 

### 19 address this adverse effect. Also no assessment of the thyroid 

### 20 is included. Either thyroid histology nor plasma thyroid 

### 21 hormones per measure. Hence both MMT and the MRT really focus 

### 22 on reproductive endpoints, but not the other adverse outcomes 

### 23 endocrine disrupters may cause, which can also have population 

### 24 effects. 

### 25 To specifically address the EDSTAC definition 

### 26 of Tier 2 again characterizing nature likelihood and dose

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1 response relationship of endocrine disruption of EAT, the 

### 2 following components were specifically addressed. 

### 3 With regards to nature, although non native 

### 4 and given the weaknesses discussed above, the biological 

### 5 advantages in Medaka as an international model for reproduction 

### 6 should allow the characterization and nature of most EAT AOPs. 

### 7 Its rapid reproductive cycle, secondary sex characteristics, 

### 8 genetic markers for gender, well study developmental and 

### 9 reproductive life history and ability to evaluate maternal 

### 10 female transport make Medaka an excellent model to characterize 

### 11 the nature of EAT endpoints. 

### 12 With regard to likelihood for the eight 

### 13 compounds evaluated, there is strong likelihood that E or A 

### 14 responses maybe detected. However, the likelihood of 

### 15 characterizing specific thyroid effects or antagonism or 

### 16 indirect effects on steroidogensis was less likely (i.e. 

### 17 certain). Disruption of the HPT access may cause atypical 

### 18 endpoints detected in this assay but the response will not 

### 19 likely be specific for thyroid disruption unless compared with 

### 20 other thyroid specific assays such as the amphibian assay. 

### 21 Now with regard to dose response relationship, 

### 22 the data provided significant uncertainty of how dose response 

### 23 relationships were considered or the meaning of what will be 

### 24 considered significant in relation to surrounding doses if a 

### 25 dose is statistically different from the control but not from a 

### 26 dose higher or a dose lower than itself.

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1 Given the variability of the inter-laboratory 

### 2 calibration studies particularly those performed by laboratories 

### 3 outside of MED and NIES obtaining significant concentration 

### 4 response data outside a single LOEC determinations may be 

### 5 limited in the MMT. 

### 6 If a multiple dose concentration response 

### 7 curve with more than two statistically significant treatment 

### 8 points, that again are different from each other is desired, 

### 9 than additional statistical power is required. 

### 10 Potential reasons for inter-laboratory 

### 11 variability were provided by the Agency and will be discussed in 

### 12 the questions subsequent to this. 

### 13 However, whatever the reason in most of the 

### 14 data provided in the ISR primarily demonstrated single point 

### 15 LOEC measurements with those concentration dependent trends in 

### 16 the MMT, which limit the causal criteria associated with a Tier 

### 17 2 paradigm. 

### 18 If the Agency is only interested in obtaining 

### 19 single point deterministic quantitative endpoints of LOEC or 

### 20 atypical endpoints, which may be impaired by EAT AOPs then MMT 

### 21 may be useful. E & A biomarker responses may also have LOEC 

### 22 measurements but specific T, EAT antagonism or steroidogenesis 

### 23 AOPs may not be as certain, which again we've already stated. 

### 24 In addition monotonic dose response is assumed 

### 25 to be dominant with the evaluations. Overall the panel strongly 

### 26 feels that caution should be used in evaluating LOECs and other

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1 statistical inferences given with this assumption since several 

### 2 ADCs such as EPA and other compounds may not exhibit typical 

### 3 sigmoidal concentration response relationships. 

### 4 Enhanced statistical power for the MRT may 

### 5 improve this relationship. However removal of F0 evaluations 

### 6 for overt toxicity particularly in females and the MRT is of 

### 7 concern which will be discussed later. We just have to get to 

### 8 the MRT. And that's all that I have. 

### 9 **DR. MARTHA SANDY:**Dr. Blazer. 

### 10 **DR. VICKI BLAZER:**I think the only thing I 

### 11 would add is something that came up earlier this morning and 

### 12 that was how does Medaka compare to the fathead minnows, since 

### 13 it's the fathead minnow that is used in Tier 1. 

### 14 **DR. MARTHA SANDY:**Dr. Friedman. 

### 15 **DR. JENNIFER FREEMAN:**I don't have any 

### 16 additional comments. 

### 17 **DR. MARTHA SANDY:**Dr. Kullman. 

### 18 **DR. SETH KULLMAN:**Sure, I'd just like to 

### 19 reiterate some of the comments based on genotype and phenotype 

### 20 for gender determination. I think that the Medaka is a really 

### 21 unique model for that and in the ISR we did not see any 

### 22 relationships to gender ratios and gender numbers. 

### 23 And I think here that since genotype is fixed 

### 24 and phenotype is possibly sensitive to environmental exposures 

### 25 or compound exposures that you have a unique opportunity to not 

### 26 only fix the idea with gender, use that as an internal control

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1 to look at your husbandry conditions to know that every 

### 2 subsequent generation of animals is giving you a consistent 

### 3 ratio of males to females, temperature being the variable that 

### 4 might effect that and then also compare that to the phenotypic 

### 5 characteristics such as the secondary sex characteristics, the 

### 6 papillae, the split anal fin as well as gonadal sex to really 

### 7 give you a nice indices of genotypic versus phenotypic or 

### 8 phenotypic versus genotypic gender. 

### 9 The second comment was or is based on the dose 

### 10 response that Dan had suggested in our summary; is that it may 

### 11 be prudent to include additional indices that we can derive from 

### 12 full dose response curves such as the NOEL and the EC-50. I 

### 13 think that was suggested in some of the public comments this 

### 14 morning. And any other determinance that can be derived from 

### 15 this. 

### 16 Also it may be again from an internal 

### 17 perspective to use some of the transformation processes that we 

### 18 have with dose response relationship such as a probit. Again to 

### 19 start comparing a cross-trials of the MMT, to compare expected 

### 20 versus observed effects across different compounds that you're 

### 21 working with. So strong agonists may have a steeper slope to 

### 22 the left versus weaker agonists and so forth, and that might 

### 23 give you a nice serial analysis on the performance of the 

### 24 assays. That's all. 

### 25 **DR. MARTHA SANDY:**Dr. PatiQo. 

### 26 Dr. PatiQo: no further comment.

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1 **DR. MARTHA SANDY:**Dr. Perkins. 

### 2 **DR. EDWARD PERKINS:**No further comment. 

### 3 **DR. MARTHA SANDY:**Dr. Johnson. 

### 4 **DR. DALLAS JOHNSON:**I have nothing to add. 

### 5 **DR. MARTHA SANDY:**Any comments from other 

### 6 members of the panel on Question 1 from Medaka. None. Okay. 

### 7 And we'll go back to the EPA staff, do they have any questions 

### 8 of clarification? 

### 9 **DR. RODNEY JOHNSON:**I have no questions for 

### 10 clarifications. I am clear on what the panel stated so far. 

### 11 **DR. MARTHA SANDY:**Thank you very much. Let's 

### 12 go to Question 2 please. 

### 13 **DR. LESLIE TOUART:**Reading the Charge 

### 14 Question 2 for the Medaka test. Test methods and their 

### 15 associated endpoints should be scientifically relevant to the 

### 16 biological processes of interest and should be demonstrated to 

### 17 be responsive to the specific type of effect/toxicity of 

### 18 interest. Each species presents unique characteristics from a 

### 19 biological perspective and allows for specialized endpoints to 

### 20 address a specific toxicological mode of action. 

### 21 Please comment on the biological and 

### 22 toxicological relevance of the assay in regards to the stated 

### 23 purpose of characterizing endocrine disruptors, as described in 

### 24 Section 3 of the MMT ISR. 

### 25 **DR. MARTHA SANDY:**And Dr. Freeman is the 

### 26 lead.

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1 **DR. JENNIFER FREEMAN:**Okay. In terms of 

### 2 biological and toxicological relevance, relevance was defined by 

### 3 the Agency as whether a test is meaningful and useful for a 

### 4 particular purpose. Given the unique characteristics of the 

### 5 Medaka life history, which was mentioned already in the first 

### 6 response, the biological and toxicological relevance of the 

### 7 assay is theoretically adequate. 

### 8 Specific responses throughout the rapid 

### 9 lifecycle allow AOP responses including vitellogenin, secondary 

### 10 sex characteristics, and gonadal histopathology to be evaluated 

### 11 and linked to atypical endpoints. The availability of genetic 

### 12 sex markers also allows the evaluation of husbandry conditions, 

### 13 which can be refined to provide better reliability and 

### 14 precision. 

### 15 Based on established AOPs that provide a 

### 16 foundation of modes of actions for defined EDCs numerous 

### 17 endpoints can be exploited to assess general toxicity including 

### 18 growth, mortality and hatching success. 

### 19 In addition to defined HPG impacted endpoints 

### 20 in Section 2.2 of the ISR these are described as either 

### 21 activational or organizational endpoints. As such assessment of 

### 22 secondary sex characteristics such as formation of papillae on 

### 23 Medaka, anal fin can be informative of either androgenic or 

### 24 antiandrogenic/estrogenic activity. Assessment of vitellogenin 

### 25 mRNA in males is known as a gold standard of estrogenic effects 

### 26 in ova paired species and is well characterized to be mediated

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1 by ligand dependent transactivation of the ER. Thus 

### 2 vitellogenin is likely to be one the most sensitive assays 

### 3 proposed for estrogenic effects. 

### 4 The fact that Medaka can be induced to spawn 

### 5 daily by controlling light and temperature is also highly 

### 6 relevant to the assay as both fertility and fecundity can be 

### 7 tightly monitored. This component of the assay must be assessed 

### 8 with significant rigor as slight modifications in regulating 

### 9 husbandry conditions may be significantly impacted on spawning 

### 10 success of this species. 

### 11 Additionally there is likely to be significant 

### 12 variability between brooding pairs as was demonstrated in their 

### 13 inter-laboratory comparison conducted during the validation 

### 14 process. 

### 15 The fact that Medaka exhibit phenotypic sexual 

### 16 dimorphic characteristics is an additional advantage relevant to 

### 17 use of the species. However this aspect of the model is 

### 18 currently underutilized in the proposed MMT and MRT assay. More 

### 19 attention should be given to the fact that there is a well 

### 20 defined mechanism to determine both phenotypic gender and 

### 21 genetic gender in this model. And I think Dr. Kullman just 

### 22 highlighted a lot of these responses but I'll just cover them 

### 23 again to highlight how we felt the importance of them. 

### 24 This is an advantage for utilization of Medaka 

### 25 model for the EDC studies that cannot be recapitulated in other 

### 26 small Aquarian fish models. For this reason greater advantage

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1 should be utilized in either the MMT or the MRT assay for 

### 2 establishing genotypic versus phenotypic gender ratios. As is 

### 3 the current MMT and MRT does not exploit this aspect of the 

### 4 model with the sole focus on genetic gender for establishing 

### 5 breeding pairs and no assessment of genotype to phenotype ratios 

### 6 within the F1 or F2 populations. 

### 7 It is also suggested that the Agency should 

### 8 pursue the development of a population estimate model that is 

### 9 similarly reported in Miller et al 2007 that has been 

### 10 established for fathead minnows and we'll include the reference 

### 11 for that particular paper in the write up for our report. 

### 12 An additional concern particularly with the 

### 13 MRT is that the assay may not differentiate endocrine responses 

### 14 with agents that cause non-endocrine responses. For example, 

### 15 severe hepatotoxicity to the reproductive females in the F0 may 

### 16 alter the endocrine development of F1 offspring providing 

### 17 endocrine response that may not be mediated directly through the 

### 18 E, A or T pathway or an endocrine like response. 

### 19 While there is adequate redundancy with regard 

### 20 to endocrine endpoints, additional evaluation of hepatic 

### 21 histopathology for severe morphological damage in whole animal 

### 22 cross-sections would allow that particular variable to be 

### 23 assessed for compounding of endocrine responses. Thus the basic 

### 24 endpoint should be collected in the parental F0 to determine 

### 25 parental toxicity. 

### 26 The full range of endpoints is needed in the

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1 F1 while assessing reproductive function and the F1 can be fully 

### 2 assessed by evaluating the F2 through hatch. It is also 

### 3 suggested that additional steroidal endpoint measurements are 

### 4 added to gain further knowledge on the impacts to the endocrine 

### 5 system. 

### 6 Overall it is initially proposed that MMT 

### 7 study has 29 weeks assessing adult parental F0. Full life of F1 

### 8 and partial F2 assessment. Based on the findings of the initial 

### 9 studies a reduced study design is proposed in the MRT ending the 

### 10 test after the hatching of the F2 for a total of 19 weeks. 

### 11 Based on the continuous exposure study design 

### 12 a longer F2 assessment does not appear to be needed. If the 

### 13 goal is no longer a multigenerational study; as such the new 

### 14 study design is more representative of a full life cycle assay 

### 15 with the focus on both general toxicity and EDC AOPs. That is 

### 16 all. 

### 17 **DR. MARTHA SANDY:**Thank you. And the 

### 18 associate discussants are Dr. Blazer. 

### 19 **DR. VICKI BLAZER:**I have nothing else to add. 

### 20 **DR. MARTHA SANDY:**Dr. Kullman. 

### 21 **DR. SETH KULLMAN:**Sure I'll add just a couple 

### 22 of things. So bear with us. So it is certainly obvious that 

### 23 the Medaka model is going to be proficient for E&A activity 

### 24 maybe a little less proficient for anti-E&A. I also wonder if 

### 25 thyroid measures could be added and that's not without precedent 

### 26 and in the fish models. So just a couple of examples of that.

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1 In the zebra fish model there has been 

### 2 established (inaudible) hormone level analysis and it's been 

### 3 done by something called the Immunofluorescence Quantitative 

### 4 Disruption Test. And I can provide a referenced for that. 

### 5 That's (inaudible) in 2009. Okay. And I think it may be just 

### 6 something worth looking into because it would increase the 

### 7 utility of the model for E, A and T. 

### 8 The second thing is it's also not without 

### 9 president again in the zebra fish using gene expression and 

### 10 transcriptional targets for thyroid hormone and other 

### 11 transcriptional targets within the HPT. And these have been 

### 12 validated, I guess validated or at least tested using 

### 13 (inaudible) as well as PTU. And it's a 20 gene PCR rating in 

### 14 zebra fish that may be adaptable to Medaka. I can also provide 

### 15 reference for that. 

### 16 **DR. MARTHA SANDY:**Dr. PatiQo. 

### 17 DR. REYNALDO PatiQo: No further comments. 

### 18 **DR. MARTHA SANDY:**Dr. Perkins. 

### 19 **DR. EDWARD PERKINS:**No further comments. 

### 20 **DR. DANIEL SCHLENK:**Just wanted to reiterate 

### 21 I really think it's important to at least do the steroid 

### 22 analysis. If you're doing it in fathead minnow I think it's 

### 23 doable with tissue aliquots and for whole tissue analysis and 

### 24 those methods are available and I can provide references if 

### 25 needed. 

### 26 **DR. MARTHA SANDY:**And Dr. Johnson.

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1 **DR. DALLAS JOHNSON:**I have nothing to add. 

### 2 **DR. MARTHA SANDY:**Other comments from members 

### 3 of the panel. And EPA staff any questions? 

### 4 **DR. RODNEY JOHNSON:**No questions I appreciate 

### 5 the recommendations and as has been mentioned earlier I really 

### 6 appreciate the idea that references or specific -- if there 

### 7 aren't references available but you have personal knowledge 

### 8 please share because it will be very helpful. 

### 9 **DR. MARTHA SANDY:**I'm sure the discussants on 

### 10 that question will do that. I think it's rather late and we'll 

### 11 call it a day you think? Yes. And so I thank everyone for all 

### 12 their hard work and attention and Dr. Matten do you have 

### 13 something to say? 

### 14 **DR. SHARLENE MATTEN:**I would just like to 

### 15 thank everyone for their good work and coming on time. The 

### 16 panel has been excellent and this microphone really works we can 

### 17 call people out from the hallway. 

### 18 So we'll start again at 9 o'clock in the 

### 19 morning and we'll continue with the Medaka multigeneration test 

### 20 charge questions. Thank you very much. 

### 21 (Whereupon a recess was taken for the day) 

### 22 23 24 25 26

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1 **DR. SHARLENE MATTEN:**Good morning. My name 

### 2 is Sharlene Matten. I am the designated federal official for 

### 3 this FIFRA Scientific Advisory Panel meeting that is reviewing 

### 4 providing advice to the EPA on the Endocrine Disruptor screening 

### 5 Programs Proposed Tier 2 Ecotoxicity Test. 

### 6 Today is Thursday, June 27th. If you're 

### 7 listening in the radio broadcast area, we are on our third day 

### 8 of our discussions. We left off yesterday with the exciting 

### 9 discussion on the Medaka Multigeneration Test on Question 3. 

### 10 I am going to turn the microphone over to Dr. 

### 11 Martha Sandy, who is the session Chair for this meeting. 

### 12 **DR. MARTHA SANDY:**Thank you, Dr. Matten. 

### 13 We've been asked to go around the room quickly, one more time, 

### 14 an identify ourselves and then we'll jump into Question 3. 

### 15 I'm Martha Sandy and I'm with the California 

### 16 Environmental Protection Agency. 

### 17 **DR. BARRY DELCLOS:**Barry Delclos from the 

### 18 FDA's National Center for Toxicological Research. 

### 19 **DR. JAMES MCMANAMAN:**Jim McManaman, 

### 20 University of Colorado. 

### 21 **DR. DANIEL SCHLENK:**Dan Schlenk, University 

### 22 of California, Riverside. 

### 23 **DR. STEPHEN KLAINE:**Steve Klaine, Clemson 

### 24 University. 

### 25 **DR. MARION EHRICH:**Marion Ehrich, Virginia 

### 26 Tech.

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1 **DR. GEOFFREY SCOTT:**Geoff Scott, National 

### 2 Oceanic and Atmospheric Administration. 

### 3 **DR. VICKI BLAZER:**Vicki Blazer, U.S. 

### 4 Geological Survey. 

### 5 **DR. REYNALDO PATINO:**Reynaldo Patino, U.S. 

### 6 Geological Survey, Texas Cooperative Fish and Wildlife Reserve 

### 7 Unit. 

### 8 **DR. CATHERINE PROPPER:**Cathy Propper, 

### 9 Northern Arizona University. 

### 10 **DR. EDWARD PERKINS:**Ed Perkins, U.S. Army 

### 11 Corps of Engineers. 

### 12 **DR. ROBERT DENVER:**Bob Denver, University of 

### 13 Michigan. 

### 14 **DR. JOHN MCCARTY:**John McCarty, University of 

### 15 Nebraska, Omaha. 

### 16 **DR. DALLAS JOHNSON:**Dallas Johnson, Kansas 

### 17 State University. 

### 18 **DR. COLIN SCANES:**Colin Scanes, University of 

### 19 Wisconsin, Milwaukee. 

### 20 **DR. SETH KULLMAN:**This is Seth Kullman, North 

### 21 Caroline State University. 

### 22 **DR. SHEA TUBERTY:**Shea Tuberty, Appalachian 

### 23 State University. 

### 24 **DR. JENNIFER FREEMAN:**Jennifer Freeman, 

### 25 Perdue University. 

### 26 **DR. MARY CHRISTMAN:**Mary Christman, MCC

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1 Statistical Consulting. 

### 2 **DR. GEORGE BENTLEY:**George Bentley, U.C., 

### 3 Berkley. 

### 4 **DR. MARTHA SANDY:**Thank you. So when we left 

### 5 off yesterday, we were in the middle of addressing questions on 

### 6 the Medaka Multigeneration Test. We have with us, EPA staff; we 

### 7 have Mary Manibusan, Dr. Les Touart, and Dr. Rodney Johnson. 

### 8 Dr. Touart, would you be reading us Charge 

### 9 Question Number 3? 

### 10 **DR. LESLIE TOUART:**Les Touart reading Charge 

### 11 Question Number 3 for the Medaka Multigeneration Test. 

### 12 The test protocol should be sufficiently 

### 13 detailed and should include a description of what is measured 

### 14 and how it is measured. The selection of endpoints within the 

### 15 assay should be reflective of the biological processes of 

### 16 interest and the endpoints should be intrinsically relevant and 

### 17 have established sensitivity. 

### 18 The test protocol should demonstrate the 

### 19 ability to measure the endpoints and provide adequate 

### 20 performance criteria for evaluation. 

### 21 Please comment on the selection, optimization 

### 22 and demonstration of the assay endpoints as outlined Section 3 

### 23 of the ISR. 

### 24 **DR. MARTHA SANDY:**Dr. Freeman is the lead 

### 25 discussant on this. 

### 26 **DR. JENNIFER FREEMAN:**Selection on the

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1 MMT/MRT endpoints exhibit biological and toxicological relevance 

### 2 with regards to EDC exposure and assessment. 

### 3 As demonstrated in Section 2.2 of the ISR, 

### 4 small aquarium fish, specifically medaka, poses unique 

### 5 biological feature that make them highly amenable to en vivo, 

### 6 EDC toxicity testing. Based on established AOPs that provide a 

### 7 foundation for modes of action for defined EDCs, numerous 

### 8 endpoints can be exploited to assess general toxicity, including 

### 9 growth, mortality hatching success in addition to defined 

### 10 HPG-impacted endpoints. 

### 11 In addition, Section 2.2 of the ISR, these are 

### 12 described as either activational or organizational endpoints. 

### 13 And we have some comment on the selection of some additional, 

### 14 secondary sex characteristics to evaluate. 

### 15 Since gender alteration is a significant 

### 16 endpoint related to endocrine disruption, additional redundancy 

### 17 with secondary sex characteristics may be an option. This is 

### 18 especially of interest, given the potential difficulties of DMY 

### 19 measurements. 

### 20 In addition to the anal papillae endpoint, 

### 21 Emma Watts, et al., 2003, and will include the reference to this 

### 22 publication, has indicated that additional morphometric 

### 23 characteristics can be recorded for each fish to generate a 

### 24 genders score. 

### 25 The morphometric characteristics include 

### 26 degree of urogenital protuberance, number of anal fin ray nodes,

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1 number of anal fin rays, number of anal fin rays for papillary 

### 2 processes, and number of dichotomously branched anal fin ray and 

### 3 degree of ventral fin length. 

### 4 Total length can be determined by a 

### 5 measurement from the most rostral point to the most caudal point 

### 6 of each fish. Degree of urogenital protuberance can be 

### 7 quantified on a scale of one to three, with 1) indicating almost 

### 8 no visible protuberance; 2) indicating slight to moderate 

### 9 urogenital protuberance development; and 3) indicating full 

### 10 urogenital protuberance that resembled a half-balloon type 

### 11 appearance. 

### 12 Number of anal fin ray nodes can be counted 

### 13 down the third anal fin ray from the fin or origin. Numbers of 

### 14 anal fin rays with papillary processes can be counted for each 

### 15 ray with any visible papillary processes. 

### 16 Degree of ventral fin length can be quantified 

### 17 on a scale of one to three, with 1) indicating a short ventral 

### 18 fin not extending to the genital core region; 2) indicating a 

### 19 ventral fin that extended to the genital core region; and 3) 

### 20 indicating a ventral fin extending to or beyond the genital 

### 21 core. 

### 22 A gendered determination score can then be 

### 23 generated for each fish in control and treatment groups. Score 

### 24 can be generated by adding degree of urogenital protuberance 

### 25 index, number of dichotomously branched anal fin rays, and 

### 26 ventral fin length index for each fish.

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1 Male fish have only one or two dichotomously 

### 2 branched anal fin rays; whereas, female fish have six to eight. 

### 3 A greater gendered determination score might be attributed to an 

### 4 individual fish exhibiting female morphometric characteristics 

### 5 and a lower score for fish with male characteristics. 

### 6 There are also automated programs that are 

### 7 available for these types of morphometric analysis to help them 

### 8 be more quickly attained. One specific software package out 

### 9 there is the BIOQUANT image analysis software that's available. 

### 10 I guess now I want to talk a little bit about 

### 11 the optimization and demonstration of assay endpoints. 

### 12 Optimization of MMT endpoints appears to be critical to 

### 13 establishing rigorous statistical inference. As such, MMT has 

### 14 been modified, reflecting several changes, increase in 

### 15 biological replication, repeatability and fecundity assessment 

### 16 in the MRT assay, other endpoints such as secondary sex 

### 17 characteristics that appear to be highly correlative to EDC AOPs 

### 18 and mode of action and indicative of ADC function. 

### 19 Overall, a relatively small number of 

### 20 compounds with differing EDC modes of action have been studied, 

### 21 using the MMT or MRT assay and thus, validation of the assay 

### 22 needs to be further substantiated. To ensure consistency in 

### 23 evaluation of individual defined endpoints, further 

### 24 standardization and possible optimization of methodology may be 

### 25 required. 

### 26 As is, Section 3 of the MMT provides a

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1 reasonable global description of the MMT/MRT assay, including 

### 2 timelines, exposure of conditions, husbandry, and overall 

### 3 measurement of defined AOPs and general toxicity endpoints. 

### 4 However, overall, the IRS and provided protocols lack sufficient 

### 5 methodology to fully recapitulate the assay. This is 

### 6 particularly true with regard to methodology and data analysis 

### 7 for each defined endpoint of the assay. 

### 8 I'm going to walk through a list of certain 

### 9 items that we think further details need to be added for 

### 10 standardization of the protocols; the first are further 

### 11 guidelines on water quality. While water quality conditions 

### 12 were briefly mentioned, water quality is a major differential 

### 13 factor among laboratories and more details should be provided in 

### 14 the research protocol in water quality requirements. This 

### 15 should also help to reduce variability among laboratories that 

### 16 would be conducting these studies. Currently, limited 

### 17 information is provided for additional water quality parameters. 

### 18 19 Most fish laboratories, especially those 

### 20 working with the small fish models, generally have an extensive 

### 21 protocol developed for water quality guidelines. These type of 

### 22 guidelines need to be shared and included in the ISR so that 

### 23 they can be undertaken by the different laboratories that work 

### 24 to perform these assays. 

### 25 The second is additional standardization on 

### 26 the assessment on the reproductive activity of the fish

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1 population before the assays have begun and detailed husbandry 

### 2 guidelines. Guidelines should be added that indicate that the 

### 3 size of the adults and reproductive capability of that 

### 4 particular fish population should be assessed before starting 

### 5 the assay. 

### 6 This is important, as these basic population 

### 7 characteristics can widely vary among the different 

### 8 laboratories. Adding standardization on minimal size and 

### 9 reproductive function of the specific fish population to be used 

### 10 in the studies should help to assist in removing some of the 

### 11 variability among the different laboratories. 

### 12 In addition, most small fish laboratories have 

### 13 extensive protocols developed for husbandry and care of the 

### 14 fish, and similar guidelines should be included in the protocol 

### 15 for guidance and optimization of the assay in all these 

### 16 laboratories. 

### 17 There is a few additional items that I'm 

### 18 going to list for optimization. I'm just going to walk through 

### 19 this list because most of these are going to be described in 

### 20 more detail when we get to Question 6. 

### 21 Items that I think need to be standardized for 

### 22 optimization of the assay include standardization of 

### 23 vitellogenin analysis, standardization of the analysis of the 

### 24 anal fin papillae, standardization of the embryo collection, 

### 25 more details on how embryo viability is determined, more 

### 26 information on the determination of the genetic sex in regards

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1 to the Vtg analysis, and also further details about an inclusion 

### 2 of the hepatotoxicity assay because these types of evaluation 

### 3 and endpoints need to be clarified. It was unclear what types 

### 4 of toxic responses are expected or how histological finding will 

### 5 inform endocrine disrupting effects of test compounds. 

### 6 It is important to establish normalization 

### 7 procedures for histopathology assays to ensure that assay 

### 8 reproduces the ability for the hatter toxicity assays. 

### 9 So overall, adding additional details and 

### 10 parameters such as those that were just described, will assist 

### 11 in optimizing the assay. More information, as I mentioned 

### 12 before, will be mentioned when we get to Charge Question 6. 

### 13 **DR. MARTHA SANDY:**Thank you. The associates, 

### 14 Dr. Blazer. 

### 15 **DR. VICKI BLAZER:**I have nothing additional 

### 16 to add. 

### 17 **DR. MARTHA SANDY:**Dr. Kullman. 

### 18 **DR. SETH KULLMAN:**As Dr. Freeman suggested, 

### 19 I'll provide more details in some of these comments in Question 

### 20 6. 

### 21 **DR. MARTHA SANDY:**Dr. Patino. 

### 22 **DR. REYNALDO PATINO:**Nothing new to add. 

### 23 **DR. MARTHA SANDY:**Dr. Perkins. 

### 24 **DR. EDWARD PERKINS:**I have nothing further to 

### 25 add. 

### 26 **DR. MARTHA SANDY:**Dr. Schlenk.

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1 **DR. DANIEL SCHLENK:**My comments are 

### 2 incorporated. 

### 3 **DR. MARTHA SANDY:**And Dr. Johnson. 

### 4 **DR. DALLAS JOHNSON:**I have nothing to add. 

### 5 I'll look up and see if Dr. Portier had anything he wanted to 

### 6 include. I kind of forgot about my job this morning. 

### 7 **DR. MARTHA SANDY:**Okay. While you're looking 

### 8 that up, I'll ask if any other members of the panel have 

### 9 additional comments on Question 3. 

### 10 (No response.) 

### 11 It doesn't look like it. We'll let you take a 

### 12 look, Dr. Johnson, at Dr. Portier's comments. 

### 13 **DR. DALLAS JOHNSON:**His comments are okay. 

### 14 There is nothing unusual here that needs to be brought out, I 

### 15 don't think. 

### 16 **DR. MARTHA SANDY:**Okay. So I take that means 

### 17 that it is along the same lines of what he mentioned about other 

### 18 assays, perhaps. 

### 19 **DR. DALLAS JOHNSON:**Okay. Thank you. So I 

### 20 will turn to the EPA staff and ask if they have any questions of 

### 21 clarification or any other issues that you don't think we've 

### 22 answered the question adequately. 

### 23 **DR. RODNEY JOHNSON:**I'd like to ask one 

### 24 clarifying question. It has to do with the statement of 

### 25 relating the genetic sex to the vitellogenin endpoint. Is that 

### 26 what you said?

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1 I'm trying to understand. It's the point that 

### 2 you made just before the conversation or the discussion on 

### 3 hepatotoxicity. 

### 4 **DR. JENNIFER FREEMAN:**There was just that 

### 5 more details needed to be added in regards to the methods if 

### 6 we're determining genetic sex. Dr. Kullman will go into detail 

### 7 about the specifics about adding those details when he gets to 

### 8 Question 6. 

### 9 **DR. RODNEY JOHNSON:**Thank you. 

### 10 **DR. MARTHA SANDY:**And that was Dr. Freeman. 

### 11 Dr. Dallas Johnson. 

### 12 **DR. DALLAS JOHNSON:**Yes. Further looking at 

### 13 what Dr. Portier had to say, I might indicate two things that he 

### 14 mentioned. On Section 3.3.3, it says the protocol for deciding 

### 15 which surviving larval fish are kept and which are culled at the 

### 16 start of week 4 is not provided. With a maximum of 10 fish per 

### 17 aquaria some surviving larval fish are discarded. How is cull 

### 18 choice made? 

### 19 And then in Section 3.3.5: A caudal fin 

### 20 sample is used to determine genotypic sex of individual which is 

### 21 then used to establish female/male breeding pairs. For some 

### 22 chemicals, some F1 subadults, XX fish have testes, female fish 

### 23 with male SSC-androgen acting chemicals, and/or some XY fish 

### 24 have decreased SSC, males with female phenotypic 

### 25 characteristics, estrogen-acting chemicals. 

### 26 The wrong choice of male and female fish, at

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1 this point, can and has led to creating non-breeding pairs. Is 

### 2 there a better protocol using other than genotype that could 

### 3 reduce the likelihood of getting non-breeding pairs? 

### 4 I assume that he'll expand on that in the 

### 5 final report. 

### 6 **DR. MARTHA SANDY:**Okay. Thank you. I'll ask 

### 7 EPA again, are there any questions of clarification on that? 

### 8 **DR. RODNEY JOHNSON:**No questions. Thank you. 

### 9 **DR. MARTHA SANDY:**I believe we're ready to 

### 10 move on to Question 4. 

### 11 **DR. LESLIE TOUART:**I'll read Question 4 as 

### 12 soon as it advances on the screen. Demonstration of the test 

### 13 method performance should be based on the testing of reference 

### 14 chemicals representative of the types of substances for which 

### 15 the test method will be used. 

### 16 Test substances should adequately represent an 

### 17 appropriate range of responses and physical chemical properties 

### 18 for which the test method is proposed to be appropriate. 

### 19 The selection of the most appropriate 

### 20 statistical approaches depends, in part, on the nature of the 

### 21 data and also on the design of the validation study. 

### 22 Statistical and non-statistical methods used to analyze should 

### 23 be described. 

### 24 Please comment on the selection test 

### 25 substances and methods, analytic and statistic, where 

### 26 appropriate, chosen for the demonstration and validation of the

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1 MMT and MRT assays. 

### 2 **DR. MARTHA SANDY:**Dr. Dallas Johnson is the 

### 3 lead on this. I presume you would like to also, as we did 

### 4 before, just split this question and you will just address the 

### 5 statistical issues at this point. 

### 6 **DR. DALLAS JOHNSON:**That is correct. I have 

### 7 emailed with the other discussants on this question and they 

### 8 have all agreed to that approach. 

### 9 This will be a lot shorter than yesterday. I 

### 10 promise you. Many of the remarks that I made yesterday could 

### 11 also be made here. In the discussion that I will do, I'm going 

### 12 to refer to the aquaria as tanks in my remarks. It seems like 

### 13 most terminologies were used in the report. 

### 14 The Table 3.1 shows the test conditions for 

### 15 the Medaka Multigeneration Test protocol and then there is a 

### 16 similar table for the revised protocol. The remarks that I have 

### 17 are probably appropriate for both protocols. 

### 18 As mentioned yesterday, and similarly here, 

### 19 Table 3.1 does a very good job of describing those conditions 

### 20 that can be controlled by the researcher and/or the lab, but it 

### 21 doesn't say anything about the randomizing process, randomizing 

### 22 fish to these conditions. 

### 23 Some of their questions that came to mind 

### 24 initially, and I'll probably add some others as the final report 

### 25 comes out, how many flow through systems are there. Is there 

### 26 one test replicate chamber size in each flow through system or

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1 all test chambers placed in the same flow through system? 

### 2 I'm thinking that there's a single flow 

### 3 through system for each tank, but I don't know whether that's 

### 4 true or not. The report leaves a lot to be described, I guess, 

### 5 in terms of how the data are actually collected and how things 

### 6 are randomized -- how the fish are randomized and so on. That 

### 7 just needs to be tightened up a bit more. 

### 8 Similarly, one of the feeds placed in the tank 

### 9 is just done in a random order or the feeds for one treated 

### 10 group, if a pair of fish are in a tank, then I assume that each 

### 11 tank gets its own feeding treatment. I don't know whether the 

### 12 feed is in the flow through thing so they all share the same 

### 13 feed or whether each tank has its own feed and where the tanks 

### 14 are placed, and are their locations randomized. 

### 15 I'm thinking there must be a lot of other 

### 16 things that could be said that would help some laboratory or 

### 17 some researcher that would try to reproduce the data that are 

### 18 collected here that they need to be made aware of and it should 

### 19 be in the protocol or in a standard operating plan. 

### 20 The types of analyses that were done here were 

### 21 similar to the types of analyses that were done on the Japanese 

### 22 quail data. A lot of the analyses were done automatically, 

### 23 using the StatCHARRMS program. 

### 24 My thinking about the -- well, my comments 

### 25 that I had yesterday about what is the appropriate probability 

### 26 distribution for each endpoint variable. I think that letting

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1 the data drive the analysis, it seems that this StatCHARRMS 

### 2 program is trying to automate things. 

### 3 Well, I know in my consulting activities in 

### 4 the past, I've had a lot of researchers, what they look for is 

### 5 some sort of PROC analysis. That they just bring their data and 

### 6 dump it into a program and it will do everything for them. 

### 7 That's a little bit about what I'm seeing about this StatCHARRMS 

### 8 program. I think that could be dangerous. 

### 9 I can see that some endpoint variables might 

### 10 be analyzed one way by some labs or researchers and it might be 

### 11 analyzed another way by other labs and researchers. So I think, 

### 12 again, the science and what you know about the endpoint 

### 13 variables that you measure should give indication at what the 

### 14 underlying probability distribution might be and then that 

### 15 appropriate distribution could help you do the best analysis for 

### 16 the data that has been collected. 

### 17 In the StatCHARRMS program, it appears that 

### 18 those tests were for equal normality and tests for equal 

### 19 variances that are performed and then the analysis just kind of 

### 20 depends on whether those tests are passed or failed. Again, as 

### 21 I mentioned yesterday, the analysis with unequal variances isn't 

### 22 much of a problem anymore. Tests for normality aren't very 

### 23 powerful or highly related to sample size. 

### 24 So again, I think that the right way to go 

### 25 about that is to let the science drive the appropriate -- what 

### 26 other people are doing and what we know about the variables that

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1 are being collected and what similar kinds of variables and 

### 2 those should drive the statistical analysis that is going to be 

### 3 performed. 

### 4 A lot of the analyses appear to be doing the 

### 5 ANOVA on ranks. And as I mentioned yesterday, the DOM 

### 6 parametrics isn't free of assumptions, it's just free of the 

### 7 normality assumption, equal variances and no interactions. You 

### 8 have interaction or you have unequal variances in non-parametric 

### 9 methods on ranks don't work very well -- work quite badly, in 

### 10 fact. 

### 11 There was a table, I guess, Table 4.3, which 

### 12 described the only two parameters in adult control fish that 

### 13 were measured the same in both MMTs, the one in the U.S. and the 

### 14 one in Japan. The report says that the MMTs are compared to 

### 15 showing similar data across the two labs. 

### 16 One of the variables that was looked at, 

### 17 fecundate in the F1 generation in the U.S., it was 17, plus or 

### 18 minus two. In Japan, it was 29, plus or minus five. So the 

### 19 difference in the means there is 12, and it looks like there are 

### 20 more than four standard deviations apart. And it would just 

### 21 wonder why you would say that that data are similar. Maybe the 

### 22 science or maybe the biological difference isn't relevant. I 

### 23 don't know, but it just seems strange. And then a similar kind 

### 24 of thing might be said about the variable SSC. 

### 25 I will say that after receiving the response 

### 26 about the correction that were made in the one way ANOVA, when

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1 the data were reanalyzed. I do believe that data now are being 

### 2 analyzed correctly. Our confusion had to do with being told 

### 3 that they were using a repeated measures analysis, with only a 

### 4 single repeated measure. 

### 5 The response that we received yesterday 

### 6 afternoon indicated that the repeated was a misnomer, as the 

### 7 program actually used a mixed model analysis with tank as a 

### 8 random affect, which is what we were proposing. And this is the 

### 9 correct way to analyze the data, with unequal numbers of 

### 10 observations per tank. 

### 11 I probably don't need to say it, but as you 

### 12 can probably tell from my previous comments regarding 

### 13 statistical analyses, I'm not very charmed with the StatCHARRMS. 

### 14 15 As I stated earlier, knowledge of the science 

### 16 should drive the appropriate ways to analyze endpoint variables. 

### 17 I don't think it's appropriate to let the data drive the 

### 18 analysis unless you're dealing with an endpoint variable that 

### 19 science knows nothing about. I don't think there are such 

### 20 variables being proposed as endpoint variables here. 

### 21 That's the close of my remarks. 

### 22 **DR. MARTHA SANDY:**Thank you. So I'm going to 

### 23 go around and ask the other discussants if they have additional 

### 24 comments related to the statistical issues part of this 

### 25 question. Dr. Patino. 

### 26 **DR. REYNALDO PATINO:**Good morning. So I

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1 think what I'll do is I'll address the question, the parts that 

### 2 remain are about the compounds that were used and then the 

### 3 methods. 

### 4 **DR. MARTHA SANDY:**Excuse me. I wanted to ask 

### 5 anyone else if there were statistical issues, first. 

### 6 **DR. REYNALDO PATINO:**Oh, I'm sorry. 

### 7 **DR. MARTHA SANDY:**Let's go around quickly and 

### 8 then we'll start with the other parts. Is that okay? 

### 9 **DR. REYNALDO PATINO:**That's fine, and I don't 

### 10 have any comments on that. 

### 11 **DR. MARTHA SANDY:**Okay. Dr. Perkins. 

### 12 **DR. EDWARD PERKINS:**No further comments. 

### 13 **DR. MARTHA SANDY:**Dr. Schlenk. 

### 14 **DR. DANIEL SCHLENK:**Actually, I guess this is 

### 15 probably the question just to reemphasize the dose dependence 

### 16 statistics. If we could actually get something that showed, 

### 17 perhaps, what the ANOVA differences were between the actual 

### 18 points in the figure that showed if there were differences 

### 19 between the points in the dose response group. I think that 

### 20 would be very helpful. 

### 21 **DR. MARTHA SANDY:**Dr. Freeman. 

### 22 **DR. JENNIFER FREEMAN:**I just want to add and 

### 23 emphasize the importance, I think, of adding those additional 

### 24 replicates in the MRT assay. I think that will help to account 

### 25 for some of the variability that you get naturally from the 

### 26 fish, with some of them breeding daily and some days they're not

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1 breeding. So I think that's a true positive to the assay. 

### 2 **DR. MARTHA SANDY:**Dr. Kullman. 

### 3 **DR. SETH KULLMAN:**I have nothing further to 

### 4 add. 

### 5 **DR. MARTHA SANDY:**Dr. Blazer. 

### 6 **DR. VICKI BLAZER:**I have nothing further to 

### 7 add. 

### 8 **DR. MARTHA SANDY:**Dr. Johnson, did Dr. 

### 9 Portier have anything? 

### 10 **DR. DALLAS JOHNSON:**I think he does. I did 

### 11 notice that I missed one thing in my report and it had to do 

### 12 with the coefficient of variation again, the CVs and in fish 

### 13 study, we have additional sources of variation. We've got 

### 14 tank-to-tank variation; we have lab-to-lab variation; we have 

### 15 fish-to-fish variation. 

### 16 So there are three places that variation could 

### 17 be described and so there are probably three different ways that 

### 18 one could calculate coefficient of variation and it's not clear 

### 19 in the report as to which way is being used, whether it's the 

### 20 standard deviation of all the fish divided by the mean or 

### 21 whether it's the tank standard deviation divided by the mean, or 

### 22 whether it's lab standard deviation divided by the mean. So it 

### 23 would be nice for that to be clarified a bit. 

### 24 And then you want me to report on Dr. 

### 25 Portier's comments? 

### 26 I think Dr. Portier like statCHARRMS better

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1 than I do. He seems to be okay with the way the analyses are 

### 2 being done. He believes that proper attention is paid to 

### 3 important issues, such as analysis model selection, inclusion 

### 4 and elimination of high order predicting factor interactions, 

### 5 model assumptions and testing that the data adequately meet 

### 6 model assumptions. The issue of accounting for experiment-wise 

### 7 error and multiple comparisons is also discussed. 

### 8 The couple of issues that he does have, 

### 9 Appendix G presents a somewhat statistically conservative, 

### 10 systematic approach to data analysis. It classifies the 

### 11 biological data generated by the MMT into one of three types: a 

### 12 continuous response that is measured once per subject, a 

### 13 continuous response that is measured multiple times per subject, 

### 14 and ordinal data, such as histopathology findings that are 

### 15 expressed as a severity score. This approach may be too 

### 16 conservative for some data that could lead to missing important 

### 17 findings. 

### 18 The StatCHARRMS tool uses a different general 

### 19 linear model for different kinds of data, including using a 

### 20 repeated measures model for the fecundity data. The general 

### 21 approach assumes normal and homogeneous data or that an 

### 22 appropriate transformation can be found that produces a response 

### 23 variable that is normal homogeneous, or if everything else 

### 24 fails, a normalized rank-order transform is used. 

### 25 This analysis approach would seem to work 

### 26 quite well for parameters such as adult bodyweight, length,

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1 vitellogenin levels, et cetera, but the approach may be more 

### 2 problematic for some variables in subadults since in some cases 

### 3 the levels tend to bunch up near zero. There's a good 

### 4 discussion of how to handle outliers on page 10. 

### 5 The fecundity data from the MMT can have 

### 6 characteristics that make the use of the repeated measures GLM 

### 7 questionable. Fecundity data are inherently counts. If the 

### 8 counts are relatively consistent and not too variable, 

### 9 transforming them by the natural log and using a GLM is not a 

### 10 bad analysis strategy. And then he says see for example, 

### 11 Appendix H. 

### 12 He talks a little bit about non-breeding pairs 

### 13 fecundity data. These kinds of data can be seen in the box 

### 14 plots presented for a number of the studies in Appendix HH, page 

### 15 56, Figure 1, Reproduction for OPDDT; page 70, Figure 1, 

### 16 Reproduction for CMP; and page 102, Figure 1, Reproduction for 

### 17 Trenbolone. 

### 18 Egg counts for breeding pairs will typically 

### 19 be centered on a non-zero value. Egg counts for non-breeding 

### 20 pairs will, by definition, be zero. It's difficult to imagine 

### 21 or find any data transformation that will make these data nice. 

### 22 That is, give it a more normal distribution and adjust for 

### 23 heterogeneity. It is not clear how the back-up plan, using of a 

### 24 normalized rank order transform produces data that have better 

### 25 properties for analysis. 

### 26 And then he says the same issues shows up in

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1 the analysis of anal fin papillae and the 4tOP subadults. See 

### 2 Appendix H, page 46, Figure 2. Although, to be fair, in this 

### 3 case, the variability is so broad that the dose response is not 

### 4 significant. A couple of other things: 

### 5 Note that the zeros for non-breeding pairs 

### 6 really point to limitations in the study design protocol and not 

### 7 a failure of reproduction between the two males and females. A 

### 8 case can be made for legitimately excluding egg count data from 

### 9 non-breeding pairs and then repeated measures analysis of 

### 10 fecundity. This could be considered a weighted analysis, see 

### 11 page 13, where the weight for the non-breeding pairs is set for 

### 12 zero. 

### 13 At a minimum, the data analysis protocol needs 

### 14 to alert researchers to the implications of non-breeding pairs 

### 15 on fecundity data. The StatCHARRMS tool may need to be modified 

### 16 to implement more appropriate analysis for these data. 

### 17 And then he has a note to the other 

### 18 statisticians on the panel. In your opinion, should we mention 

### 19 here more complex models such as a Bernoulli-Poisson generalized 

### 20 linear model or should we push to exclude zeros from the 

### 21 analysis under the assumption that these zeros come from 

### 22 non-breeding pairs? 

### 23 If the zeros are excluded, the StatCHARRMS 

### 24 model approach is likely not that bad. I assume that we 

### 25 statisticians will have to discuss that a little bit in the 

### 26 final report.

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1 He talks more about analysis of fertility. 

### 2 There is very little discussion about how fertility is analyzed. 

### 3 Fecundity and fertility are shown on the same graphs as counts. 

### 4 In this case, fertility would be analyzed in the same way as 

### 5 fecundity, but now zero fertility in the presence of non-zero 

### 6 fecundity is a true treatment effect on reproduction. 

### 7 Fertility could also be analyzed as the 

### 8 proportion of eggs that are viable. In this case, the arc-sin 

### 9 square root transformation could be used as the response 

### 10 variable in the StatCHARRMS tool. Note that for non-breeding 

### 11 pairs, fertility is zero divided by zero and, hence, is 

### 12 undefined. 

### 13 Fertility as a proportion could become a 

### 14 problem as the average fertility percent approaches zero or one. 

### 15 There is a similar issue with analysis of hatching rate and 

### 16 hatching survival. 

### 17 The assumption of monotonic treatments 

### 18 effects, he has a comment or two. StatCHARRMS uses a novel 

### 19 approach termed "RSCABS" (Rao-Scott Cochran-Armitage by Slices) 

### 20 developed by John Green and DuPont; Timothy Springer, Wildlife 

### 21 International, Easton, MD), and Amy Saulnier, the Franklin 

### 22 Company. 

### 23 RSCABS incorporates the replicate vessel 

### 24 aquarium experimental design and the biological expectation that 

### 25 severity of effect will tend to increase with increasing 

### 26 concentrations or reduced. Clearly, making this assumption

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1 facilitates identifying an LOEC or NOEC. 

### 2 There does not seem to be general agreement 

### 3 that severity of effect will always tend to increase with 

### 4 increasing concentrations for EDCs. Appendix G discusses this 

### 5 on page 8, deciding between pair-wise and trend-based 

### 6 comparisons, but all of the citations for expecting and using a 

### 7 test based on monotone dose response seems to precede those 

### 8 arguing against such tests when there is evidence of a 

### 9 non-monotonic response. It looks likes it can, though, in this 

### 10 verbose here. 

### 11 The monotonic response pattern assumption is 

### 12 appropriate for traditional toxins, but there has been much 

### 13 discussion before the SAP and other toxicology panels over the 

### 14 years as to whether EDC can produce U-shaped dose responses that 

### 15 are not strictly monotonic. There are hints of non-monotonic 

### 16 dose response patterns in the data reported in Appendix H. 

### 17 Although the data are definitive and the case 

### 18 could be made that the response was flat for the first couple of 

### 19 doses before the effect increase is observed. Examples include 

### 20 page 6, F1 and F2 fecundity for E2; page 10, F1 subadult 

### 21 vitellogenin -- well, you know what I mean. Page 24, F1 

### 22 fecundity for 4tOP; and page 26, F1 adult SSC, anal fin papillae 

### 23 for 4tOP 

### 24 The points made on page 9 of Appendix G in 

### 25 particular that Both formal tests and visual inspection are 

### 26 recommended to determine whether there is significant departure

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1 from monotonicity needs greater visibility in the protocol 

### 2 document. 

### 3 The discussion in Appendix G suggests that the 

### 4 EPA rule is to assume monotonicity, with strong evidence needed 

### 5 before one can depart from this rule. The StatCHARRMS tool does 

### 6 provide safeguards, in that the program will not perform the 

### 7 Jonckheere test, a test for monotonic trend, in situations where 

### 8 a linear contrast of treatment trend is not significant but the 

### 9 quadratic contrast is significant. 

### 10 He has some remarks on power, the 

### 11 sensitivity of the power analysis. The power analysis assumes 

### 12 that the rate of creating non-mating pairs is the same across 

### 13 all three generations. There does not seem to be any data for 

### 14 or against this assumption. 

### 15 The power analysis assumed that average 

### 16 fecundity is normally distributed with a mean of 21 and a 

### 17 standard deviation of 7. While the likelihood of simulating a 

### 18 negative mean is small, what does the program do if a simulated 

### 19 mean is less than zero or equal to zero? 

### 20 In the power analysis for SCC, are zeros, 

### 21 actual measurements or simply coded to zero when the assessment 

### 22 could not be scored. In the latter case, the zero implies 

### 23 censored data suggesting a different estimation approach may be 

### 24 needed. The sensitivity of the power calculation to the percent 

### 25 censored should be examined. 

### 26 The power analysis for VTG examines the raw

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1 data responses for each treatment group for normality. Since 

### 2 the analysis found a significant study effect, the two-way ANOVA 

### 3 model, two-factor GLM, should be fit and then the residuals 

### 4 examined for normality. And that concludes his comments. 

### 5 **DR. MARTHA SANDY:**Thank you very much. So 

### 6 I'm going to ask the statisticians here, Mary and Dallas, if you 

### 7 have any thoughts now. We need some discussion in the meeting 

### 8 if you're going to comment on the proposal that Dr. Portier has 

### 9 put forward for alternative analysis when you have non-breeding 

### 10 pairs. So either now or later. Would you like to discuss it? 

### 11 **DR. DALLAS JOHNSON:**Yeah. We probably better 

### 12 discuss it and talk about it later. 

### 13 **DR. MARTHA SANDY:**Okay. So we'll come back 

### 14 to that later. I'll ask all the Panel members, do you have 

### 15 anything else to comment on for the statistical issues? 

### 16 (No response.) 

### 17 No. Okay. That was a lot of information. 

### 18 Maybe we'll go EPA staff and ask, regarding the statistical 

### 19 issues that were just discussed, do you have any clarifying 

### 20 questions? 

### 21 **DR. RODNEY JOHNSON:**I have no clarifying -- I 

### 22 do not need any answers or comments clarified. Thank you very 

### 23 much. Why should that be so hard to say? 

### 24 **DR. MARTHA SANDY:**So now, shall we take the 

### 25 rest of this question all together, the test substances? The 

### 26 selection of test substance and methods, analytical methods?

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1 **DR. REYNALDO PATINO:**I think so. 

### 2 **DR. MARTHA SANDY:**That's fine. So I'll let 

### 3 you, Dr. Patino, start off. 

### 4 **DR. REYNALDO PATINO:**What we'll do is we'll 

### 5 address the part of the question that has to do with the test 

### 6 chemicals. The methods are probably the way we did it, they are 

### 7 addressed partly in Question 5 and then probably in a little 

### 8 more detail in Question 6, during the discussion on 

### 9 transferability. So that's the way we organized this answer and 

### 10 hopefully that will be okay with you. 

### 11 So it would be primarily test chemicals and 

### 12 it's going to be a very short presentation. For this assay 

### 13 there were a number of compounds that were used that were tested 

### 14 in the assay. It included four ER agonists, and that included a 

### 15 strong compound. It's the same thing beta-estradiol, a medium 

### 16 potency compound, DDT, and too weak -- well, maybe 

### 17 differentially weak compounds of octylphenol and 

### 18 4-Chloro-3-methlyphenol. 

### 19 It also included in the assays, Tamoxifen, 

### 20 which is an ER antagonist with some agonist activity. 

### 21 Trenbolone, which is a strong AR agonist; Prochloraz, which was 

### 22 in the report that listed an aromatase inhibitor with AR 

### 23 antagonist activity, but we will discuss more later when we deal 

### 24 with the LAGDA assay, but there is evidence that it is also a 

### 25 thyroid active compound. But we'll deal with that in the next 

### 26 assay.

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1 And then the last one that was used here was 

### 2 Vinclozolin, which is listed in the report as an AR antagonist, 

### 3 but there's also evidence for this compound that it may be a 

### 4 thyroid active compound with possible thyroid -- potentially 

### 5 with activity to inhibit thyroid peroxidase. So I'll deal with 

### 6 that a little bit more here and that will probably be the end of 

### 7 my comments. 

### 8 But the Vinclozolin was used in the MMT assay 

### 9 on the basis of the presumptive mode of action that is initiated 

### 10 by binding to the AR without activating anergy-dependent genes. 

### 11 But there is a recent study with rats, where evidence was 

### 12 presented, suggesting that Vinclozolin is also a thyroid active 

### 13 compound that affects TH production and/or metabolism. And I'll 

### 14 provide that reference in the written report. 

### 15 And there is also another study using human 

### 16 recombinant TPO that show that or suggested that Vinclozolin is 

### 17 able to suppress thyroid peroxidase activity. 

### 18 Although there is no information concerning 

### 19 the ability of Vinclozolin to act as a thyroid endocrine 

### 20 disruption in fishes, the Agency or the Panel would encourage 

### 21 the Agency to review the data for Vinclozolin that was presented 

### 22 in the ISR, in the context of potential effects of both the 

### 23 androgen and thyroid endocrine systems. 

### 24 Also, given the report of association between 

### 25 -- and there is evidence for fish, as well as amphibians, but no 

### 26 direct evidence for Medaka, but there is evidence for fish and

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1 amphibians that disruption of the thyroid endocrine system can 

### 2 also skew sex ratios in populations. 

### 3 Again, I'll provide some references for that. 

### 4 So given this, even though there is no direct evidence for 

### 5 Medaka, it is notable that the Agency study with Vinclozolin 

### 6 reported mismatches between the genotypic sex or the genetic sex 

### 7 and the gonadal sex in some of their fish treated with 

### 8 Vinclozolin, apparently not in their control fish. 

### 9 Again, for two of the compounds that were 

### 10 listed, using assay, Prochloraz and Vinclozolin, there may be 

### 11 additional modes of action, if considered, may help explain, at 

### 12 least conceptually, some of the results, the unexpected results 

### 13 that were reported by the Agency. 

### 14 We'll go a little bit more into this when we 

### 15 are addressing Question 5 as well, but for now, those are my 

### 16 comments on the test chemicals that were chosen. I should say 

### 17 that probably the rationale, to add, the rationale for using 

### 18 Vinclozolin was not very clear in the report. We had some 

### 19 discussion about this when the Agency presented the report. 

### 20 Other than that, those are my comments. If anybody has any 

### 21 input or change, please do. Thank you. 

### 22 **DR. MARTHA SANDY:**So I will ask each of you 

### 23 individually. Dr. Perkins. 

### 24 **DR. EDWARD PERKINS:**I would just like to 

### 25 reiterate about the choice of Vinclozolin, similar, I think we 

### 26 talked about that before. It would be better for validation

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1 studies to have something that has a stronger effect for 

### 2 interlaboratory effects. So you can look at assay performance 

### 3 and endpoint perform and reproducibility in a more predicable 

### 4 manner. 

### 5 **DR. MARTHA SANDY:**Thank you. We have some 

### 6 noise outside. It would be nice if people directly into the 

### 7 microphone. I'll remind everyone again. Dr. Schlenk. 

### 8 **DR. DANIEL SCHLENK:**Yeah, just a couple of 

### 9 comments. I think as we met the other night, I think what came 

### 10 across was that I think you've got the estrogen and androgen 

### 11 side covered pretty well. Those seem to be fairly potent 

### 12 compounds for those particular AOPs. 

### 13 The rationale for the other AOPs was fairly 

### 14 weak. I don't think the documentation really supported why 

### 15 those particular compounds were chosen. One would think you'd 

### 16 want something a little stronger. I know MEDs don't work with 

### 17 flutamide, so I was actually kind of shocked that that wasn't 

### 18 used as the anti-androgen for the Medaka, since you have such 

### 19 pretty good data and Fatheads on that. 

### 20 The thyroid, the quail studies use 

### 21 perchlorate. That would be sort of an obvious sort of thyroid 

### 22 or PBDE. Something like that that would sort of target thyroid 

### 23 endocrine function. Something like would be a little stronger, 

### 24 I guess I would recommend in that particular case. 

### 25 The steroidogenesis inhibitor was also 

### 26 somewhat unclear why Prochloraz was selected. If you want to

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1 look at that, why not pick a stronger Ketoconazole, for example, 

### 2 or some stronger azoles, aromatase inhibitor, I guess would be 

### 3 another recommendation for that. 

### 4 I guess this is my own personal thing here, 

### 5 but I think a non-endocrine hepatoxicant would be a really good 

### 6 sort of negative control in a way, just to rule out the 

### 7 non-endocrine pathway. So something like ANIT or something like 

### 8 that that actually targets the liver that doesn't necessarily 

### 9 have a endocrine mode of action that you could base the assay 

### 10 against, would be strong. 

### 11 Overall, I think we're consistent with what 

### 12 the Japanese quail folks were saying too. I think you really 

### 13 think you need a reference toxicant. Any other assay that was 

### 14 ever run, at least that I've ever ran, usually have a reference 

### 15 toxicant that you run alongside it, periodically or every once 

### 16 in awhile just to make sure that you're producing the results 

### 17 you need to produce. 

### 18 So again, depending on what you AOP is, I 

### 19 would say that probably one of these compounds would be a nice 

### 20 reference toxicant that you would want to run alongside that; 

### 21 maybe one or two doses, something like that. But at least you 

### 22 know what you're getting. 

### 23 As we all know, it's very difficult to publish 

### 24 negative results and it's also very hard to prove a negative 

### 25 result. It's harder to prove a negative result than is a 

### 26 positive result. So again, the Prochloraz results, I think,

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1 were somewhat felt not be very strong. Even though there wasn't 

### 2 any effect, it nice to actually have a compound that you do see 

### 3 in effect, to make sure the assay is valid. So those are my 

### 4 comments related to the compounds. 

### 5 **DR. MARTHA SANDY:**Dr. Freeman. 

### 6 **DR. JENNIFER FREEMAN:**Most of my comments 

### 7 have already been covered, but I just wanted to add one other 

### 8 thing that we also talked about was that if the MRT assay is 

### 9 going to be implemented, we felt that needed to be assayed with 

### 10 the fuller panel of chemicals to get the effects and more to be 

### 11 understood from that assay. 

### 12 **DR. MARTHA SANDY:**Dr. Kullman. 

### 13 **DR. SETH KULLMAN:**The same. Most of the 

### 14 comments have been covered. One thing that basically has not 

### 15 been put out to the Panel or to the EPA then is, however. How 

### 16 many compounds -- well, I'm put in a form of a question and in 

### 17 essence. How many compounds do we need to test to unequivocally 

### 18 determine expected versus observed results in any of the AOPs 

### 19 tested? 

### 20 So at this point, you've got one -- 

### 21 **DR. MARTHA SANDY:**Can you speak into the 

### 22 microphone? 

### 23 **DR. SETH KULLMAN:**I'm sorry. You've got one 

### 24 strong estrogen and a couple of weak estrogens or moderate, one 

### 25 AR and anti-AR. So the question, essentially, is how many 

### 26 compounds do we need to use in order to assess the

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1 reproducibility, liability validation of the assay? 

### 2 **DR. MARTHA SANDY:**Dr. Blazer. 

### 3 **DR. VICKI BLAZER:**All my comments have been 

### 4 covered. 

### 5 **DR. MARTHA SANDY:**And Dr. Johnson. 

### 6 **DR. DALLAS JOHNSON:**I have nothing to add. 

### 7 **DR. MARTHA SANDY:**And nothing from Ken on 

### 8 these? 

### 9 DR. DALLAS JOHNSON: No. 

### 10 **DR. MARTHA SANDY:**Thank you. Any other 

### 11 comments from the Panel on any other issue related to Question 

### 12 4, selection of test substances and analytic methods? 

### 13 (No response.) 

### 14 So seeing none from the Panel, I see Dr. 

### 15 Bradbury has something to say. 

### 16 **DR. STEVEN BRADBURY:**More of a clarification 

### 17 as you put together the report. I'm trying to figure out how to 

### 18 say this carefully because you can go lots of different 

### 19 directions, in terms of how much is enough to know to go 

### 20 forward. 

### 21 So I'm not asking to get into policy, but I am 

### 22 asking if you can help clarify some of the degrees of 

### 23 uncertainty and what kinds of uncertainty, in terms of 

### 24 understanding how well a protocol could be used in 

### 25 cross-laboratories. How much of that uncertainty could be 

### 26 reduced by doing some of the things that you're all describing?

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1 2 So it's not to push back on what you're 

### 3 suggesting, but it's understanding, if you will, what will be 

### 4 the return on investment, in terms of reduced uncertainty, as we 

### 5 think about the cost and time, in terms of gathering information 

### 6 to make informed risk assessments, and if needed, regulatory 

### 7 decisions. 

### 8 So again, I'm not asking you to go into that, 

### 9 but knowing what the uncertainties are, degrees to which the 

### 10 uncertainty could be reduced, for example, by doing one chemical 

### 11 more, two chemicals more, three chemicals. Each chemical may 

### 12 reduce uncertainty. It may create more questions too, but it is 

### 13 understanding where you see the biggest sources of uncertainty 

### 14 that additional endpoints or whatever it may be, what's it 

### 15 tackling, by how much so that we could sort of understand the 

### 16 policy choices we have to make, which would be informed by the 

### 17 science peer review you're providing us. 

### 18 **DR. MARTHA SANDY:**Dr. Rodney Johnson, do you 

### 19 have any areas where clarification is needed? 

### 20 **DR. RODNEY JOHNSON:**Just one small question 

### 21 with respect to the comments that Dr. Schlenk made. Towards the 

### 22 end of your comments you mentioned the lack of response with 

### 23 Prochloraz. I'm thinking that perhaps you meant Vinclozolin or 

### 24 did you mean Prochloraz? 

### 25 **DR. DANIEL SCHLENK:**I think it's Vinclozolin. 

### 26 I think you're right. Yes. Sorry.

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1 **DR. MARTHA SANDY:**Okay. I believe we're 

### 2 ready for Question 5 to be read into the record. 

### 3 **DR. LESLIE TOUART:**Yes. I can read the 

### 4 question as soon as it's advanced. This is Les Touart to read 

### 5 Charge Question Number 5. Considering the variability inherent 

### 6 in biological and chemical test methods, a test method needs to 

### 7 be repeatable and reproducible. 

### 8 A test is robust and reliable if the results 

### 9 are repeatable and reproducible within a laboratory and between 

### 10 different laboratories, respectively. At-test protocol should 

### 11 provide sufficient guidance to ensure proper and consistent 

### 12 performance across labs and chemicals. 

### 13 Please comment on the test method robustness 

### 14 and reliability and the repeatability and reproducibility of the 

### 15 results obtained with the MMT and MRT assays. 

### 16 **DR. MARTHA SANDY:**Dr. Patino, you're the lead 

### 17 discussant on this. 

### 18 **DR. REYNALDO PATINO:**What I would like to do 

### 19 first is define how we defined the terms on the questions that 

### 20 you posed to the Panel so that you understand when we use the 

### 21 terms here, what exactly do we mean. 

### 22 Reliability, we define as, as was defined in 

### 23 the ISR as the reproducibility of results from an assay within 

### 24 and between laboratories. For robustness, there was no specific 

### 25 definition that was provided in the ISR, so we looked on the 

### 26 internet and found one that we liked.

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1 We put the full citation here, but in general, 

### 2 being robust, according to Investopedia, means that a system can 

### 3 handle variability and remain effective under a variety of 

### 4 different conditions. So you can just take it; took it this 

### 5 way, took it the other way and if you get results that make 

### 6 sense, then that's a robust system. So that's the context that 

### 7 we'll be using in this answer. 

### 8 For each of the reference chemicals used for 

### 9 the validation, the assay validation was associated with one or 

### 10 more endocrine-dependent modes of action that were summarized in 

### 11 the integrated -- in the ISR. 

### 12 At one level, at a basic level, the general 

### 13 reliability of the ISR can at least be initially evaluated by 

### 14 comparing the effects of each test chemical with what's known in 

### 15 the literature. What do all the people have found, 

### 16 historically. 

### 17 With the exception of Vinclozolin, the MMP 

### 18 assay, generally performed as expected for the reference 

### 19 materials. To provide a little more specific information, all 

### 20 the ER agonists typically resulted in reduced fecundity. And in 

### 21 males, also, decreased secondary sex characteristics as CC, OP 

### 22 regulation of hepatic vitellogenin, messenger RNA (mRNA). So 

### 23 that was a typical result when you use the ER agonist. 

### 24 Tamoxifen, which has both ER antagonist and 

### 25 some agonist activity, also reduced fecundity. In females, 

### 26 Tamoxifen reduced vitellogenin production or mRNA same as above.

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1 This result is expected in view of this ER antagonist activity. 

### 2 In males, Tamoxifen reduced SSC and increased vitellogenin to 

### 3 some degree, and that is expected in view of the compounds ER 

### 4 agonist activity. So in this regard, the assay performed as 

### 5 expected. 

### 6 The androgen agonist resulted in decreased 

### 7 fecundity, and in females, also caused development of 

### 8 male-specific secondary sex strains and also reduced hepatic 

### 9 mRNA. It probably would be a disruption of the HPG axis and 

### 10 also cause sex reversal and masculinization of XY fish. So 

### 11 that's expected. 

### 12 Concerning Prochloraz, in fish, unlike the 

### 13 situation in many others, aromatase inhibitors in Medaka did not 

### 14 result in sex reversal of genetic males. I'm sorry, of genetic 

### 15 females. But they did disrupt -- I'm sorry. 

### 16 In Medaka, the aromatase inhibitors do not 

### 17 result in sex reversal of genetic females, but they did disrupt 

### 18 their normal environment development, and therefore, it is still 

### 19 expected to lead to exposure to aromatase inhibitor, it's still 

### 20 expected to lead to reproductive impairments. 

### 21 Indeed, in the MMT assay, the Prochloraz, 

### 22 which is an aromatase inhibitor, caused a reduction in fecundity 

### 23 and also inhibition of hepatic vitellogenin, especially in 

### 24 female. And had no clear or consistent effects on SSC in males. 

### 25 Again, these observations are generally as expected for Medaka, 

### 26 given what is known about the effects of aromatase inhibitors.

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1 For androgen antagonist, the ISR noted that 

### 2 the effects of androgen antagonist have been inconsistent or a 

### 3 little ambiguous in the literature, and therefore, no expected 

### 4 result for this compound was clearly defined in the document; 

### 5 therefore, an assessment of this performance cannot be fully 

### 6 made. 

### 7 So again, with the exception of Vinclozolin, 

### 8 everything else kind of performed as expected, and therefore, 

### 9 that's one measure of reproducibility, a general measure of 

### 10 reproducibility. To more specifically address the question of 

### 11 reproducibility in a little more in depth, two sets of 

### 12 interlaboratory assays were discussed in the document in the 

### 13 first set. The Agency, in a Japanese laboratory, independently 

### 14 examined the effects of octylphenol which is a weak ER agonist, 

### 15 using the full MMT, the full format. 

### 16 Although the test format was not identical 

### 17 between the two labs, the Panel feels that the differences are 

### 18 small enough that the results can be compared, validly compared. 

### 19 In general, the results between the two labs 

### 20 with the MMT, using octylphenol were consistent. Both reported 

### 21 impaired reproduction, reduced fecundity, reduced SSC and 

### 22 induction of hepatic vitellogenin in males, and sex, either sex 

### 23 reversal or intersex condition in XY individuals. 

### 24 For fecundity, SSC and hepatic vitellogenin, 

### 25 the LOEC values were also fairly comparable between the two 

### 26 laboratories. They were in good agreement. So overall, these

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1 observations suggest that when using a test chemical with fairly 

### 2 defined, established AOP, in the hands of experienced 

### 3 laboratories, with extensive experience in the test protocols, 

### 4 then you can expect a high level of reproducibility from this 

### 5 assay. 

### 6 Such was not the case for the second round of 

### 7 ring test. In the second round, there were three contract 

### 8 laboratories involved in that ring test. And this is actually 

### 9 the formal ring test for the assay. This test used Vinclozolin 

### 10 as test chemical and an abbreviated MMT format. It was 24 weeks 

### 11 instead of 29. The results of this ring test were not very 

### 12 encouraging. Several major inconsistencies or technical 

### 13 difficulties were noted. I have a few here as an example to 

### 14 highlight what the Panel means. 

### 15 On the technical side, the measured 

### 16 concentrations of Vinclozolin were apparently difficult to 

### 17 achieve by some of the labs. There were inconsistencies, either 

### 18 achieving or maintaining the concentrations. 

### 19 Second, another inconsistency was several of 

### 20 reproductive endpoints in the F1, as well as endpoints in the 

### 21 control F1 and F2 were significantly different between these 

### 22 three laboratories. 

### 23 The ISR attributed this variability, or at 

### 24 least part of it, to the unusually small size that was achieved 

### 25 by fish in one of the labs. And in fact, correcting for size 

### 26 generally eliminated were differences. So that may be part of

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1 the reason why there was a lot of variability between the 

### 2 laboratories. But the fact still remains that there were 

### 3 differences in size, major differences in performance of the 

### 4 fish itself. 

### 5 Third, because there were problems with the 

### 6 measurement of SSC in one of the labs -- and this was an 

### 7 important endpoint -- three-way comparisons for the F1 for this 

### 8 particular endpoint were not possible. 

### 9 Although the ISR states that when data were 

### 10 available for this endpoint, they were consistent, we actually 

### 11 looked into the report and it appeared that only one lab showed 

### 12 a change in the SSC in F1 males that the other lab actually did 

### 13 not report any changes. 

### 14 So, you know, for that, even if you exclude 

### 15 the lab where there was a problem measuring that, the results 

### 16 were still inconsistent between laboratories, or at least that's 

### 17 how the data appeared to us. 

### 18 Another point is that the Agency study with 

### 19 Vinclozolin, using the full MMT, as well as those of the 

### 20 contract laboratories, which used the abbreviated MMT, had a 

### 21 problem in that a number of their fish showed genetic sex that 

### 22 did not match their gonadal sex. This problem was much more 

### 23 serious for the contract laboratories, which reported high 

### 24 levels of both types of mismatches in XX males and XY females 

### 25 and also in their controls. 

### 26 The ISR ascribed this problem to potential

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1 artifacts of contamination during sample collection or 

### 2 processing. And this is probably a correct assessment, at least 

### 3 for the case of the contract laboratories, because as I said, 

### 4 they also had high levels in their control animals. 

### 5 I already said this, but I need to point out 

### 6 that in the Agency study, their controls, apparently there was a 

### 7 perfect match there. It was no problems with matching their 

### 8 animals, genotype and phenotype in the controls. 

### 9 As we noted already, because Vinclozolin may 

### 10 have alternative AOPs that were not considered in the IRS that 

### 11 it could potentially cause sex reversal in fish. And by that, I 

### 12 mean the thyroid axis. At least some of the mismatches, both in 

### 13 the Agency study, as well as the contract labs, may potentially 

### 14 could have been true biological responses to Vinclozolin. 

### 15 Overall, the Panel feels that an evaluation of 

### 16 group possibility from the ring test, using Vinclozolin is 

### 17 difficult because, 1) there was relatively undefined AOP for 

### 18 this compound, and therefore, all the expected outcomes; 2) the 

### 19 technical problems that were encountered by the performing 

### 20 laboratories, the contract laboratories, and also because of the 

### 21 largely negative results that this compound had in the assay. 

### 22 So it's kind of difficult to assess reproducibility under these 

### 23 conditions. 

### 24 But to possibly shed some light on the reasons 

### 25 for the relatively poor performance of the ring test, the Panel 

### 26 recommends that the Agency considers, at least conceptually, the

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1 possibility, at least for some of the observations, of 

### 2 alternative AOPs for this test compound. 

### 3 Overall, while the interlaboratory study 

### 4 between the agencies and the Japanese labs with octylphenol show 

### 5 similar data responses between labs, and that indicated, at 

### 6 least the assay itself, is reproducible when you competent or 

### 7 experienced laboratories with a well-defined chemical. The 

### 8 Vinclozolin results were very inconsistent or not very clear at 

### 9 all. 

### 10 The ring test with Vinclozolin that was 

### 11 conducted by the contract laboratories is probably more relevant 

### 12 to assess the reproducibility of this assay because it is 

### 13 laboratories like those that will be doing the testing in the 

### 14 future. 

### 15 For practical purposes, the Panel feels that 

### 16 the reliability and the robustness of the assay have not been 

### 17 fully demonstrated for this assay. And again, I'm defining 

### 18 robustness as the performance of the assay on the conditions of 

### 19 variable levels of experience among the laboratories conducting 

### 20 the assay. In that regard, robustness was poor because you 

### 21 tweak it here, tweak it there, in terms of technical expertise, 

### 22 and the assay kind of fell apart. 

### 23 So to improve both the reliability and 

### 24 robustness of the assay, the Panel recommends that the Agency 

### 25 develop more specific guidelines on how to conduct the assay and 

### 26 in greater detail. More of this will come up in more detail

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1 under Question 6. 

### 2 For both the MMT and the MRT assays, greater 

### 3 emphasis needs to be placed on the overall health of fish 

### 4 populations being used for these studies, specifically the 

### 5 problem with growth that was noted among the labs. 

### 6 In particular, procedures and conditions for 

### 7 the adequate care in husbandry of the animals, and this includes 

### 8 the specific ranges for pollution water quality variables such 

### 9 as hardness and micronutrients also need to be better 

### 10 highlighted in the guidance. So those are my comments. I'm 

### 11 sure all those would have more things to add. Thank you. 

### 12 **DR. MARTHA SANDY:**We'll turn now to Dr. 

### 13 Kullman. 

### 14 **DR. SETH KULLMAN:**I think everything has been 

### 15 covered. Thank you. 

### 16 **DR. MARTHA SANDY:**Dr. Perkins. 

### 17 **DR. EDWARD PERKINS:**I think everything has 

### 18 been covered for me. 

### 19 **DR. MARTHA SANDY:**Dr. Schlenk. 

### 20 **DR. DANIEL SCHLENK:**Just one thing to add, 

### 21 with Dr. Patino's comments about Vinclozolin. This probably 

### 22 should've actually probably gone in Question 4, but in terms of 

### 23 choice of compounds, again, I wouldn't select a compound that 

### 24 degrades as readily as Vinclozolin. If you're going to pick a 

### 25 compound to run through a ring test, it probably would be 

### 26 something a little easier, analytically, to evaluate.

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1 So I just wanted to add that for Question 4, 

### 2 as well, but nothing further to add for Question 5. 

### 3 **DR. MARTHA SANDY:**Dr. Blazer? 

### 4 **DR. VICKI BLAZER:**Nothing further to add. 

### 5 **DR. MARTHA SANDY:**Dr. Freeman. 

### 6 **DR. JENNIFER FREEMAN:**I don't have anything 

### 7 additional to add. 

### 8 **DR. MARTHA SANDY:**And Dr. Dallas Johnson. 

### 9 And he can comment for Dr. Portier. 

### 10 **DR. DALLAS JOHNSON:**Right. I don't have much 

### 11 to say for myself, except some of the comments that I made in 

### 12 Question 4 could also probably be put into here. Indicated, it 

### 13 seems like some of the things experimenters have control over is 

### 14 pretty well laid out in the protocol, but things that they may 

### 15 not have control over, the randomization process, there is 

### 16 nothing really about it in the protocol. 

### 17 So you're trying to develop a protocol that 

### 18 might be reproducible and robust. There needs to be a lot more 

### 19 said about how tanks are randomized, how fish are randomized to 

### 20 tanks and so on and so on. 

### 21 Dr. Portier also defined robustness. I don't 

### 22 think it's contradictory to the way it's been defined here, but 

### 23 the ability -- he defined it as the ability to produce results 

### 24 with deviations from protocol. And reliability, he defined as 

### 25 the ability of a procedure. I guess it's defined in the report, 

### 26 the ability of a procedure to yield the same results on repeated

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1 trials, within and between laboratories. 

### 2 Repeatability: the ability of the protocol to 

### 3 be repeated. And reproducibility: the ability of results from 

### 4 one run of the protocol to be reproduced by others running under 

### 5 the same protocol conditions. And then with respect to the test 

### 6 method, robustness, he says I don't think we have sufficient 

### 7 data to address this characteristic. 

### 8 There are four sources of variation that must 

### 9 be accounted for to address reliability, repeatability and 

### 10 reproductive ability. These are among individuals within 

### 11 replicates, among replicates within studies, among studies 

### 12 within laboratories, and among laboratories. 

### 13 Section 6.1 controlled data correlation 

### 14 endpoint variability of Vinclozolin interlaboratory report is 

### 15 closest to what I was looking for in terms of having data 

### 16 capable of estimating these sources of variation. At least for 

### 17 Vinclozolin, there are sufficient data to estimate all these 

### 18 sources of variation for the zero control animals. 

### 19 Test method reliability, he says an assay will 

### 20 -- well, I think the report says an assay will generally be 

### 21 considered to be reliable by EPA if its overall variability is 

### 22 low enough to give a level of sensitivity or power consistent 

### 23 with the purpose of the assay as intended to serve; page 11, 

### 24 lines 14 to 16. 

### 25 He says here, the variability among 

### 26 individuals within replicates could be pooled across studies

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1 and/or laboratories as used in the power analysis to address 

### 2 whether a reasonable number of replicates can be used to find 

### 3 biologically significant differences in key endpoints. With 

### 4 respect to test method repeatability, he indicates repeatability 

### 5 can be assessed by examining control endpoint measurements from 

### 6 study to study within a lab, both within generation and between 

### 7 generations. 

### 8 For fecundity, the only endpoint I attempted 

### 9 to examine, it looks like at least within one lab the control 

### 10 endpoint measurements are repeatable. From what I conclude, 

### 11 there is some degree of ability to repeat the protocol. 

### 12 With respect to test method reproducibility, 

### 13 it says there are clearly some unresolved issues with test 

### 14 method repeatability that can be identified with various 

### 15 interlaboratory reports, and refers then to the Vinclozolin 

### 16 interlaboratory report, Section 6.1, control data correlation 

### 17 and endpoint variability. 

### 18 I believe the report says comparison in 

### 19 control data from each of the laboratory studies summarized in 

### 20 Table 3, and then he adds, this analysis is indicated that for 

### 21 most endpoints, the control response was not consistent between 

### 22 laboratories and two endpoints that showed greater consistency, 

### 23 which were not statistically different between labs of F1 and 

### 24 F2, anal fin processes and hepatic expression of VTG1; however, 

### 25 it should be noted that successful measurement of anal fin 

### 26 processes only occurred for two labs in both the F1 and F2

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1 generation, limiting interlab comparisons. 

### 2 In Appendix H, MMT Summary Report, on page 2 

### 3 of the report, he says again, there were some unexplained 

### 4 differences between weekly egg outputs within treatments, 

### 5 including the controls. And then Dr. Portier says for the DDT 

### 6 data, the write-up says there are six replicates, but the 

### 7 control for F0 displays 10 dots. 

### 8 For the CMP study, fecundity means were 50 

### 9 percent higher than all other chemical tests. No explanation is 

### 10 given. This produced more variability across all treatments 

### 11 making differences much more difficult to be significant. Did 

### 12 something change in the test protocol? 

### 13 At the same time, it seems that the test fish 

### 14 are about twice as heavy and much longer. 

### 15 For the Prochloraz, several of the treatments 

### 16 were lower than controls in the fifth week; however, controls 

### 17 seemed to be decreasing as well, suggesting a general trend of 

### 18 ever decreasing reproductive output as the reproductive 

### 19 assessment progressed. The mean number of eggs spawned daily in 

### 20 the 41-milligram leader group was 17.3 in the first week, and 

### 21 this declined to just three eggs per day, with several of the 

### 22 pairs not producing any eggs in the fifth week. 

### 23 These data suggest a strong trend of 

### 24 decreasing reproduction in F0, however, because of the generally 

### 25 low statistical power of the MMT, it was not detected by our 

### 26 statistical tests until the last week. Since this MMT was

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1 completed, a power analysis has shown that the power to see a 30 

### 2 percent reduction in fecundity is only about 0.5 and to see a 40 

### 3 percent reduction is still only 0.7; therefore, it is not 

### 4 surprising to see trends in the data that are not statistically 

### 5 significant. 

### 6 Study protocol control/unexplained 

### 7 variability: Looking at F0 fecundity, replicate ranges were 

### 8 generally 18 to 25, but occasionally you would get a 28 to 48 in 

### 9 the CMP study or a 6 to 22, Trenbolone, F1 bodyweight and 

### 10 lengths for control group subadults ranged from an overall 

### 11 average low of 130 milligrams, 23 milliliters (Tamoxifen) to a 

### 12 240 milligrams, 27 milliliter (DDT). These findings suggest 

### 13 that the level of control is less than good. 

### 14 MMT ISR final report, Section 5.1.4. and 

### 15 Figure 5.4., examining the subadult endpoints we see differences 

### 16 in subadult bodyweight/length that could be simply one week 

### 17 difference in when measured. This could indicate a weakness in 

### 18 the protocol. 

### 19 And then note in Appendix H, all of the box 

### 20 plot footnotes indicate that the box represents one standard 

### 21 deviation and the whisker represents one standard error. He 

### 22 says since in all cases, the whisker half-length is greater than 

### 23 the box half-length it is not clear to what these two terms 

### 24 actually refer. If both refer to the estimate of the mean, the 

### 25 footnotes are all in error. 

### 26 That concludes what Dr. Portier has to say.

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1 **DR. MARTHA SANDY:**Thank you very much. Are 

### 2 there other comments from anyone else on the Panel on Question 

### 3 5? 

### 4 (No response.) 

### 5 Seeing none, I'll turn to Dr. Rodney Johnson 

### 6 with the EPA. 

### 7 **DR. RODNEY JOHNSON:**I have no questions for 

### 8 clarification. 

### 9 **DR. MARTHA SANDY:**I believe we're scheduled 

### 10 for a break. Let's take a 10-minute break and be back at what 

### 11 this clock says would be 10:35. Thank you. 

### 12 (Whereupon, a break was taken) 

### 13 **DR. MARTHA SANDY:**Thank you. I believe we 

### 14 are on Question 6. The slides have been advanced, so Dr. 

### 15 Touart. 

### 16 **DR. LESLIE TOUART:**The test protocol should 

### 17 be descriptive enough to be fully transferable to a functional 

### 18 laboratory. The protocol should describe the methodology of the 

### 19 assay in a clear and concise manner so that a laboratory could 

### 20 comprehend the objective, conduct the assay, observe and measure 

### 21 prescribed endpoints, compile and prepare data for statistical 

### 22 analyses and report the results. 

### 23 Section 4 of the MMT ISR outlines the process 

### 24 of and challenges experienced in the interlaboratory studies, 

### 25 and Section 5 presents the optimization and proposed Medaka 

### 26 Reproduction Test protocol.

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1 Please comment on the transferability across 

### 2 labs and provide any suggestions or recommendations for 

### 3 improvement of the MMT and MRT assays. 

### 4 **DR. MARTHA SANDY:**Dr. Kullman, you are the 

### 5 lead discussant. 

### 6 **DR. SETH KULLMAN:**Okay. I'll see if I can 

### 7 talk clearly into this. So I think we formulated our response 

### 8 to this question based on looking at some of the ICCVAM 

### 9 requirements for validation criteria throughout the ISRs. So 

### 10 the first ICCVAM definition was within tests, intralaboratory 

### 11 and interlaboratory variability and how these parameters vary 

### 12 with time should be evaluated. 

### 13 Here, we may substitute the idea of time with 

### 14 individual trials. As Dr. Schlenk alluded to before, there's 

### 15 going to be some redundancy in this answer to some of the other 

### 16 answers because we felt it was appropriate. 

### 17 So with this in mind, to evaluate 

### 18 transferability, we felt it can be evaluated following the 

### 19 criteria of the trials that were conducted either within the 

### 20 lab, within the EPA lab or across the different labs that were 

### 21 working on this. There were sort of three scenarios that we 

### 22 were able to pull from the ISR based on that. 

### 23 The data presented in the MMT ISR suggested 

### 24 that there is some indication of moderate -- and I'm using these 

### 25 as sort of soft terms -- moderate agreement between defined 

### 26 assay parameters between labs. And the first instance of that

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1 was the comparison of the trial for 4-t-octylphenol in the U.S. 

### 2 EPA 2007 trial and NIES Japan, where they reported similar 

### 3 values for fecundity for the LOEC, and the same for other 

### 4 components of the assay for the secondary sex characteristics. 

### 5 And that there was moderate agreement within those two trials 

### 6 between those two labs. 

### 7 The Panel felt, basically that the strongest 

### 8 agreement, however, for transferability would be made by 

### 9 assessing the control parameters across each of the trials. 

### 10 If you look at Section 4.1.4, it illustrates 

### 11 that comparisons between trials 4-t-octylphenol, that between 

### 12 the U.S. EPA lab and the NIES Japan lab, there was this fair 

### 13 amount of consistency and reproducibility for fecundity and SSC 

### 14 in both F1 and F2 populations. And this section also continues 

### 15 to state successful reproducibility by comparison of additional 

### 16 endpoint LOEC data for 4-t-octylphenol. 

### 17 So we felt like that was a very good indicator 

### 18 of agreement between the two different labs; however, let me 

### 19 clarify this a little bit. I think the Panel felt that that was 

### 20 more indicative of reproducibility rather than transferability. 

### 21 And that since the Japanese laboratory was highly trained in 

### 22 medaka techniques and already had existing protocols that only 

### 23 needed slight modifications to comply with the MMT assay, that 

### 24 that might have played into the ability for these two labs to 

### 25 have such close concordance with the ability to run the trial. 

### 26 And that maybe, as what we saw with some of the contract labs

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1 and Vinclozolin, that that may not necessarily be the case. 

### 2 So onto the interlab tests that were conducted 

### 3 to assess basically the AOP of AR antagonism with the 

### 4 Vinclozolin study, I think as addressed in Question 5, the Panel 

### 5 really found that this ring test was highly variable and 

### 6 suffered from a lack of consistency and assay performance. 

### 7 The ring test also suffered from a lack of, 

### 8 essentially, the compound tested; Vinclozolin is a difficult 

### 9 compound. The mechanism of Vinclozolin is somewhat 

### 10 questionable, and certainly the fate the parent compound is 

### 11 questionable as the M1 and M2 metabolites are active. And that 

### 12 just makes comparison of that compound very difficult and I 

### 13 think it was more detailed in Question 5, so I won't belabor 

### 14 that. 

### 15 So I guess in conclusion, the Panel felt that 

### 16 based on the Vinclozolin study that the data at this time 

### 17 suggests that the assay is not 100 percent transferable. 

### 18 Next, let's detail or go over another 

### 19 component of the ICCVAM validation criteria. And that's the 

### 20 necessity for a formal detailed protocol and that protocol must 

### 21 be provided and be available in the public domain. And mostly, 

### 22 the second part is that it should be sufficiently detailed to 

### 23 enable the user to adhere to it and should include data analysis 

### 24 and decision criteria. 

### 25 So for the first part of that is the detailed 

### 26 protocol. I think in total, the Panel felt that the MMT and the

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1 MRT protocol should include refined methods for sections of all 

### 2 analysis conducted. 

### 3 As present, we found that there is an 

### 4 inadequate, essentially, description of the assays and that the 

### 5 defined components of general experimental principles including 

### 6 experimental design, biological treatments, technical 

### 7 replicates, husbandry conditions, assay performance, test agents 

### 8 used and concentration range. And definition of the primary and 

### 9 secondary endpoints of the assay, such as water chemistry and 

### 10 overall timing of multigenerational approach could be improved. 

### 11 Okay. 

### 12 So we did identify noticeable components of 

### 13 the MRT and the MMT assay that were not present in provided 

### 14 documentation and that we felt that it would aid in reducing 

### 15 data variability, the assay results, the overall concordance and 

### 16 transferability of the assays. That's essentially what we're 

### 17 assessing in this particular question, with both the inter and 

### 18 intralaboratory trials. 

### 19 We felt that it would be expected that most 

### 20 laboratories running these trials would have some working 

### 21 knowledge of Medaka biology and/or fish biology, but we felt 

### 22 that further standardization of the protocol would really help 

### 23 in the ideas of transferability. 

### 24 And this is where some redundancy will come in 

### 25 from Question 3 and a little bit from Question 5. I'm going to 

### 26 one of these in great detail and then the rest will just sort of

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1 be inferred for similar detail. 

### 2 For example, vitellogenin: we noticed across 

### 3 the two different labs that one lab was using ELISA and another 

### 4 lab was using sort of an RT-PCR method to do it. So as an 

### 5 example, if we simply chose an RT-PCR method to assess 

### 6 vitellogenin, what are we looking at as far as all the 

### 7 components that are associated simply to run a qPCR analysis and 

### 8 how can we make this qPCR analysis consistent across all labs 

### 9 that will conduct all the analysis? 

### 10 So 1) we have to collect tissue. How is the 

### 11 liver dissected? What washes are used? How is the liver 

### 12 stored? For how long? Under what conditions? Are substances 

### 13 like RNA later used, right, or is this tissue flash frozen. 

### 14 Okay. So these are the levels of detail that 

### 15 I think are necessary to be defined in the protocol to really 

### 16 ensure consistency across all these different laboratories that 

### 17 would be doing it. 

### 18 So let's stay with vitellogenin. Standardize 

### 19 RNA extraction processes. You know, what reagents are used? 

### 20 What methods? What QA/QC is done on RNA extraction? Do you use 

### 21 concentration and quality? Do you use a bioanalyzer to assess 

### 22 that? Do you run a gel, you know, to look at 18S RNA? 

### 23 cDNA synthesis and amplification reagents and 

### 24 methods: what reagents are used for cDNA synthesis? Is it 

### 25 random priming or poly A priming? What concentration of RNA is 

### 26 used in the reaction? What type of RT incubation time? What

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1 type of RT enzyme, et cetera? So all of these components. 

### 2 qPCR primers: I think a standard set of qPCR 

### 3 primers should be used and it should be clearly defined in what 

### 4 those are, what their concentrations are, what their 

### 5 purification is. 

### 6 Also, it's not even noted which VTG is 

### 7 targeted. Is it VTG1 or VTG2; there are two forms in Medaka. 

### 8 What reference genes was evaluated for this? Is it 18S? Is it 

### 9 tubulin acting as something else, as far as comparing? 

### 10 All qPCR conditions need to be determined. 

### 11 What amplification conditions. What is your annealing 

### 12 temperature? What's your association temperature? What's your 

### 13 extension temperature? How many amplification runs are you 

### 14 doing? 

### 15 Concentrations of your buffer; concentration 

### 16 of your dNTPs, technical replicates, so on and so forth. I know 

### 17 I'm belaboring this, but I just want to put this in for the 

### 18 record. 

### 19 Also, qPCR analysis: what is your data 

### 20 analysis? There are a number of different ways to crunch qPCR 

### 21 data. What is your data analysis protocol 

### 22 Reference standards; I think this will be 

### 23 important. We can easily produce a reference standard for VTG, 

### 24 especially when you're measuring copy number. Simply have a 

### 25 vector with a VTG copy in it and have that expressed and you can 

### 26 make VTG and know exactly what your copy number is.

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1 Overall guidelines should make sure that MIQE 

### 2 (Minimum Information for Public Quality of real-time PCR 

### 3 Experiments). There is a reference for this and it's Bustin, et 

### 4 al., 2009. This is similar to what happened with the microarray 

### 5 data and everything should be qualified and the standard should 

### 6 be put into a database where it is publically available. 

### 7 So with this in mind, I'm just going to 

### 8 mention other things that could be added with this degree of 

### 9 detail. I'm certainly not going to go with this degree of 

### 10 detail into each of these things to make sure that we don't stay 

### 11 here all day. 

### 12 As was mentioned before, there are a number of 

### 13 secondary sex characteristics that can be determined, and 

### 14 particularly, the anal fin papillae, which is used now. But in 

### 15 Question 3, we addressed some other issues and some other assays 

### 16 that could be used or measurements, if you will, that could be 

### 17 used. 

### 18 Let's have sufficient detail on how to measure 

### 19 those. I think it was suggested before, having YouTube videos 

### 20 or some sort of web type of thing. It would be the Journal of 

### 21 Observational Visualization (JOV), has detailed protocols where 

### 22 there is sufficient webcasts associated with these that can be 

### 23 done. 

### 24 Embryo collection: there is no mention to how 

### 25 embryo is collected. There are several methods to collect 

### 26 embryos. Some labs use scissors to remove all the threads

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1 between the embryos. Other labs use sandpaper and they roll it. 

### 2 Using that sandpaper method is very effective, but it also has 

### 3 the disadvantage of possibly crushing embryos, since you could 

### 4 lose embryo viability simply by doing that. 

### 5 With embryo viability, how is it determined? 

### 6 Are you looking at opaqueness of the embryos? Are you using 

### 7 methylene blue? So if you're going to be counting survival of 

### 8 embryos, we should have a standardized method in how we're 

### 9 actually determining viability. 

### 10 It was suggested before that a detailed 

### 11 protocol be presented for genetic sex. Rodney, I'll say that 

### 12 I'm very familiar with your publication from Padilla, et al., 

### 13 where you were a co-author in 2009, where you have a detailed 

### 14 description on how to determine genetic sex. I'd imagine there 

### 15 is some modification since then, but I think that's already 

### 16 available. 

### 17 Water quality parameters were presented 

### 18 earlier, so I won't reiterate those, but we definitely, as a 

### 19 Panel, agree that strict adherence to defined water quality 

### 20 parameters hardness solemnity, conductivity, pH, temperature, is 

### 21 necessary to ensure generalized success of breeding and 

### 22 reliability of results that will definitely translate towards 

### 23 transferability. 

### 24 Reference materials for the DMY, again, and 

### 25 the VTG, I suggested this. We thought that maybe they should be 

### 26 deposited with NIST. So again, readily available. And then

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1 certainly fish husbandry characteristics, not only for the 

### 2 animals that are in the assay, but certainly fish husbandry 

### 3 conditions for animals that are used as brood stock for the 

### 4 assay. 

### 5 And within that, we noticed that there is no 

### 6 assessment of fish health and quality within. Some general 

### 7 histopathology, some other types of general assessment of fish 

### 8 health that could be made prior to initiating each of the 

### 9 experiments may provide really beneficial to ensure that there 

### 10 is consistent quality of animals across all trials of these 

### 11 assays. We do it regularly in our lab. 

### 12 So it was generally decided that all these 

### 13 guidelines should be as detailed as possible to communicate the 

### 14 importance of proper QA/QC parameters, as needed for the assay. 

### 15 And that QA/QC parameters be incorporated into the assay and 

### 16 that the labs that are going to be running these assays have a 

### 17 mechanism to assess quality and assurance for running there. 

### 18 Consequently, the Panel found that the 

### 19 protocol should be reevaluated to determine if clarity could be 

### 20 improved. And it is recommended that both the MMT and MRT 

### 21 should incorporate reference toxicants and materials that can be 

### 22 used for internal assessment of responses. Reference toxicants 

### 23 include EAT, anti-E, anti-A, anti-T should be used if disruption 

### 24 of these pathways are identified in Tier 1. 

### 25 It was also thought that a positive control be 

### 26 run in each of these trials, or maybe part of the QA/QC, where

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1 every third trial, or something like that, a contract lab runs a 

### 2 positive control to ensure that all the parameters of the assay 

### 3 are performing correctly. 

### 4 There was also some comment on whether 

### 5 unresolved assays should really undergo any type of data 

### 6 manipulation. I think that came about in the Vinclozolin ring 

### 7 test. I think it was Lab 2, where the data was manipulated to 

### 8 normalize for the lack of size. We really wondered if that's an 

### 9 appropriate method to sort of qualify whether a trial has been 

### 10 run appropriately or not. And it may be that, simply, if the 

### 11 data does not look appropriate, that that trial should be 

### 12 nullified and that no manipulation should occur to try and get 

### 13 that within a reasonable stretch to sort of justify the data. 

### 14 And then lastly, there was some mention that 

### 15 there was no justification for a non-solvent control, and it 

### 16 doesn't seem to be clear in the documentation. So I think in 

### 17 conclusion, the Panel believes that the MMT and the MRT assay is 

### 18 sound, but it has the potential to detect EDC toxicity and 

### 19 inform MOA. 

### 20 But the incorporation of recommended 

### 21 refinements and strict adherence to designated, MMT and MRT 

### 22 assays protocols are likely to demonstrate a stronger 

### 23 correlation between labs and demonstrate that at least under 

### 24 most defined conditions to perform with a degree of 

### 25 repeatability and reliability, which would relate to 

### 26 transferability of the assay.

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1 It was suggested that subsequent assessments 

### 2 should then be made with test agents that are less definitive 

### 3 AOPs and assess apparent sensitivity of assay conditions across 

### 4 labs. So certainly, start this and I think this will be, in 

### 5 summary, for Question 9, start the assay ring test with some 

### 6 strong agonists, antagonists, for each of the AOPs and then 

### 7 maybe once that's been validated, move to some compounds that 

### 8 may test the sensitivity of the assays and give you, in essence, 

### 9 of transferability across a range of sensitivities. 

### 10 I'll stop there. 

### 11 **DR. MARTHA SANDY:**Thank you. I'll turn to 

### 12 the associates, Dr. Perkins. 

### 13 **DR. EDWARD PERKINS:**I guess it's similar to 

### 14 the quail. I would also recommend consideration of some 

### 15 alternative methods for providing clear guidance on methods and 

### 16 maintenance and other issues like that, such as alternative 

### 17 media, YouTube videos, things like that, that might be able to 

### 18 give a little clearer guidance for people on exactly you want 

### 19 things performed. 

### 20 **DR. MARTHA SANDY:**Dr. Freeman. 

### 21 **DR. JENNIFER FREEMAN:**I don't have anything 

### 22 additional to add. 

### 23 **DR. MARTHA SANDY:**Dr. Patino. 

### 24 **DR. REYNALDO PATINO:**Nothing new to add. 

### 25 **DR. MARTHA SANDY:**Dr. Blazer. 

### 26 **DR. VICKI BLAZER:**Nothing additional to add.

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1 **DR. MARTHA SANDY:**Dr. Schlenk. 

### 2 **DR. DANIEL SCHLENK:**My comments are 

### 3 incorporated in Dr. Kullman's. 

### 4 **DR. MARTHA SANDY:**And Dr. Dallas Johnson and 

### 5 Ken Portier. 

### 6 **DR. DALLAS JOHNSON:**Okay. During the break, 

### 7 I mentioned something to one of the panelists, and they didn't 

### 8 laugh me out of the room, so I'll bring it up here. I assume 

### 9 that a small part of the total cost for these studies involving 

### 10 Medaka is the cost of acquiring the Medaka and feeding the 

### 11 Medaka. 

### 12 So given that this is true, would it be 

### 13 possible and/or make sense to include several pairs of adult 

### 14 Medaka within each tank? 

### 15 That is, currently, each tank at the beginning 

### 16 contains one male and one female Medaka, and then at each stage, 

### 17 there is again, only one male and one female Medaka that goes 

### 18 into the tank. It seems to give a fair chance, then, of having 

### 19 pairs that are not able to breed and not able to produce any 

### 20 results. 

### 21 So I was just wondering, as a dumb 

### 22 statistician, whether or not it would be possible to say, 

### 23 include five pairs, five female and five male Medaka within each 

### 24 tank at each of those stages. You wouldn't need to analyze all 

### 25 of them or select all of them for analysis, but it might, at 

### 26 least in terms of egg production and so on, it might make sure

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1 that you at least have a chance of getting some eggs that might 

### 2 be analyzed. So I just throw that out there as something that 

### 3 could be considered. 

### 4 Ken -- Dr. Portier responded to this charge 

### 5 and says I keep coming back to the quote in the summary report 

### 6 of Task Order Number 32. Comparison of control data from each 

### 7 of the laboratory studies provides a measure of the natural 

### 8 variation for each of the variables and is summarized in Table 

### 9 3. 

### 10 This analysis indicated that for most 

### 11 endpoints, the control response was not consistent between 

### 12 laboratories. I worry that inconsistency in control endpoint 

### 13 responses are indicative of the protocol being not fully 

### 14 transferable. Inconsistencies are different from variability. 

### 15 I recognize that some lab-to-lab variability 

### 16 will occur in some parameters. For a fully transferable 

### 17 protocol, I would expect lab-to-lab control animal variability 

### 18 to be of a similar order of magnitude as study-to-study control 

### 19 animal variability within one lab. 

### 20 **DR. MARTHA SANDY:**Thank you. Did any of the 

### 21 discussants want to respond to Dr. Johnson's suggestion of 

### 22 adding more breeding pairs? 

### 23 Yes. Dr. Kullman. 

### 24 **DR. SETH KULLMAN:**Yes, Rodney. I think there 

### 25 is a number of labs using Medaka when they do reproductive 

### 26 studies. There is sort of a pretest period, where you take

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1 breeding pairs and you put them together and you assess 

### 2 viability or reproductive capability for, say, a week and you 

### 3 assess the number of embryos produced prior to treatment. That 

### 4 might be a suggestion to make, along Dr. Johnson's 

### 5 recommendations so you do identify breeding pairs that are 

### 6 performing well that feed into the study. 

### 7 **DR. MARTHA SANDY:**I'll open it up to other 

### 8 members of the panel for any comments on this question. I'll 

### 9 turn to the EPA, Dr. Rodney Johnson. Do you have questions of 

### 10 clarification? 

### 11 **DR. RODNEY JOHNSON:**I have no clarification 

### 12 questions. 

### 13 **DR. MARTHA SANDY:**All right. Thank you. Dr. 

### 14 Touart. 

### 15 **DR. LESLIE TOUART:**Just some clarification. 

### 16 I mean, clearly, here we need to provide more detail that would 

### 17 go into a test guideline, but also recognizing that an SOP is a 

### 18 lot more specific than a test guideline where they might be some 

### 19 flexibility. 

### 20 This is more of, I guess a plea to the Panel, 

### 21 since I'd likely be one of those involved in revising a test 

### 22 guideline, is the more you can identify of those elements, you 

### 23 know, procedures or methodologies that need to be more 

### 24 prescriptive than flexible, would be of help because with 

### 25 guidelines, you know, we don't necessarily have to detail every 

### 26 specific thing.

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1 I also recognize and I think it's a good 

### 2 suggestion that we have some auxiliary materials for guidance. 

### 3 You know, things like videos or other information, but I think 

### 4 that when you identify some of the elements that need more 

### 5 detail, how much of that needs to be very prescriptive, versus, 

### 6 you know, just some general guidance and maybe additional 

### 7 materials that would be associated with the test guideline but 

### 8 maybe not part of the test guideline. 

### 9 **DR. MARTHA SANDY:**So the discussants can add 

### 10 the detail, such as specify that you have one set of primers 

### 11 that are used to target one of the vitellogenin genes. Things 

### 12 like that. 

### 13 Are we ready for Question 7? 

### 14 **DR. LESLIE TOUART:**I'll read Charge Question 

### 15 7. The purpose of the validation process is to determine the 

### 16 readiness of a test for inclusion in a testing program. A 

### 17 component of readiness of a test is the evaluation of the 

### 18 usefulness and limitations of the test, including the classes 

### 19 and types of test substances that can and cannot be tested. 

### 20 Please comments on the strengths and/or 

### 21 limitations of the MMT and MRT assays. 

### 22 **DR. MARTHA SANDY:**Dr. Kullman. 

### 23 **DR. SETH KULLMAN:**Essentially, we just have a 

### 24 list. So this will not be too embellished. I'm just going to 

### 25 basically read off a list and you guys can reference that in the 

### 26 final summary report.

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1 For strengths, we kind of broke it up into two 

### 2 different forms, model and assay. So let me speak first to the 

### 3 advantage of the model. I think it was, again, described in 

### 4 Question 1 and the initial sections of the ISR, but certainly, 

### 5 the advantage of using this particular model, where the 

### 6 husbandry models are well-established and there is a general 

### 7 ease of manipulation of handling these animals. 

### 8 There is a rich history available with the 

### 9 resources for endocrine toxicity. So certainly reference to 

### 10 that can be made. They have a short generation time. It 

### 11 decreases your assay time. There is routine external spawning 

### 12 with daily spawn, which really makes this a nice animal for your 

### 13 embryo collection. 

### 14 And then certainly, external embryonic 

### 15 development occurs, which I think a comment was made before that 

### 16 if there is going to be any early life stage toxicity 

### 17 assessment, it certainly makes that assessment much easier. And 

### 18 then lastly, there is a well-sequenced genome, again, for things 

### 19 like assay endpoints such as vitellogenin being able to readily 

### 20 assess the genome or looking at primer sequences and such rated. 

### 21 Also, from the EDC perspective, the ability to 

### 22 assess EDC AOP diagnostics, certainly the capability for 

### 23 determining genetic sex, as been mentioned before, is a real 

### 24 strong, strong advantage of this model and then the ability to 

### 25 make a comparison to phenotypic sex is also well-established in 

### 26 this model and makes it very strong. So again, the comparison

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1 of genotypic and phenotypic sex is capable here, which is a real 

### 2 strong advantage. 

### 3 Well-established and readily available 

### 4 biomarkers for the EDC effects, such as VTG and those applying 

### 5 to the phenotypic sex, such as secondary sex characteristics and 

### 6 then the ability to use a large number of animals and make this 

### 7 a statistically robust assay really helps this assay. 

### 8 And again, the same thing speaking to that 

### 9 from the perspective of performing multiple generation assays. 

### 10 The assay itself, the MMT assay includes an assessment of an F0 

### 11 through F2 to reproduction. This was seen as a full assessment 

### 12 of reproduction through the second generation that allows the 

### 13 ability to evaluate epigenetic effects that may occur as a 

### 14 result of EAT disruption. This was seen as a strong component 

### 15 of the assay, particularly for the MMT and not necessarily as 

### 16 much for the MRT. 

### 17 The strengths and usefulness of this assay 

### 18 include inclusion of chemical exposure in F0 to assess parental 

### 19 transfer. I know there has been some, I'd say disagreement 

### 20 among the Panel to how much parental transfer occurs or maternal 

### 21 transfer occurs with different chemicals. That's to be 

### 22 determined, but certainly the parental transfer and maternal 

### 23 transfer of endogenous factors are also important. 

### 24 The MRT assay is much improved as the 

### 25 replicate nature of the experimental design significantly 

### 26 improves power for both the fecundity and fertility tests. And

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1 then the strengths, specifically to the MRT assay include 

### 2 reduction in time in performing the assay, as well as sort of 

### 3 the number of human hours necessary to perform that. I think 

### 4 that was a big added bonus for the MRT. 

### 5 Limitations; there are numerous limitations as 

### 6 with any assay. The MMT has a lower power than the MRT assay 

### 7 for statistical comparison. And this is important, given 

### 8 limited causal dose concentration dependence in documented 

### 9 studies. 

### 10 The MMT assay has higher labor requirements, 

### 11 as I just suggested, time, animal use, and overall cost than the 

### 12 MRT assay. So the Panel was excited about the reduction of 

### 13 those components for the MRT assay. 

### 14 To date, there are a limited number of trials 

### 15 with either assay, particularly the MRT and we still felt as if 

### 16 there are a limited number of trials with the MMT with 

### 17 representative camels -- camels (sic) -- representative 

### 18 chemicals for each of the EDC AOPs. And as mentioned before, a 

### 19 nice comparison to make would be with a strong reference of 

### 20 hepatic toxicant such ANIT that does not act as an EDC. 

### 21 So maybe a really good inference for just 

### 22 looking at general toxicity and how generalized toxicity may 

### 23 impact some of the overall apical and/or biomarker endpoints of 

### 24 the assay. Certainly, the strength lies in the strong agonists, 

### 25 the E, the A, and possibly the steroidogenesis compounds, but 

### 26 certainly the anti-E and anti-A was not as strong in this

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1 particular assay. 

### 2 And again, sort of the lack of standardized 

### 3 protocols, as we've mentioned previously, we wonder if some of 

### 4 the reproductive endpoints really may be sensitive to animal 

### 5 husbandry conditions. 

### 6 So that really, again, drives some of the 

### 7 aspects of Question 6 and really being able to define those 

### 8 husbandry conditions very well because this organism has been 

### 9 known to be sensitive to some husbandry conditions with 

### 10 fecundity and fertility. 

### 11 As stated for the quail assay, there was no 

### 12 information on the strain used. So maybe there is a number of 

### 13 Medaka strains that are there out there that the Japanese tend 

### 14 to use, a northern versus southern, versus CAB. 

### 15 There are a number of different types of 

### 16 strains that are available to run the assay. And it's known 

### 17 that these strains differ in their polymorphisms, and thus, 

### 18 could potentially differ in responses to the different AOPs. As 

### 19 of yet, unproven interlaboratory reproducibility need for 

### 20 greater replication MMT, thus, considered MRT. 

### 21 Let's see. I'm going to pass that one because 

### 22 we've talked about it already, and the same with this, full 

### 23 potential for phenotypic sex is not necessarily utilized in the 

### 24 assay as is. We talked about the statistical power, the lack of 

### 25 proper training in assays or guidance to other laboratories. 

### 26 So certainly not just the protocols, but the

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1 necessity to make sure that the people running these assays can 

### 2 actually conduct them. We felt that that was not necessarily 

### 3 stated. Animal health was stated. Then they felt as if the 

### 4 assessments of steroid concentrations may also -- could be added 

### 5 to the assay. 

### 6 And then certainly last but not least is the 

### 7 need to correlate and integrate possible transfer between Tier 1 

### 8 Fathead Minnow studies and MMT and MRT assay. 

### 9 **DR. MARTHA SANDY:**Could you repeat that last 

### 10 phrase, last recommendation? 

### 11 **DR. SETH KULLMAN:**Sure. So the last 

### 12 recommendation was the need to correlate and/or integrate 

### 13 between Tier 1 Fathead Minnow studies and the MMT and MRT. 

### 14 **DR. MARTHA SANDY:**So I'll now turn to the 

### 15 associate discussants. Dr. Perkins. 

### 16 **DR. EDWARD PERKINS:**I have nothing further to 

### 17 add. 

### 18 **DR. MARTHA SANDY:**Dr. Freeman. 

### 19 **DR. JENNIFER FREEMAN:**All of my comments have 

### 20 been incorporated. 

### 21 **DR. MARTHA SANDY:**Dr. Patino. 

### 22 **DR. REYNALDO PATINO:**I'm good. Thank you. 

### 23 **DR. MARTHA SANDY:**Dr. Blazer. 

### 24 **DR. VICKI BLAZER:**I think the only one that 

### 25 wasn't mentioned and maybe it was and I missed it was that we 

### 26 discussed that in MRT, the loss of any endpoints for the F0 and

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1 F2 could result in some loss of essential data for AOP and 

### 2 toxicity. 

### 3 **DR. MARTHA SANDY:**Dr. Schlenk. 

### 4 **DR. DANIEL SCHLENK:**Nothing to add. 

### 5 **DR. MARTHA SANDY:**Dr. Dallas Johnson. 

### 6 **DR. DALLAS JOHNSON:**I have nothing to add and 

### 7 Dr. Portier had nothing for this charge. 

### 8 **DR. MARTHA SANDY:**Other Panel members, any 

### 9 comment? 

### 10 (No response.) 

### 11 Seeing none, I'll turn to Dr. Rodney Johnson 

### 12 of the EPA. 

### 13 **DR. RODNEY JOHNSON:**I have no clarifying 

### 14 questions. 

### 15 **DR. MARTHA SANDY:**Thank you. Can we have the 

### 16 next question, Question 8 read, please? 

### 17 **DR. LESLIE TOUART:**Les Touart, and I'm 

### 18 reading Charge Question 8. There is sufficient evidence to 

### 19 indicate that endocrine disrupting chemicals can disrupt normal 

### 20 development and reproduction success, however, the value added 

### 21 if the F2 generation compared to the P0 or F1 is less clearly 

### 22 defined. The EPA is proposing the Medaka Reproduction Test as 

### 23 an EDSP Tier 2 assay. 

### 24 The MRT-test terminates after the F2 embryo 

### 25 hatch, and a rationale for the proposed protocol is provided in 

### 26 Section 5 of the MMT ISR.

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1 Please comment on the Agency's rationale that 

### 2 the value added by the F2 generation is not sufficient to 

### 3 warrant its inclusion in the Tier 2 fish test protocol. 

### 4 **DR. MARTHA SANDY:**Dr. Perkins, you're the 

### 5 lead discussant. 

### 6 **DR. EDWARD PERKINS:**The current structure of 

### 7 the MMT assay appears to be a true multigeneration assay with 

### 8 reproduction testing of F1 and F2 fish examined. Removal of the 

### 9 F2 generation of the MRT then begs the question if the MRT is 

### 10 truly a multigenerational test. 

### 11 As designed, the P0 or F0 generation is solely 

### 12 utilized for production of F1 animals putative maternal transfer 

### 13 of test agent. While chemical exposure in the P0 or F0 may 

### 14 likely have impact F1 embryos by maternal transfer modulation of 

### 15 maternal factors, information regarding activation and some 

### 16 organizational impacts of EDCs may be lost, such as effects on 

### 17 mating behavior in the F2 or F1. This is particularly true for 

### 18 inductive responses observed in F0 adults, such PTP expression. 

### 19 However, comparison of data across MMT trials 

### 20 suggest that the analysis of biomarker and histopathology data 

### 21 for the F2 generation may be redundant, as no significant 

### 22 difference in these endpoints is observed in between F1 and F2 

### 23 generations. 

### 24 This hypothesis is illustrated in Tables 5.7 

### 25 and 5.8 in the summary review that demonstrate concordance 

### 26 between the F1 and F2 LOEC values or fecundity and biomarker

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1 data, respectively. 

### 2 The authors provide a strong case for removing 

### 3 the F2 reproduction portion of MRT to create the MMT to create 

### 4 the MRT. That's assuming that EDSTAC doesn't really require a 

### 5 multigeneration test. 

### 6 So the rationale for these modifications are 

### 7 to increase statistical power, reduce animal number, reduce 

### 8 person hours to perform the assay, reduce overall assay time and 

### 9 reduce possible variability of data, due to increased risk of 

### 10 experimental incidence resulting in test failure. 

### 11 Overall, the generational rationale suggests 

### 12 that additional generational data would not increase the quality 

### 13 of risk assessment. Assessments of the F1 generations were 

### 14 likely to formative for both activational organizational 

### 15 responses and assessments of F1 reproductive capacity, will 

### 16 likely reflect both, thus, satisfying these associations to EDC 

### 17 toxicity in a single generation. 

### 18 Exclusion of F2 generational effects thus 

### 19 makes this test the single generation, or at most, one and a 

### 20 half generation assay. One possible modification that could be 

### 21 made is to allow F2 animals to reach sexual maturity about nine 

### 22 weeks post fertilization so that additional data could be 

### 23 derived regarding both phenotypic and genetic gender 

### 24 development. 

### 25 As currently described, neither the MMT assay 

### 26 nor the MRT assay exploit the ability to establish genotypic or

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1 phenotypic gender ratios. Establishing this analysis with data 

### 2 already derived from existing assay structure will further 

### 3 substantiate modifications in phenotypic genders or genetic 

### 4 state will change the exposure. 

### 5 Given the current dataset determination of the 

### 6 Medaka assay, FF2 hatch, appears warranted without significant 

### 7 loss of sensitivity for endocrine points. Animal number and 

### 8 labor costs will also be minimized; however, it is recommended 

### 9 that not all F0 endpoints should be discarded. In particular, 

### 10 endpoints that evaluate organ histopathology or overt toxicity 

### 11 should not be removed due to potential confounding of 

### 12 non-endocrine maternal effects. 

### 13 It should be noted that the dataset that this 

### 14 decision has been made, has been based upon as limited to fewer 

### 15 than 10 compounds with only comparison of MMT with MRT using 

### 16 octylphenol. While the eight compounds represent a range of 

### 17 endocrine responses, there were still significant uncertainty 

### 18 with AR antagonism and steroidogenesis AOPs. 

### 19 It is unclear, based upon this current data 

### 20 whether more potent air antagonist aromatase inhibitor might 

### 21 affect F2 in the chronology. With the continuous exposure 

### 22 design in the MRT assay, it is expected that results will be 

### 23 similar in F1 and F2, but we also suggest the addition of tissue 

### 24 chemical analysis to aide in determining if bioaccumulation of 

### 25 chemical in tissues is occurring. Although it's realized that 

### 26 this would lead to an increase in the overall number of animals

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1 for the study. 

### 2 **DR. MARTHA SANDY:**Thank you. And now turning 

### 3 to the associates. Dr. Blazer. 

### 4 **DR. VICKI BLAZER:**I have nothing additional 

### 5 to add. 

### 6 **DR. MARTHA SANDY:**Dr. Freeman. 

### 7 **DR. JENNIFER FREEMAN:**All my comments have 

### 8 already been addressed. 

### 9 **DR. MARTHA SANDY:**Dr. Kullman. 

### 10 **DR. SETH KULLMAN:**I'll just state one thing, 

### 11 essentially that I talked about initially when we had the first 

### 12 round of discussions is that sort of the loss of the F2 and the 

### 13 new assays within the design of the endpoints in the F1 really 

### 14 limits the ability to make comparisons between gonadal 

### 15 histology, vitellogenin and reproduction. 

### 16 I would argue for inclusion of histopath, 

### 17 vitellogenin and reproductive measures at nine weeks and 

### 18 termination of the F1. 

### 19 **DR. MARTHA SANDY:**Dr. Patino. 

### 20 **DR. REYNALDO PATINO:**Nothing further to add. 

### 21 **DR. MARTHA SANDY:**Dr. Schlenk. 

### 22 **DR. DANIEL SCHLENK:**Nothing to add. 

### 23 **DR. MARTHA SANDY:**And Dr. Dallas Johnson. 

### 24 **DR. DALLAS JOHNSON:**I have nothing, but Dr. 

### 25 Portier indicated that the answer to this question depends on a 

### 26 clear statement of the key endpoints for each EDC study and

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1 this, on the hypothesized AOP. 

### 2 If the endpoint is bodyweight and the issue is 

### 3 increasing bodyweight in subsequent generations, the EDC link to 

### 4 obesity issue, it would not be beneficial to not take the F2 

### 5 generation to adulthood to allow adult weights to be collected. 

### 6 I realize that the focus of this test is 

### 7 reproduction and bodyweight other than as a measure of vigor and 

### 8 readiness to reproduce is typically not considered a 

### 9 reproductive endpoint of concern. I am not certain that there 

### 10 are no other reproductive endpoints that may require assessment 

### 11 through the F2 adult stages. 

### 12 **DR. MARTHA SANDY:**I'll open it up to the 

### 13 entire Panel for any additional thoughts. 

### 14 (No response.) 

### 15 Seeing none, I'll turn to Dr. Rodney Johnson 

### 16 with the EPA. 

### 17 **DR. RODNEY JOHNSON:**I once again have no 

### 18 clarifying questions. I appreciate all the comments. 

### 19 **DR. MARTHA SANDY:**Thank you. Shall we move 

### 20 on to Question 9? 

### 21 **DR. LESLIE TOUART:**This is Charge Question 9 

### 22 for the Medaka. It is the Agency's opinion that the outcomes of 

### 23 the various MMT trials had provided enough information to 

### 24 recommend a Medaka Reproduction Test for use as a fish test in 

### 25 Tier 2 of the EDSP. 

### 26 Two major changes from the MMT are proposed,

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1 an increase in the number of replicates per treatment for 

### 2 evaluating effects on reproduction, and terminating the test 

### 3 after the embryos hatch in F2. Other proposed changes include 

### 4 minimizing the collection of endpoint data from the F0 and 

### 5 evaluating pathology in only the F1 adults sampled after the 

### 6 assessment of reproduction. 

### 7 Overall, the authors conclude that both the 

### 8 MMT and MRT are transferable methods and are capable of 

### 9 adequately characterizing potential disruption of the endocrine 

### 10 system by putative endocrine disrupting chemicals; however, the 

### 11 MRT is recommended as a preferred EDSP Tier 2 test method for 

### 12 fish because is it less resource intensive with improved 

### 13 statistical power, appears to be as sensitive, and is better 

### 14 able to ensure consistent findings when performed routinely by 

### 15 testing laboratories. 

### 16 Please comment on the scientific rationale of 

### 17 the Agency's proposed Tier 2 fish test, the MRT, with respect to 

### 18 statistical power, sensitivity, and consistency in performance 

### 19 across laboratories. 

### 20 In addition, please comment on the adequacy of 

### 21 the MRT to characterize potential endocrine disruption 

### 22 requirement for the Tier 2 of the EDSP. 

### 23 **DR. MARTHA SANDY:**Dr. Blazer is the lead 

### 24 discussant on this. 

### 25 **DR. VICKI BLAZER:**So the majority of the ISR 

### 26 focuses on the MMT with Section 5 providing a rationale for the

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1 MRT. As stated, the MRT differs from the MMT with two 

### 2 fundamental modifications; 1) increased replication of breeding 

### 3 pairs for evaluation of reproduction in the F1 generation, and 

### 4 2) the termination of the test after hatching of F2 embryos with 

### 5 no assessment of the F2 individuals. 

### 6 So based on statistical considerations, 

### 7 evaluations of reproductive parameters will be much improved 

### 8 with the increase of biological replication and are likely to 

### 9 enhance the accuracy of acquiring precise LOEC data from 

### 10 reproductive endpoints in the F1 population. 

### 11 Detailed modeling of reproductive replicates 

### 12 strongly supported the modification and trial experiments with 

### 13 only one compound, being octylphenol, demonstrated increased 

### 14 sensitivity, enabling detection of a lower reproductive LOEC, 

### 15 which was 23 micrograms per liter compared to the MMT, which was 

### 16 100, depending on the lab, but approximately 100 micrograms per 

### 17 liter. 

### 18 However, if you look at the MMT, the LOEC is 

### 19 based on biomarker assays, many of which will not be completed 

### 20 for more than one of the lifestages or generations in the MRT or 

### 21 lower. So again, it gets back to what was discussed in Question 

### 22 Number 8, as far as sensitivity of other endpoints. 

### 23 The MRT assay has many less endpoints than the 

### 24 MMT, and hence, there was some concern about the impact of these 

### 25 modifications to assess multigenerational effects, as well as to 

### 26 assess non-reproductive parameters.

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1 In the MRT, no assessments are made on the F0 

### 2 animals. Vitellogenin is only conducted in the subadults at 

### 3 nine weeks and histopathology only in the F1 adults at week 15 

### 4 after spawning. While the rationale for these modifications is 

### 5 given, we do feel like exclusion of some of these endpoints may 

### 6 reduce the overall assay performance. 

### 7 So for instance, pathology should be included 

### 8 at nine weeks, as there is significant variability in growth and 

### 9 development in Medaka at this stage. Given that vitellogenin 

### 10 will be assessed at this stage only, some support of assessment 

### 11 of gonad development and maturation should also be determined. 

### 12 A similar argument can be made for 

### 13 determination of vitellogenin in the adult F1 generation, as 

### 14 these animals are expected to have fully matured gonads. And 

### 15 it's currently unknown if correlations can be demonstrated 

### 16 against the vitellogenin MRNA levels, gonad morphology, degree 

### 17 of intersex or other gonadal abnormalities and fecundity. 

### 18 Hence, important data may be lost by only 

### 19 having one of these endpoints in a particular generation. So 

### 20 some effort should be made to coordinate the endpoints. 

### 21 Additionally, it was felt that the F0 animals 

### 22 should be evaluated for histopathology to determine whether 

### 23 non-endocrine effects or overt toxicity, particularly at the 

### 24 maternal stage, are not confounding the assay and perhaps, 

### 25 indicating endocrine AOPs in offspring when it may not be an 

### 26 endocrine effect.

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1 So for instance, overt toxicity may diminish 

### 2 blood flow to ovaries or cause liver damage which may lead to 

### 3 responses in reproduction that are not mediated through the EAT 

### 4 AOPs. Along these lines, as has already been stated, I think a 

### 5 couple of times non-endocrine toxicants that may indirectly 

### 6 effect reproduction should be evaluated during validation of the 

### 7 MRT assay. 

### 8 So while there have certainly been a 

### 9 significant amount of work evaluating the MMT, additional 

### 10 confirmatory studies, using the specific and more detailed 

### 11 guidelines are necessary before the MRT can be adopted as a Tier 

### 12 2 assay. Overall, it appears that global validation of both the 

### 13 MMT and MRT is rather subjective. 

### 14 So on a global scale, there is little 

### 15 statistical indication of performance parameters needed to 

### 16 establish assay reproducibility and reliability. Additional 

### 17 validation tests would address this issue, and additional 

### 18 interlaboratory studies are needed to demonstrate 

### 19 transferability, reliability and reproducibility. 

### 20 So in conclusion, it was felt that the MRT 

### 21 clearly showed promise with regard to statistical power, 

### 22 sensitivity, and consistency, and this was shown in the 

### 23 performance of the octylphenol test. So the rationale for 

### 24 modification is adequate; however, given the uncertainties 

### 25 inherited in fish husbandry and endocrine variability with this 

### 26 species, additional confirmation with other chemicals is

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1 necessary for validation. 

### 2 It was observed that initial EDSTAC panels 

### 3 recommended 50 to 100 compounds go through Tier 1, but it's 

### 4 unclear how many should go through Tier 2. So we had discussion 

### 5 about recommending what might be needed both for adoption of 

### 6 this, as far as performance, but also ring testing. And we 

### 7 recommend that representative compounds, both low and high 

### 8 potency of at least seven endocrine AOPs. So in other words, 

### 9 the estrogen, androgen, thyroid, both agonists, and antagonists, 

### 10 and steroidogenesis inhibitors and a non-endocrine AOP are 

### 11 evaluated for MRT assay performance prior to adoption of as a 

### 12 Tier 2 assay. And then ring testing with, perhaps, one AOP. 

### 13 Using the MRT assays is also recommended. And that's all I 

### 14 have. 

### 15 **DR. MARTHA SANDY:**And now we turn to the 

### 16 discussants. Dr. Schlenk. 

### 17 **DR. DANIEL SCHLENK:**Nothing to add. 

### 18 **DR. MARTHA SANDY:**Dr. Dallas Johnson. 

### 19 **DR. DALLAS JOHNSON:**I have nothing to add and 

### 20 I think Dr. Portier's comments have been covered. 

### 21 **DR. MARTHA SANDY:**Dr. Freeman. 

### 22 **DR. JENNIFER FREEMAN:**All of my comments have 

### 23 been addressed. 

### 24 **DR. MARTHA SANDY:**Dr. Kullman. 

### 25 **DR. SETH KULLMAN:**Nothing to add. 

### 26 **DR. MARTHA SANDY:**Dr. Patino.

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1 **DR. REYNALDO PATINO:**Nothing to add. Thank 

### 2 you. 

### 3 **DR. MARTHA SANDY:**And Dr. Perkins. 

### 4 **DR. EDWARD PERKINS:**I have nothing to add. 

### 5 **DR. MARTHA SANDY:**Do other members of the 

### 6 Panel have any comments in response to this charge question? 

### 7 (No response.) 

### 8 Seeing none, I'll turn to the EPA staff. Ms. 

### 9 Mary Manibusan. 

### 10 **MS. MARY MANIBUSAN:**Just for clarification 

### 11 from Dr. Blazer on your comment, it was indeed the 1999 SAB SAP 

### 12 joint panel that made the recommendation to the Agency with 

### 13 regard to the Tier 1 data, that after validation and recognition 

### 14 that the validation process would only be limited to a few 

### 15 laboratories for interlab testing that we gain additional 

### 16 experience after having issued test orders for 50 to 100 

### 17 chemicals to bring those back to give the Agency a reflection of 

### 18 what the real world laboratory performance would be for the Tier 

### 19 1 assays for review, and that review was had just last month. 

### 20 Is that your recommendation for Tier 2 with 

### 21 respect to the test methodologies that once validated to go out 

### 22 and wait for 50 to 100 chemicals to come back to the Agency for 

### 23 review? 

### 24 So just clarification on what the intention of 

### 25 that comment was. 

### 26 **DR. VICKI BLAZER:**No. It was just

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1 recognizing that 50 to 100 was what was recommended in the Tier 

### 2 1 and that, as far as we know, there is no recommendation for 

### 3 how many should be looked at in Tier 2. So then we came up with 

### 4 that number, which would be more like 14 if you did a low and a 

### 5 high potency of each of those AOPs. Is that what you're asking 

### 6 or did I misunderstand? 

### 7 **MS. MARY MANIBUSAN:**So the clarification 

### 8 was that for the 50 to 100 Tier 1, that was post-validation. So 

### 9 the clarification, I'm asking from you, again, just for 

### 10 understanding, is that post-validation that you're asking for 

### 11 the Agency to collect that experience to bring back to a panel 

### 12 for review? 

### 13 **DR. VICKI BLAZER:**I think what we were 

### 14 suggesting is that those 14 should be used for validation. 

### 15 **MS. MARY MANIBUSAN:**Yes. Okay. And then 

### 16 following up to a previous comment made to the overall panel, as 

### 17 we move forward on the Tier 2 validation effort, discussion of 

### 18 what -- how many test chemicals would increase X number of 

### 19 certainty or confidence as we move forward. So is it 14? Is it 

### 20 15? What's the difference between 16? That clarification would 

### 21 be very helpful to the Agency. 

### 22 **DR. MARTHA SANDY:**Okay. Dr. Schlenk. 

### 23 **DR. DANIEL SCHLENK:**Mary, I think we outlined 

### 24 which particular AOP and how many chemicals we would recommend 

### 25 for each particular AOP. So you should have, I think it's 14 or 

### 26 something, is what we came up with. So justification for those

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1 high potency, low potency at each of those different AOPs is the 

### 2 justification for that. 

### 3 **DR. MARTHA SANDY:**Are there any other 

### 4 comments from the Panel in response to that? Any questions of 

### 5 clarification from Dr. Rodney Johnson? 

### 6 **DR. RODNEY JOHNSON:**My clarification question 

### 7 regards the issue of validating what would be the AOP pathways 

### 8 as a discreet part of validation compared to what would be 

### 9 reproduction. A few of the identified apical endpoints and 

### 10 would like some sort of clarification of whether or not you 

### 11 believe that the apical endpoints, perhaps, are validated. 

### 12 So the apical endpoints in this case being 

### 13 reproduction, growth, and gonadal sex phenotype as it relates to 

### 14 intersex conditions and sex reversal. I mean, those are 

### 15 identified in the ISR by us as being the apical endpoints that 

### 16 are part of either the MMT or the MRT and then we can discuss 

### 17 the AOP or biomarker endpoints as indicative of, in fact, the 

### 18 AOP. 

### 19 But I think of them as distinct as compared to 

### 20 the apical endpoints for risk assessment, which I think if it is 

### 21 being primarily reproduction growth and the sex reversal, sex 

### 22 phenotype outcome. So sex reversal on intersex as distinct from 

### 23 let's say, secondary sex of vitellogenin. 

### 24 So all I'm getting at is clarification on that 

### 25 would be helpful in the Panel's review. 

### 26 **DR. DANIEL SCHLENK:**I, for one, think the

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1 apical endpoints are definitely justified for risk assessment. 

### 2 I would totally agree with that. I don't know what the rest of 

### 3 the panel thinks, but I would agree those are definitely 

### 4 justified in terms of endpoints you'd want to use in a risk 

### 5 assessment paradigm, those apical endpoints. 

### 6 **DR. MARTHA SANDY:**That was Dr. Schlenk. Dr. 

### 7 Patino. 

### 8 **DR. REYNALDO PATINO:**I probably repeat a 

### 9 little bit here, but just to make sure it's clear, I agree with 

### 10 that. I think the apical endpoints are okay. My take on why we 

### 11 made those recommendations on the 14 chemicals is to demonstrate 

### 12 that if you tweak those AOPs that you've identified as leading 

### 13 to the apical endpoints, then you will actually get a change in 

### 14 the -- it will validate the assay in the context of the AOPs. 

### 15 That's why I think we recommended that. 

### 16 **DR. MARTHA SANDY:**Dr. Kullman. 

### 17 **DR. SETH KULLMAN:**So, Rodney, one thing that 

### 18 comes to mind about this is, you know, whether we're validating 

### 19 the individual endpoints of the assay or we're validating the 

### 20 assay as a whole. So certainly the fecundity growth survival, 

### 21 those types of things can be validated as individual endpoints, 

### 22 but as a whole, I believe the assay is supposed to be indicative 

### 23 of an AOP. 

### 24 So I think in total, AOP combined with all the 

### 25 components that are measured needs to be validated. The ring 

### 26 test essentially says no impact on reproduction. I don't think

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1 that's a reasonable measure of whether reproduction is 

### 2 functioning. I think the control values are a good measure of 

### 3 that. I think some of the trials with octylphenol are a good 

### 4 measure of that, but overall validation of the assay as is, 

### 5 probably is not -- from a ring test perspective, has not been 

### 6 substantiated. 

### 7 So let's make sure that we talk about the 

### 8 validation of the assay, versus the validation of the individual 

### 9 components of the assay. 

### 10 **DR. RODNEY JOHNSON:**Thank you. 

### 11 **DR. MARTHA SANDY:**Dr. Bradbury. 

### 12 **DR. STEVEN BRADBURY:**As you can tell, we're 

### 13 asking for additional clarification on this and part of it is 

### 14 that some of the endpoints like vitellogenin, measuring 

### 15 different species and works through the Tier 1 and some of the 

### 16 secondary sex characteristics in a different species through 

### 17 Tier 1. 

### 18 Having said that, the Medaka is used in Japan 

### 19 for a similar kind of program. So there is some historical 

### 20 information. 

### 21 So I'm just kind of doing the math and 14 

### 22 chemicals, maybe a half a year to do the assay, probably several 

### 23 months to analyze X number of labs. We could be talking four 

### 24 years, five years from now before you go through this, which 

### 25 could be a path forward. But just sort of curious if the Panel 

### 26 has any suggestions on how existing information or information

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1 that could be generated in different ways to help get at the 

### 2 attributes of the different endpoints. 

### 3 I think I'm hearing from the Panel that the 

### 4 apical endpoints, just thinking about the hundreds of 

### 5 reproductive studies that go to Europe and Japan and the United 

### 6 States and Canada to, I guess, your pesticides, maybe we know 

### 7 more about those than we do than maybe some of the genome typing 

### 8 of the fish going in. 

### 9 So if there is just some suggestions on how to 

### 10 tackle the issue in as an efficient way as possible. Any 

### 11 suggestions you have to balance time with the ability to start 

### 12 answering questions to evaluate the chemicals properly. 

### 13 **DR. MARTHA SANDY:**Okay. Any comments? 

### 14 (No response.) 

### 15 No? Okay. I believe we have gone through all 

### 16 the charge questions in the Medaka test. Should we take a break 

### 17 for lunch now and then come back and start the amphibian 

### 18 questions? 

### 19 I'd like us to come back from lunch at a 

### 20 quarter to 1:00. We'll give ourselves an hour. Thank you. 

### 21 (Whereupon, a lunch break was taken) 

### 22 **DR. SHARLENE MATTEN:**Good afternoon. We're 

### 23 back in action. It's about 10 minutes to 1:00. We're going to 

### 24 start with the questions on the Larval Amphibian Growth and 

### 25 Development Assay (LAGDA). 

### 26 So with that, I will turn the microphone back

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1 over to Dr. Martha Sandy. 

### 2 **DR. MARTHA SANDY:**Thank you, Dr. Matten. So 

### 3 I welcome everyone back to the meeting. If EPA, is it Dr. 

### 4 Touart again, will be Charge Question 1 for the LAGDA assay. 

### 5 **DR. LESLIE TOUART:**Yes. I need to get the 

### 6 slide projected and it will be the first question for the LAGDA. 

### 7 This is Charge Question Number 1 for the LAGDA. A rationale 

### 8 for the test methods should be available, including a clear 

### 9 statement of scientific basis and the regulatory purpose and 

### 10 need for the test method. 

### 11 The EDSTAC introduced the Tier 2 test as 

### 12 having the purpose to characterize the nature, likelihood and 

### 13 dose-response relationship of endocrine disruption of estrogen, 

### 14 androgen, and thyroid in humans and wildlife. 

### 15 Tier 2 tests were designed to be definitive 

### 16 tests which generate sufficient data to characterize the 

### 17 specific hazard of the substance and provide sufficient 

### 18 information and dose response and adverse effects to permit risk 

### 19 decisions. 

### 20 Please comment on the rationale and purpose of 

### 21 the assay as part of the Tier 2 testing in the EDSP as described 

### 22 in Section 3 of the LAGDA ISR. 

### 23 **DR. MARTHA SANDY:**Dr. Propper is the lead 

### 24 discussant on this. 

### 25 **DR. CATHERINE PROPPER:**So our group got 

### 26 together and we've all formulated a fairly common opinion, but

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1 I'll encourage everybody if I miss something to please step in. 

### 2 Amphibian development can be used as assays, 

### 3 both for gonadal development-related issues associated with 

### 4 androgen and estrogen disruption and for thyroid and hormone 

### 5 disruption. The development of validated consistent techniques 

### 6 is critical to being able to interpret individual laboratory 

### 7 findings and understand where sources of variation may occur. 

### 8 The rationale behind the testing was well 

### 9 developed and built upon earlier EDSP protocol development. It 

### 10 includes the ease of monitoring morphological and growth 

### 11 endpoints in amphibians, the ease of exposure -- relative ease 

### 12 of exposure -- and the background knowledge and availability of 

### 13 sensitive endpoints for both the gonadal and thyroid hormone 

### 14 with the gonadal endpoints being things like testicular oocytes 

### 15 or overt pathologies. 

### 16 Gonadal duct, as well, seems to become a 

### 17 fairly sensitive and probably one of the more consistent 

### 18 endpoints, and then time to development is potentially 

### 19 significant. Thyroid endpoint, especially when it's coupled 

### 20 with the histopathology; however, we'll address some issues 

### 21 between trying to identify direct endocrine disruption versus 

### 22 overt toxicity as we move through our discussion. 

### 23 Vitellogenin levels also, and possibly T4 

### 24 levels, and we'll talk about that more. Furthermore, the 

### 25 ability to genetically and phenotypically sex the animals does 

### 26 make this a stronger model, as was seen with the Medaka assay.

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1 So having the capacity to know whether or not 

### 2 you have sex reversed your individuals by the treatments with 

### 3 estrogenic or anti-estrogenic compounds is very valuable. So 

### 4 these are important strengths of the assay. All of these 

### 5 endpoints, although subject to overt toxicity, may be valuable 

### 6 as endpoint measures that can address estrogen, androgen or 

### 7 thyroid hormone disruption. And again, we'll discuss that a 

### 8 little more. 

### 9 The criteria used to develop the validation 

### 10 methodology we found to be sound and it was based on ICCVAM and 

### 11 OECD published criteria, so it wasn't starting from scratch. 

### 12 Amphibians were recognized by the EPA to have limited utility 

### 13 for long term, multigenerational studies and this is because 

### 14 they take so long to get to that developmental stage that the 

### 15 cost of growing them out to do those studies becomes really 

### 16 pretty prohibitive in time to an answer with the data under 

### 17 those circumstances also becomes very long. So we appreciated 

### 18 that and that was explained very clearly in the integrated 

### 19 summary report. 

### 20 They also adequately addressed the issues of 

### 21 the differences between the commonly two used amphibian species 

### 22 for these kinds of ecotoxicity tests, Xenopus laevis and Xenopus 

### 23 tropicalis, and we believed addressed why they went with laevis 

### 24 rather than tropicalis. 

### 25 We still have some concerns with understanding 

### 26 negative results because of the risk of false negatives, just

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1 utilizing this assay alone because you don't have those really 

### 2 strong apical endpoint outcomes. 

### 3 As was shown on page 24, in Table 3-4 in the 

### 4 ISR for the Medaka assay, there was a very nice contingency 

### 5 table of expected outcomes for the different modes of action of 

### 6 each of the compounds that they chose to use for the validation. 

### 7 We really like that table and wish we had had one similar to 

### 8 that in the amphibian ISR. 

### 9 But the functions of the assays to determine 

### 10 -- I hit that point before so I'm going to move on. We do 

### 11 overall feel that as an assay, whether or not the mode of action 

### 12 is overt toxicity or endocrine disruption, the assay itself has 

### 13 potential to evaluate at least surrogates for adverse outcomes, 

### 14 if not real adverse outcomes. And that's going to depend on the 

### 15 endpoints. 

### 16 **DR. MARTHA SANDY:**Discussants. We have Dr. 

### 17 Denver. 

### 18 **DR. ROBERT DENVER:**I have nothing to add. I 

### 19 concur. 

### 20 **DR. MARTHA SANDY:**Dr. Patino. 

### 21 **DR. REYNALDO PATINO:**Nothing new to add. 

### 22 **DR. MARTHA SANDY:**Dr. Blazer. 

### 23 **DR. VICKI BLAZER:**Nothing more to add. 

### 24 **DR. MARTHA SANDY:**Dr. Christman. 

### 25 **DR. MARY CHRISTMAN:**I have nothing to add, 

### 26 but Dr. Portier does. So Dr. Portier says that he felt that the

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1 rationale for the use of Xenopus laevis was a bit weak, since 

### 2 most of the discussion was on why they wouldn't use tropicalis. 

### 3 So he thought there must be other things they could bring out to 

### 4 explain why Xenopus was a better choice. I mean laevis was a 

### 5 better choice. I apologize. 

### 6 The other thing he wanted to comment on was in 

### 7 a previous SAP, concern was expressed the Xenopus laevis might 

### 8 be too robust. That is, might not be very sensitive to EDCs. 

### 9 Plus, laevis is not native to the U.S. (I may be wrong here). 

### 10 And it was thought that a native U.S. species such as Rana 

### 11 should be recommended. What happened to this recommendation? 

### 12 That was his comment. 

### 13 **DR. MARTHA SANDY:**Does that prompt anyone 

### 14 else to have any other comments? 

### 15 Yes, Dr. Propper. 

### 16 **DR. CATHERINE PROPPER:**We'll be addressing 

### 17 that a bit more later. 

### 18 **DR. MARTHA SANDY:**So I'll ask other members 

### 19 of the Panel. Anyone have anything to add? 

### 20 (No response.) 

### 21 No. Then we're turn to EPA staff, Dr. Degitz. 

### 22 **DR. SIGMUND DEGITZ:**I have no clarifying 

### 23 questions and appreciate the comments. 

### 24 **DR. MARTHA SANDY:**All right. Thank you. 

### 25 Shall we move on to Charge 2? 

### 26 **DR. LESLIE TOUART:**Les Touart reading Charge

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1 Question 2 of the LAGDA. Test methods and their associated 

### 2 endpoints should be scientifically relevant to the biological 

### 3 processes of interest and should be demonstrated to be 

### 4 responsive to the specific type of effect toxicity of interest. 

### 5 6 Each species presents unique characteristics 

### 7 from a biological perspective and allows for specialized 

### 8 endpoints to address a specific toxicological mode of action. 

### 9 Please comment on the biological and 

### 10 toxicological relevance of the assay in regards to the stated 

### 11 purpose of characterizing endocrine disrupters as described in 

### 12 Sections 3 and 4 of the LAGDA ISR. 

### 13 **DR. MARTHA SANDY:**Dr. Denver is the lead 

### 14 discussant. 

### 15 **DR. ROBERT DENVER:**Good afternoon. The 

### 16 purpose of the assay is to detect effects of chronic exposure to 

### 17 potential endocrine disrupting chemicals identified in the Tier 

### 18 1 assays on some aspects of the amphibian development. And 

### 19 exposure begins during embryogenesis and continues throughout 

### 20 metamorphosis, a period of postembryonic development, and then 

### 21 into the juvenile stage. 

### 22 Now, if it can be executed in a reproducible 

### 23 way the assay can have a high degree of biological and 

### 24 toxicological relevance for understanding chemical impacts on 

### 25 amphibian development that could also have relevance to other 

### 26 animal taxa.

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1 It may also suggest endocrine modes of action, 

### 2 but it is uncertain whether the LADGA will be able to clearly 

### 3 distinguish endocrine from non-endocrine modes of action, or 

### 4 possibly toxicity as was recognized in the ISR. 

### 5 Whole organism developmental progression 

### 6 expressed as the time to reach metamorphic climax is measured in 

### 7 the LAGDA as a proxy for thyroid hormone production and/or 

### 8 action. 

### 9 Concordance among measures of developmental 

### 10 timing, thyroid histopathology and possibly plasma T4 

### 11 concentration could be a powerful means to support that a 

### 12 chemical has thyroid active properties; however, results of the 

### 13 validation tests with Prochloraz suggests that an effect on 

### 14 thyroid histopathology that was seen in three laboratories, 

### 15 although the LOEC ranged from 6.7 micrograms per liter to 180 

### 16 micrograms per liter, and in one lab was correlated with an 

### 17 increase in T4 was not expressed as a change in development 

### 18 rate. 

### 19 So while the potential exists for the LAGDA to 

### 20 be a powerful diagnostic for thyroid disruption that can impact 

### 21 animal development. No data from the interlaboratory analysis 

### 22 support that it has been validated for this purpose. 

### 23 One might expect that compounds that are 

### 24 identified as thyroid disrupters in the amphibian metamorphosis 

### 25 Tier 1 assay will also be identified as such in the LAGDA. 

### 26 However, because chemical exposure begins during embryogenesis

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1 in the LAGDA, the effects on development could be manifest as a 

### 2 failure of organogenesis, independent of thyroid hormone, and 

### 3 any thyroid effects that are seen may be secondary to the 

### 4 teratogenic effects. 

### 5 So distinguishing between an endocrine and a 

### 6 non-endocrine mode of action, even if the chemical results in 

### 7 altered thyroid gland development and function may actually be 

### 8 quite difficult. This is also true for the measures of 

### 9 reproductive development. 

### 10 The LAGDA also monitors gonadal and urogenital 

### 11 ductal morphology and plasma vitellogenin as evidence for 

### 12 disruption of estrogen or androgen production and/or action. 

### 13 Altered reproductive system development is intended to be a 

### 14 proxy for impacts on fertility and fecundity, which are not 

### 15 directly measured in the LAGDA. 

### 16 The oviductal morphology is likely to be the 

### 17 best diagnostic endpoint for predicting effects on reproduction 

### 18 if there is no oviduct the female cannot oviposit her eggs. It 

### 19 will not be possible to definitively link alterations in 

### 20 testicular morphology or elevated plasma vitellogenin with 

### 21 impacts on fertility or fecundity. Vitellogenin is low or 

### 22 nondetectable in 10-week old female frogs, and so while an 

### 23 elevation in vitellogenin may be diagnostic of an estrogenic 

### 24 action, this endpoint cannot detect an anti-estrogenic effect. 

### 25 However, anti-estrogenic effects may be 

### 26 detected by comparing genetic and phenotypic sex. Frogs are a

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1 good model for this type of outcome since estradiol is necessary 

### 2 for ovarian organogenesis. Male-biased sex ratios can support 

### 3 an anti-estrogenic mode of action. 

### 4 Effects on growth may be due to endocrine or 

### 5 non-endocrine mechanisms; that is, such effects should not be 

### 6 dismissed as simply representing toxicity, unless they are seen 

### 7 only at the highest concentrations and exhibit clear liver 

### 8 histopathology. They could represent endocrine or non-endocrine 

### 9 modes of action that may or may not have been represented in the 

### 10 Tier 1 assay. 

### 11 While it may be difficult to distinguish overt 

### 12 toxicity from endocrine disruption, it does not compromise the 

### 13 ability to identify adverse outcomes at apical endpoints. It 

### 14 does, however, compromise the ability to predict mode of action 

### 15 with certainty. 

### 16 Overall, the biological and toxicological 

### 17 relevance of the LAGDA for detecting developmental abnormalities 

### 18 caused by chemical exposure is strong. Some endpoints may be 

### 19 good measures of endocrine disruption; for example, thyroid 

### 20 hypertrophy can support a goitrogenic action; elevated plasma 

### 21 vitellogenin can signal an estrogenic action; failure in oviduct 

### 22 formation can signal an androgenic action and sex reversal 

### 23 compared to the control may indicate either steroidal or 

### 24 anti-steroid action. But many effects may not be directly 

### 25 related to endocrine disruption, per se, but instead due to a 

### 26 failure in organogenesis.

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1 So distinguishing a non-endocrine from an 

### 2 endocrine mode of action will be difficult and assessing kidney 

### 3 and liver histopathology will be important to help support an 

### 4 endocrine mode of action versus toxicity. 

### 5 **DR. MARTHA SANDY:**I'll turn to the associate 

### 6 discussants. Dr. Patino. 

### 7 **DR. REYNALDO PATINO:**Nothing need to add. 

### 8 **DR. MARTHA SANDY:**Dr. Propper. 

### 9 **DR. CATHERINE PROPPER:**Only one more thing to 

### 10 add and that is just a reminder than nonmontonic dose responses 

### 11 may be important and can't be completely ignored here as just 

### 12 statistical anomalies because of the nature of the feedback 

### 13 loops. 

### 14 **DR. MARTHA SANDY:**Dr. Blazer. 

### 15 **DR. VICKI BLAZER:**The only thing I'd like to 

### 16 add is just that for the liver and the kidney histopathology, I 

### 17 wonder if some of those can't also be induced by husbandry or 

### 18 water quality and that might have to be differentiated between 

### 19 toxic. 

### 20 **DR. MARTHA SANDY:**And Dr. Christman. 

### 21 **DR. MARY CHRISTMAN:**Dr. Christman has no 

### 22 comments, but Dr. Portier does. Point of order, can I 

### 23 paraphrase his statement or do you want me to read it, 

### 24 specifically? 

### 25 I can paraphrase it? Good. Because 

### 26 otherwise... Essentially, his concern is that there are some

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1 pieces of information missing in the description for biological 

### 2 and toxicological relevance because they are a little too, my 

### 3 word: "squishy." 

### 4 For example, what is meant by adverse 

### 5 consequences or interferers with the normal development. So he 

### 6 feels that it is hard to justify relevance without having more 

### 7 quantitative measures and basically he goes on to say he cannot 

### 8 really interpret the tables that show some of the outcomes 

### 9 because there was no power analysis to determine whether they 

### 10 were statistically relevant or measurable, given the sample 

### 11 sizes. 

### 12 So for example, he says is a 15 percent 

### 13 difference in body weight under Prochloraz actually biologically 

### 14 important, relevant measure of an effect. So he goes on to talk 

### 15 about how he feels that from a statistical point of view, the 

### 16 protocol is incomplete until we see a power analysis that would 

### 17 help us identify biologically important differences for 

### 18 endpoints. 

### 19 **DR. MARTHA SANDY:**Thank you. Any other 

### 20 comments from Panel members? 

### 21 (No response.) 

### 22 Seeing none, I'll turn to EPA for questions of 

### 23 clarification. 

### 24 **DR. SIGMUND DEGITZ:**I have no clarifying 

### 25 questions, and I appreciate the comments. 

### 26 **DR. MARTHA SANDY:**Thank you. Dr. Touart,

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1 would you read Question 3, please. 

### 2 **DR. LESLIE TOUART:**Les Touart on Charge 

### 3 Question 3, and this is the first part, Question 3.1. The test 

### 4 protocol should be sufficiently detailed and should include a 

### 5 description of what is measured and how it is measured. 

### 6 The selection of endpoints within the assay 

### 7 should be reflective of the biological processes of interest and 

### 8 the endpoints should be intrinsically relevant and have 

### 9 established sensitivity. The test protocol should demonstrate 

### 10 the ability to measure the endpoints and provide adequate 

### 11 performance criteria for evaluation. 

### 12 Please comment on the selection, optimization 

### 13 and demonstration of the assay endpoints, as outlined in 

### 14 Sections 3 and 4 of the LAGDA ISR. 

### 15 **DR. MARTHA SANDY:**Dr. Denver. 

### 16 **DR. ROBERT DENVER:**I'm going to address each 

### 17 of the point selection, optimization and demonstration 

### 18 individually. So for selection, the assay endpoints are 

### 19 appropriate for evaluating chemical effects on growth and 

### 20 development of Xenopus larvae, and on gonad and thyroid gland, 

### 21 and urogenital tract organogenesis that may reflect endocrine 

### 22 disruption. 

### 23 Alterations in growth of larvae combined with 

### 24 clear liver and possibly also kidney histopathology may be 

### 25 scored as toxicity. Effects on growth, particularly at lower 

### 26 concentrations of test chemicals, and possibly without clear

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1 liver or kidney histopathology could indicate disruption of 

### 2 specific cellular signaling pathways that could be either 

### 3 endocrine or non-endocrine. 

### 4 A change in the timing of metamorphosis 

### 5 combined with thyroid histopathology and possibly also changed 

### 6 plasma T4 can signal chemicals that alter thyroid activity. 

### 7 Elevations in plasma vitellogenin in males or females, since 

### 8 females are immature and have low or nondetectable circulating 

### 9 vitellogenin can signal estrogenic activity as can the presence 

### 10 of testicular oocytes. 

### 11 Abnormal oviduct development can indicate 

### 12 androgenic activity. The oviduct morphology may be, as I said 

### 13 earlier, the best diagnostic endpoint for potential effects on 

### 14 reproduction. Comparison of phenotypic sex ratios with the 

### 15 genotypic sex ratios is potentially a very powerful endpoint for 

### 16 assessing effects compounds that may alter sex steroid 

### 17 signaling. 

### 18 The assay endpoints can support hypotheses for 

### 19 modes of action that involve disruption of the endocrine system, 

### 20 but may not be able to distinguish endocrine from non-endocrine 

### 21 modes of action, or disruption of specific cellular signaling 

### 22 pathways from overt toxicity. 

### 23 Optimization: the variability in results among 

### 24 laboratories that participated in the interlaboratory study 

### 25 suggests that some or all of the methods are not optimized, 

### 26 and/or that the guidelines for their execution can be improved.

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1 Assay endpoints appear to be very sensitive to 

### 2 animal husbandry and rearing parameters. The husbandry 

### 3 guidelines for these assays must be established by controlled 

### 4 studies and clearly defined in the protocols. Deviations from 

### 5 the husbandry protocols may disqualify the results, as we 

### 6 discussed yesterday. 

### 7 A source of variation that can impact 

### 8 optimization of the protocol is variation in the rearing water; 

### 9 using local water sources without standardizing for hardness, or 

### 10 iodine content, and there was wide range that was considered 

### 11 allowable under the guidelines of iodine, may lead to variation 

### 12 in the response to thyroid hormone disruption and growth 

### 13 parameters. 

### 14 Furthermore, potential endocrine disruptors in 

### 15 source water, if not removed by charcoal filtration, could lead 

### 16 to variation across testing regimes even if other issues like 

### 17 the feeding regime are standardized. Some of the 

### 18 interlaboratory variation in the LOECs may be related to water 

### 19 quality; for example, mineralization in kidney tubules seen 

### 20 occasionally may be related to water quality. 

### 21 There was a range of animal densities used in 

### 22 the different laboratories. This may not be an important issue, 

### 23 since as the EPA indicated, the flow rate is a more important 

### 24 variable than stocking density, but it would be good if the EPA 

### 25 could provide data to support this conclusion. 

### 26 Impaired growth, combined with liver and

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1 kidney histopathology may be robust endpoints for detecting 

### 2 toxicity; however, there are issues related to the 

### 3 histopathology that were identified in the ISR. 

### 4 Gonadal histopathology, particularly testes 

### 5 appears particularly robust. Ductal histopathology is a 

### 6 particularly important endpoint for supporting that a chemical 

### 7 can impact fertility and/or fecundity, since the potential for 

### 8 population level effects will not be directly addressed in the 

### 9 LAGDA. There are significant issues with the plasma 

### 10 vitellogenin and T4 assays that are addressed in subparts 2 and 

### 11 3 of this charge question. This is Part 3.1. 

### 12 Similar for the quail and Medaka assays, we 

### 13 considered that the lack of positive controls is a significant 

### 14 limitation to the LAGDA. Positive controls are important for 

### 15 evaluating whether the assay is behaving the way that it should 

### 16 be in the contract labs. However, there are many potential 

### 17 modes of action represented in the Tier 1 assays, and therefore, 

### 18 selecting an appropriate positive control may be challenging in 

### 19 many cases. 

### 20 In some cases it may be straightforward; for 

### 21 example, a compound that appears as a goitrogen in tier 1 can be 

### 22 compared to a well-known goitrogen such as methimazole or 

### 23 thiourea in the LAGDA. An apparent estrogenic compound can be 

### 24 compared to estradiol. 

### 25 The positive control chosen will help 

### 26 demonstrate that the assay is working for the predicted mode of

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1 action, but may not account for modes of action that are not 

### 2 predicted in the Tier 1 assays. So the positive control may not 

### 3 always be useful in the analysis. 

### 4 Demonstration: there were four chemicals 

### 5 analyzed in the interlaboratory validation studies. For some of 

### 6 the endpoints there was consistency across labs, at least for 

### 7 the presence of an effect, but the LOECs typically showed great 

### 8 variation among labs. For each of the four chemicals analyzed 

### 9 there were significant issues related to reliability and 

### 10 reproducibility of assay endpoints. 

### 11 Prochloraz is predicted to inhibit aromatase 

### 12 and act as an androgen receptor antagonist. There are recently 

### 13 published data that support that it can impact thyroid function 

### 14 in anuran amphibians. There was large variability among labs in 

### 15 the LOEC for thyroid histopathology. With Prochloraz, the LOECs 

### 16 ranged from 6.7 to 180 micrograms per liter. 

### 17 The effects on gonad and ductal morphology 

### 18 were more consistent among the labs, at least for the males, 

### 19 less so for the females. Plasma vitellogenin could not be used 

### 20 since it was below the limits of detection of the assay. 

### 21 4-tert-octylphenol is predicted to be a weak 

### 22 estrogen receptor agonist. Effects on thyroid endpoints were 

### 23 seen in only one lab, Lab A. Lab C reported an effect opposite 

### 24 to Lab A on time to metamorphic climax, possibly due to problems 

### 25 with animal husbandry. 

### 26 Most labs found effects on measures of

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1 reproductive development and function, but these were variable 

### 2 among labs; some found effects, some not, and the LOECs varied 

### 3 among labs when an effect was found. For example, effects on 

### 4 duct morphology in females varied from 6.25 to 50 micrograms per 

### 5 liter. That is, each lab found a different LOEC. 

### 6 17B Trenbolone is predicted to be a strong 

### 7 androgen receptor agonist. It was tested only in lab A and they 

### 8 found some effects on the testes and male and female ducts at 

### 9 the higher doses tested. 

### 10 Benzophenone-C is predicted to be an estrogen 

### 11 receptor agonist and inhibitor of thyroid peroxidase. It was 

### 12 tested only in Lab E. They reported dramatic effects on thyroid 

### 13 histology at the lowest dose, but increased time to metamorphic 

### 14 climax only at the highest dose, which was 6 milligrams per 

### 15 liter which exhibited significant toxicity. 

### 16 As for Prochloraz, these results question the 

### 17 relationship of thyroid histopathology to thyroid hormone 

### 18 production and the apical endpoint metamorphosis. There were 

### 19 clear effects of the lowest dose of Benzophenone-C on 

### 20 reproductive measures, supporting its predicted estrogenic 

### 21 activity. 

### 22 And I have some general comments on the 

### 23 histopathology which were raised with the other assays. 

### 24 Automated morphometric analysis could allow for quantitation of 

### 25 some, but perhaps not all histological features. It would also 

### 26 provide for greater objectivity in the analysis provided that

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1 the choice of histological planes; that is, their position and 

### 2 the number of representative sections in relation to the extent 

### 3 of the organ is standardized. 

### 4 For example, the size of organs like the 

### 5 thyroid, the testes, and the oviduct, and thyroid follicular 

### 6 cell height are potentially amenable to morphometric analysis 

### 7 that could be automated to some extent. 

### 8 Now, the interlaboratory analysis showed that 

### 9 there could be divergent results with liver histopathology among 

### 10 labs; this was seen in the Prochloraz trial. The basis for 

### 11 these differences may be that two histopathologists conducted 

### 12 the analyses, or that there were animal husbandry issues in the 

### 13 lab with divergent results, that is Lab C. The ISR authors 

### 14 suggested that this could be due to disease in Lab C animals. 

### 15 The two possible explanations illustrate that 

### 16 a better method, or better guidelines are needed for 

### 17 histopathological analyses, and that more comprehensive 

### 18 guidelines are needed for animal husbandry. The interlaboratory 

### 19 analysis intended to rely on one histopathologist to analyze 

### 20 tissue sections from each of the participating labs. 

### 21 In validating a method that will be applied in 

### 22 different settings it is important to demonstrate that it 

### 23 performs similarly across laboratories. To assess the validity 

### 24 of the assay, it is important that several histopathologists 

### 25 score the same sets of slides and that the results be compared. 

### 26 So given the uncertainty in the reason for the anomalies, it is

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1 not currently possible to determine if the liver histopathology 

### 2 is sufficiently robust and repeatable to be used in the Tier 2 

### 3 assay. 

### 4 **DR. MARTHA SANDY:**Thank you. The associates, 

### 5 Dr. Patino. 

### 6 **DR. REYNALDO PATINO:**Nothing needed to add. 

### 7 **DR. MARTHA SANDY:**Dr. Propper. 

### 8 **DR. CATHERINE PROPPER:**Nothing to add. 

### 9 **DR. MARTHA SANDY:**Dr. Blazer. 

### 10 **DR. VICKI BLAZER:**Nothing to add. 

### 11 **DR. MARTHA SANDY:**Dr. Christman. 

### 12 **DR. MARY CHRISTMAN:**I concur and just wanted 

### 13 to point out that part of the problem with trying to identify 

### 14 optimal endpoints is that we don't know if the lack of results 

### 15 or the inconsistent results are that they're not important 

### 16 endpoints or were simply underpowered. That was all I had to 

### 17 say. 

### 18 Now, Dr. Portier, his comments, he says 

### 19 upfront that he assumes that optimization refers not to the 

### 20 endpoints, but to whether the test concentrations were chosen 

### 21 appropriately, assuming those were appropriate endpoints. 

### 22 So he discusses the fact that he is not 

### 23 particularly sure that the choice of concentrations were, in 

### 24 fact, the best choices for biologically important differences. 

### 25 That they should cover maximum concentrations that would be 

### 26 expected to be seen under environmental contamination. That

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1 they should also elicit responses if there are responses to be 

### 2 seen. And he does not think that the choice of equal distance 

### 3 on a power or log scale is necessarily the best choice because 

### 4 you would not necessarily be able to identify non-monotonic 

### 5 responses. 

### 6 **DR. MARTHA SANDY:**Are there any other 

### 7 comments on Charge Question 3.1 from the Panel? 

### 8 (No response.) 

### 9 Seeing none, I'll turn it over to the EPA 

### 10 staff. Dr. Degitz. 

### 11 **DR. SIGMUND DEGITZ:**I have one clarifying 

### 12 question from some of the comments that were made in the 

### 13 previous charge questions. The point that reoccurs is this 

### 14 overt toxicity versus specific endocrine. I'm asking would the 

### 15 Panel provide us recommendations in later charge questions on 

### 16 methods to discern overt toxicity from endocrine toxicity. I 

### 17 guess I'm asking could they provide recommendations. 

### 18 **DR. ROBERT DENVER:**Short answer: "No." I 

### 19 mean, this point has come up various times throughout this Panel 

### 20 and we've discussed it individually. I don't think there are 

### 21 any good ways to distinguish that in these Tier 2 assays. 

### 22 As you pointed out yesterday, there is overlap 

### 23 in what might be considered an endocrine disruption in toxicity 

### 24 in many of the doses that are used for a lot to the chemicals 

### 25 that are being tested. Sorry. 

### 26 **DR. MARTHA SANDY:**That was Dr. Denver. I'll

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1 turn to Dr. Propper. 

### 2 **DR. CATHERINE PROPPER:**We discussed this 

### 3 quite a bit last night. Certainly if you see no liver 

### 4 histopathology, no kidney histopathology and then no ovarian 

### 5 ducts, it might be suggested of an endocrine mechanism, but the 

### 6 problem is going to be when you probably will not often get that 

### 7 kind of clean cut result. 

### 8 So again, it's more of a continuum kind of 

### 9 question and I don't have a full recommendation for how to sort 

### 10 that continuum out, but the other side of that, and I think 

### 11 you're the one that brought it up yesterday, is if you're 

### 12 looking at adverse outcome potential endpoints, it may be that 

### 13 figuring out that mode of action from a regulatory standpoint 

### 14 isn't as relevant as knowing that you have an adverse outcome 

### 15 and that that's really what needs to be focused on, is the 

### 16 potential for adverse outcome that has population level effects, 

### 17 which is really what the Tier 2 assays are supposed to support. 

### 18 So knowing exactly what the mode of action is, 

### 19 isn't as relevant as knowing that you have a potential adverse 

### 20 outcome. 

### 21 **DR. MARTHA SANDY:**Dr. Denver. 

### 22 **DR. ROBERT DENVER:**Could I just follow-up? I 

### 23 see the Tier 2 assays as a means to evaluate potential endocrine 

### 24 disruptive chemicals identified the Tier 1 assay for effects on 

### 25 development, physiology and reproduction. Period. 

### 26 I don't see these Tier 2 assays as necessarily

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1 providing any clear information on mode of action of endocrine 

### 2 disruption. 

### 3 **DR. SIGMUND DEGITZ:**I appreciate your 

### 4 comments and encourage you to -- in terms of this issue, any 

### 5 guidance you could provide to the Agency would be helpful. 

### 6 **DR. MARTHA SANDY:**That was Dr. Degitz. Are 

### 7 we ready to move on to Question 3.2? I believe so. 

### 8 **DR. LESLIE TOUART:**Les Touart reading the 

### 9 second part of Question 3, 3.2. Three of the four labs in the 

### 10 interlaboratory validation of the LAGDA were unsuccessful in 

### 11 properly performing the thyroxine (T4) analyses, using 

### 12 commercially available, enzyme-linked Immunosorbent assay 

### 13 (ELISA) kits with antibodies specific for the human or canine 

### 14 T4. 

### 15 Subsequently, the EPA developed an extraction 

### 16 method, Section 5.3.2, and presents a revised ELISA method for 

### 17 measuring T4 in amphibian samples in Appendix 7 of the LAGDA 

### 18 ISR. 

### 19 Please comment on the technical feasibility, 

### 20 reproducibility, and accuracy of the revised ELISA T4 

### 21 measurement method. Provide any recommendations regarding 

### 22 additional guidance to ensure the reproducibility of T4 

### 23 measurements. 

### 24 **DR. MARTHA SANDY:**Dr. Denver. 

### 25 **DR. ROBERT DENVER:**The contract labs were 

### 26 unable to accurately determine plasma T4 concentrations in

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1 metamorphic climax stage tadpoles using commercially available 

### 2 ELISAs. 

### 3 The ARMY lab reported plasma T4 concentrations 

### 4 in tadpole and juvenile frogs; however, the measures were highly 

### 5 variable and the values, at least for the juveniles, are much 

### 6 higher than plasma T4 concentrations reported in the literature 

### 7 in which radioimmunoassay was used. 

### 8 The Fort Lab also measured plasma T4 in 

### 9 tadpoles and juvenile frogs. The values were much lower than 

### 10 those from the ARMY lab, but were closer to the range reported 

### 11 in the literature for Xenopus laevis, I think. 

### 12 The Mid-Continent EPA lab in Duluth has 

### 13 developed an ethanol extraction protocol to remove interfering 

### 14 substances like plasma proteins that may enhance the ability to 

### 15 measure plasma T4 in tadpoles using ELISA. However, this method 

### 16 is still under development, as I understand, and therefore, 

### 17 insufficient information is currently available to determine the 

### 18 technical feasibility, reproducibility and accuracy of the 

### 19 revised extraction and T4 ELISA method. So that's answering the 

### 20 question, but I just want to add one other thing. 

### 21 Members of the SAP questioned the rationale, 

### 22 and thus the need for determining plasma T4 in the LAGDA. 

### 23 Beyond the technical difficulties of obtaining sufficient blood 

### 24 from small tadpoles and measuring plasma T4 by ELISA, published 

### 25 findings show that there is often large inter-individual 

### 26 variation in plasma and whole body thyroid hormone levels at

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1 metamorphic climax. Thus, with the small sample size it may be 

### 2 difficult to detect treatment effects. 

### 3 More importantly, assessing plasma T4 at 

### 4 metamorphic climax may not provide information on impacts on the 

### 5 thyroid function. That the animals have reached metamorphic 

### 6 climax; that is Nieuwkoop and Faber stage 62, generally 

### 7 indicates that they have produced sufficient thyroid hormone to 

### 8 support tissue transformations. They could have achieved this 

### 9 stage of development through compensatory mechanisms that may be 

### 10 evident in the thyroid histopathology, but maybe not in plasma 

### 11 T4. 

### 12 Hypertrophy of the thyroid may reflect 

### 13 compensation such that sufficient thyroid hormone was made to 

### 14 get to NF stage 62. It is also possible that a chemical will 

### 15 alter tissue sensitivity to thyroid hormone. It could affect 

### 16 thyroid hormone receptor levels or tissue monodeiodinases, as 

### 17 has been shown for hormones in some chemicals without affecting 

### 18 thyroid hormone production. 

### 19 Therefore, given the technical challenges in 

### 20 measuring plasma T4 and the limited interpretative power of 

### 21 measurements at the single stage of metamorphosis; that is, NF 

### 22 stage 62, the SAP recommends that the EPA consider eliminating 

### 23 plasma T4 measures from the LAGDA. 

### 24 **DR. MARTHA SANDY:**Dr. Patino. 

### 25 **DR. REYNALDO PATINO:**No further comments. 

### 26 **DR. MARTHA SANDY:**Dr. Propper.

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1 **DR. CATHERINE PROPPER:**No further comments. 

### 2 **DR. MARTHA SANDY:**Dr. Blazer. 

### 3 **DR. VICKI BLAZER:**No further comments. 

### 4 **DR. MARTHA SANDY:**Dr. Christman. 

### 5 **DR. MARY CHRISTMAN:**No further comments from 

### 6 myself or Dr. Portier. 

### 7 **DR. MARTHA SANDY:**Any other comments? Dr. 

### 8 Scott. 

### 9 **DR. GEOFFREY SCOTT:**Just a general comment 

### 10 about ELISAs and ethanol or solvent extractions. There is quite 

### 11 a bit of literature on other ELISA-based assays for contaminant 

### 12 measurements and other endpoints which have used ethanol or 

### 13 other solvent extraction methods. I would only point that you 

### 14 could look in the literature and see that that's not specific to 

### 15 this one, but in general. 

### 16 **DR. MARTHA SANDY:**Dr. Scanes. 

### 17 **DR. COLIN SCANES:**A couple of very minor 

### 18 points. The question has a problem within it. And that is, it 

### 19 says, "Antibodies specific for canine or human T4." T4 has the 

### 20 same structure in dogs, frogs, chickens, quail. It's really 

### 21 antibodies specific for the measurement of T4 in human and 

### 22 canine samples. I raise that just to be helpful to the EPA. 

### 23 **DR. MARTHA SANDY:**Any other comments from 

### 24 Panel members? I'll remind Panel members, if you've made a 

### 25 comment, you'll need to send that in writing to the lead 

### 26 discussant so he can capture that.

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1 Does the EPA have any questions of 

### 2 clarification? 

### 3 **DR. SIGMUND DEGITZ:**I have no further 

### 4 questions for clarification, and appreciate the well-constructed 

### 5 and thorough response. 

### 6 **DR. MARTHA SANDY:**Thank you. Dr. Touart, 

### 7 would you read Question 3.3 for us, please? 

### 8 **DR. LESLIE TOUART:**Charge Question 3.3. 

### 9 Vitellogenin (VTG) is an established biomarker of estrogenic 

### 10 exposure and is used as a key endpoint in endocrine disruptor 

### 11 testing. There is currently not a standardized commercial 

### 12 source of Xenopus laevis VTG antibodies for an ELISA. 

### 13 Please comment on the protocol for measuring 

### 14 and reporting VTG levels. Provide any recommendations regarding 

### 15 additional guidance to ensure the consistency, repeatability and 

### 16 reproducibility of VTG measurements. 

### 17 **DR. MARTHA SANDY:**Dr. Denver. 

### 18 **DR. ROBERT DENVER:**Plasma vitellogenin or 

### 19 liver vitellogenin messenger RNA are useful diagnostic tools for 

### 20 disruption of sex steroid signaling, particularly estrogenic 

### 21 signaling. 

### 22 There was large variation in the control 

### 23 plasma vitellogenin concentration measurements among the 

### 24 contract laboratories that conducted the assays. The reasons 

### 25 for the variation are unknown. Most were unable to detect 

### 26 vitellogenin, and this is likely due to the immaturity of the

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1 frogs at 10 weeks post-metamorphosis. 

### 2 So there are two issues to be addressed: 1) 

### 3 the performance of the vitellogenin ELISA, and the other is the 

### 4 inability to detect plasma vitellogenin in immature frogs. 

### 5 Now, regarding issue number two, the low 

### 6 circulating plasma vitellogenin at 10 weeks post-metamorphosis 

### 7 may allow for sensitive detection of estrogenic compounds 

### 8 compared with mature females, but this needs to be demonstrated. 

### 9 10 Regarding issue number one, the performance of 

### 11 the vitellogenin ELISA, it's difficult to know why the contract 

### 12 labs had trouble with the method, since assay performance 

### 13 metrics, for example, data on the standard curves, quality 

### 14 control samples were not provided; however, there are some 

### 15 ambiguities in the protocol that could have contributed to the 

### 16 problems, so I'll just list a few of those. 

### 17 The protocol says that at the end of the 

### 18 sampling, tubes should be centrifuged to remove the blood cells 

### 19 from the plasma. Guidance should be given on the g-force 

### 20 generated during centrifugation to avoid hemolysis. It's 

### 21 possible that some of the variance in the assays is due to 

### 22 hemolysis caused by centrifugation of the blood sample. 

### 23 The pre-absorption of the primary vitellogenin 

### 24 antiserum with control male serum could have introduced a 

### 25 variable that may have led to variation in the assay. It was 

### 26 not clear if this was done at the EPA Duluth lab or in the

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1 contract labs. 

### 2 Other potential sources of variability include 

### 3 preparation of collection tubes with aprotinin which requires 

### 4 lyophilization in a Speedvac. The normal goat serum used for 

### 5 blocking; different lots may give different results. 

### 6 Generating serial dilutions of the top 

### 7 standard as opposed to providing sets of prediluted standards as 

### 8 is typically done in commercial kits; inaccuracies caused by 

### 9 dilution of the samples, particularly the one to 300 (1:300) 

### 10 dilution that relied on pipetting only one microliter of plasma, 

### 11 which is not very accurate. 

### 12 The ABC Laboratories used a fortified 

### 13 interassay quality control sample which was freshly prepared 

### 14 each day of analysis by spiking pooled male Xenopus plasma with 

### 15 vitellogenin from the same source as was used to prepare the 

### 16 standards. And this was done to a nominal concentration of two 

### 17 micrograms per deciliter. So such quality control samples could 

### 18 be incorporated into the assay. 

### 19 However, this same lab accepted a coefficient 

### 20 of variation of up to 20 percent between assays, the interassay 

### 21 coefficient of variation, which is quite high. Most samples 

### 22 analyzed by this lab were nondetectable, although they are 

### 23 reported as half the minimum detectable value. And that is 

### 24 presumably to provide a number for statistical analysis; 

### 25 although, in fact, they are nondetectable. 

### 26 The minimum and maximum detection limits on

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1 the different plates varied widely, and samples from a single 

### 2 LAGDA trial were run on different plates on different days. 

### 3 Now, this could introduce an important confound if the samples 

### 4 were detectable since there was wide variation in the general 

### 5 parameters of the assay and just how it was set up. So I think 

### 6 more guidelines on how the assay is actually set up, especially 

### 7 in labs that don't have experience running these types of assays 

### 8 is important. 

### 9 However, members of the SAP agree that 

### 10 measuring plasma vitellogenin is an important endpoint for 

### 11 detecting estrogenic activity, but this requires a robust assay 

### 12 method that laboratories contracted to conduct the LAGDA can 

### 13 perform. 

### 14 Alternatively, a contract assay laboratory 

### 15 should be identified with expertise to process samples from the 

### 16 contract LAGDA labs, which might be preferable. The SAP 

### 17 recommends that the EPA consider identifying a contract assay 

### 18 laboratory to run the vitellogenin ELISA, or alternatively, 

### 19 identify a company willing to develop and market a vitellogenin 

### 20 ELISA kit with a standardized protocol. 

### 21 **DR. MARTHA SANDY:**Any additional comments 

### 22 from the associates? Dr. Patino. 

### 23 **DR. REYNALDO PATINO:**No additional comments. 

### 24 **DR. MARTHA SANDY:**Dr. Propper. 

### 25 **DR. CATHERINE PROPPER:**An alternative might 

### 26 be real-time PCR for liver vitellogenin as well.

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1 **DR. MARTHA SANDY:**Dr. Blazer. 

### 2 **DR. VICKI BLAZER:**I have no additional 

### 3 comments. 

### 4 **DR. MARTHA SANDY:**Dr. Christman. 

### 5 **DR. MARY CHRISTMAN:**I have no additional 

### 6 comments. Dr. Portier does, though. I'm just going to read 

### 7 what he says. VTG, at least as measured in Xenopus laevis, 

### 8 seems to have nice statistical characteristics, making it easy, 

### 9 within the size protocol used in these studies to demonstrate a 

### 10 dose response. 

### 11 I did notice a slight increase in variances as 

### 12 the mean increased, suggesting that the data may need to be 

### 13 transformed or the transformation used was not adequate to 

### 14 adjust for this pattern in the data. 

### 15 **DR. MARTHA SANDY:**Dr. Propper. 

### 16 **DR. CATHERINE PROPPER:**I thought of one more 

### 17 thing. This assay in particular does have value as being on 

### 18 that's probably a nontoxic and a direct endocrine measure. So 

### 19 for that, I think this is one of those places where it will have 

### 20 value. So it just needs to be gotten right. 

### 21 **DR. MARTHA SANDY:**Dr. Christman. 

### 22 **DR. CHRISTMAN:**Actually, that does make me 

### 23 think of one more thing. If in fact this particular endpoint 

### 24 has responses as sort of on a threshold level, then some serious 

### 25 thought needs to be given to how to optimize the experiment to 

### 26 capture exactly that effect.

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1 **DR. MARTHA SANDY:**Any other comments from the 

### 2 Panel? 

### 3 No. Dr. Degitz, any questions? 

### 4 **DR. SIGMUND DEGITZ:**Yes. I have one 

### 5 clarifying question. You made mention of evaluating gene 

### 6 expression. Does the Panel intend to provide more detailed 

### 7 advice on that in your report? 

### 8 **DR. CATHERINE PROPPER:**In regards to that, I 

### 9 did look into the literature to see if there has been 

### 10 well-documented development of vitellogenin real-time PCR assay 

### 11 for Xenopus because these do have to be done fairly species 

### 12 specifically, and there was not. So no, at the moment, I don't 

### 13 have a recommendation for how to go about that, other than with 

### 14 very standard mechanisms for developing those assays. But Bob 

### 15 may know something I don't know. 

### 16 **DR. ROBERT DENVER:**In fact, at a conference I 

### 17 was at just last month, there was a lab that reported 

### 18 vitellogenin real-time qPCR assay. We develop them in my lab 

### 19 all the time, so I think it is fairly straightforward, as do 

### 20 also. 

### 21 **DR. SIGMUND DEGITZ:**I guess I should clarify. 

### 22 Is the Committee going to recommend that we do that? 

### 23 **DR. CATHERINE PROPPER:**So we haven't spoken 

### 24 as a group about that as a recommendation. And maybe we can do 

### 25 so later today and come back to you tomorrow, unless my opinion 

### 26 is agreed upon here and now which is my recommendation is to

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1 choose one or the other and go for it and just do it well. And 

### 2 which one, they should both give you an assay that's viable. It 

### 3 would take a lot of validation to see if one was more sensitive 

### 4 than the other. 

### 5 So at this point, I'm reluctant to make a 

### 6 recommendation. If other folks would like to discuss this 

### 7 later, we can, and then come back to you. 

### 8 **DR. ROBERT DENVER:**I agree. We'll discuss it 

### 9 and get back to you because I understand it would require, 

### 10 perhaps additional animals to collect the tissues, given what's 

### 11 in the assay right now. 

### 12 **DR. SIGMUND DEGITZ:**Thank you. 

### 13 **DR. MARTHA SANDY:**Dr. Touart, I believe we're 

### 14 ready for Charge Question 4. 

### 15 **DR. LESLIE TOUART:**Les Touart reading Charge 

### 16 Question 4. Demonstration of the test method performance should 

### 17 be based on the testing of reference chemicals representative of 

### 18 the types of substances for which the test method will be used. 

### 19 Test substances should adequately represent an appropriate range 

### 20 of responses and physical chemical properties for which the test 

### 21 method is proposed to be appropriate. 

### 22 The selection of the most appropriate 

### 23 statistical approach depends, in part, on the nature of the data 

### 24 and also on the design of the validation study. Statistical and 

### 25 non-statistical methods used to analyze should be described. 

### 26 Please comment on the selection of test

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1 substances and methods, analytical and statistical where 

### 2 appropriate, chosen for the demonstration and validation of the 

### 3 LAGDA assay. 

### 4 **DR. MARTHA SANDY:**Dr. Christman is the lead. 

### 5 **DR. MARY CHRISTMAN:**Yes. Hello. As was done 

### 6 for the other two assays, I will defer for the test substances 

### 7 in the analytical methods to my esteemed colleagues. 

### 8 Secondly, I'm very visual. So I have a 

### 9 PowerPoint. There are no pictures, though. I apologize; it's 

### 10 all words, which is really boring, but it helps me to remember 

### 11 things. 

### 12 I would like to start by reiterating 

### 13 everything that has been said to date about the statistical 

### 14 methodologies as valid here as well. There are, of course, 

### 15 minor differences. This is not a multigenerational study, so 

### 16 some of those questions are irrelevant. 

### 17 I also have some additional points I want to 

### 18 bring out, on top of what's already been discussed. And in some 

### 19 cases, they just remind you of what's been discussed. So I'm 

### 20 boring you, just take a nap for a minute and then come back. 

### 21 I apologize. This is probably not as well 

### 22 organized as it ought to be, but it was 11:00 last night when I 

### 23 decided that I needed to have a PowerPoint, so bear with me. 

### 24 I'm representing comments from me, discussions 

### 25 with Dallas and Dr. Portier. So Dr. Johnson and Dr. Portier, 

### 26 I'm simply doing a presentation. There are some things, though,

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1 as I said, that are very specific to LAGDA and there are some 

### 2 things that are more generic for statistical methodology. 

### 3 Just as a reminder, there are three different 

### 4 kinds of data types in this analysis, in this particular assay 

### 5 rather. There are the truly continuous ones, like length or 

### 6 weight, even though we may measure them on units, like 

### 7 centimeters or something. Then there are truly discreet 

### 8 variables as well, which are things like counts or the number 

### 9 that are dead out of end observed. And then finally, there is 

### 10 the histopathology ordinal categorical. 

### 11 Now, these should all be handled distinctly. 

### 12 The continuous variable should be analyzed according to 

### 13 probability distributions that are continuous. The discreet 

### 14 ones should be analyzed according to the distribution that 

### 15 discreet random variables take on. 

### 16 And finally, ordinal categorical should be 

### 17 handled by categorical techniques, such as the (inaudible) that 

### 18 has been described by Dr. Green in the documentation provided, 

### 19 or other models such as Cumulative Logit Models or what are also 

### 20 known as Proportional-Odds Models. 

### 21 Part of the problem is that the current 

### 22 statistical methods, as you've heard repeatedly, have a tendency 

### 23 to treat the data, any quantitative data as continuous and its 

### 24 default is to look for transformations to force things to be 

### 25 cookie-cutter normal homogeneous variance. Statistical 

### 26 methodology has moved beyond that nowadays. We have the

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1 computing capability to handle data as it really should be 

### 2 handled. That optimizing power. The same reason why you use a 

### 3 normal distribution when things aren't normally distributed 

### 4 because it optimizes power. 

### 5 The way it's set up, what happens is that when 

### 6 normality fails, the next-test is to go to something like a 

### 7 non-parametric, but the problem is that the decision tree that 

### 8 is described by, say, StatCHARRMS, one differs among labs and 

### 9 among assays. What some people do as the decision tree isn't 

### 10 even the same. So you've got people who are using completely 

### 11 different approaches. It's not unlike the one lab that decided 

### 12 to feed their Xenopus twice as much as all the other labs. I 

### 13 mean, it's the same kind of thing where you have variations in 

### 14 the protocol. 

### 15 This leads to loss of power. The other thing 

### 16 is, you're not using your biological expert opinion and 

### 17 knowledge and expertise to inform your ability to analyze the 

### 18 statistics today. This is the same thing that Dallas Johnson 

### 19 was saying that you should be taking advantage of the historical 

### 20 knowledge of the distribution of these endpoints and using that 

### 21 information in the methods. It's so much more powerful when you 

### 22 know what you're doing. Sorry. 

### 23 Part of the problem too is that, you know, we 

### 24 all have our own terminology. Statisticians have a terminology 

### 25 and non-statisticians have a terminology. The way the 

### 26 statistics are described in the ISR, I'm sorry; in some cases

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1 they're just downright wrong. I think what it is, is that they 

### 2 don't understand that this test does this as opposed to doing 

### 3 that and it's just a matter of -- so if a statistician is 

### 4 reading it, I'm sitting here saying, you can't possibly be doing 

### 5 the right thing. 

### 6 So how can you understand whether these 

### 7 endpoints or useful or not or whether the study has been 

### 8 validated or not if you can't even understand what the 

### 9 statistical methods are? 

### 10 I've noticed this for the LAGDA. I've only 

### 11 really read two of them, but in the case of the LAGDA, the ISR, 

### 12 at least, I found confusing. I don't mean this in a negative 

### 13 way. It's literally just a matter of my language versus 

### 14 somebody else's language. It would be like me trying to speak 

### 15 French; it definitely would not work. And I give an example 

### 16 there. 

### 17 I have a whole list of those sorts of things 

### 18 in the write-up, so I'm not going to go into them. But the 

### 19 other thing I want to point out that the way that the 

### 20 methodologies are described varies among the various documents 

### 21 that were provided, which makes it that much more difficult for 

### 22 me to understand what's being done. And that's an example of 

### 23 where we're using different decision trees. 

### 24 You have one lab that decides that they'll go 

### 25 non-parametric if at least half of the treatments are not 

### 26 normally distributed, which is different than statCHARRMS's

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1 decision to go to non-parametrics. So this yields all sorts of 

### 2 problems, I think. Basically, how do you compare results among 

### 3 labs when one lab is using a really liberal test that tends to 

### 4 reject more easily than the stated alpha and another lab is 

### 5 using a real conservative test? 

### 6 That might be one reason why you're seeing 

### 7 differences in the LOECs among labs. One is like, oh, yeah, 

### 8 sure. That's good. And the other one is going oh, no. We have 

### 9 to have really strong evidence. So I find that it's difficult 

### 10 to compare among labs as a result. 

### 11 The other thing I wanted to point out is that, 

### 12 you know, we think that by having an auto option in StatCHARRMS, 

### 13 where you simply let it default to the decision tree is 

### 14 objective. But it's not. It's completely data-driven, which 

### 15 means that every dataset you get is going to be a whole new 

### 16 route for the decision tree, which makes it extremely difficult 

### 17 to determine what the true Type I error is for this particular 

### 18 method. 

### 19 Type I error is based on the idea of repeated 

### 20 long-term repetitions of the same experiment with new data each 

### 21 time. That assumes you're using the exact same methodology as 

### 22 well. If you start varying methodology, depending on what your 

### 23 data happen to show you, one data point, all it needs is to be 

### 24 over one thing and you're suddenly in the non-parametrics. You 

### 25 don't know what your Type I error is anymore. That causes all 

### 26 sorts of problems I think.

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1 This is one thing I find a little confusing 

### 2 and I'm sure you guys can help me. There is no statistical 

### 3 interlab comparisons done, except for the mysids. It's the only 

### 4 I've seen this. All of the study to determine whether there is 

### 5 reliability and robustness among laboratories is based on 

### 6 comparing CVs, visually, descriptively, or looking at the means 

### 7 or looking at whether they reject or accept and what direction 

### 8 their rejection or acceptance is for the various tests. 

### 9 From a statistician's point of view, that's 

### 10 not evidence. Evidence is if I actually test whether the labs 

### 11 are reproducing the same sorts of results. So it may not be 

### 12 possible for you to do that with these particular procedures 

### 13 because you don't necessarily have enough information to be able 

### 14 to do it, but I urge that you think in future that if there were 

### 15 going to be redesigns of these standardized techniques and 

### 16 experimental designs that some thought be given to that ultimate 

### 17 goal of actually testing whether the labs are showing 

### 18 reproducibility. 

### 19 Rather than simply describing the control 

### 20 result and saying oh, look, all the labs stay within 20 percent 

### 21 CV or their range of CVs is nice. I would instead argue that 

### 22 you need something a little more statistically relevant. 

### 23 You've heard this repeatedly. I think you 

### 24 guys are underpowered with your sample sizes. That may not be 

### 25 something that you can correct by just adding additional 

### 26 animals. If there is no density dependence, then maybe you

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1 could add a few more into the tanks or something, but if there 

### 2 is, then we have to think of other ways to increase our power. 

### 3 I've mentioned one already, which is to use the best test for 

### 4 the data, which means you have to bring your expert knowledge to 

### 5 identify what that best-test is. 

### 6 The easy one is we'll do a power analysis and 

### 7 just add some more replicates. And of course, that's the one 

### 8 that you all cringe at because it's expensive to add replicates, 

### 9 or you have to add lab facilities or whatever. So we want to 

### 10 think about other ways we can increase power here. 

### 11 Well, it may be that you need to consider -- 

### 12 and this was brought up earlier -- that you need to consider all 

### 13 the sources of variance that are being introduced and then the 

### 14 experimental design, that has already been said, among 

### 15 individuals. So that's biological variance, among tanks, tanks 

### 16 within treatments among the labs. So you want to think of those 

### 17 four sources. But there is additional variance that's 

### 18 introduced by the lack of the statistical test -- all right. So 

### 19 I did that already. 

### 20 One of the things that I'm surprised is 

### 21 everything is analyzed independently. If it's a gender-specific 

### 22 endpoint, we analyze it for that gender. Female lengths are 

### 23 analyzed separately from male lengths. One of the things you 

### 24 learn in an experimental design class is this technique or this 

### 25 idea of what's known as hidden replication. If you use a more 

### 26 complete model, like include gender as an effect in the model,

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1 you actually gain power in some ways because what you have now 

### 2 is if you have homogeneous variances, even if the means are 

### 3 different. 

### 4 If you have homogeneous variances, your 

### 5 estimate error of variance, the error, is increased because you 

### 6 have more replication. Because you have both the males and the 

### 7 girls in there as part of the leftover noise. You have more 

### 8 tanks to get tank variability, so you increase your replication, 

### 9 even though your testing the female length in Treatment 1 

### 10 against the female length in Treatment 2. It's the denominator 

### 11 of your test that you're getting more precision. 

### 12 So you might want to consider altering your 

### 13 statistical methodologies to capture some of that. That's an 

### 14 easy way to possibly -- I mean, I'm not saying that his has to 

### 15 be done; I'm saying that this is something that if you have a 

### 16 statistical intern sitting around, put them on this task of 

### 17 taking the data and playing with it, basically, to see if you 

### 18 can gain some power this way. 

### 19 Ultimately, what this is it's a plea to 

### 20 rethink your statistics, basically. Other things you could do 

### 21 is, as I said, identify the sources of uncertainty. I've 

### 22 already talked about what we think of as the experimental design 

### 23 sources of uncertainty, but there are additional sources. There 

### 24 is the lack of comparability of statistical methodology among 

### 25 the labs when they do it independently. That needs to be 

### 26 corrected to reduce that uncertainty. You're introducing added

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1 uncertainty by doing that. 

### 2 Another thing is, as has been pointed out 

### 3 repeatedly, the protocols are not always followed. Maybe you 

### 4 need to send something emphasizing that you're not going to get 

### 5 good reproducibility if you don't put more emphasis on the need 

### 6 to follow the protocols more explicitly. 

### 7 One of the things -- sorry. I don't know how 

### 8 important this is because I know the tendency is to use things 

### 9 like the having of the dose response and the choice of the 

### 10 maximum dose is based on range-findings studies and things of 

### 11 that nature, but if you have a priori belief of a monotonic, 

### 12 linear increase in the dose response, then you should set your 

### 13 choice of concentrations appropriately. 

### 14 If you believe that is it sigmoidal curve, 

### 15 then you should set your concentrations accordingly. You could 

### 16 probably save, if you could decrease the number of treatment 

### 17 levels, you could probably put those replicates to one of the 

### 18 other treatments and increase your power to see a difference. 

### 19 Of course, it relies on this a priori belief, which you need to 

### 20 be fairly strong about, otherwise you've just wasted a lot of 

### 21 time on your experiment. But it's something to think about. 

### 22 And this is actually a question I have, which I think might be a 

### 23 source of variation. 

### 24 In our discussions with the mysids, someone 

### 25 commented about how even just 24 hours can change the response 

### 26 of the animal because they molt in that particular case. Well,

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1 in the case of the frogs, other than going from a tadpole to a 

### 2 frog, I don't think there is any great -- I don't know. I'm not 

### 3 a biologist. I'll let you guys figure it out. 

### 4 But the way the experiments are set up is that 

### 5 you have things happening at different rates for the different 

### 6 animals and is it possible that that's causing some of the 

### 7 uncertainty and should the covariate be included in the models 

### 8 to somehow capture that source of noise and adjust it out? Just 

### 9 something to think about. 

### 10 This has been mentioned repeatedly, I don't 

### 11 completely understand the description of the protocol of how it 

### 12 should be performed. If I had to follow that, I'd probably 

### 13 screw it up. Let's put it that way. So I think it needs to be 

### 14 made more clear. 

### 15 One of the problems, and you saw yesterday 

### 16 when I asked about the experimental units is clearly, the 

### 17 protocol wasn't very clear about what the experimental units 

### 18 were, what the measurement units were. This is Ken's comment. 

### 19 What was the measurement being made on? 

### 20 Was it being made on an individual or a tank? 

### 21 What is your ultimate goal with respect to the ecological risk 

### 22 assessment? What is your population unit that you want to 

### 23 describe the behavior for? It's not a tank. It's usually an 

### 24 individual animal. 

### 25 So you want to think in terms of what you're 

### 26 measuring, what you're analyzing, and what is your replicate

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1 with respect to data analysis. 

### 2 So we saw that that was part of the problem 

### 3 with StatCHARRMS; the problem with going to the tank replicate 

### 4 was because this wasn't explicitly stated or analyzed then 

### 5 either. 

### 6 There are so many pages of methodology and 

### 7 then the statistics that's half-a-page. Well, it's not an 

### 8 afterthought, guys; it's a major part of the study. At least I 

### 9 think it is. Being a statistician, I want to say that. 

### 10 As I said, there are some issues about the 

### 11 methods being incorrectly applied. They're not accounting for 

### 12 the nesting structure correctly to get some additional sources 

### 13 of information. 

### 14 Oh, this is one that hasn't been mentioned 

### 15 yet, which I think is a problem. When you're doing the time to 

### 16 event, in this particular case, they're looking at time to stage 

### 17 NF 62, I think it is, the way the method is described is you do 

### 18 a Kaplan-Meier product estimator, which you look for the 

### 19 survival curve, essentially. 

### 20 In this case, "survival" means survival to 

### 21 stage 62. It doesn't mean they died. And you use this curve, 

### 22 you pull the median off of it and you then treat that as your 

### 23 response variable and you analyze it. Well, that's like using 

### 24 the tank mean without recognizing that that mean is itself, 

### 25 variable because there is some -- it's based on a set of data. 

### 26 So the appropriate test is actually the Wallis

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1 (phonetic) rank test; not pretending that that median is a 

### 2 number that I can throw into an ANOVA. So that's an example of 

### 3 where there's misuse of the analysis. 

### 4 Just to reiterate: randomization, 

### 5 randomization, randomization. Very important. One of the 

### 6 things is the power analyses that I've seen done, I didn't see 

### 7 one for LAGDA, but the power analyses I've seen done have talked 

### 8 about the percent difference detected with the given data. 

### 9 Instead, I would argue that you need to actually be trying to 

### 10 determine what the appropriate sample size is and whether you 

### 11 meet that condition or not. 

### 12 And that if you do a power analysis, please do 

### 13 the right one. The power analysis I saw assumes that all you're 

### 14 doing is a two-sample T-test, but that's not what you're doing. 

### 15 You're doing a Jonckheere-Terpstra Trend Test or a Dunnett, 

### 16 which is totally different. And as a consequence, it has a 

### 17 difference source of error in the denominator. It has a 

### 18 different-testing structure that's not being accounted for in 

### 19 the power analysis. 

### 20 The power analysis is pretending and all 

### 21 you're doing is comparing two means, which is not even remotely 

### 22 what you're really doing. You're trying to get this overall 

### 23 picture of behavior. And if you're trying to get an overall 

### 24 picture of behavior, you have think in terms of all the tests 

### 25 that go into that overall picture. 

### 26 So I would argue that if you are going to do a

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1 power analysis, do it for the actual testing structure that 

### 2 you're planning on performing; base it on that. So as an 

### 3 example, if I'm doing a t-test of two means, I use the typical 

### 4 alpha .05, power of .8, I get an "n" of 17 in each of my two 

### 5 groups. But if I'm doing a one-way ANOVA with three levels and 

### 6 I want the same -- I want to know how many observations do I 

### 7 have to have in order to see an difference of this amount, the 

### 8 same amount as my two sample t-test, because I'm using -- is 

### 9 there a pointer on this? Oh, wait; this might be the pointer. 

### 10 You know that Dunnett's or Bonferroni-Holm 

### 11 step-down procedure, or any of those, adjusts the alpha that you 

### 12 are testing at. So even to adjust a simple thing like this, 

### 13 adjust the alpha, I went from 17 to needing 20 to see a 

### 14 difference of 10. And if I'm doing something like an LOEC, 

### 15 based on this kind of simple test, to see a difference between 

### 16 control and Dose 1, when they're apart by five units, I need 78 

### 17 observations. And I'm not even getting into nesting structure 

### 18 here. This is just the simplest. 

### 19 So my point here is that you need to be 

### 20 calculating power based on your actual methodology, not on a 

### 21 simple t-test that's a hand waiver. And that's it. That's 

### 22 enough. I apologize for taking so long. 

### 23 Oh, and just quickly, Ken's comments are 

### 24 incorporated. His only other comment was "See everything I said 

### 25 about the MMT." 

### 26 **DR. MARTHA SANDY:**Thank you very much.

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1 Rather than going all through the names, if anyone has any 

### 2 additional comments on the statistical methods, raise your hand. 

### 3 (No response.) 

### 4 None seen. All right. Oh, yes? You're 

### 5 welcome to comment. 

### 6 **DR. DALLAS JOHNSON:**Thank you. I don't know 

### 7 whether you are going to be able to put up a slide that I 

### 8 prepared or not, but I'm not ready for it yet, anyway. 

### 9 I just had a few additional comments, I think. 

### 10 Again, they're kind of in the order of the report. One thing I 

### 11 wanted to mention is when I teach classes, in terms of designing 

### 12 and experiments, there has been a lot of confusion between what 

### 13 is the treatment structure and what is the way the 

### 14 randomizations are taking care of. 

### 15 So the terminology here is an experimental 

### 16 design is not my definition of experimental design. 

### 17 Experimental design is used in the document is really the 

### 18 treatment design; how the treatments are going to be compared. 

### 19 What things we're going to look at and what the analysis units 

### 20 are going to be and so on, and there is nothing about the design 

### 21 itself. 

### 22 So I would like to encourage people to think 

### 23 of design structure and treatment structure. And you can almost 

### 24 use any treatment structure with almost any design structure. 

### 25 So they're really separate. I think if you try to separate 

### 26 those and think of them in that way, what are we going to study,

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1 is treatment structure; how are we going to study is design 

### 2 structure. So I would just encourage people to think about that 

### 3 when you write up a plan or a standard operating procedure or a 

### 4 guideline for people to follow. 

### 5 I did notice that in this particular report, 

### 6 there is a bit more attention paid to the type of data that is 

### 7 being collected and the analysis that is going to be done. It 

### 8 said that there were statements in there that indicated that 

### 9 mortality data that is expressed as a percentage that died 

### 10 should be arc-sine square root transformed. Time to 

### 11 metamorphosis data should be treated as time to event data. And 

### 12 then some other things, liver weight was taken as a proportion 

### 13 of the total bodyweight. So there was some things in this ISR 

### 14 that showed the science, it sort of gave a little bit of a lead 

### 15 into the way the data should be analyzed. That's the kind of 

### 16 thing that I've been trying to encourage when I was talking 

### 17 yesterday about -- and what Mary and I has been talking about 

### 18 today to think about how the data are collected and how it's 

### 19 historically analyzed and try to pick the different analyses 

### 20 that are similar so that the analysis is associated with the 

### 21 type of data that is collected and not the data that's actually 

### 22 collected in a particular study. 

### 23 **DR. MARY CHRISTMAN:**Dallas, this Mary 

### 24 Christman. Peer review. 

### 25 **DR. DALLAS JOHNSON:**Oh, yeah. I guess Mary 

### 26 and I talked about this over lunch is whether the methodology --

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1 my understanding is, at least, is that StatCHARRMS is sort of a 

### 2 series of SAS Macro. So SAS Macro that you sort of run data 

### 3 through and it actually uses SAS programs to do the analysis. 

### 4 So we had a question about whether that 

### 5 process has been peer reviewed or not. And if it hasn't, 

### 6 perhaps it should be peer reviewed. And particularly, if you're 

### 7 going to create a new package in AR, that it would be important 

### 8 to have that peer reviewed before you put too much weight in the 

### 9 use of it. I wanted to see if you could put up my slide. 

### 10 **DR. MARTHA SANDY:**I think they have it up. 

### 11 We'll get it. 

### 12 **DR. DALLAS JOHNSON:**Yesterday we talked about 

### 13 the corrected analysis that was being done, the Medaka used a 

### 14 tank as a random effect and did the analysis that way. And I 

### 15 mentioned this morning that that analysis appears to be correct. 

### 16 And in this case, they did an analysis on the tank means, but 

### 17 they corrected it by doing weights. And it's the DEJ comments 

### 18 -- well, that's the one that's there. 

### 19 I just wanted to say that the analysis, using 

### 20 weights is not correct and I wanted to try to give an indication 

### 21 of why. Is it going to open? 

### 22 **DR. MARTHA SANDY:**I'm understanding that they 

### 23 cannot open up a WordPerfect file. 

### 24 **DR. DALLAS JOHNSON:**It's not WordPerfect. It 

### 25 should be Word. It's got some formulas in it, probably. 

### 26 Well, let me describe it. If I describe it

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1 well enough, maybe it'll be in the document. 

### 2 **DR. MARTHA SANDY:**Excuse me, Dr. Johnson. If 

### 3 you could give it to Sharlene and she'll try and convert it to 

### 4 something else. 

### 5 **DR. DALLAS JOHNSON:**It's in Word. 

### 6 **DR. SHARLENE MATTEN:**Yeah, but I have to 

### 7 convert it to .pdf. 

### 8 **DR. DALLAS JOHNSON:**Pardon? I'll put it up 

### 9 as a .pdf file. 

### 10 **DR. SHARLENE MATTEN:**Well, if you want to 

### 11 convert it, we can put it up as a .pdf or we can cut it and put 

### 12 it in a PowerPoint. 

### 13 **DR. DALLAS JOHNSON:**We can come back to this 

### 14 later if you want. During the break we can fix it up. 

### 15 **DR. SHARLENE MATTEN:**Right. Well, this 

### 16 particular computer was out of commission for a few months. 

### 17 **DR. DALLAS JOHNSON:**I'll create a .pdf file 

### 18 and we can come back to it later. 

### 19 **DR. MARTHA SANDY:**So let's take a short break 

### 20 for five minutes so that we can get this converted to a .pdf so 

### 21 we can all see it. 

### 22 (Whereupon, a break was taken.) 

### 23 **DR. MARTHA SANDY:**Okay. Can we reconvene? 

### 24 The dozen or so Panel members that need to come back in the 

### 25 room, please come and we will start. We have our quorum. So we 

### 26 left off on Question 4 for the LAGDA assay. We have Dr. Dallas

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1 Johnson's calculations on the screen because we had to wait for 

### 2 the Word updates and now we've converted it to .pdf. 

### 3 **DR. DALLAS JOHNSON:**Thank you. I wanted to 

### 4 put this up there so you could see what us statisticians really 

### 5 like to do. We have our own little nerdy, geeky things. 

### 6 So I wanted to sort of describe, I guess, why 

### 7 the weighted analysis is wrong. If we have a model for the 

### 8 data, we have the response that has taken on an individual 

### 9 animal, in this case a tadpole or a frog, and then it could be 

### 10 modeled by some overall mean which changes, depending on the 

### 11 dosage included. And then there is an error component given by 

### 12 the tank that it has to be in. 

### 13 So delta IJ ($ij) represents the variability 

### 14 in the tank and then we have variability with respect to the 

### 15 animal's stealth that is denoted by the Eijk. So we have I 

### 16 equal one, two up to the number of doses, J one, two up to the 

### 17 number of tanks and then K is the sample size that's within a 

### 18 tank. It goes from one, two up to NIJ. 

### 19 So delta IJ is a random tank effect. Eij is a 

### 20 random error component associated with the animal. So if you 

### 21 look at the tank mean, which I denoted as Yij dot bar and follow 

### 22 this through, it's equal to the overall mean plus the delta IJ, 

### 23 plus the average of the Eijs. K is the error. And if you look 

### 24 at the variance of one of these, in the variance it's sigma 

### 25 squared delta (32$), to branch the delta term, plus the variance 

### 26 of the error divided by the sample size.

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1 So when you do a weighted analysis and you use 

### 2 the sample size as a weight, then it only affects one of these 

### 3 components of variance. It only effects the E sigma squared E 

### 4 ([(32E)]E) part. So if sigma squared delta is zero [(32$)= 0], 

### 5 then the weighted analysis is going to be correct. But if is 

### 6 sigma squared delta (32$) is not zero, which it very seldom will 

### 7 be, then the weighted analysis would be incorrect. 

### 8 The analysis that was used in the Medaka -- I 

### 9 can't even think of how you pronounce it anymore. In the little 

### 10 fish study, the reanalysis there did take into account, used 

### 11 this model up here by putting in the tanks, effects as random. 

### 12 So that analysis ended up being correct, but this analysis if 

### 13 you use weights, will only be correct if sigma squared delta 

### 14 (32$) is zero. 

### 15 Now, will it change any of your conclusions? 

### 16 I don't think it probably does. So I'm guessing that the report 

### 17 that was given and the conclusions that were made in it are 

### 18 probably okay, but if you're going to rewrite or adjust the ISR 

### 19 for this study, you ought to go ahead and reanalyze again, using 

### 20 the appropriate model and the appropriate analysis. Thank you. 

### 21 **DR. MARTHA SANDY:**Any additional comments on 

### 22 the statistical analysis from the Panel? 

### 23 (No response.) 

### 24 No? Then I'll turn it over to the EPA for any 

### 25 clarifying questions. 

### 26 **DR. SIGMUND DEGITZ:**Clarifying question, huh?

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1 I have no clarifying questions. Look forward 

### 2 to the advice that we get in the report. 

### 3 **DR. MARTHA SANDY:**Okay. 

### 4 **DR. SIGMUND DEGITZ:**I have no clarifying 

### 5 questions. 

### 6 **DR. MARTHA SANDY:**That's great. Thank you. 

### 7 I will turn to the other Panel members then for comments on the 

### 8 other parts of Question 4; is that correct, Mary? 

### 9 Yes. Comments on the issues related to the 

### 10 selection of test substances and the analytical methodology 

### 11 chosen for this assay. Dr. Patino. 

### 12 **DR. REYNALDO PATINO:**Like I did with the 

### 13 Medaka assay discussion, I'm going to limit my comments to the 

### 14 selection of the test compounds. The analytic methodology, some 

### 15 of it has already been discussed and maybe in subsequent 

### 16 questions some of it may come again. So we'll limit this, at 

### 17 least in my discussion, to the selection of the test compounds. 

### 18 Four test compounds were used to validate this 

### 19 assay; Prochloraz was one. This was listed in the ISRs and 

### 20 aromatase inhibitor with AR inhibitory activity. This has 

### 21 already been mentioned by Bob, but there is at least one 

### 22 previous study that I'm aware of with amphibians, anuran 

### 23 amphibians, a Rana, where they tested Prochloraz in this 

### 24 organism and found that it disrupted the thyroid endocrine axis 

### 25 and delayed metamorphosis. And I'll provide a reference for 

### 26 that in the report.

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1 My suggestion is that this AOP, a thyroid 

### 2 active dependent AOP should be added to the list of AOPs that 

### 3 the report had originally associated with this compound. That 

### 4 may help you. I guess we'll visit a little bit about this more 

### 5 on Question 5, interpret some of the results that you had with 

### 6 this compound. 

### 7 So this was one of the compounds. The second 

### 8 compound on the list was octylphenol. That's a weak ER agonist. 

### 9 Trenbolone was used as well. That's a strong AR agonist, then 

### 10 Benzophenone-2 BB2 has characteristics of an ER agonist and also 

### 11 thyroid peroxidase inhibitor. 

### 12 The Panel did not have concerns about the 

### 13 specific compounds on the list. The logic behind the choice of 

### 14 these absences was clear and each of them can provide -- are 

### 15 good for proof of concept outcomes for the assay. 

### 16 However, because the purpose of these various 

### 17 studies is to validate the LAGDA assay and at least to our 

### 18 understanding, also the AOPs in which they are based. And this 

### 19 may be my misunderstanding, but it's not just the apical 

### 20 endpoints but in the context of the AOPs, the Panel wondered why 

### 21 it was not as strong and well-characterized thyroid active 

### 22 compound not included on the list for the validation of 

### 23 development endpoints. 

### 24 A strong AR agonist was used. That was in the 

### 25 validation that was Trenbolone, but there was no strong ER 

### 26 agonist used. The issue of positive control is to validate the

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1 assay is important and it has already been pointed out for a 

### 2 number of the other assays as well. Just a more general comment 

### 3 was that it would've been nice, for comparison purposes, just to 

### 4 compare on assay against the other in terms of which one is 

### 5 better to do what. 

### 6 If there would've been more overlap between 

### 7 the test compounds that were used for the validation assays, for 

### 8 all the -- at least the vertebrate models. So that's all I have 

### 9 as far as comments on the test compounds. If other members have 

### 10 comments, they'll add them themselves. Thank you. 

### 11 **DR. MARTHA SANDY:**Dr. Denver. 

### 12 **DR. ROBERT DENVER:**I have no further 

### 13 comments. 

### 14 **DR. MARTHA SANDY:**Dr. Propper. 

### 15 **DR. CATHERINE PROPPER:**I just have a couple 

### 16 of things to add, and again, one is emphasizing that positive 

### 17 control because I have seen studies before, especially when you 

### 18 get no effect outcome without that positive control. It becomes 

### 19 very difficult to interpret that negative outcome and it puts it 

### 20 at risk as being potentially considered a false negative. 

### 21 Whereas, if you have the positive control in there, it does add 

### 22 validity to the assay. 

### 23 And then second, and this is a bit of a 

### 24 different issue, but this was just on establishing the highest 

### 25 test concentration, I wasn't able to read through the protocol 

### 26 and decide that I had confidence that I would be able to repeat

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1 that, and I read through it several times. 

### 2 I wanted to know what were the endpoints that 

### 3 were being used in the initial range-finding study. Is it LD50? 

### 4 Is it a shift in weight? I wasn't altogether clear. What will 

### 5 the labs be looking at when they do that test to determine that 

### 6 high end of your dosing that you're then going to go down from 

### 7 for the EDC testing? 

### 8 It may well be that it's in the AMA protocol 

### 9 and I just don't remember seeing it because it's been years 

### 10 since I read that through from beginning to end, but if it is, 

### 11 you can just reference those pages. 

### 12 The protocol itself says that if 50 percent 

### 13 dilution series would be used for at least three test 

### 14 concentrations, and I want to know, does this start at 50 

### 15 percent as the highest no effect level in your initial study, or 

### 16 does it start at the no effect level of the initial study? 

### 17 And then again, there was kind of 

### 18 inconsistency on using either 50 percent dilution or it went to 

### 19 .33 percent dilution. So that kind of consistency needs to be 

### 20 in there for the range-finding parts of the study. 

### 21 Overall, I just think a little more detail in 

### 22 those protocols would be a real useful guideline. 

### 23 **DR. MARTHA SANDY:**Dr. Blazer. 

### 24 **DR. VICKI BLAZER:**I have no further comments. 

### 25 **DR. MARTHA SANDY:**Any other members of the 

### 26 Panel? No? Dallas.

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1 **DR. DALLAS JOHNSON:**I remembered a couple of 

### 2 other things I wanted to say, if I may. In the ISR, there is a 

### 3 statement made that the step down Jonckheere-Terpstra Trend Test 

### 4 should be applied to the data as long as the monotonic response 

### 5 was observed. 

### 6 I wonder if that shouldn't be changed to as 

### 7 long as a monotonic response is expected. I think there's a 

### 8 difference between what you observe and what you expect and 

### 9 again, it's a situation where I don't think you want to let the 

### 10 data drive the analysis. You should let the science drive the 

### 11 analysis. 

### 12 Another thing I noticed, in Table 1 was giving 

### 13 a listing of when certain things should be collected. The 

### 14 timing of apical endpoint measurements on the course of the 

### 15 study. Two of the things that were in there were mortality and 

### 16 clinical signs of disease and said that observations should be 

### 17 done daily. 

### 18 I guess I wonder, I'm curious as to whether 

### 19 daily means that you do it on Saturday and Sunday as well. And 

### 20 then I think also the data is probably not descriptive enough. 

### 21 Maybe it should be at the end of the day or a certain time of 

### 22 the day so that's it done consistently over time. That again 

### 23 would perhaps remove some of the variability that might be in 

### 24 the data that you're trying to get analyses on. 

### 25 **DR. MARTHA SANDY:**Dr. Propper. 

### 26 **DR. CATHERINE PROPPER:**One more point along

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1 there. It did say the animals should be checked daily for 

### 2 mortality and abnormal behavior. While those of us that work 

### 3 with Xenopus may be able to recognize abnormal behavior pretty 

### 4 quickly, a contract lab just starting out with this might need 

### 5 further guidelines for that as well. 

### 6 **DR. MARTHA SANDY:**Okay. Last call for 

### 7 comments from the Panel. 

### 8 (No response.) 

### 9 Seeing none, we'll go to the EPA. 

### 10 **DR. SIGMUND DEGITZ:**I do not have any 

### 11 clarifying questions. Thanks for the comments. 

### 12 **DR. MARTHA SANDY:**Dr. Touart, if you would be 

### 13 kind enough to read Charge Question 5. 

### 14 **DR. LESLIE TOUART:**Les Touart reading Charge 

### 15 Question 5. Considering the variability inherent in biological 

### 16 and chemical test methods, a test method needs to be repeatable 

### 17 and reproducible. 

### 18 A test is robust and reliable if the results 

### 19 are repeatable and reproducible within a laboratory and between 

### 20 different laboratories, respectively. A test protocol should 

### 21 provide sufficient guidance to ensure proper and consistent 

### 22 performance across labs and chemicals. 

### 23 Please comment on the test method for 

### 24 robustness and reliability and the repeatability and 

### 25 reproducibility of the result obtained with the LAGDA assay. 

### 26 **DR. MARTHA SANDY:**Dr. Patino is the lead

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1 discussant. 

### 2 **DR. REYNALDO PATINO:**I also lead this 

### 3 question for the Medaka and I refer to some definitions of the 

### 4 terms in the question. I refer you to our answer to that 

### 5 question for definitions to save time here. 

### 6 Each of the reference chemicals used for the 

### 7 assay validation was associated with one or more endocrine 

### 8 dependent AOPs in the ISR. Again, at a basic level, the general 

### 9 reliability of the assay can be initially at least evaluated by 

### 10 comparing the effects of pain with each test chemical against 

### 11 the expected endpoint results of their respective AOPs. 

### 12 With the exception of Prochloraz, which had 

### 13 some unexpected effects on the thyroid on the basis of the AOPs 

### 14 originally described, the assay generally performed as 

### 15 anticipated, for the reference materials. 

### 16 To go one-by-one, perhaps, specifically, 

### 17 Prochloraz was listed as aromatase inhibitor and with AR 

### 18 antagonist activity. We've already mentioned that there is an 

### 19 additional AOP for this compound that has been reported in the 

### 20 literature that could potentially explain the unexpected results 

### 21 of thyroid hypertrophy and hyperplasia that were noted in three 

### 22 of the four studies where this was measured in the ring study, 

### 23 where they measured -- determined effects of Prochloraz. 

### 24 It's likely that that unexpected finding may 

### 25 be explained by the additional AOP and makes that result more 

### 26 consistent with what one would expect. And again, I said

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1 already, I'll provide the reference that I have for that in the 

### 2 report. 

### 3 4-octylphenol listed as a weak ER agonist. 

### 4 This compound did not yield consistent observations with plasma 

### 5 vitellogenin, but when the effects were reported, they were 

### 6 positive and that is an anticipated outcome. 

### 7 Similarly, also, effects on duct development, 

### 8 especially in females, were noted that are consistent with the 

### 9 ER agonistic activity of the phenol. So there is concordance 

### 10 there. 

### 11 Trenbolone is a strong AR agonist, resulted in 

### 12 reproductive effects, duct development in particular in both 

### 13 males and females that were consistent with its AR agonist 

### 14 activity. For Benzophenone-2, it strongly induced vitellogenin 

### 15 in both males and females and had gonadal and duct pathologies 

### 16 consistent with this ER agonist activity, including 100 percent 

### 17 sex reversal of the highest concentration in genetic males. It 

### 18 also delayed metamorphosis and effected thyroid endpoints in a 

### 19 manner that was consistent with the TPO inhibitor activity. 

### 20 So generally speaking, this passes the initial 

### 21 level of determined reliability. It kind of consistently 

### 22 reproduced, based on the expected of a piece of what's known in 

### 23 the literature that you have. 

### 24 To more specifically address the question of 

### 25 reproducibility, the report, the ISR presented the results of 

### 26 two sets of ring tests; one with Prochloraz involving four

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1 different labs and another with octylphenol with three labs. 

### 2 In a test with Prochloraz, there was one, if I 

### 3 read the report correctly, only one individual endpoint that 

### 4 showed a consistent positive response across the four 

### 5 laboratories and that was a concentration-dependent delay in the 

### 6 involution of the oviduct in juvenile males. 

### 7 Also, below X or across the labs were fairly 

### 8 similar, so this one probably passed the test. Among the 

### 9 consistent negative results with this compound, all laboratories 

### 10 recorded no delays in time to NF stage 62. No effects on 

### 11 oviduct development in females. And in all cases, the genotypic 

### 12 sex matched the gonadal phenotype. So those are consistently 

### 13 negative across labs. 

### 14 Technical issues, however, concerning 

### 15 histological sample processing and reading and also with the 

### 16 thyroxine analysis, prevented a full assessment of the 

### 17 interlaboratory variability for many of the apical endpoints 

### 18 associated with endocrine axis. So in this context, it was kind 

### 19 of a mixed result in that regard. Some individual endpoints 

### 20 performed well. Others didn't. 

### 21 In the second set of assays using octylphenol, 

### 22 observations of thyroid histopathology or metamorphoses yielded 

### 23 inconsistent results among laboratories. T4 comparisons could 

### 24 not be conducted because of technical problems, as has been 

### 25 noted in some of the laboratories. 

### 26 Female duct development, there was one

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1 endpoint that was consistent among, laboratories and in a 

### 2 concentration-dependent manner. But the LOEC, even thought it 

### 3 was a consistent result in the same direction, 

### 4 concentration-dependent, the LOEC was about a 10-fold difference 

### 5 between the lowest value and the largest value that was reported 

### 6 by the different labs. 

### 7 Among the consistent negative results that 

### 8 genetic sex matched gonadal phenotype, made a result there, in 

### 9 terms of sex reversal. 

### 10 So overall, the interlab test yielded mixed 

### 11 results. There were a few endpoints that showed consistent 

### 12 results, especially for the thyroid and (inaudible) endpoints. 

### 13 And among the reproductive endpoints, the oviduct, both in males 

### 14 and females, generally, was the only endpoint that where a level 

### 15 of consistency was demonstrated. 

### 16 The ISR noted that some of the variability end 

### 17 results may have been due to technical difficulties with 

### 18 procedures or in some cases by the lack of complete adherence to 

### 19 the guidelines, but this could indicate a problem with the 

### 20 transferability of the assay in its present form. 

### 21 So overall, the Panel feels that the 

### 22 reliability and robustness of the assay, for practical purposes, 

### 23 has not been fully demonstrated for this assay. And again, the 

### 24 same definition we used for the Medaka, robustness here is one 

### 25 of the performance of the assay under the conditions of a 

### 26 relatively inexperienced labs. Those are my comments. Thank

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1 you. 

### 2 **DR. MARTHA SANDY:**Thank you. I'll go to the 

### 3 associate discussants. Mary Christman. 

### 4 **DR. MARY CHRISTMAN:**I concur with what Dr. 

### 5 Patino said. I would add or emphasize that I am not sure that 

### 6 reliability or robustness has been demonstrated yet. And Ken's 

### 7 comment, if I may. Dr. Portier's comments are: nothing to add. 

### 8 **DR. MARTHA SANDY:**Okay. Dr. Denver. 

### 9 **DR. ROBERT DENVER:**I concur and have nothing 

### 10 to add. 

### 11 **DR. MARTHA SANDY:**Dr. Blazer. 

### 12 **DR. VICKI BLAZER:**I have nothing to add. 

### 13 **DR. MARTHA SANDY:**Dr. Propper. 

### 14 **DR. CATHERINE PROPPER:**I concur and I only 

### 15 have one small point to add, which is standing alone negative 

### 16 data on gonadal or gonadal duct findings does not necessarily 

### 17 mean there isn't some biochemical shift that's going on that 

### 18 could affect long-term fitness outcomes in this assay because it 

### 19 does not explicitly hit that adverse outcome potential unless 

### 20 you have no gonadal duct. It makes it difficult to evaluate for 

### 21 population level types of effects and will need to be coupled 

### 22 with an assay that does make those outcomes -- or does evaluate 

### 23 those outcomes. 

### 24 **DR. MARTHA SANDY:**Any comments from other 

### 25 members of the Panel? 

### 26 (No response.)

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1 None. All right. Dr. Degitz from EPA. 

### 2 **DR. SIGMUND DEGITZ:**I appreciate your 

### 3 comments and I have no clarifying questions. 

### 4 **DR. MARTHA SANDY:**Shall we read Question 6, 

### 5 Dr. Touart. 

### 6 **DR. LESLIE TOUART:**This is Charge Question 6. 

### 7 The test protocol should be descriptive enough to be fully 

### 8 transferrable to a functional laboratory. The protocol should 

### 9 describe the methodology of the assay in a clear and concise 

### 10 manner so that a laboratory could comprehend the objective, 

### 11 conduct the assay, observe and measure prescribed endpoints, 

### 12 compile and prepare data for statistical analyses and report the 

### 13 results. Section 5 and 6 of the LAGDA ISR outline the process 

### 14 of and challenges experience in the interlaboratory studies. 

### 15 Please comment on the transferability across 

### 16 labs and provide any suggestions or recommendations for 

### 17 improvement of the LAGDA assay. 

### 18 **DR. MARTHA SANDY:**Dr. Propper. 

### 19 **DR. CATHERINE PROPPER:**So we've covered a lot 

### 20 of the issues associated with improvement of the assays, so I'll 

### 21 try to hit on those briefly. The first one being there is a 

### 22 need for positive controls. 

### 23 Again, this has been brought up across all the 

### 24 different assays and we've brought it up several times. We did 

### 25 see a lot of variability in the outcomes and some of that could 

### 26 be taken care by very straightforward and directed protocols.

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1 Consistency or figuring out a way to have more 

### 2 consistent sources of control water may be very useful in 

### 3 limiting some of that type of variation because that's not 

### 4 following the protocol kind of variation; consistency in iodine; 

### 5 availability is important and density of the animals being 

### 6 treated. 

### 7 One suggestion, and I don't know if the EPA is 

### 8 planning on incorporating this into the weight-of-evidence SAP 

### 9 that's coming, but figuring out how to incorporate all of the 

### 10 data in a kind of weight-of-evidence approach may also 

### 11 ultimately be useful, rather than evaluating all of these 

### 12 different endpoints as their own individual outcome, might 

### 13 provide some usefulness and actually reduce the potential for 

### 14 variation across laboratories. 

### 15 So really, most of our suggestions have now 

### 16 been incorporated into a lot of our other responses to 

### 17 questions. So I don't have much more to add beyond that. The 

### 18 transferability, I think we've also pretty much addressed, in 

### 19 that there are problems with transferability, but maybe these 

### 20 can be addressed through these mechanisms we've already 

### 21 mentioned. 

### 22 **DR. MARTHA SANDY:**Dr. Propper, if you could 

### 23 just clarify in your comments, I'll just reiterate, the 

### 24 weight-of-evidence panel that's coming up is discussing 

### 25 weight-of-evidence for the Tier 1 assays, and this is a Tier 2 

### 26 assay. I believe I heard on another question, another assay, a

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1 recommendation to integrate the results from Tier 2. Do you 

### 2 want to elaborate a bit on what you just said? 

### 3 **DR. CATHERINE PROPPER:**That's exactly what I 

### 4 am suggesting is that find a way to integrate all of the 

### 5 outcomes from the Tier 2 measures, either within one assay or 

### 6 potentially across all assays to help support whether or not 

### 7 there's evidence for the potential for adverse outcomes that can 

### 8 have population level effects. 

### 9 **DR. MARTHA SANDY:**Thank you. And now we'll 

### 10 turn to the associates from Question 6. Dr. Denver. 

### 11 **DR. ROBERT DENVER:**I agree with everything 

### 12 that Dr. Propper said. I just want to reiterate that from 

### 13 previous recommendations that modification of the histopathology 

### 14 to provide for quantitative measures should be investigated. 

### 15 The plasma vitellogenin ELISA should be analyzed in a single 

### 16 contract lab with appropriate expertise or a commercial kit 

### 17 should be developed. These are just ways to improve the assay. 

### 18 We did briefly mention measuring liver 

### 19 vitellogenin mRNA as a possible sensitive measure and we'll 

### 20 revisit that and provide some recommendations there. The 

### 21 thyroxine assays, we recommend that they be dropped. And 

### 22 lastly, that there be clear guidelines for statistical analysis 

### 23 that should be established and adhered to. 

### 24 **DR. MARTHA SANDY:**Dr. Patino. 

### 25 **DR. REYNALDO PATINO:**Nothing needed to add. 

### 26 Thank you.

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1 **DR. MARTHA SANDY:**Dr. Blazer. 

### 2 **DR. VICKI BLAZER:**I guess the only thing I'd 

### 3 like to add is, as we mentioned in the Medaka, evaluating 

### 4 general health in some way because a number of the frogs had 

### 5 granulomatous inflammation and other inflammation which might 

### 6 suggest infectious disease. 

### 7 **DR. MARTHA SANDY:**Dr. Christman. 

### 8 **DR. MARY CHRISTMAN:**I concur with Dr. 

### 9 Propper, Dr. Denver, and Dr. Blazer's comments. Dr. Portier's 

### 10 comments are: one gets the impression that the LAGDA assay has 

### 11 higher likelihood of being transferable among different labs 

### 12 than was observed for the JQTT or the MMT. 

### 13 This may be because of the robust nature of 

### 14 Xenopus laevis, which means that even with lab-to-lab 

### 15 variability and adherence to the protocol, this animal should 

### 16 perform and respond consistently to similar test concentrations 

### 17 if the protocol problems are worked out. 

### 18 **DR. MARTHA SANDY:**Are there any other 

### 19 comments from Panel members on Question 6? 

### 20 (No response.) I see none. I'll turn to Dr. 

### 21 Degitz. 

### 22 **DR. SIGMUND DEGITZ:**I have no clarifying 

### 23 questions. 

### 24 **DR. MARTHA SANDY:**I think we'll move on to 

### 25 Charge Question 7, please. 

### 26 **DR. LESLIE TOUART:**This is Charge Question 7

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1 for the LAGDA. The purpose of the validation process is to 

### 2 determine the readiness of a test or inclusion in a testing 

### 3 program. A component of readiness of a test is the evaluation 

### 4 of the usefulness and limitations of the test, including the 

### 5 classes and types of test substances that can and cannot be 

### 6 tested. 

### 7 Please comment on the strengths and/or 

### 8 limitations of the LAGDA assay. 

### 9 **DR. MARTHA SANDY:**Dr. Propper is the lead. 

### 10 **DR. CATHERINE PROPPER:**I apologize, but this 

### 11 also includes summary, so it may be a little long. Amphibians, 

### 12 generally, and Xenopus laevis in particular have been strong 

### 13 developmental models for vertebrates in general, especially with 

### 14 relationship to thyroid hormone-related issues associated with 

### 15 development. And these animals have been used as a model for 

### 16 endocrine disruption, specifically because of the readiness, 

### 17 both gonadal and thyroid hormone outcomes, which is a particular 

### 18 strength of this assay. 

### 19 Although it may be difficult in this assay to 

### 20 determine whether the endocrine endpoints are a result of direct 

### 21 endocrine mode of action or toxicological endpoint, 

### 22 nevertheless, thyroid and gonadal and gonadal duct in 

### 23 particular, histopathologies will be indicative of adverse 

### 24 endocrine action following exposure. 

### 25 The exact mode of action may not be 

### 26 determinable, but nevertheless, the adverse outcome potential

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1 can be evaluated such that the assay is a Tier 2 measure of 

### 2 outcome on growth development and reproduction as endpoints that 

### 3 can be necessary towards an AOP evaluation. 

### 4 Because this assay ends in the juvenile stage, 

### 5 however, a significant limit of this assay includes the 

### 6 inability to determine what the adult outcome of developmental 

### 7 exposure will be. And that's the point I brought up in the last 

### 8 question as well. So it needs to be coupled with some kind of 

### 9 adverse outcome with respect to fitness and potential population 

### 10 level effects. 

### 11 Because of the inability to determine 

### 12 fecundity and fertility issues, the lack of overt histological 

### 13 changes in the gonads during development -- and I said this 

### 14 earlier -- doesn't necessarily mean there won't be adult 

### 15 outcomes. Careful comparisons between this, and in particular, 

### 16 the Medaka assay will be needed to really evaluate those kinds 

### 17 of outcomes. 

### 18 The one problem with Xenopus as a model, and I 

### 19 believe that Ken Portier was the one that first brought this up, 

### 20 is that it is a rather unusual amphibian, as far as amphibians 

### 21 goes because in the adult animal, it's still completely aquatic. 

### 22 So physiologically, it is a bit different from other 

### 23 amphibians. There also already been documented some sensitivity 

### 24 between strains in response to endocrine disruptors. 

### 25 So extrapolating the data from Xenopus out to 

### 26 all amphibians needs to be done very carefully and in the

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1 context of both the literature and in perhaps, having an error 

### 2 -- a level of buffer when setting LOEC levels in the regulatory 

### 3 context. 

### 4 Ultimately, the EPA charge question document 

### 5 specifically states that the purpose of the Tier 2 assays is to 

### 6 characterize the nature of likelihood in dose response 

### 7 relationship of endocrine disruption of the estrogen, androgen, 

### 8 and thyroid in humans and wildlife. 

### 9 While we find the logic, development, and 

### 10 implementation of this assay has attempted to meet the nature 

### 11 and likelihood of endocrine disruption through these modes of 

### 12 actions, and this can occur through the toxicity or endocrine 

### 13 disruption, the demonstration of the assay can determine the 

### 14 dose response relationships and ultimately, an LOEC for 

### 15 regulatory purpose has not yet been demonstrated and that's 

### 16 because of all of the variation across laboratories that we saw. 

### 17 18 However, a few endpoints that are related to 

### 19 AOPs were remarkably consistent -- and Reynaldo brought that up 

### 20 during his evaluation -- and they do provide specifically useful 

### 21 information in a Tier 2 approach. 

### 22 Many of the other endpoints, there was a lot 

### 23 of variation among labs with some labs finding effects and 

### 24 others not. This variation suggests there will be considerable 

### 25 risk of either false negatives or positives if this assay is 

### 26 implemented in a single laboratory kind of context.

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1 So therefore, in discussing the readiness of 

### 2 this test for inclusion in a testing program, we believe that 

### 3 with further protocol development, the assay may perform at 

### 4 least as a yes/no outcome for some of these endpoints, but that 

### 5 the use for it with LOECs may still be difficult. 

### 6 So as a tool for risk assessment, applicable 

### 7 to other wildlife and/or humans, which more the mammalian assay 

### 8 that was evaluated previously, again, the endpoint outcomes are 

### 9 suggested of toxicity or endocrine disruption. They will 

### 10 provide valuable information with regards to risk assessment and 

### 11 potentially regulatory action, but the interassay variation, 

### 12 especially in the LOECs, suggests that extrapolating out to 

### 13 other species is going to be pretty complicated because if you 

### 14 can't even get the same results with one species across 

### 15 laboratories, how do you even begin to evaluate this across all 

### 16 wildlife. Again, I suggest that if it is implemented that a 

### 17 buffer needs to be put in there when making a regulatory 

### 18 decision. 

### 19 In summary, once the protocols are clear and 

### 20 the QA/QC parameters are stringently outlined and met by the 

### 21 contract companies running the assays, the LAGDA assay will 

### 22 function towards the stated purpose of a Tier 2 assay. It has 

### 23 potential for understanding the risk of adverse outcomes, 

### 24 however, the variation across laboratories shows that the 

### 25 validation to date has not demonstrated the capacity for 

### 26 consistent dose outcomes leading to clear LOECs at this time.

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1 **DR. MARTHA SANDY:**And now we turn to the 

### 2 associates. Dr. Denver. 

### 3 **DR. ROBERT DENVER:**No further comments. 

### 4 **DR. MARTHA SANDY:**Dr. Patino. 

### 5 **DR. REYNALDO PATINO:**No additional comments. 

### 6 **DR. MARTHA SANDY:**Dr. Blazer. 

### 7 **DR. VICKI BLAZER:**No additional comments. 

### 8 **DR. MARTHA SANDY:**Dr. Christman. 

### 9 **DR. MARY CHRISTMAN:**I concur, and Dr. Portier 

### 10 requests a power analysis. 

### 11 **DR. MARTHA SANDY:**Comments from other members 

### 12 of the Panel on this last question on the LAGDA? 

### 13 (No response.) 

### 14 Seeing none, I turn to Dr. Degitz of EPA. 

### 15 **DR. SIGMUND DEGITZ:**I have no clarifying 

### 16 questions. Thank you. 

### 17 **DR. MARTHA SANDY:**This is a good stopping 

### 18 point, I think, to take another break. Let's convene back and 

### 19 ten after 3:00. 

### 20 (Whereupon, a break was taken) 

### 21 **DR. SHARLENE MATTEN:**Hello. We are ready to 

### 22 reconvene. We're now about to begin the charge question 

### 23 discussion on the aquatic and vertebrate assays. I am going to 

### 24 turn the microphone over to Dr. Martha Sandy. 

### 25 **DR. MARTHA SANDY:**Thank you. We are going to 

### 26 address Charge Questions 1 and 2, we hope, today. We'll start

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1 tomorrow with Question 3. That's the plan. So I'll ask Dr. 

### 2 Touart to read Charge Question 1, please. 

### 3 **DR. LESLIE TOUART:**Thank you. This will be 

### 4 the first charge question for the Mysid Toxicity Test. A 

### 5 rationale for the test method should be available, including a 

### 6 clear statement of scientific basis and the regulatory purpose 

### 7 and need for the test method. 

### 8 The EDSTAC introduced the Tier 2 test as 

### 9 having the purpose to characterize the nature, likelihood and 

### 10 dose response relationship of endocrine disruption of estrogen, 

### 11 androgen, and thyroid in human and wildlife. 

### 12 Although the hormones produced and used by 

### 13 invertebrates are not directly analogous to those of 

### 14 vertebrates, example: estrogen, androgen, and thyroid, growth, 

### 15 reproduction, development and other aspects of invertebrate 

### 16 physiology and life cycle are known to be under endocrine 

### 17 control. 

### 18 EDSTAC went on to note that chemicals that 

### 19 affect these vertebrate hormones may also effect invertebrate 

### 20 hormones, resulting in altered reproduction development and 

### 21 growth. 

### 22 Tier 2 tests were designed to be definitive 

### 23 tests which generate sufficient data to characterize the 

### 24 specific hazard of the substance and provide sufficient 

### 25 information on dose response and adverse effects to permit risk 

### 26 decisions.

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1 Please comment on the rationale and purpose of 

### 2 the assay as part of the Tier 2 testing in the EDSP, as 

### 3 described in Sections 2.1 and 2.2 of mysid ISR. 

### 4 **DR. MARTHA SANDY:**And the lead discussant on 

### 5 this is Dr. Scott. 

### 6 **DR. GEOFFREY SCOTT:**Thank you. I'd like to 

### 7 thank my colleagues. We have put together sort of a summary 

### 8 statement and I will start by going through that statement and 

### 9 then we will let individual Panel members come back. Let me 

### 10 just go ahead and get started. 

### 11 The rationale and purpose defined for 

### 12 inclusion of invertebrate or endocrine disrupting chemical 

### 13 effects is clearly defined in the ISR document. Invertebrates 

### 14 are significant parts of global ecosystems and thus, they must 

### 15 be protected from the adverse effects of chemicals that disrupt 

### 16 their endocrine system. 

### 17 Invertebrate endocrine systems will respond 

### 18 differently -- actually quite differently than vertebrate 

### 19 endocrine systems, and thus, they need to be addressed in terms 

### 20 of ecotoxicological risk assessment through and EDC assessment 

### 21 process. Both the mysid and the harpacticoid copepod described 

### 22 in the document are relevant assays for addressing endocrine 

### 23 effects in invertebrates. 

### 24 While the initial EDSTAC charge for developing 

### 25 the Tier 2 assays was focused on characterizing the nature, 

### 26 likelihood, and dose response relationships of endocrine

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1 disruption of either estrogen, androgen or thyroid 

### 2 invertebrates, including humans and wildlife, EDSTAC clearly 

### 3 defined the linkage between that endocrine disruption in 

### 4 vertebrates and the importance of invertebrates by further 

### 5 stating, as is stated in the question, assessing the effects of 

### 6 chemicals that disrupt the ANT, invertebrates should be further 

### 7 invertebrates in invertebrates for disruption of growth 

### 8 development and reproduction. 

### 9 The current mysid and copepod bioassays 

### 10 described in the ISR are designed specifically to address 

### 11 alterations in survival, growth, development, and reproduction 

### 12 in a way, and when taken in toto, to define endocrine effects in 

### 13 invertebrates. 

### 14 What is missing in these current invertebrate 

### 15 bioassay approaches, however, is a clearly defined EDC mechanism 

### 16 of action test endpoint or the hormone systems in invertebrates 

### 17 for either ecdysone or other important controlling hormones that 

### 18 control growth development and reproduction. 

### 19 And this would be similar to the E, A and T 

### 20 approaches that we have invertebrate testing. What is unclear 

### 21 in the initial EDSTAC charge and in the ISR is the linkage 

### 22 between the disruption of E, A, and T in vertebrates within 

### 23 vertebrate testing and the linkage with the disruption of 

### 24 invertebrate hormones. This is particularly important in seeing 

### 25 how disruption of invertebrate hormones not found in 

### 26 invertebrates will work in accompanying adverse outcome pathway

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1 approaches unless parallel and diverging AOPs for invertebrate 

### 2 endocrine disruption are developed. 

### 3 If an invertebrate adverse outcome pathway 

### 4 approaches develop that will compliment the current A, E, and T 

### 5 of AOP approaches for vertebrates, it will be important for EPA 

### 6 to consider the large variety of invertebrate EDC pathways that 

### 7 must be protected, given that crustaceans are only one member, 

### 8 in addition to mollusk and other classes of invertebrates that 

### 9 we are trying to protect in this approach. 

### 10 This is particularly important, given the 

### 11 ecological -- not only their ecological importance as 

### 12 invertebrates in ecosystems, but also the large commercial and 

### 13 recreational importance of many of these invertebrate species as 

### 14 fishery species around the world. 

### 15 As a result, it would be very important to 

### 16 consider multiple invertebrate species for possible 

### 17 consideration as EPA has done. The ISR clearly defines why 

### 18 vertebrate protection from EDC effects is important, and that is 

### 19 the significant ecological linkage between invertebrates and 

### 20 vertebrates in terrestrial, aquatic, estuarine, and marine 

### 21 ecosystems. 

### 22 The ISR indicated the importance of the 

### 23 linkage of the mysid test, for example, to estuarine ecosystems 

### 24 for environmental protection. For example, in estuarine 

### 25 systems, most of the important estuarine-dependent and 

### 26 commercially important fin fish species -- in fact, in the

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1 southeast, over 86 percent of the fin fish species begin their 

### 2 earliest life history stage in the estuary, consuming copepods 

### 3 and later consuming arthropods and mysid shrimp and grass 

### 4 shrimp, and then eventually penaeid shrimp as they mature and 

### 5 then move out to the ocean. 

### 6 This crustacean cascade is vital to the 

### 7 functioning of estuaries as nursery grounds for the vertebrate 

### 8 fin fish species that utilize those for protection and growth. 

### 9 Since 53 percent of the U.S. population lives 

### 10 in only 17 percent of the land area of coastal areas around the 

### 11 U.S., this results in more than 5.2 times more people per square 

### 12 mile and 5.87 times more gross domestic product per land area 

### 13 and the attending pollution that goes with economic development 

### 14 is thus much larger in these area. Thus, providing species that 

### 15 afford protection, not only to all invertebrates, but to these 

### 16 coastal areas, we think, from ANOVA perspective particularly, 

### 17 here, I'll speak, is critical. 

### 18 As a result, there may be greater potential 

### 19 for impact in these coastal ecosystems, as I've said, as a 

### 20 result. The current battery of invertebrate Tier 2 tests are 

### 21 crustaceans, and indeed based upon their ecological importance, 

### 22 but also their toxicological sensitivity. 

### 23 Crustaceans are generally more sensitive than 

### 24 other invertebrates in terms of their conventional acute and 

### 25 chronic toxicity, as this was brought out in an earlier SAP 

### 26 Panel, i.e., harmonization of FIFRA and Clean Water Act

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1 protocols. The FIFRA 5 was good and the crustaceans drove that. 

### 2 3 So we really think tying this invertebrate 

### 4 protection to crustaceans is logical and is sound. By 

### 5 extension, the EDC testing crustaceans have played a critical 

### 6 and effective role already in protecting the environment for 

### 7 known endocrine disrupting chemicals. By Office of Pesticides 

### 8 has used these current assays with the mysid test to safely and 

### 9 effectively regulate insect growth regulating pesticides with 

### 10 known endocrine properties for invertebrate adverse outcome 

### 11 pathways; compounds such as Methoprene, Dimilin and Fenoxycarb, 

### 12 just to name a few. 

### 13 The conventional one-generation mysid test 

### 14 played a vital key role and effectively defined a risk-based 

### 15 approach for regulating these EDCs, establishing clear dose 

### 16 response relationships as prescribed by the EDSTAC process. The 

### 17 mysid test provides a clear assessment of the ecological effects 

### 18 to aquatic organisms. We also have to be concerned about 

### 19 settlement dwelling organisms. 

### 20 Having assessment endpoints that can assess 

### 21 sediment toxicity as well may be very important for certain 

### 22 classes of chemical in certain particular pesticides, since the 

### 23 fate of many chemical contaminants in aquatic and marine and 

### 24 estuarine ecosystems may be sediment exposure. 

### 25 The addition of the harpacticoid copepod assay 

### 26 provides an additional assay for the assay for the assessment of

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1 EDC effects and invertebrates, which addresses not only aquatic 

### 2 effects through the current pathways, but provides the potential 

### 3 to predict effects in benthic communities and also can be 

### 4 modified, as we'll get into later, into a sediment assay. 

### 5 Although that's not under consideration, but I'll simply point 

### 6 that out. 

### 7 I will also point out, in addition, the 

### 8 copepod assay was developed, was assessed in study that we did 

### 9 for EPA and found to be one of the most sensitive test species 

### 10 for superfund site testing in the southeast, U.S. So there's a 

### 11 clear and established role of a sensitivity of that assay. 

### 12 Invertebrates are a critical part of the 

### 13 ecosystem, and as such, need to be included in an endocrine 

### 14 disrupting screening protocol. It is unclear, however, that the 

### 15 rationale for inclusion of this assay in Tier 2 is adequate. 

### 16 According to the EPA Tier 1 test should screen for the potential 

### 17 to interact with estrogen, androgen, and thyroid systems. 

### 18 Further, they should be shorter term and lower 

### 19 cost test with minimal chances of false negatives. The Tier 2 

### 20 test should determine interactions with the endocrine system and 

### 21 quantify dose response relationships. Further, it is 

### 22 anticipated that these would be longer term and capable of 

### 23 detecting modes of action. We interpret that to mean modes of 

### 24 actions for invertebrates. 

### 25 The Panel endorses the use of an invertebrate 

### 26 assay, but as prescribed in the protocols. But feels that

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1 without some Tier 1 invertebrate assay that focuses on a 

### 2 chemical's potential to interact with the hormone system in 

### 3 invertebrates or other appropriate invertebrate hormone metrics, 

### 4 either ecdysone or what-have-you, that this Tier 2 test, in some 

### 5 ways, is out of place without having some way to define the 

### 6 potential to effect endocrine systems; otherwise, we're left 

### 7 with testing all compounds with A, E, and T to be tested for an 

### 8 invertebrate endocrine effects. 

### 9 The Panel has raised the issue of a lack of 

### 10 the Tier 1 screen for invertebrates in the current EDSTAC 

### 11 process and the importance in how EPA would address the 

### 12 identification of E, A, and T active compounds in the current 

### 13 vertebrate screenings for selection of testing in the Tier 2 

### 14 invertebrate protocol. So that's really where we spent a lot of 

### 15 time talking. 

### 16 Public comments by the Endocrine Policy Forum 

### 17 raised this same issue and obviously, we're not the only ones 

### 18 asking that question. The process at this point is we've asked 

### 19 some question and EPA has said there will be a process. 

### 20 I know we have a weight-of-evidence panel and 

### 21 a lot more can become apparent after that, so I don't want to 

### 22 put the cart before the horse, but in the absence of that today, 

### 23 I would just say that we would ask the EPA to consider if any of 

### 24 the current invertebrate Tier 2 screenings might be effectively 

### 25 modified to develop into a possible Tier 1 or a pre-Tier 2 

### 26 prescreen to help us in this process.

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1 For example, the copepod bioassay, given that 

### 2 it is miniature and small, may be a minimal. Similarly, the 

### 3 current mysid test that we do with the conventional mysid F1 

### 4 generation, not multigeneration, could also be considered for 

### 5 modification. And we'll have some more information on that as 

### 6 we go through and discuss this. As possible, either Tier 1 

### 7 screenings or Tier 2 prescreens. Okay. 

### 8 In the ISR, it is stated that Tier 2 tests 

### 9 were defined to be definitive tests which generate sufficient 

### 10 data to characterize the specific hazard of the substance and 

### 11 provide sufficient information on dose response and adverse 

### 12 effects to permit risk decisions. 

### 13 Both bioassays address these tenets of Tier 2 

### 14 test requirements, both the mysid and the copepod. But will be 

### 15 dependent, presently on a weight-of-evidence approach in 

### 16 differentiating between overt toxicity versus endocrine-specific 

### 17 effects, at least as we see it right now, given the non-EDC 

### 18 specific nature of many of the test endpoints for growth, 

### 19 survival, development and reproduction. 

### 20 We also in public comment had similar concerns 

### 21 about this expressed in some of the comments by the Endocrine 

### 22 Policy Forum. This lack of consistency in response between labs 

### 23 and between F1 and F2 generations and interlab testing 

### 24 undermines not only the complexity of these assays to be able to 

### 25 maybe move a spore as a test-to-test in a multigenerational 

### 26 aspect, but also on the ability to define dose response

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1 effectively. 

### 2 Additionally, the consistency in response 

### 3 among many of the labs, in some instances, was low for certain 

### 4 aspects of this. We'll get into a lot more of that in the 

### 5 subsequent questions. So many of these issues about specifics 

### 6 will be addressed in the subsequent questions. 

### 7 In conclusion, let me just say the Panel 

### 8 strongly appreciates the importance of including invertebrates 

### 9 in the battery of tests included in the EDSTAC testing 

### 10 requirements and acknowledges that some compounds, generally 

### 11 anti-steroidogenic compounds such Prochloraz, could have a broad 

### 12 affect on both invertebrates and vertebrates. 

### 13 The use of the Tier 2 mysid test, without a 

### 14 representative Tier 1 or Tier 2 prescreen to determine endocrine 

### 15 disruptive dose response effects on estrogen, androgen, thyroid 

### 16 controlling processes in human and wildlife has questionable 

### 17 validity and is counterintuitive, considering the current EDSTAC 

### 18 framework. 

### 19 This is due both to the lack of guidance of 

### 20 EPA decision-making as to how the Tier 2 will be required 

### 21 following the Tier 1 battery of tests. Due to the differences 

### 22 in the actual hormones and endocrine receptor controls of 

### 23 reproduction and development invertebrates compared to 

### 24 vertebrates, this really underlies a complexity of this issue. 

### 25 Therefore, the SAP suggests that a goal of EPA 

### 26 should be to gain inclusion of invertebrate or ecdysone or the

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1 other steroid hormone processes in invertebrates that should be 

### 2 added to these requirements, along with the estrogen, 

### 3 testosterone -- excuse me -- androgen or thyroid, in order to 

### 4 help build consistency between Tier 1 and Tier 2. This would 

### 5 also require possibly a Tier 1 or a Tier 2 prescreen as we sit. 

### 6 7 Let me just real quickly, potential to use the 

### 8 copepods for Tier 1 as a Tier 1 screen or other Tier 2 assays, 

### 9 or other Tier 1 assays such as daphnids, using ecdysone steroid 

### 10 receptors, juvenile hormone receptors or other types of assays 

### 11 would also provide and invertebrate mediated process assessment. 

### 12 And finally, the Panel approves the rationale 

### 13 and purpose of the mysid to generation test as a Tier 2 screen. 

### 14 It believes that it is a very sensitive test to measure the 

### 15 possible environmental impacts of endocrine disrupting chemicals 

### 16 on invertebrates, but we do need that linkage with the 

### 17 invertebrate endocrine system. 

### 18 With that, I'm going to stop and we'll 

### 19 conclude my comments and see if any of the other panelist want 

### 20 to jump in. 

### 21 **DR. SHARLENE MATTEN:**I wanted to clarify the 

### 22 objective of this particular meeting versus -- because you've 

### 23 been to the previous meeting, you have a flow-through system of 

### 24 knowledge that perhaps mixes the apples and the oranges. 

### 25 What I'm trying to say is the response that 

### 26 related to Tier 2 for these charge questions is independent of

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1 what, at least for this charge, is independent of what leads to 

### 2 that decision to have a Tier 2. It's as if you were given tests 

### 3 for assays and you're evaluating the assays in how they formed 

### 4 and could be transferred to other laboratories. 

### 5 While it's very exciting and very interesting, 

### 6 it gets a bit confusing since the information that was provided 

### 7 as background doesn't include all or a portion of the Panel's 

### 8 responses to that particular charge question. 

### 9 So at least from the role of the Panel to 

### 10 focus on those points that led to the evaluation of the 

### 11 rationale and choice of those assays. And the other things that 

### 12 you mentioned, save them. They are useful to consider, but I'm 

### 13 not sure how to package them into the report other than some 

### 14 kind of appendix that might be useful as points to consider for 

### 15

-- 

### 16 **DR. GEOFFREY SCOTT:**Maybe I misunderstood the 

### 17 question. The question said "rationale and purpose." I think 

### 18 that's what we focused on. So if that's not what we're supposed 

### 19 to focus on, my apologies. But that was what we focused on is 

### 20 whether there is a rationale for including these assays and 

### 21 whether they have a purpose. 

### 22 The other parts of what you're asking for are 

### 23 more specifically spelled out, as far as evaluating the 

### 24 individual assays in the subsequent questions. So we did not 

### 25 want to mix apples and oranges in the charge on this question 

### 26 about rationale and purpose.

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1 So if we should've done that, we'll go back 

### 2 and can do that, but at this point, all we were trying to do is 

### 3 talk about the rationale of having an invertebrate assay that 

### 4 essentially has no adverse outcome pathway that is being 

### 5 identified through a screening process for vertebrate assays and 

### 6 whether or not that makes sense. 

### 7 Now, if that wasn't our charge, my apologies, 

### 8 but that's what we did and I thought that's what the question 

### 9 asked. 

### 10 **DR. MARTHA SANDY:**I appreciate what you said 

### 11 that this is your interpretation of the charge question. I 

### 12 think for now we'll leave it at that. I'd like to hear if there 

### 13 are any comments from the associate discussants. Dr. Tuberty. 

### 14 **DR. SHEA TUBERTY:**So many of my comments were 

### 15 included in Dr. Scott's comments already. I think it's 

### 16 problematic approaching invertebrates as if they are going to 

### 17 have the same axis, reproductive axis, thyroid axis as 

### 18 vertebrates. And we all, I think, can appreciate that they're 

### 19 very different in many ways. We've already acknowledged that 

### 20 they make up the great majority of biomass of animals on earth 

### 21 and are often overlooked. 

### 22 As an invertebrate zoologist, I'm used to it. 

### 23 And I have to convince my students that they've chosen the right 

### 24 class to take when they take invertebrate zoology and then I do 

### 25 that. But choosing one invertebrate to represent one species of 

### 26 invertebrate to represent 95 percent or more, it's probably more

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1 like 99 percent of animals on earth is already an 

### 2 underrepresentation, but we appreciate that they were selected 

### 3 for inclusion in the Tier 2 assays. We appreciate the 

### 4 opportunity to make a chance to evaluate the appropriateness of 

### 5 these assays. And I think they have a lot of value and there is 

### 6 value added in adding them to Tier 2. 

### 7 Having said that, linking them to the few 

### 8 existing compounds that pass through the Tier 1 assay battery 

### 9 creates problems with deciding mode of action, if that's even 

### 10 important anymore. One of the reasons why our group has 

### 11 struggled with moving forward quickly is because of our trouble 

### 12 in grasping what approach to take this issue. 

### 13 Do we just assume that invertebrates are never 

### 14 going to be added to Tier 1, even though they're a huge 

### 15 representation in other groups that are involved in testing? 

### 16 As an invertebrate zoologist, I feel it's my 

### 17 duty to all those invertebrates out there that I say they really 

### 18 do need to be on Tier 1 tests. I think the two assays that we 

### 19 were given to evaluate are both very good. Either one can be 

### 20 used. And I understand the problems with adopting the copepod 

### 21 for some groups that have never adopted it before, however, I 

### 22 don't think that's a reason why they shouldn't be adopted and we 

### 23 never move forward just because we're not there already. It 

### 24 doesn't make sense to me, but we'll come to those questions 

### 25 later. 

### 26 I understand Dr. Matten's issue with the

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1 direction we took, but I think it's one that we put a lot of 

### 2 effort and time and thought into, and we thought it was worth 

### 3 saying on the record. 

### 4 Having said that, there are lots of 

### 5 opportunities to using cutting edge techniques, all of which 

### 6 have been represented in the other three assays to date. I 

### 7 think it's been kind of a patent answer that non-invertebrate 

### 8 folks, and even us invertebrate folks have had to say we don't 

### 9 know enough to do anything about this yet, but I think we're 

### 10 going to push forward a lot of evidence. 

### 11 Some of it is very, very recent, as recent as 

### 12 just a month or two ago that screening assays have been 

### 13 developed that would give us insight into mechanisms of actual 

### 14 endocrine disruption effects in invertebrates. Measurable ones, 

### 15 mode of action and POAs that are very similar to those for 

### 16 vertebrates, but probably not going to be the same compounds 

### 17 recognized through the E, A and T axis tests that are done now. 

### 18 Having said that, it seems like the small 

### 19 number that, Mary, you gave us that moved up from the 10,000 

### 20 screening, which is good there's only that many, but it might be 

### 21 a different group altogether if we had a Tier 1 assay with 

### 22 invertebrates represented there. So it's problematic in trying 

### 23 to show a cause and effect when we have thyroid compound or 

### 24 anti-thyroid compound that somehow is being toxic to 

### 25 invertebrate and gives us crazy result in the F0 and F1 

### 26 generations. We can't make sense of it and it clouds our

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1 judgment and it flies in the face of the science we have 

### 2 available to us to try to do this kind of thing. 

### 3 But again, as Les has mentioned, the apical 

### 4 endpoints are still really critical and they're very important. 

### 5 So if we show effects on reproduction and development and 

### 6 growth, for me, that's good enough at this point, just that it's 

### 7 there. Thank you. 

### 8 **DR. MARTHA SANDY:**Dr. Perkins. 

### 9 **DR. EDWARD PERKINS:**I just wanted to support 

### 10 what everyone else has said and also reiterate that I thought 

### 11 the rationale for the purpose of putting a chemical through the 

### 12 EAT AOP Tier 2 screening and also looking for effect on 

### 13 invertebrates, the rationale is very good for that. 

### 14 However, given the caveats that have been 

### 15 given, it's going to be very difficult to provide a rationale 

### 16 for effects on endocrine systems of invertebrates just because 

### 17 we don't have enough data on that. 

### 18 However, the endpoints that are being looked 

### 19 at, they are very important for risk assessment. And the data 

### 20 has already shown that the vertebrate endocrine disruptors have 

### 21 effects on invertebrates. So it's clearly a useful assay. 

### 22 Whether or not when the assay need to be applied and how it 

### 23 should be applied is a different beyond the scope of this Panel, 

### 24 clearly. 

### 25 However, we do think -- I think we're at a 

### 26 consensus that it is a very useful assay and it has shown a lot

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1 of different effects for it. 

### 2 **DR. MARTHA SANDY:**Dr. Klaine. 

### 3 **DR. STEPHEN KLAINE:**Thanks. I'm not going to 

### 4 go and reiterate what all my colleagues have said, but I would 

### 5 like to say that we took this really seriously and irregardless 

### 6 of the entire scheme, you asked us to look at this assay. So we 

### 7 felt that this is a good opportunity to bring some really 

### 8 current science to this assay. 

### 9 So yes, there is some connection issues and 

### 10 yes, there are some other issues in terms of what triggers a 

### 11 test, but that's not what we're focusing on. So some of those 

### 12 things may have come out in the rationale, but we have focused 

### 13 on this test. So in deference to Dr. Matten's comments, I think 

### 14 we focused on what we needed to focus on. So I hope you will 

### 15 agree when you hear everything. 

### 16 **DR. MARTHA SANDY:**Dr. Scott. 

### 17 **DR. GEOFFREY SCOTT:**And again, if you read 

### 18 through the question, it really talks about the EDSTAC charge 

### 19 and the EDSTAC charge for linking EAT effects with 

### 20 invertebrates. That's what we did. 

### 21 While we may have stepped beyond the 

### 22 assessment of just the Tier 2 test, we have kept the Tier 2 test 

### 23 as the basis of looking at the recommendations I've alluded to. 

### 24 And the specific comments we're going to make on the Tier 2 

### 25 test, I think are pragmatic and are designed to stay within the 

### 26 context of what this SAP is about.

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1 We certainly are not cavalier enough to think 

### 2 we can come in the room and change the rules. Rather, we're 

### 3 trying to answer the question and use as much innovation as we 

### 4 could and the cumulative knowledge around the table to do that. 

### 5 And I will apologize if we overstepped, out of bounds. We'll 

### 6 back in bounds. Hopefully, we'll decide that as we go through 

### 7 the rest of our questions and decide whether it was a charge or 

### 8 a block and fail, one of the two, because that's always hard to 

### 9 call. 

### 10 Anyway, we'll move forward. 

### 11 **DR. MARTHA SANDY:**I'll see if Dr. Christman 

### 12 has anything. 

### 13 **DR. MARY CHRISTMAN:**I have no additional 

### 14 comments. 

### 15 **DR. MARTHA SANDY:**Any comments from anyone 

### 16 else on the Panel? I'll turn over to EPA. Yes, Mary Manibusan. 

### 17 **MS. MARY MANIBUSAN:**Dr. Scott, I do hope to 

### 18 clarify my sensitivities as I've articulated them to Dr. Matten. 

### 19 I was very concerned with the bleeding over of topics. You had 

### 20 several SAPs very close in timing. So I wanted to be careful 

### 21 that this meeting wasn't particularly focused on the performance 

### 22 of the Tier 2 methods and their validation process. 

### 23 I do understand the interest and excitement 

### 24 over rethinking the Tier 1 so that it matches with Tier 2, but 

### 25 because of where we are as a program in honoring a lot of the 

### 26 recommendations that have been made by previous panels and where

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1 we are today with our test order issuance and evaluating of the 

### 2 initial list of chemicals for the 11 set of assays for Tier 1, I 

### 3 want to make sure that we're holding that steady as we move 

### 4 forward into Tier 2. But we do understand the interest in 

### 5 trying to correlate the Tier 1 information with how we make 

### 6 decisions on which Tier 2 to trigger, if you will, when we do 

### 7 see an interaction. 

### 8 I have been often very parochial, perhaps, in 

### 9 this meeting in establishing that the clear line between Tier 1 

### 10 and Tier 2 because of the issue of the difference in certainty 

### 11 and the difference in questions that we're asking. So Tier 1, 

### 12 we're always asking, does it even have the potential, not for 

### 13 different species, not for all modes of action, but for E, A and 

### 14 T, recognizing that we're looking across different biological 

### 15 levels of organization and trying to present a good enough 

### 16 screen, not the best. Certainly not the most perfect, but a 

### 17 good enough screen to allow the Agency to determine that of the 

### 18 10,341 chemicals in our combined universe of FIFRA and SDWA 

### 19 chemicals, how do we begin to prioritize which chemicals to move 

### 20 forward for Tier 2, the more expensive longer-term testing? 

### 21 So that's a challenge that we're facing. If 

### 22 you're hearing this punctuated sensitivity, it's because of 

### 23 that. Just recognizing where we are in the SAP process. I 

### 24 wanted to make sure they were walking through each of those 

### 25 steps incrementally and not bleeding topics over so that we're 

### 26 confusing this data for science. That's it. Thank you.

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1 **DR. GEOFFREY SCOTT:**Thank you for that. We 

### 2 certainly will try to maintain our focus as we can. Again, I 

### 3 just go back, the question was rationale and purpose - not 

### 4 specifics on the assay. 

### 5 So we tried to get into the philosophy of the 

### 6 assay. I read that to be philosophy of why we're doing the 

### 7 assay. So if we over-stepped, my apologies. And I understand 

### 8 the sensitivity because we don't want to put the cart before the 

### 9 horse. There is a process you all are following to do that and 

### 10 all the Panel recognizes that. 

### 11 **DR. SHARLENE MATTEN:**I have talked to one of 

### 12 our leaders in our office, deputy office director, and one of my 

### 13 colleagues just to see a way to figure out a tactful way to 

### 14 include all the scientific information, which is what I was 

### 15 trying to explain, but I got a little crazy and sounded a little 

### 16 too strident what I was trying to explain. 

### 17 I have this problem of the over-exuberant and 

### 18 enthusiastic panel in January that had a specific question, but 

### 19 they wanted to answer a whole bunch of other things. So the way 

### 20 the report was designed was answer directly the question related 

### 21 to Tier 2 first and then everything else that you said, these 

### 22 were the other consideration and then because if you interpret a 

### 23 question, you interpret a question. 

### 24 I was only trying to say separate out into the 

### 25 bits that are Tier 2 and then take the rest of your bits all 

### 26 nicely organized: and these are the other considerations that we

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1 want to suggest. That's what I was trying to point out. 

### 2 **DR. GEOFFREY SCOTT:**Sharlene, I think we can 

### 3 certainly do that in our final written report. I've captured it 

### 4 the way I've captured it today, and we certainly will work with 

### 5 you to make sure it's in the right format. 

### 6 **DR. MARTHA SANDY:**Dr. Touart, do you have a 

### 7 clarifying question? 

### 8 **DR. LESLIE TOUART:**Not a clarifying question, 

### 9 just a comment just to say we appreciate that kind of 

### 10 thoroughness in thinking that the Panel had considered here and 

### 11 the opinions you all provided. I also appreciate some of the 

### 12 comments you heard that really our primary objective is the 

### 13 focus on the test method in terms of its ability to look at the 

### 14 apical endpoints and that the invertebrate AOPs are a little bit 

### 15 outside the scope of what was intended. And I think that's 

### 16 understood. 

### 17 So I appreciate that and I look forward to the 

### 18 comments and opinions on the other areas as they would apply, 

### 19 especially in terms of some of the new science, in terms of 

### 20 other recommendations that may be made. I think, as indicated, 

### 21 as long as those are captured or caveated in the context of 

### 22 those recommendations. I think that's appreciated. 

### 23 **DR. MARTHA SANDY:**I think we're ready to move 

### 24 to Charge Question 2 for the Mysid Two-Generation Toxicity Test. 

### 25 **DR. LESLIE TOUART:**This is Les Touart and 

### 26 this is Charge Question 2 for the invertebrate test. Test

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1 methods and their associated endpoints should be scientifically 

### 2 relevant to the biological processes of interest and should be 

### 3 demonstrated to be responsive to the specific type of effect 

### 4 toxicity of interest. Each species presents unique 

### 5 characteristics from a biological perspective and allows for 

### 6 specialized endpoints to address a specific toxicological mode 

### 7 of action. 

### 8 Please comment on the biological and 

### 9 toxicological relevance of the assay in regards to the stated 

### 10 purpose of characterizing endocrine disruptors as described in 

### 11 Section 2 of the Mysid Two-Generation ISR. 

### 12 **DR. MARTHA SANDY:**The lead is Dr. Scott. 

### 13 **DR. GEOFFREY SCOTT:**Thank you. According to 

### 14 the EPA guidelines in the presentation, relevance is defined as 

### 15 whether a test is meaningful and useful for a particular 

### 16 purpose. The Panel members felt that both the mysid and copepod 

### 17 assays described in the ISR have high biological and 

### 18 toxicological relevance for helping define EDC relevancy as 

### 19 stated in the MTTT ISR. 

### 20 Both assays have multiple and interrelated 

### 21 survival growth development and reproductive endpoints that are 

### 22 useful in describing whether or not a compound identified in 

### 23 Tier 1 screening is altering A, E, and T will affect 

### 24 invertebrates. 

### 25 Given that A, E, and T are lacking in a 

### 26 vertebrate endocrine systems, there are several possible

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1 outcomes which can occur in Tier 2 screening in the invertebrate 

### 2 assays, including 1) the compounds may affect some invertebrate 

### 3 hormone mediated pathway; 2) the compound may cause overt 

### 4 toxicities through a non-invertebrate endocrine mediated mode of 

### 5 action, or 3) the compound may affect both an invertebrate 

### 6 hormone mediated pathway and cause overt toxicity through some 

### 7 non-invertebrate ECD-mediated pathway. 

### 8 The mysid shrimp bioassay has been used in 

### 9 regulatory testing for pesticides and other chemicals for more 

### 10 than 30 years. The current mysid shrimp standard testing 

### 11 protocol; that is, the conventional test, have been developed, 

### 12 which assess a variety of toxicity, growth, development and 

### 13 reproductive endpoints and provide an integrated evaluation of 

### 14 chemicals by assessing these endpoints and relating them back to 

### 15 the neuroendocrine control, at least under the current 

### 16 guidelines for Tier 2 testing. 

### 17 Again, we'll just say, the Panel felt one 

### 18 shortcoming of this approach for the chronic assessment is that 

### 19 these combination endpoints of growth development and 

### 20 reproduction may be affected by chemicals that have both EDC 

### 21 effects and non-EDC effects. 

### 22 And again, the Panel felt linking those back 

### 23 to some juvenile hormone or invertebrate hormone-mediated 

### 24 pathway is important. The current test guidelines underway do 

### 25 measure growth, survival, and reproduction, but do not 

### 26 specifically measure molting. In discussing this among the

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1 panelist, since molting directly controls the endocrine pathways 

### 2 in the organisms, while our indirect measures of time and growth 

### 3 and development reproduction try to capture that we may want to 

### 4 go back and rethink a little bit about how we further could 

### 5 assess molting. I think that was one feeling we had since there 

### 6 is that direct linkage for invertebrate hormones. 

### 7 The addition of the multigenerational assay to 

### 8 the current mysid assay was designed to enhance the 

### 9 effectiveness of the traditional use one-generation approach, 

### 10 using this assay to be more predictive of endocrine effects by 

### 11 including the potential for maternal transfer to offspring as an 

### 12 additional and significant exposure route. I think that is a 

### 13 very good thing to look at, given research that we see in many 

### 14 places on this. 

### 15 The conventional mysid test has been used 

### 16 safely and effectively as we sat and answered a question for 

### 17 Question 1 to evaluate a number of compounds and in the current 

### 18 test has been also used to look at a number of additional 

### 19 compounds. 

### 20 We really believe that the program to expand 

### 21 and look at this assay, this new assay in the current protocols 

### 22 for Test 2 were warranted. And then what we have done under the 

### 23 mysid test is list a number of what we feel are strengths and 

### 24 weaknesses of the mysid assay. 

### 25 I'm not going to read every one those; they 

### 26 will be in the written because I've said many of these already.

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1 I'm just going to try to make a couple of points. We felt the 

### 2 test endpoints are simple and easy to measure. Males and 

### 3 females are distinguishable by conventional microscopy. These 

### 4 are under "strengths." 

### 5 It is a standardized EPA protocol and we're 

### 6 adding additional duration of exposure to it with some more 

### 7 endpoints. And it is a test that looks at growth development 

### 8 and reproduction. The test allows comparisons between F0 and F1 

### 9 -- excuse me, F1 and F2, and we feel that is very important. 

### 10 Currently, the test is recommended as part of the OECD 

### 11 guidelines as, I think Les said, a Level 5 test. And we think 

### 12 that's consistent with this being part of our battery here in 

### 13 the U.S. 

### 14 Under "weaknesses," we just listed a couple. 

### 15 The lack of a specific measure of endocrine disruption in 

### 16 vertebrate systems. The flow through nature of the test does 

### 17 generate more waste; for example, the copepod test. 

### 18 The EDC effects can be confounded by possible 

### 19 overt toxicity. I feel this way, I don't think we're able, 

### 20 necessarily, to distinguish intersex and things like that, 

### 21 currently, with the assay. Now, if I'm wrong, you all correct 

### 22 me on that, but that was my impression. 

### 23 Let's talk a little bit about the harpacticoid 

### 24 copepod since that is also listed in the protocol. It is an 

### 25 additional assay that has been developed to assess endocrine 

### 26 effects, similar to the mysid test, has been used to address

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1 multigenerational effects. It's quite complimentary to the 

### 2 mysid test and it is also currently included under the Phase 5 

### 3 testing in OECD. 

### 4 As was stated in discussions earlier in the 

### 5 Panel this week, the copepod test has the ability to possibly, 

### 6 in some ways, discern endocrine effects slightly differently 

### 7 than the mysid test, since you are able to follow individuals 

### 8 more directly by the nature of the design in a 96-well plate. 

### 9 The copepod species addresses aquatic risk, 

### 10 but is a sediment-dwelling organism, but it has been modified 

### 11 for this. The test can be run in plates, generates smaller 

### 12 amounts of waste. The disadvantage is that it is not widely 

### 13 used and that was another advantage of the mysid test was that 

### 14 the mysid test has been widely used in pesticide testing for 

### 15 over 30 years. 

### 16 The copepod test will require this additional 

### 17 training. There were problems with test delivery of dose with 

### 18 the small nature of the wells and there were some problems in 

### 19 distinguishing between life history stages reported by 

### 20 participants in the assay. 

### 21 The Panel feels the Tier 2 test should 

### 22 quantify, when we can, the interactions between the vertebrate 

### 23 endocrine system to help better discern the measurements that we 

### 24 have in the current mysid and copepod assay. I know we've 

### 25 already said that, so I won't dwell on it as to why. 

### 26 One other aspect I should mention is the

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1 ability to do population modeling with the copepod assay and I 

### 2 think also with the mysid assay. I think we should really try 

### 3 to look at how, particularly in some of our discussions, we were 

### 4 looking at the mysid assay, the current mysid assay, to see if 

### 5 we had actually applied modeling to outputs from that in a 

### 6 predictive way. So we just simply had that comment. 

### 7 I think we heard from the public response 

### 8 also, from the endocrine policy forum, the need to look at where 

### 9 we have existing mysid data and how we might use that. I think 

### 10 the Panel also feels that way and I think adding modeling to 

### 11 that would certainly seem to possibly be a way to help move the 

### 12 process along, although that was not part of the protocol, but 

### 13 we simply add that. 

### 14 Finally, the last comment I'll make, and then 

### 15 I'll leave it to my colleagues here, the Panel felt that the 

### 16 current OECD approach of using a toolbox approach with the 

### 17 testing at different levels, which includes both the mysid and 

### 18 copepod assay, Level 5 as a wise and warranted approach, given 

### 19 the large diversity of biological diversity in the invertebrate 

### 20 pathway in trying to protect these with an assay. This issue 

### 21 will be further addressed in subsequent charge questions. 

### 22 With that, I will stop, other than saying that 

### 23 we felt that the addition of molecular endpoints, as we stated 

### 24 in Question 1, that are endocrine-specific, may provide us 

### 25 guidance on better interpreting data out of either the copepod 

### 26 or mysid assay. Thank you.

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1 **DR. MARTHA SANDY:**Dr. Tuberty. 

### 2 **DR. SHEA TUBERTY:**As a test method being 

### 3 pushed forward for the assay, I think it's very good. I have 

### 4 some experience myself. It does test very well for growth 

### 5 reproduction and developmental effects. As a Tier 2 test, of 

### 6 course, it's not looking for the mechanisms and they're not 

### 7 supposed to. 

### 8 So as far as the demonstrating responsiveness 

### 9 to endocrine disruption compounds, it's hard to know that's 

### 10 happening, for the reasons we've talked about already. But as 

### 11 important endpoints for success of populations of invertebrates, 

### 12 they're all critical. 

### 13 So I think that the assay is sensitive to 

### 14 those measures and can be used for risk assessment and 

### 15 population growth or loss, evaluations as they have been done 

### 16 already with this assay. So I'm very supportive of this assay 

### 17 pushing forward. 

### 18 I think this might be an appropriate time to 

### 19 mention some of the things that Dr. Scott has mentioned for me 

### 20 and that is for some of the endpoints that could be added. Like 

### 21 the other assay, I think these are going to be things that we 

### 22 give to you as a list of resources to look into. Mary, you've 

### 23 mentioned you want references and whatnot, and we'll be doing 

### 24 that too. But the ecdysteroid and the retinoid X receptors in 

### 25 the last two years and in the last two months in one case, for 

### 26 mysids, both neomysis and America mysis have their ecdysteroid

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1 receptor and RXR receptors, the heterodimer, fully cloned and 

### 2 made into a screening assay and it's even been screened with 

### 3 some of the endogenous hormones to look for positive control. 

### 4 It's very reactive and also screened with a number of, actually, 

### 5 almost the same compounds used for both mysids, so you've got 

### 6 some crosstalk there. And comparability with some steroid 

### 7 agenesis inhibitors, also a non-steroidal ecdysone inhibiter. 

### 8 I'm curious as to what the mechanism is there, 

### 9 which does not bind at all to the receptor or with low affinity. 

### 10 So it seems to have a very good applicability here. 

### 11 Considering the techniques that are being adopted in the other 

### 12 assays, it might be something worth looking into that could 

### 13 serve as just another endpoint to a Tier 2. It could be using 

### 14 the material at the end of the test or once the material in the 

### 15 animals not chosen for the F1 pairings would give you a nice 

### 16 endpoint there. Thank you. 

### 17 **DR. MARTHA SANDY:**Dr. Perkins. 

### 18 **DR. EDWARD PERKINS:**I have nothing further to 

### 19 add. 

### 20 **DR. MARTHA SANDY:**Dr. Klaine. 

### 21 **DR. STEPHEN KLAINE:**I have nothing further to 

### 22 add. 

### 23 **DR. MARTHA SANDY:**Dr. Christman. 

### 24 **DR. MARY CHRISTMAN:**I have no additional 

### 25 comments. 

### 26 **DR. MARTHA SANDY:**Any other members of the

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1 panel have comments on Charge Question 2? Dr. Scott. 

### 2 **DR. GEOFFREY SCOTT:**I would just add one 

### 3 additional comment that we will be addressing in subsequent 

### 4 questions and you've heard this with a number of other 

### 5 discussions on the other test protocols and that's the use of 

### 6 positive controls that would possibly be known invertebrate 

### 7 endocrine disruptors and positive controls. You will be hearing 

### 8 from us on that as a way to help augment the current 

### 9 measurements of growth, development, and reproduction that we 

### 10 look at, which in addition to having specific measures of 

### 11 invertebrate hormone, may provide a combination of approaches to 

### 12 help better define the assay, in terms of discerning endocrine 

### 13 effects from non-endocrine effects. 

### 14 That's a subsequent question, but I just 

### 15 thought I would mention that because I do think what we're after 

### 16 is helping you assure that what we call an effect is an 

### 17 endocrine effect. And I think that's what the four of us have 

### 18 been focused on like a laser beam because I think that's 

### 19 hopefully what you wanted us to focus on. So we'll do that in 

### 20 the subsequent discussion. 

### 21 **DR. MARTHA SANDY:**Any other comments? I'll 

### 22 turn to EPA staff. Dr. Tuberty, do you have any questions of 

### 23 clarification? 

### 24 **DR. LESLIE TOUART:**This is Les Touart. Thank 

### 25 you for the comments and also thank you for signaling some of 

### 26 the comments to come that are going to be helpful as well. I

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1 think I can appreciate and look forward to those. 

### 2 **DR. MARTHA SANDY:**And Mary Manibusan. 

### 3 **MS. MARY MANIBUSAN:**I just want to thank 

### 4 this subset of the Panel for their thoughtfulness in engaging 

### 5 with the Agency to push forward and advance in this area of 

### 6 technology in looking at invertebrates. This certainly is a new 

### 7 area for us too. 

### 8 So Dr. Tuberty, any of the papers that you 

### 9 have that you could share with the Agency, that would be greatly 

### 10 appreciated. I'm also interested if you have any information -- 

### 11 and I'm going back to Dr. Scott, your thinking about a Tier 1 or 

### 12 a prescreen -- and I'm saying it, not you -- but if you have 

### 13 information that would help the Agency think about a prescreen 

### 14 or a predictive tool or technology that would help us kind of 

### 15 decide how to prioritize chemicals for this particular Tier 2 

### 16 assay, that would be very much appreciated. Thank you. 

### 17 **DR. MARTHA SANDY:**I'm getting tired. I think 

### 18 we're going to stop here on this set of charge question. We'll 

### 19 take up Charge Question 3 tomorrow morning. I wanted to circle 

### 20 back with the first set of assays that we had started with and 

### 21 turn to Dr. Scanes to see if you had some comments on the dose 

### 22 selection for the Japanese quail. 

### 23 **DR. COLIN SCANES:**The group got together last 

### 24 night and came up with a consensus, and that was, first of all, 

### 25 we were talking about dose response relationships LOEC to the 

### 26 base 2, 2.5, 3, and also the top dose. We came back with the

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1 conclusion, which is that this needs to be a judgment call based 

### 2 upon the chemical and the anticipated results. 

### 3 So I think we've been learning quite a bit 

### 4 from our statisticians, in terms of not having this one size 

### 5 fits all. So coming back with the recommendation to use 

### 6 judgment call, top dose could be based upon the LOEC or the NOEC 

### 7 dose, but there does need to be significant discussion and 

### 8 looking at the individual chemical. 

### 9 **DR. MARTHA SANDY:**I'll ask if any of the 

### 10 other discussants on the Japanese quail assay had anything else 

### 11 they wanted to say? Dr. McCarty? 

### 12 **DR. JOHN MCCARTY:**I think the only other 

### 13 addition to that that came out of our discussion was that 

### 14 thoughtfulness should be reflected when its reported. I think a 

### 15 lot of what caught our attention in the first place was no 

### 16 explanation. 

### 17 **DR. MARTHA SANDY:**Dr. Touart, do you have any 

### 18 questions? 

### 19 **DR. LESLIE TOUART:**Well, a comment, I think, 

### 20 more than a question. In terms of the dose, I appreciate the 

### 21 comments. That's very helpful. But if you have something more 

### 22 specific in terms of how to phrase that within the test 

### 23 guideline would be helpful, and also in the context of with 

### 24 judgment, you know, would there be a recommendation that 

### 25 range-finding would be necessary or other alternative steps than 

### 26 range-finding test may be useful in terms of the judgments to

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1 establish what doses would be appropriate for the chemical to be 

### 2 used. 

### 3 **DR. COLIN SCANES:**We will have discussions on 

### 4 that to try and come up with a consensus recommendation. By 

### 5 bringing it out now, it gives us a chance to put additional 

### 6 thought into this and really come up with a very thoughtful and 

### 7 consensus response. 

### 8 **DR. LESLIE TOUART:**That's helpful. Thank 

### 9 you. 

### 10 **DR. MARTHA SANDY:**Any other comments from the 

### 11 Panel? No? I see Dr. Scanes has something to say. 

### 12 **DR. COLIN SCANES:**Based upon listening to the 

### 13 other species, the quail group wanted to put something on the 

### 14 table to enable them to have further discussions afterwards and 

### 15 then look at a recommendation. 

### 16 I think summarizing review of the Japanese 

### 17 quail to a three-generation test, conclusions were to recommend 

### 18 validity, but with a series of caveats. That's not necessarily 

### 19 the same approach as other groups and we stick to our 

### 20 recommendation; however, one thought that might be helpful to 

### 21 the Agency or to the industry or to the public, would be the 

### 22 possibility of beta testing. 

### 23 Now, we have not discussed this and come to a 

### 24 recommendation, but we wanted to put it on the table to allow us 

### 25 to, perhaps have a series of emails back and forth between the 

### 26 three of us on that issue of beta testing.

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1 **DR. SHARLENE MATTEN:**Just from a process 

### 2 standpoint, once the meeting is over tomorrow, you can't really 

### 3 go about having a consensus. All you can do is say that beta 

### 4 testing is, whatever, an option or an issue or a something, but 

### 5 you can't have these ongoing discussions and develop additional 

### 6 conclusions. That's just more of a Federal Advisory Committee 

### 7 Act process that we need to follow. 

### 8 **DR. MARTHA SANDY:**I'll say that we don't 

### 9 require consensus. If you would like to put on the record that 

### 10 at this point you are recommending that they consider something 

### 11 -- Mary Manibusan would like to speak. 

### 12 **MS. MARY MANIBUSAN:**Clarification question. 

### 13 It's unclear to the Agency what you mean by beta testing. What 

### 14 does that look like? What type of test design are you proposing 

### 15 for beta testing? And to whom should the beta testing be 

### 16 available to? So it's a lot more specifics behind just beta 

### 17 testing? 

### 18 **DR. COLIN SCANES:**This is where we were 

### 19 hoping we would have the opportunity to spend a little bit of 

### 20 back and forth after the meeting, but if it's not going to be 

### 21 possible, then we raise the issue of were the caveats 

### 22 incorporated into the protocol, including the training programs, 

### 23 including the positive control? 

### 24 In order to test whether a contract lab could 

### 25 deliver reasonable results, at least one of the members of the 

### 26 Panel believe that this is something which may be helpful to the

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1 Agency and helpful to the regulatory process. 

### 2 **DR. MARTHA SANDY:**Dr. Bentley. 

### 3 **DR. GEORGE BENTLEY:**I would like to put on 

### 4 the record that I agree with Dr. Scanes on this issue. I think 

### 5 it would be very helpful to establish any contract labs or any 

### 6 labs that are contracted to perform this particular assay. It 

### 7 can demonstrate that they can provide expected results from a 

### 8 positive control in looking at different endpoints in the assay. 

### 9 **MS. MARY MANIBUSAN:**Clarification. 

### 10 Sorry. I'm still confused about beta testing and how that 

### 11 differs from interlab validation. I'm just still confused about 

### 12 what beta testing looks like in reference to that. And then are 

### 13 you speaking to demonstrating laboratory proficiency, which is 

### 14 part of a certification-like program? 

### 15 **DR. GEORGE BENTLEY:**Yes, that's my 

### 16 understanding of it. Having small studies involving positive 

### 17 control with a small number of animals, looking at specific 

### 18 endpoints that are described in this assay, demonstrating that 

### 19 different labs can come to a consensus on the finding they have 

### 20 and that they can actually produce the expected results. 

### 21 **DR. MARTHA SANDY:**And your use of the term 

### 22 "beta testing," are you referring to with a more complete 

### 23 protocol and study design parameters that the U.S. EPA would 

### 24 provide to the performing labs? That you would see if that was 

### 25 enough? 

### 26 **DR. COLIN SCANES:**That's exactly what we're

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1 suggesting, is that if the protocol is refined, the training 

### 2 programs brought forward, among the other recommendations was a 

### 3 positive control, to beta test that in the contract labs exactly 

### 4 in the way that Dr. Bentley mentioned, such that not necessarily 

### 5 they got a consensus in terms of the results, but they got what 

### 6 was expected. 

### 7 And if one found that a contract lab, for the 

### 8 sake of argument, had no effect of were Trenbolone to be used as 

### 9 a positive control and they saw no effects at a level where 

### 10 other labs have seen them. That gets into an issue of lab 

### 11 certification and it also may get into an issue of was the 

### 12 protocol clarified sufficiently that contract labs can execute 

### 13 the protocol in a satisfactory manner. 

### 14 **DR. MARTHA SANDY:**Dr. Klaine. 

### 15 **DR. STEPHEN KLAINE:**What I'm interpreting you 

### 16 saying is that you're thinking of something akin to a reference 

### 17 toxicant test in which a laboratory would perform a ref-tox 

### 18 test. That's what you guys are familiar with, a ref-tox test. 

### 19 And you know what the expected results are going to be and that 

### 20 way you can gauge the performance. Is that what you're 

### 21 describing? 

### 22 **DR. COLIN SCANES:**Correct. 

### 23 **MS. MARY MANIBUSAN:**Sorry. I apologize 

### 24 again. Third time asking. Let me see if I can repeat back what 

### 25 I heard to be part of this beta testing approach. 

### 26 So the beta test would be after we've

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1 established the final study protocol, along with all the 

### 2 supplemental training materials, as well as a much more refined 

### 3 test guideline. We would allow laboratories, contract labs to 

### 4 come in and participate in a beta testing using a reference 

### 5 known compound that we know the expected results and see if they 

### 6 can demonstrate proficiency in performing that particular study 

### 7 protocol for that known chemical. And that would be a part of, 

### 8 in my mind envisioning, is an extension of an interlab 

### 9 combination of having a positive control as well as 

### 10 demonstrating lab proficiency in a certification process. 

### 11 **DR. COLIN SCANES:**I would say the answer to 

### 12 what you said is yes, with one little wrinkle, and that is, were 

### 13 there to be found to be a lack of clarity in the revised 

### 14 protocol, that gives EPA a chance to revisit the protocol or the 

### 15 training program. 

### 16 **DR. MARTHA SANDY:**Dr. Bradbury. 

### 17 **DR. STEVEN BRADBURY:**I think we're getting 

### 18 convergence. Well because there is sort of this spectrum of 

### 19 ongoing validation to the one extreme of doing it again to see 

### 20 if you get a consensus because the consensus is around whether 

### 21 or not the endpoint itself is reproducible as opposed to moving 

### 22 into did we explain how to do this clearly and getting some data 

### 23 back and going, yep, it looks like we explained how to do this 

### 24 clearly, which is a little different than continuing the 

### 25 validation process, per se. It's more like did we explain it 

### 26 clearly.

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1 And then the other point, which I think came 

### 2 up in all -- well, we'll see about the fourth assay -- but the 

### 3 first three assays was a robustness. I think Dr. Portier's 

### 4 thoughts about what robustness meant, is it some aspects of 

### 5 running an assay. You don't have to go right at 55 miles an 

### 6 hour. You could go 50 or you can go 65 and it's going to work 

### 7 pretty good, but there is some components of these four assays 

### 8 where you really need to go 55 miles an hour because if your car 

### 9 won't run 55 miles an hour, i.e., you can't do the genotyping. 

### 10 You're going to have a heck of a time pulling this off. You get 

### 11 the idea. 

### 12 So to the extent there is some insight on what 

### 13 attributes of pulling off the four assays, you really need to 

### 14 demonstrate you can do that. So that when you go forward with 

### 15 the assays, you're likely to execute them reasonably. That 

### 16 would be really helpful. 

### 17 So what are the key parameters where that 

### 18 capability is really important to document. How we would 

### 19 actually implement that, there are all sorts of options in doing 

### 20 that. But for sure from the Panel, for me at least, and my 

### 21 other colleagues can tell me he's nuts, but to at least know, 

### 22 like out of the Medaka assay, these three things are really 

### 23 critical, whatever number you all think and what they are is 

### 24 really critical to pull these off carefully. 

### 25 You need to document you've got the diet 

### 26 right, the genotyping is right, whatever the keys ones are.

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1 That would be really helpful to get some feedback a) on making 

### 2 sure we explain this really, really clearly and b) it may be a 

### 3 way to factor that into a lab, feeling like they can do it 

### 4 right. And that could include a positive control or something 

### 5 so that they can document it. 

### 6 **DR. COLIN SCANES:**It seems very reasonable 

### 7 what you just said. 

### 8 **DR. MARTHA SANDY:**Dr. Ehrich. 

### 9 **DR. MARION EHRICH:**I work in a toxicology 

### 10 diagnostic lab and we do this all the time. We get a sample to 

### 11 come out to test. It's a protocol that is sent from the FDA or 

### 12 the USDA and we have to see if we fit within a certain range. 

### 13 So this would be no different than doing what we do for 

### 14 diagnostic labs to make sure you're fitting standards. 

### 15 **DR. MARTHA SANDY:**Dr. Propper. 

### 16 **DR. CATHERINE PROPPER:**I want to be clear 

### 17 because that is part of an internal kind of control within any 

### 18 assay you run, is to have those diagnostic criteria there and be 

### 19 met. 

### 20 Let's say tadpoles and the controls always 

### 21 weigh one gram at stage 62, but what Dr. Scanes is saying is 

### 22 even before the labs are allowed to run these assays, they go 

### 23 through a protocol of certification demonstrating they can do it 

### 24 as a first step and then they must still have those diagnostic 

### 25 criteria in there. Am I understanding it correctly? 

### 26 **DR. COLIN SCANES:**From my point of view, yes.

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1 **DR. MARTHA SANDY:**Dr. Bentley. 

### 2 **DR. GEORGE BENTLEY:**I was just going to say 

### 3 that that's also true from my point of view. I think it allows 

### 4 for lab certification without using a large number of animals in 

### 5 the first place where a lab might not be certified or the 

### 6 protocol might not be clear enough. So it allows for a 

### 7 reevaluation and retinkering of the protocol, per se, before a 

### 8 full blow test is performed. 

### 9 **DR. MARTHA SANDY:**Dr. Propper. 

### 10 **DR. CATHERINE PROPPER:**So I'm very glad you 

### 11 all brought this up because I almost brought it up during our 

### 12 discussion and then I wasn't sure whether it was really within 

### 13 the purview of the questions. So then I was quiet about it, but 

### 14 I do just want to go on the record in saying that I do agree 

### 15 with some kind of certification as a recommendation, but I 

### 16 understand that that's not really within our questions. 

### 17 **DR. COLIN SCANES:**I hope I wasn't jumping in 

### 18 too fast. And that was to reiterate something that George 

### 19 Bentley just said. This is testing not only the contract labs 

### 20 and their ability to do the protocol, it is also testing with 

### 21 the clarified protocol, is that doable? 

### 22 So I think it's a two-way test. 

### 23 **DR. MARTHA SANDY:**Yes. So it's not just 

### 24 certification. It's also allowing the EPA to see if the 

### 25 protocol is clear enough or if they have to tinker with it. 

### 26 Mary, did you have thing?

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1 **DR. MARY CHRISTMAN:**I actually have a 

### 2 question. I'm not 100 percent sure of what you guys mean by 

### 3 positive control, but if you were to perform such an analysis, I 

### 4 would recommend that you blind it to the expected result so that 

### 5 they don't know what the outcome should be; otherwise, you 

### 6 cannot guarantee objectivity. I just wanted to point that out. 

### 7 **DR. MARTHA SANDY:**Dr. Scott. 

### 8 **DR. GEOFFREY SCOTT:**As an analogous 

### 9 comparison, your chemistry, you have to have certification in 

### 10 chemistry, the same way like the border horizon. You have to go 

### 11 through a certification process before, you know, you ran 

### 12 samples. We had to go through NIST and our lab validation 

### 13 before we started running samples. So to me, this is being 

### 14 consistent with this approach, even though chemistry is only 

### 15 part of it, but certainly this is consistent with other 

### 16 performance-based metrics. 

### 17 **DR. MARTHA SANDY:**Dr. McCarty. 

### 18 **DR. JOHN MCCARTY:**I just wanted to comment on 

### 19 the statement that this might not fall under our questions. In 

### 20 my opinion, this falls directly under our Question Number 6, 

### 21 talking about transferability of this to other labs and part of 

### 22 what we're saying if this is going to be successfully 

### 23 transferred, it has to be a back and forth. 

### 24 Maybe this is my bias as an educator, but part 

### 25 of transferring is demonstrate that I've transferred it to you 

### 26 through certification or passing the test. So it does fall

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1 under our questions. 

### 2 **DR. MARTHA SANDY:**Dr. Bentley. 

### 3 **DR. GEORGE BENTLEY:**I agree with that. And 

### 4 we were also asked to comment on how to improve the assay and I 

### 5 think that this is a good way to improve the assay. 

### 6 **DR. MARTHA SANDY:**Dr. Ehrich. 

### 7 **DR. MARION EHRICH:**I don't know if formal 

### 8 certification is something that you really need. You send it 

### 9 out and you don't know what's going to happen and you get it 

### 10 back and then you know because you sent it out. 

### 11 I don't know that you need a formal, you know, 

### 12 I've got a certificate I can hang on the wall for this 

### 13 laboratory that I can do this test. I think that's going a 

### 14 little farther than necessary. But that's my opinion. 

### 15 **DR. MARTHA SANDY:**Dr. Bradbury. 

### 16 **DR. STEVEN BRADBURY:**This was interesting. 

### 17 As the DFO said, you can write things down that you want to 

### 18 write down. I would encourage you to think about the places 

### 19 you're going to write some of this down because, frankly, we 

### 20 don't certify laboratories to provide the registrant's data to 

### 21 us. There are GOP labs that need to follow test guidelines. 

### 22 There are things we look at when a study is 

### 23 submitted to ensure we consider that study acceptable. If each 

### 24 test guideline, blah, blah, blah, say for the current Fish 

### 25 Early-life Stage Test, it's been validated and we what a study 

### 26 looks like if it's good enough to accept or we're going to

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1 reject it and make them do it over again. And we can look 

### 2 across control data across many labs. We can look at control 

### 3 data within that lab. Sometimes things happen and you make 

### 4 adjustments. Like I said, at the first stage, but we don't 

### 5 certify labs. 

### 6 Now, having said that, you also have talked 

### 7 about within each, I'm assuming all four assays, there are going 

### 8 to be certain attributes of those assays that you feel are 

### 9 really critical to have a high likelihood that the overall assay 

### 10 can be executed well. I think giving feedback on that is very 

### 11 helpful. And that could be used in a variety of ways. 

### 12 So my careful request would be to focus on 

### 13 what are the key parameters that you need to be performed really 

### 14 well - really well. You know what I mean. Because if you can't 

### 15 do that part right, you're unlikely to pull off the rest of the 

### 16 assay and giving some advice about how to communicate clearly, 

### 17 what that needs to be so that if somebody is skilled and they 

### 18 read the instruction, there is a high likelihood they could at 

### 19 least execute it the way we intended so we can go yeah, it looks 

### 20 like that lab has got attributes that suggests that they can 

### 21 pull the assay off well. And it can even be something that 

### 22 needs to be periodically documented within a laboratory or 

### 23 something like that. 

### 24 But I just wanted to let you know that some of 

### 25 that policy outcomes -- I mean, you can say what you want to 

### 26 say, but just wanted to be frank with you that there are certain

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1 things that we just don't do in this program. 

### 2 Dr. Scott talked about certification and 

### 3 enforcement. If you're an enforcement lab and the data coming 

### 4 out of your lab may be used in an enforcement case, that's a 

### 5 different place, but that's not what we are in the registration 

### 6 program. 

### 7 **DR. COLIN SCANES:**While it may not be 

### 8 something that is done, listening to the last two days, it's 

### 9 very clear that many contract labs are missing the boat on 

### 10 certain parameters or it may be that the protocol is unclear, 

### 11 not allowing the contract labs to come up with a reasonable 

### 12 response. And that's why we suggested a beta test, which was an 

### 13 opportunity for the lab to try to replicate reasonable data. 

### 14 Now, that progressed into a certification 

### 15 program. You know, one can get into a situation of definitions, 

### 16 but if a contract lab cannot, for the sake of argument, 

### 17 determine whether it's a male quail or a female quail, 

### 18 genetically, then we've got a problem with all of the results 

### 19 that come out of that lab. And I think that might be true in 

### 20 other situations. 

### 21 So that's why we're suggesting that the notion 

### 22 of a beta test, beta testing the protocol one because there may 

### 23 be clarity issues beta testing the training programs, but having 

### 24 the labs beta tested to see if they are coming out -- that they 

### 25 can detect; for instance, maleness, femaleness, genetically. 

### 26 **DR. MARTHA SANDY:**I was going to suggest

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1 everyone remember what they said and capture it in the 

### 2 appropriate charge question. I'm now turning to Dr. McCarty. 

### 3 **DR. JOHN MCCARTY:**I guess I want to turn 

### 4 around and ask a clarifying question to you guys because we seem 

### 5 to be falling into this semantics morass, and I've got to help 

### 6 write this section. You don't like certification. What do you 

### 7 want us to call this in the report? If you understand what 

### 8 Colin is saying, what do you want us to call it? 

### 9 **MS. MARY MANIBUSAN:**I think as Dr. Bradbury 

### 10 has suggested, we move away from terminologies like "lab 

### 11 certification" or "beta testing" and focus on what we're really 

### 12 after, which is demonstrated performance from proficiency within 

### 13 the laboratory for the study protocol. So what do we absolutely 

### 14 need to make clear? 

### 15 What do these labs have to demonstrate so that 

### 16 we know that our protocol is clear and it's understandable. 

### 17 It's doable. I would just focus on the attributes rather than 

### 18 give it a name or requirement or numbers or labs or any kind 

### 19 naming clichI like that. Just move away from that and just 

### 20 focus on concepts. 

### 21 **DR. MARTHA SANDY:**That might be good. 

### 22 Instead of using a term that might be interpreted differently, 

### 23 say more clearly what exactly you mean. And I think it's been 

### 24 stated by the Panel members multiple times. Dr. Propper. 

### 25 **DR. CATHERINE PROPPER:**So my understanding is 

### 26 that the emphasis we placed as several or all of the groups did

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1 on positive controls always being run with each exposure may, in 

### 2 fact, help with that kind of -- I won't call it beta testing or 

### 3 certification -- but may at least give you that performance 

### 4 criteria that's consistent and may allow for the kinds of 

### 5 outcomes that Dr. Scanes is asking for. Does the Panel feel 

### 6 that that would be something that would function or would you 

### 7 want more than that? 

### 8 **DR. COLIN SCANES:**I think it would be helpful 

### 9 that the positive controls was one of the strong recommendations 

### 10 from the quail panel. But also to see that the first time it's 

### 11 run to see if it works in the lab where they can demonstrate, 

### 12 using Mary's term, "demonstrate proficiency." Demonstrate the 

### 13 clarity of the protocol and the effectiveness of the training 

### 14 program. 

### 15 **DR. MARTHA SANDY:**I think we've covered this 

### 16 well enough. Before we close today, I would like to give the 

### 17 folks, the few Panel members that will not be here tomorrow 

### 18 morning and would like to give them a chance to make any other 

### 19 comments that they would like to. 

### 20 We typically go around and let everyone do 

### 21 that at the close of the meeting. Dr. McCarty, I believe you're 

### 22 one of the individuals. 

### 23 **DR. JOHN MCCARTY:**I think I've said what I 

### 24 needed to say. 

### 25 **DR. MARTHA SANDY:**Dr. Bentley, am I correct 

### 26 that you won't be here tomorrow morning?

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1 **DR. GEORGE BENTLEY:**I won't be here tomorrow, 

### 2 but yeah, I've said enough. Thanks. 

### 3 **DR. MARTHA SANDY:**Dr. Kullman. 

### 4 **DR. SETH KULLMAN:**I had one point on Question 

### 5 4 with the Medaka assay, and it's really just a technicality, 

### 6 but one thing that wasn't said and I didn't catch it at the time 

### 7 was looking back at the ICCVAM criteria for validation purposes, 

### 8 Criteria 5 suggests that a test method performance must have 

### 9 been demonstrated to use a series of reference chemicals, 

### 10 preferably coded to exclude bias. 

### 11 I just want to put on the record that in any 

### 12 of the assays that I saw today that none of these compounds have 

### 13 be coded. So there may be inherent bias. I think that comment 

### 14 was just made as well in this last round of discussion. 

### 15 **DR. MARTHA SANDY:**Is there anyone else that 

### 16 won't be here tomorrow morning? 

### 17 (No response.) 

### 18 Okay. I thank you all for all your hard work, 

### 19 to the Panel, and I thank the EPA staff and all the audience. 

### 20 We will be convening tomorrow at 9:00 a.m. I would like to ask 

### 21 the Panel members to join us in the back room as soon as we're 

### 22 adjourned. Dr. Matten, do you have anything else to say? 

### 23 **DR. SHARLENE MATTEN:**Thank you for the lively 

### 24 discussion. I'm sure that as the report comes together that the 

### 25 organization will be an effective way to address the charge 

### 26 questions and provide the other additional comments in a way

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1 that is very illustrative. 

### 2 Most everything that could have been loaded 

### 3 into the docket has already been loaded. I'm just waiting for 

### 4 the quality assurance process to be completed before it's 

### 5 available to the public through the open portal, under 

### 6 regulations.gov. 

### 7 So I thank you again for your attentiveness 

### 8 and appreciate the fact that everyone is leaving but me. I 

### 9 know; my children just say the same thing. With that, I'll 

### 10 close the meeting for today and I'll see everyone at 9:00 

### 11 tomorrow morning. 

### 12 (Whereupon a recess was taken for the day) 

### 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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1 **DR. SHARLENE MATTEN:**Good morning. Greetings 

### 2 Panel. My name is Sharlene Matten. I am the designated federal 

### 3 official working with the Panel and the Agency on the 

### 4 Independent Peer Review of the Issues Concerning the Proposed 

### 5 Endocrine Disruptor Screening Program, Tier 2 Ecotoxicity Tests. 

### 6 This is June 28th. You are listening to a 

### 7 live broadcast of the FIFRA Scientific Advisory Panel from 

### 8 Arlington, Virginia. Thank you very much. I will turn the 

### 9 microphone over to Dr. Martha Sandy. 

### 10 **DR. MARTHA SANDY:**Good morning and welcome to 

### 11 everyone. This is Martha Sandy. This is our fourth day and we 

### 12 are discussing the Mysid Two-Generation Toxicity Test and a 

### 13 Copepod Development and Reproduction Test. Before we get into 

### 14 the charge questions for this set of assays, we still need to go 

### 15 around the room and introduce ourselves for the folks that are 

### 16 on the webcast. So again, my name is Martha Sandy and I am a 

### 17 toxicologist. I work at the office of Environmental Health 

### 18 Hazard Assessment, part of California EPA. 

### 19 **DR. K. BARRY DELCLOS:**Barry Delclos from the 

### 20 FDA's National Center for Toxicological Research. 

### 21 **DR. JAMES MCMANAMAN:**Jim McManaman, 

### 22 University of Colorado. 

### 23 **DR. DANIEL SCHLENK:**Dan Schlenk, University 

### 24 of California Riverside. 

### 25 **DR. STEPHEN KLAINE:**Steve Klaine, Clemson 

### 26 University.

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1 **DR. MARION EHRICH:**Marion Ehrich, 

### 2 toxicologist, Virginia Tech. 

### 3 **DR. GEOFFREY SCOTT:**Geoff Scott, National 

### 4 Oceanic and Atmospheric Administration. 

### 5 **DR. VICKI BLAZER:**Vicki Blazer, U.S. 

### 6 Geological Survey. 

### 7 **DR. REYNALDO PATINO:**Reynaldo Patino, US 

### 8 Geological Survey, Texas Cooperative Fish and Wildlife Research 

### 9 Unit. 

### 10 **DR. CATHERINE PROPPER:**Cathy Propper, 

### 11 Northern Arizona University. 

### 12 **DR. EDWARD PERKINS:**Ed Perkins, U.S. Army 

### 13 Corps of Engineers. 

### 14 **DR. ROBERT DENVER:**Bob Denver, University of 

### 15 Michigan. 

### 16 **DR. DALLAS JOHNSON:**Dallas Johnson, Kansas 

### 17 State University, statistician. 

### 18 **DR. COLIN SCANES:**Colin Scanes, University of 

### 19 Wisconsin, Milwaukee. 

### 20 **DR. SHEA TUBERTY:**Shea Tuberty, Appalachian 

### 21 State University, ecotoxicologist. 

### 22 **DR. JENNIFER FREEMAN:**Jennifer Freeman, 

### 23 Purdue University. 

### 24 **DR. MARY CHRISTMAN:**Mary Christman, MCC 

### 25 Statistical Consulting. 

### 26 **DR. MARTHA SANDY:**We left off, we had

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1 finished responding to charge question two, and now we are ready 

### 2 to move to charge question three. So if EPA staff, Dr. Touart, 

### 3 wouldn't mind reading question three into the record please. 

### 4 **DR. LESLIE TOUART:**Mysid Charge Question 

### 5 number 3; the test protocol should be sufficiently detailed and 

### 6 should include a description of what is measured and how it is 

### 7 measured. The selection of endpoints within the assay should be 

### 8 reflective of the biological processes of interest and the 

### 9 endpoints should be intrinsically relevant and have established 

### 10 sensitivity. The test protocol should demonstrate the ability 

### 11 to measure the endpoints and provide adequate performance 

### 12 criteria or evaluation. 

### 13 Please comment on the selection, optimization 

### 14 and demonstration of the assay endpoints, as outlined in 

### 15 Sections 3 and 4 of the mysid ISR. 

### 16 **DR. MARTHA SANDY:**Dr. Perkins is the lead on 

### 17 this. 

### 18 **DR. EDWARD PERKINS:**During the course of two 

### 19 lifecycles, over 60 days, over 20 endpoints are collected with 

### 20 optional biochemical measurements suggested. In general, we 

### 21 felt the test protocol for the MTTT is described in sufficient 

### 22 detail the requirements for test acceptability presented well. 

### 23 However, additional details on animal 

### 24 husbandry and maintenance should be provided as the mysid can be 

### 25 easily disturbed during testing through lights and other 

### 26 factors. The Panel will provide further details on animal

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1 husbandry that can be helpful if included in the protocol. 

### 2 However, the endpoints are also reflective of general toxicity 

### 3 that might be caused by nonendocrine disruptors. 

### 4 The endpoints are not only reflective of 

### 5 mysid, crustacean endocrine impact such as growth, molting, 

### 6 reproduction, but they are also reflective of general toxicity 

### 7 and might be caused by non-crustacean endocrine disruptors. 

### 8 Therefore, the selected endpoints are appropriate for detecting 

### 9 the apical adverse effects. There is regulatory concern that 

### 10 vertebrate EAT AOP act of chemicals may have on invertebrates, 

### 11 regardless of whether or not they directly disrupt the 

### 12 crustacean endocrine system. 

### 13 The Agency should consider inclusion of assays 

### 14 for additional molecular endpoints such as activation of 

### 15 ecdysteroid receptor, which have recently been developed as was 

### 16 mentioned in an earlier response to question two. These 

### 17 endpoints will provide more mechanistic information on effects 

### 18 to be used in interpreting apical effects seen in the assay. 

### 19 In the absence of a direct measure of 

### 20 invertebrate EDC effects, such as changes in endogenous 

### 21 ecdysteroid or juvenile hormones, EcR/USP or RXR receptor ligand 

### 22 complexing, inferences indirect based on these EDC-specific test 

### 23 endpoints. In none of the optimization or demonstration tests 

### 24 was it possible with a mysid or copepod assays discern specific 

### 25 invertebrate endocrine effects per se in a mechanistic manner. 

### 26 A large number of endpoints results in many

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1 endpoints being interrelated, given that none individually are 

### 2 definitive for endocrine disruption, the use of multiple 

### 3 endpoints allows for a weight of evidence approach to draw 

### 4 conclusions from the assay. The integration of these endpoints 

### 5 is critical when attempting to understand cumulative effects of 

### 6 a potential endocrine disruptor using these test endpoints. 

### 7 More importantly, inconsistencies among endpoints can be 

### 8 examined for indication of other modes of action. 

### 9 In the ISR, the responses of the endpoints 

### 10 were noted, but no attempt was made to characterize 

### 11 interrelatedness or lack thereof of responses. This issue was 

### 12 noted for endpoints measured in both generations during the 

### 13 assay. 

### 14 One Panel member did question whether all the 

### 15 endpoints are in fact needed for characterizing the effects of 

### 16 endocrine disruptors on invertebrates. They suggested that it 

### 17 would seem, for example, that weight and length measurements may 

### 18 not be needed to be taken at three time points, rather two might 

### 19 be sufficient and that the first brood size rather than the size 

### 20 of the first two broods may be sufficient. 

### 21 The optimization demonstration of the 

### 22 endpoints appears sufficient. However, the amount of 

### 23 variability and general disagreement seen in inter-laboratory 

### 24 testing of what endpoints are effected at which dose suggests 

### 25 that either the protocols for carrying out the tests or 

### 26 determining when an endpoint is impacted need further

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1 optimization and demonstration before the test can be reliably 

### 2 reproduced or used in non-expert laboratories. 

### 3 In the multigenerational testing approach, 

### 4 some concern is placed on the loss of highly sensitive 

### 5 subpopulations of males and females in the F0 population, which 

### 6 may result in have an F1 population that is less sensitive to a 

### 7 chemical, thereby resulting in a lower reproductive impact than 

### 8 seen in the F0. 

### 9 Given the known potential for many pesticides 

### 10 that cause increased tolerance in development of pesticide 

### 11 resistance, this is a valid concern. Public comments by the 

### 12 Endocrine Policy Forum Presentation indicated concerns about the 

### 13 higher NOEC in the F1 versus the F0 for the mysid test. In 

### 14 additional studies using the copepod bioassay, development of 

### 15 pesticide tolerance and resistance has been shown for some 

### 16 pesticides such as chlorpyrifos. 

### 17 The Panel encourages discussions between the 

### 18 EPA and Pesticide Registrants to use any known information on 

### 19 development of pesticide resistance for a compound that may add 

### 20 value to our conduct of these Tier 2 tests. 

### 21 **DR. MARTHA SANDY:**I will ask the discussants 

### 22 on the question, the associate discussants if they have anything 

### 23 to add. Dr. Tuberty? 

### 24 **DR. SHEA TUBERTY:**I have nothing further to 

### 25 add. 

### 26 **DR. MARTHA SANDY:**Dr. Scott?

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1 **DR. GEOFFREY SCOTT:**Just a reference of 

### 2 Gardner et al. that will be added as the reference to what an 

### 3 example of one of the molecular endpoints that was referred to. 

### 4 **DR. MARTHA SANDY:**Dr. Klaine? 

### 5 **DR. STEPHEN KLAINE:**My comments have been 

### 6 incorporated. 

### 7 **DR. MARTHA SANDY:**Dr. Christman? 

### 8 **DR. MARY CHRISTMAN:**At first, I took this 

### 9 question to refer specifically just to the endpoints, in which 

### 10 case Dr. Perkins' comments include anything I might have said, 

### 11 but in thinking about this more, I thought if you are looking at 

### 12 validation studies and you are interested in protocol, that 

### 13 includes the statistical parts. What do you do once you have 

### 14 obtained this information from these endpoints? And in that 

### 15 sense, I do believe that the protocol is terribly incomplete. 

### 16 Let me give you an example. The test 

### 17 guidelines document that was provided to us is supplementary 

### 18 information says, page five, "The design of materials used for 

### 19 the exposure system are optional. It is strongly recommended 

### 20 that a statistician be involved in both the design and analysis 

### 21 of the test results since the guideline allows for variation in 

### 22 experimental design." It also goes on to say in the ISR, the 

### 23 ISR recognizes that there are some problems here because it goes 

### 24 on to say statistical guidance provided by the draft guidelines 

### 25 is minimal, recommending that a laboratory include a 

### 26 statistician in the process.

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1 There is no guidance about appropriate 

### 2 experimental design that should be used. The correct 

### 3 comparisons are not addressed with respect to vehicle control or 

### 4 regular controls, and the lack of guidance causes confusion over 

### 5 the correct design, what types of tests should be performed, et 

### 6 cetera, et cetera. So I would just concur with that statement 

### 7 and make sure that it is clearly indicated that this protocol is 

### 8 missing a lot of that information. 

### 9 **DR. MARTHA SANDY:**Other comments from other 

### 10 members of the Panel on this question? Dr. Klaine? 

### 11 **DR. STEPHEN KLAINE:**I've got a question for 

### 12 Mary. If you follow those guidelines and two different 

### 13 laboratories ended up selecting statisticians to help them with 

### 14 the design of the experiments, is it likely they'll come up with 

### 15 the same design? 

### 16 **DR. MARY CHRISTMAN:**That is an interesting 

### 17 question. I think it depends on how the problem is posed to 

### 18 them, so how the laboratory sees the problem posed. 

### 19 **DR. STEPHEN KLAINE:**I was just curious -- 

### 20 that might explain some of the inter-laboratory variability. 

### 21 **DR. MARY CHRISTMAN:**Well in this particular 

### 22 case, in the mysid cases, I will mention later, Battelle did a 

### 23 reanalysis of all of the data. They all used the same design as 

### 24 it turned out. So it was not a problem, but more generally, it 

### 25 could be an issue. That doesn't mean you can't compare across 

### 26 labs even if they use different designs. You just have to make

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1 sure that the analyses were appropriate for the design that was 

### 2 used. 

### 3 So I'm not claiming that it can't be done. I 

### 4 mean, I like the statement about the involving a statistician, 

### 5 of course. But the reality is that there needs to be more 

### 6 guidelines as to what should be an optimal design for this 

### 7 problem. 

### 8 **DR. MARTHA SANDY:**Other comments from other 

### 9 members of the Panel? Yes, Dr. Scott? 

### 10 **DR. GEOFFREY SCOTT:**Just a followup to the 

### 11 comment on the statistics for this assay, but also in light of 

### 12 the presentation we saw yesterday on the statistics for really 

### 13 looking at all the assays. I believe let's just don't pick on 

### 14 this one, let's pick on them all. It would seem like we heard 

### 15 yesterday the need for what I almost heard like a staff meeting, 

### 16 not exactly, but something like that to guide something this 

### 17 important. I don't know how the rest of the Panel feels about 

### 18 that, but while we're picking on this particular assay on this, 

### 19 I would bring up that broader issue across all the assays, more 

### 20 specificity, like a statistics manual. I don't know how the 

### 21 rest of the Panel -- if that's out of order, we can come back 

### 22 and talk about it later. 

### 23 **DR. MARTHA SANDY:**Dr. Scanes? 

### 24 **DR. COLIN SCANES:**I fully agree with Geoff 

### 25 Scott's comments. 

### 26 **DR. MARTHA SANDY:**Other comments? Dr.

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1 Touart, any things that need to be clarified? 

### 2 **DR. LESLIE TOUART:**I guess some 

### 3 clarifications in terms of some of the recommendations that are 

### 4 heard, like on the weight measurements, to perhaps reduce those, 

### 5 the more specific the recommendations could be made in terms for 

### 6 our writing the guideline, if you could recommend which time 

### 7 points may be better than other time points, like drop day zero 

### 8 and only look at day seven or 14 or whichever you could do would 

### 9 be helpful. 

### 10 With the statistical comment, I know there are 

### 11 some other questions that deal with the statistics and 

### 12 recognizing that designs that were used by the laboratory, say 

### 13 in the inter-lab, and if those approaches were appropriate, then 

### 14 those could form the basis for this additional, more 

### 15 prescriptive guidance in terms of the statistical designs if 

### 16 that is good. If not, then reconsider, so we would look for 

### 17 some advice in terms of better guidance, recognizing that to 

### 18 consult a statistician is a little weak in terms of specifics. 

### 19 But in terms of what more specific can be put into the guideline 

### 20 in terms of the design and treatment level procedures would be 

### 21 helpful for us. 

### 22 **DR. MARY CHRISTMAN:**I completely agree. I 

### 23 don't know how specific it could be. It might not -- if we 

### 24 wanted to do this across assays, it would probably not be too 

### 25 specific. It wouldn't say, well clearly here is your major 

### 26 source of variation. So you ought to put more animals per tank

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1 as opposed to number of tanks or something. But we can 

### 2 certainly make recommendations about exploring that information 

### 3 and then how you might use that information to optimize design. 

### 4 5 In other words, I can't get too specific just 

### 6 because I don't have the raw data, but I can certainly, and same 

### 7 with Dr. Johnson, I'm sure I'll let him comment separately. But 

### 8 I do believe that we can provide guidance as to the approaches 

### 9 that ought to be taken. 

### 10 **DR. LESLIE TOUART:**And that is fair and thank 

### 11 you for that. I think the recommendation in terms of overall 

### 12 for statistical workshop or discussions would be helpful to us. 

### 13 So we appreciate that recommendation. Thank you. 

### 14 **DR. MARTHA SANDY:**All right, I think we're 

### 15 ready for question 4.1, if Dr. Touart would read that in. 

### 16 **DR. LESLIE TOUART:**Reading the first part of 

### 17 the question 4.1 -- demonstration of the test method performance 

### 18 should be based on the testing of reference chemicals 

### 19 representative of the types of substances for which the test 

### 20 method will be used. Test substances should adequately 

### 21 represent an appropriate range of responses and 

### 22 physical/chemical properties for which the test method is 

### 23 proposed to be appropriate. The selection of the most 

### 24 appropriate statistical approaches depends in part on the nature 

### 25 of the data and also on the design of the validation study. 

### 26 Statistical and non-statistical methods used to analyze should

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1 be described. 

### 2 Please comment on the selection of test 

### 3 substances and methods (analytical and statistical where 

### 4 appropriate) chosen for the demonstration and validation of the 

### 5 mysid assay. 

### 6 **Dr. MARTHA SANDY:**Dr. Christman has the lead, 

### 7 and I assume as we have done before, we'll split this question 

### 8 into a couple parts, the statistical parts and the other. 

### 9 **DR. MARY CHRISTMAN:**That is correct. So we 

### 10 will start with the statistical procedures. I want to start 

### 11 with what turned out to be a major question the other day 

### 12 concerning blocking, if you recall. Dr. Touart in his 

### 13 presentation had said that blocking was often ineffective, and 

### 14 in the ISR itself, it says, "The design of the experiment should 

### 15 be completely randomized, unless blocking is required. In that 

### 16 case, blocks should be appropriately characterized by 

### 17 environmental qualities." 

### 18 It then went on to say, "Blocking makes the 

### 19 analysis of the data more difficult, especially if the 

### 20 non-parametric approach is required, and so the preference would 

### 21 be for a completely randomized design." In addition, the ISR 

### 22 also said on page 30, and this is what motivated me to worry 

### 23 about blocking, "Done its tests conducted after removing the 

### 24 block effect indicates that there were significant differences 

### 25 between treatment and controls," and then in parentheses says, 

### 26 "the significance of these responses may have been over

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1 estimated due to removal of the block effect." 

### 2 So there is a concern there from a statistical 

### 3 point of view that you're not modeling correctly. So EPA 

### 4 provided an additional document that goes back to something that 

### 5 was reported in 2008 about determining whether blocks were a 

### 6 problem or not. They had done a randomized block design, in 

### 7 which they blocked -- at least as -- Dr. Johnson and I, when we 

### 8 read it, believed that what they did was a true blocking design 

### 9 and then went on to incorrectly analyze it to decide that there 

### 10 was not block effect. 

### 11 So that was where the concern was, was 

### 12 blocking being done and then being ignored as described by this 

### 13 description in the ISR. We will just remove it, by the way, 

### 14 here's a problem. If you go to the actual documents by the 

### 15 individual labs, it turns out not one of them used a randomized 

### 16 block design. They all used a completely randomized design, so 

### 17 it's a nonissue now with respect to it, but I would recommend 

### 18 the ISR be fixed because that is what generated the problem. I 

### 19 don't know if you can fix a summary that is already written. 

### 20 Now with respect to other aspects, because of 

### 21 the lack of detail about the statistical methodology that was 

### 22 provided in the test guidelines, this particular assay was 

### 23 probably the best analyzed of the bunch with respect to there's 

### 24 still the issues of how to handle normality moving onto 

### 25 heterogeneous variants, et cetera, et cetera, that we've seen 

### 26 for all the other assays. So I'm not going to dwell on that.

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1 But instead, I'd like to point out that what they did do was one 

### 2 of the companies, Battelle, went ahead and reanalyzed all the 

### 3 data using exactly the same methods. They did do them 

### 4 independently, though. In other words, they treated each lab 

### 5 separately, so the decisions about normality and non-normality 

### 6 are still problematic. 

### 7 They also separated F1 and F2, or is it F0 and 

### 8 F1? I'm not sure. I had problems keeping that straight all the 

### 9 time. But the separated the two generations and analyze them 

### 10 separately, et cetera, and I would still argue that this is 

### 11 incorrect, that there are better ways to analyze these data to 

### 12 increase your power to discern differences and take advantage of 

### 13 some of the analyses instead of analyzing everything as its own 

### 14 little data set. 

### 15 But they did do that. They did have one place 

### 16 analyze it all, which was really nice because it allowed me to 

### 17 see how it was done more clearly. They also very clearly 

### 18 identified for every endpoint, they identified the experimental 

### 19 unit that was used in the modeling, which was thank you, I 

### 20 appreciate that. 

### 21 The other thing that was done here was EPA 

### 22 requested Battelle to do what we've been recommending be done 

### 23 all along, that there be an inter-laboratory comparison, which 

### 24 they did. They also did a variance components analysis, tried 

### 25 to identify where the sources of variation were, using the 

### 26 controls among the labs, which was a very good thing to do, and

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1 I'm very glad to see that. I'm not 100 percent sure it was 

### 2 completely done correctly, but it looks like it was. As far as 

### 3 I can tell, it does look like it was done correctly. So it was 

### 4 easy to follow. 

### 5 It was well written. They did do that 

### 6 analysis, which was very nice to see. I'm not sure that they 

### 7 followed up and actually drew conclusions out of it. They may 

### 8 have simply provided results to allow EPA to draw conclusions 

### 9 because I really didn't see any conclusions in there, but I just 

### 10 thought I'd mention that. 

### 11 The other thing they did was a power analysis. 

### 12 But once again, they still did the power analysis from, my 

### 13 point of view, incorrectly because they used simple T test 

### 14 comparisons as opposed to using the experimental design as part 

### 15 of the analysis. So I have specific comments for particular 

### 16 pages, but they all relate to these overview comments that I 

### 17 just made that it was nice to see the inter-lab comparison. It 

### 18 was nice to see the power analysis. It could have been done a 

### 19 little better. It was nice to see the variance components, so I 

### 20 was very pleased. I would have liked to have seen that kind of 

### 21 thing done for all of the assays that I work on at least. So in 

### 22 future, keep that in mind. 

### 23 Let me take a quick gander and see if there 

### 24 was anything else. Yes, there was one more thing that came up 

### 25 when Dr. Perkins just mentioned in question number three about 

### 26 trying to synthesize the information about the endpoints. One

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1 of the things I would like to encourage thought would be to use 

### 2 some sort of multivariant approach that does synthesize the 

### 3 information from all of the endpoints and would allow you to 

### 4 discern differences among treatments in a more holistic way. In 

### 5 other words, in an animal level not at this endpoint level, but 

### 6 to move onto that kind of more complete analysis in the future 

### 7 and the recommendations that we heard for question three are 

### 8 completely appropriate. 

### 9 So that was all I had to say about the 

### 10 statistical methods. I will allow my esteemed colleagues to 

### 11 discuss test substances and analytical methods. 

### 12 **DR. MARTHA SANDY:**Is there anyone else on the 

### 13 panel that would like to comment on the selection of statistical 

### 14 methods? Yes, Dr. Johnson? 

### 15 **DR. DALLAS JOHNSON:**Perhaps we could have 

### 16 said this several days ago, but it occurred to me last night and 

### 17 I have been sitting here typing a little bit up about it. I 

### 18 think it would be interesting or helpful to have it in the 

### 19 report. 

### 20 A lot of the methods that are being used right 

### 21 now are based just on statistical tests. I had a major 

### 22 professor that always said confidence intervals had uniformly 

### 23 more information in them than test statistics or hypothesis 

### 24 tests. In this case, there is an interest in finding the NOECs 

### 25 and the LOECs. I think it would be helpful to provide 

### 26 confidence intervals for the mean differences between control

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1 and each of the dose levels. These confidence intervals should 

### 2 provide a lot more information about the true differences 

### 3 between controls and doses. These confidence intervals could be 

### 4 based on Dunnett's procedure so that you can take into account 

### 5 the multiple testing and the fact that you are testing 

### 6 everything to control. If one provides confidences intervals 

### 7 for the true mean differences, then it would seem that a power 

### 8 analysis and coefficients of variations may not be as necessary 

### 9 to consider. 

### 10 **DR. MARY CHRISTMAN:**I complete concur with 

### 11 Dr. Johnson, and I just wanted to mention one other thing that I 

### 12 forgot to mention earlier was when you do read the statistical 

### 13 protocol aspect in most of the assays, there is quite a bit of 

### 14 effort expended on identifying what is an outlier. But it 

### 15 doesn't say what to do except we send that information to EPA 

### 16 and they decide what to do. In most cases, if it truly is a 

### 17 recording error, then of course it is appropriately removed. 

### 18 If it is not a recording error, I just want to 

### 19 encourage thought about using statistical methods that are less 

### 20 sensitive or more robust to outliers. If you have significant 

### 21 outliers and you are not modeling the data according to a 

### 22 probability distribution that recognizes that you sometimes have 

### 23 extreme values in the tails. Examples include, a really simple 

### 24 example is replacing the normal distribution with the T 

### 25 distribution, which is a heavier tail distribution. So it 

### 26 captures those more unusual observations at the two ends or

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1 moving to a statistical method that is robust to outliers. It 

### 2 is specifically designed to handle outliers. So I just wanted 

### 3 to bring up outliers as well. 

### 4 **DR. MARTHA SANDY:**Are we ready to move onto 

### 5 the second part of this question 4.1 on the selection of test 

### 6 substances and analytical methods? I'll turn to Dr. Tuberty to 

### 7 lead off that discussion, 4.1. Maybe there is someone else that 

### 8 has been designated or has comments ready? 

### 9 **DR. MARY CHRISTMAN:**Well in our discussion 

### 10 the other day, it was Dr. Tuberty was going to discuss the 

### 11 appropriateness of the test substances and the analytic methods. 

### 12 I don't remember who was going to take the lead, though. 

### 13 **DR. MARTHA SANDY:**Would it be easier for Dr. 

### 14 Scott to take the first crack at this? 

### 15 **DR. SHEA TUBERTY:**Most of the comments came 

### 16 from Dr. Scott. I have them here. I can read them if you'd 

### 17 like. 

### 18 So the test substances used for the mysid test 

### 19 include a large number of compounds for both the demonstration 

### 20 and inter-lab testing. The compounds tested for demonstration 

### 21 purposes, therefore Fenoxycarb, DCP and Fipronil include 

### 22 compounds that were known invertebrate IGRs, especially 

### 23 Fenoxycarb and they are very appropriate. 

### 24 With known impacts on crustacean hormone 

### 25 mediated systems, I would include there that this mysid test was 

### 26 in fact, developed specifically with many of these compounds in

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1 mind when designed. This was not the case for the optimization 

### 2 with lindane, prochloraz, atrazine, which were estrogen, 

### 3 androgen or weak estrogen androgen compounds. Perfluorodecanoic 

### 4 acid, which is a thyroid disruptor, trials as these compounds 

### 5 tested did not include known invertebrate and EDC effects, but 

### 6 were in part based on EAT invertebrate effects and screening as 

### 7 preliminary tests. This is way the Tier 1 screening will work 

### 8 with future selection for Tier 2 testing as we understand it. 

### 9 Future testing should include the use of known 

### 10 positive controls, which are known invertebrate endocrine 

### 11 disruptors with new compounds as was described in more detail in 

### 12 question two yesterday. That's all I have. 

### 13 **DR. MARTHA SANDY:**Additional thoughts, Dr. 

### 14 Scott? 

### 15 **DR. GEOFFREY SCOTT:**I think we had quite a 

### 16 bit of discussion. I'll just add a couple. I think Shea 

### 17 captured everything right there in our discussions. 

### 18 The one comment I would make, and we will have 

### 19 more discussion I think in the next question or two on positive 

### 20 controls, I think it's very important with this particular 

### 21 protocol lacking a screening endpoint specific to endocrine 

### 22 disruption as we talked about yesterday, to include it's more 

### 23 important probably for this assay than any of the others to have 

### 24 positive controls. I think as you reform more assays with 

### 25 positive controls and perhaps a range of positive controls, and 

### 26 somebody said yesterday blinded positive controls, that really

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1 makes the labs have to perform. 

### 2 But I really believe you begin to develop 

### 3 growth development and reproductive profiles that give you 

### 4 specificity in deciding whether a compound that is an A, E or T 

### 5 compound, perhaps most resembles an integrated growth 

### 6 development and reproductive response that is most like an 

### 7 ecdysone inhibitor or some other hormonal pathway. Now that to 

### 8 me would be ideal in an ideal world. Whether in practice that 

### 9 would actually occur, I'm not sure, but at least I think if you 

### 10 include those, you do have that benchmark with which to say this 

### 11 compound has 80 percent similarity, and I believe there are 

### 12 similarity analysis that can be statistically run to identify 

### 13 the degree of similarity. So at least in my mind, that's how I 

### 14 would try to link up some of this with the positive controls. 

### 15 **DR. MARTHA SANDY:**Dr. Tuberty? 

### 16 **DR. SHEA TUBERTY:**I guess I would like to add 

### 17 that in the one example of Fenoxycarb as an IGR that was thrown 

### 18 in here, the F1 generation was quite lacking and did not give us 

### 19 a lot of statistical analysis to work with. I can't be sure, 

### 20 but it seems like I know there are studies out there that would 

### 21 have been better to use. I guess this is the demonstration 

### 22 study that was reported with this particular group in mind, but 

### 23 it seems like there would've been more applicable data available 

### 24 out there. 

### 25 But that would have been the one time we could 

### 26 have used the data to compare the other results to, as Dr. Scott

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1 mentions here, to determine if similar outcomes were found that 

### 2 we could link them just by weight of evidence to endocrine 

### 3 disrupting compound effect, not just generalized toxicity -- 

### 4 over toxicity. 

### 5 **DR. GEOFFREY SCOTT:**Just to make really clear 

### 6 what I was trying to ramble and say here, I think the similarity 

### 7 analysis leads you down the road toward helping with weight of 

### 8 evidence. I think that is not intuitively obvious to everybody, 

### 9 but let me just state that. 

### 10 **DR. MARTHA SANDY:**Dr. Perkins? 

### 11 **DR. EDWARD PERKINS:**I have nothing further to 

### 12 add. 

### 13 **DR. MARTHA SANDY:**Dr. Klaine? 

### 14 **DR. STEPHEN KLAINE:**Yeah, just to add onto 

### 15 the previous discussion. In this particular assay, it seems 

### 16 that we're really asking an organism to be very sensitive and be 

### 17 consistent among all the different tests from lab to lab and 

### 18 from test to test and from chemical to chemical. So the 

### 19 incorporation of a positive control not only gives you the 

### 20 ability to test how a lab is performing, but also allows you to 

### 21 test the consistency in the culture. Because as I am sure you 

### 22 are well aware, even when you do a simple acute aquatic 

### 23 invertebrate bioassay, the sensitivity varies tremendously based 

### 24 on the health of the culture, which isn't always apparent unless 

### 25 you are doing something like a ref-tox test regularly. So I 

### 26 think it's really critical here, even more critical than in the

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1 standard acute assays. 

### 2 **DR. MARTHA SANDY:**Are there any other 

### 3 comments on the Panel? I'll turn to EPA and Dr. Touart, any 

### 4 issues that need clarifying? 

### 5 **DR. LESLIE TOUART:**I appreciate the 

### 6 statistical comments. Those are helpful. But I do have a 

### 7 question, when I hear the positive control, especially when I 

### 8 hear controls -- plural -- and the context in terms of the 

### 9 purpose of the positive control as I am understanding that you 

### 10 are suggesting is that the positive control chemical that would 

### 11 be chosen would be a known invertebrate-relevant compound that 

### 12 effects ecdysteroid or some other feature, depending upon if you 

### 13 can recommend a specific compound for that, but again the 

### 14 purpose is in to evaluate or EAT active compound that is being 

### 15 tested and the results seen and the pattern seen for that 

### 16 compound correlates in some way in some way to the pattern seen 

### 17 for the invertebrate-relevant endocrine-active compound. Is 

### 18 that what's being suggested? 

### 19 **DR. GEOFFREY SCOTT:**Les, that is what I would 

### 20 suggest. I really think that's what I would -- if I was king 

### 21 for a day, that's I would do. In fact, I think we heard a 

### 22 comment it would be good to have blinded reference toxicants or 

### 23 positive controls. I think if you are only going to end up 

### 24 selecting one as opposed to going with multiple hormonal routes, 

### 25 I think you probably could look at what you distinctly know 

### 26 about known IGRs and pick the one pathway that appears to be

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1 most protective. 

### 2 That's an idea. That may not be right and I 

### 3 don't know if the other panel members agree with that, but you 

### 4 have to have some rationale to pick it if you're going to pick 

### 5 one. I guess I would simply say that's consistent with the 

### 6 logic in this assay in that we are using crustaceans because of 

### 7 their, I believe in part because of their sensitivity in 

### 8 protecting other invertebrates. 

### 9 So it would seem to me if you're using that 

### 10 logic in the selection of which invertebrate, then you can also 

### 11 use the logic of the pathway that protects the best to also be 

### 12 perhaps used in the selection. We didn't really talk about 

### 13 this, but I'll throw that out in my two cents. 

### 14 **DR. SHEA TUBERTY:**I would just add that I 

### 15 agree with Dr. Scott, but I think that the two things that need 

### 16 to be weighed against one another is the benefit of knowing 

### 17 whether or not the outcomes are in fact due to EDCs when we 

### 18 don't have the Tier 1 screening first to identify that 

### 19 initially. So if you want to separate the effect of just over 

### 20 toxicity versus EDC-specific compounds in invertebrates, then 

### 21 this is a way of doing it. 

### 22 So it would be the same assay with just 

### 23 another cohort exposed to either before or after initially or 

### 24 even just providing the data, so that when you look at the 

### 25 results, you know that these are the types of endpoints that 

### 26 should be affected. Not all of them, maybe just this one for

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1 this compound or this type of compound. In a few minutes, we 

### 2 will talk about some of the data that were available to us for 

### 3 assessment here. We put together some tables and tried to do 

### 4 that thing given the minimal number that were run already. 

### 5 Actually, it wasn't that middle. It's about 10. 

### 6 But I feel the same concerns that this is not 

### 7 giving us a whole lot of information about endocrine disruption. 

### 8 That is what this Panel is here to talk about. We could talk 

### 9 about toxicity effects and again, the apical endpoints are 

### 10 really critical for population growth in these crustaceans and 

### 11 very sensitive organisms that will in fact not just 

### 12 invertebrates but protect vertebrates as well. I think that's 

### 13 probably why they were put on here initially. 

### 14 **DR. MARTHA SANDY:**Any other thoughts on this? 

### 15 So we move on to question 4.2 please. 

### 16 **DR. LESLIE TOUART:**Yes, we can move on and 

### 17 thanks for the comments and clarifications. That's helpful for 

### 18 me. I understand now what you're suggesting. Reading the 

### 19 second part of question 4, 4.2 -- the proposed mysid study 

### 20 design uses three replicates per control and treatment group and 

### 21 additional replicates require greater resources. 

### 22 Please comment on the proposed level of 

### 23 replication and the subsequent statistical analysis of the data. 

### 24 25 **DR. MARY CHRISTMAN:**The short answer is no 

### 26 that three is not enough. Even Battelle's power analysis, the

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1 not-quite-correct power analysis indicates that three is 

### 2 insufficient. There are several options. If it's just too 

### 3 expensive to add much more replication is to reconsider the 

### 4 treatment levels, the choices of treatment level. For example, 

### 5 do you really need six levels if the behavior is in fact a 

### 6 monotonic response? If it's highly nonlinear and you're not 

### 7 sure what the form of that is, than you have a problem. You may 

### 8 have to add replication. 

### 9 But if you have some idea of a threshold 

### 10 behavior or something, then you may not need the lowest dose. 

### 11 You may be able to combine that into the control, for example, 

### 12 because this was the only study, the only assay of the four in 

### 13 which the control had the same number of replicates as the 

### 14 treatment levels. There is a reason why there were increased 

### 15 controls. That aids in precision in the Dunnett's test, for 

### 16 example. 

### 17 So trying to change treatment levels might be 

### 18 an option. Running a different power analysis to identify how 

### 19 much of a difference you actually can discern. Using the 

### 20 correct statistical methods, I think, is an important aspect 

### 21 that you also ought to consider. So those are the main 

### 22 comments. I think you know you have too few. 

### 23 **DR. MARTHA SANDY:**Dr. Tuberty? 

### 24 **DR. SHEA TUBERTY:**Outside the three block 

### 25 replicate issues, there were some noticeable and understandable 

### 26 issues with the design in regard to number of animals available

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1 for the F1 pairing and reproduction portions of the assay. The 

### 2 tradeoff between mortality and loss of statistical power in the 

### 3 F1 generation endpoints and measuring effects of the toxicant is 

### 4 an enduring issue amongst EDC toxicologists. 

### 5 During the initial demonstration optimization 

### 6 studies, there were many issues apparent with number of F1 

### 7 available following reproduction in the F0, which is obviously 

### 8 affected by the toxicity in chosen concentration ranges of the 

### 9 toxicant. It seemed that for most toxicants, the highest 

### 10 exposure concentration was chosen by taking anywhere from 

### 11 one-third to one-one hundredth of the 21 day LC50. Sometimes, 

### 12 that was a number chosen form a 96 hour LC-50, I believe. This 

### 13 is a difficult decision to make. We all face it, and it must be 

### 14 done cautiously because it will have significant impact, 

### 15 obviously, on the outcome of the assay. 

### 16 Specifically in regard to the F1 generation 

### 17 endpoints, due to the length of time for the exposure and the 

### 18 number of things that can go wrong for those organisms along the 

### 19 way, I would like to dis-acknowledge that in the comments by the 

### 20 previous reviewers of the assays on page 43 of the mysid ISR, I 

### 21 think hit it on the head when they state that higher 

### 22 concentrations may be used due to lack of responsiveness and 

### 23 this could be due to the chosen compounds as well. 

### 24 But higher concentrations might need to be 

### 25 used, seeing that there was very low mortality throughout all 

### 26 the studies. I think there was an average of about 88 percent

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1 survivorship, which is very good. So they suggested that maybe 

### 2 an LC20 value would increase the likelihood of measuring an 

### 3 effect while still preventing losses to the F1. That's all I 

### 4 have to say. 

### 5 **DR. MARTHA SANDY:**Dr. Scott? 

### 6 **DR. GEOFFREY SCOTT:**I think Shea got most of 

### 7 my thoughts and I agree with everything that he and Mary have 

### 8 said. I think this question about toxicity in the first 

### 9 generation is important if we assume the individuals that die 

### 10 are the most sensitive. Because that then means what we're 

### 11 looking at in the next generation may be more resistant, or 

### 12 let's just say sensitive individuals for lack of a better term. 

### 13 14 So I really think one thing that would be 

### 15 important in looking at the data we do have would be 

### 16 potentially, and this is more a question for Mary and Dr. 

### 17 Johnson, in looking at the results or those doses where there is 

### 18 no mortality versus those doses where there are and comparing 

### 19 those results in the two generations. While I realize you still 

### 20 have the effect of dose that you do have in a lower number of 

### 21 individuals contributing in the subsequent generation that could 

### 22 be -- it may give you a way to hen at that question of concern 

### 23 about sensitivity between generations. 

### 24 I think you obviously do not want to run a 

### 25 test where you don't get a response. So I think this is one of 

### 26 those fine juggling things you have to do in devising an

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1 exposure scheme that gives you the responses, then you want to 

### 2 minimize and I would say with the survival you have -- what did 

### 3 you say it was 88 percent I think Shea? To me, that's the range 

### 4 you would really want to evaluate something like that at if you 

### 5 lose 12 percent of your population. Remember, we don't have to 

### 6 -- for nonhumans we do on population basis. It's a healthy and 

### 7 well-balanced ecosystem, I believe is the term. That doesn't 

### 8 mean every individual survives. 

### 9 So I guess my point it that at least allows 

### 10 you to look at that question a little bit, maybe further with 

### 11 some of the data you already have. 

### 12 **DR. SHEA TUBERTY:**Let me add one more thing 

### 13 and that was a comment about the F1. I know Mary's hit on this 

### 14 already. Mary has hit on the number of replicates, but the 

### 15 target parings in F1 I believe was about seven. More often than 

### 16 not, the comments at the bottom of the table said that the N was 

### 17 three unless otherwise designated, and a few times it would be 

### 18 between eight all the way to zero. 

### 19 So it would seem like we were making a lot of 

### 20 inferences based on an N of three, two, four, something like 

### 21 that, which is awfully small. As Geoff has already mentioned, 

### 22 that's calling out the most sensitive individuals, at least in 

### 23 the parental population, assuming that the genetics would be 

### 24 passed on. 

### 25 In the inter-laboratory statistical analysis 

### 26 report that was generated from mysids, the CV that were produced

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1 for F0 versus F1 were quite different, further indicating, as we 

### 2 might know already, that this is indicating these populations 

### 3 are different. So 15 of the 25 F1 CVs exceeded the 

### 4 corresponding the F0 CVs. What does that tell us? That there 

### 5 is more variability in that population when exposed to the same 

### 6 concentrations as the F0. 

### 7 It's quite telling. We'll talk about this 

### 8 more later on, but the number of times the F0 population was as 

### 9 sensitive or more sensitive than the F1 generation, if I was 

### 10 someone that made decisions with the money and how it should be 

### 11 spent, I might make the argument that maybe it's not necessary, 

### 12 but as an ecologist that wants to be more conservative about our 

### 13 effects of toxins the couple of times, and this was to say it 

### 14 happened 20 percent of time out of 10, so two out of 10, if we 

### 15 multiple it by 1000 compounds, then we are talking about a lot 

### 16 more, were actually found to be more effective at lower 

### 17 concentrations in that F1 generation. 

### 18 Regardless of our low statistical power, we 

### 19 still were able to capture that. I just wanted to say we're 

### 20 picking this assay apart, but having done it myself when you can 

### 21 get that kind of number anyway, what does that tell us? If we 

### 22 could be protective even 10 or 20 percent of the time in that 

### 23 second generation, then I would argue that it is in fact 

### 24 necessary. Again, when you're looking at just 10 compounds or 

### 25 seven compounds and you only get one that hits on that second 

### 26 one, that's fine but we're talking about 67 compounds, we're

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1 talking about six or seven, how many of them have to be out 

### 2 there affecting our ecology before we start seeing loss of a 

### 3 healthy environment. 

### 4 So I just want to make sure that we're not 

### 5 just looking at 10 and 20 percent as insignificant numbers. 

### 6 We're talking about whole populations. Again, one assay 

### 7 representing a million and one, 1.1 million invertebrate species 

### 8 out there, we need to be cognisant that that number is not 

### 9 insignificant. 

### 10 **DR. MARTHA SANDY:**Dr. Perkins? 

### 11 **DR. EDWARD PERKINS:**The thing I want to add 

### 12 sort of expands -- follows onto what Shea said is that in the 

### 13 relating to the great variability seen in the inter-lab testing 

### 14 that the low number of replicates also might play into some of 

### 15 the variability seen between lab testing. As you've shown in 

### 16 other assays where you increase the number of replicates, you 

### 17 get much better results. And this also may come into a great 

### 18 deal of play showing any consistency between labs. 

### 19 **DR. MARTHA SANDY:**Dr. Klaine? 

### 20 **DR. STEPHEN KLAINE:**I have nothing further to 

### 21 add. 

### 22 **DR. MARTHA SANDY:**Anything from other members 

### 23 of the Panel? Yes, Dr. Johnson? 

### 24 **DR. DALLAS JOHNSON:**I just wanted to add that 

### 25 in this case where you have a small number of replicates, it 

### 26 might be even more important to look at confidence intervals

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1 than in those cases where you have large sample sizes. So it 

### 2 really -- a confidence interval will tell you what you know and 

### 3 what you don't know. If the interval goes from minus 400 to 

### 4 plus 675, you probably don't know much. Otherwise, if the 

### 5 interval was a lot shorter than that, then you probably know a 

### 6 lot. 

### 7 Thinking about this, I'm reminded about one of 

### 8 my favorite statistical jokes about the scientist that came into 

### 9 a friend of mine and said I got this data, I just don't know how 

### 10 to analyze it. He says, well how much do you have? He says I 

### 11 got lots. I got 80,000 observations. He said where did they 

### 12 come from? Well they all came from this one cow. 

### 13 So you can have a lot of data and there's not 

### 14 much information in it if you don't have very many replicates. 

### 15 I think also in reading this, it seemed -- and I don't know 

### 16 about the science, so this could be completely off the wall, but 

### 17 I noticed that in this case, each replicate could have, I think 

### 18 30 to 40 invertebrates involved in that replicate. So the 

### 19 question I have, I guess, is could I increase the number of 

### 20 replicates if I decreased the number of invertebrates that are 

### 21 in each replicate. And there might be some ways to think about 

### 22 how one might do that. It probably depends on -- I know some 

### 23 endpoints are measured on each invertebrate. Some endpoints are 

### 24 measured on the tank. So anyway, it's something that I'll throw 

### 25 out there that one can think about. 

### 26 **DR. MARTHA SANDY:**Any other comments from the

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1 Panel? Dr. Touart, any questions? 

### 2 **DR. LESLIE TOUART:**I appreciate the comments 

### 3 and just one clarifying question back with increasing 

### 4 replicates. I think what I understood was that we first start 

### 5 to look at controls, increasing the number of control replicate 

### 6 tanks, that that would provide some advantage, but also consider 

### 7 increasing the replicates and changing physical design and what 

### 8 not to improve treatments and perhaps the value of replicates 

### 9 may be more than number of treatments itself. Is that my 

### 10 understanding? 

### 11 **DR. MARY CHRISTMAN:**I'm sorry, could you 

### 12 repeat your question? 

### 13 **DR. LESLIE TOUART:**Again, just to kind of 

### 14 cover what I think I heard you recommending was that in moving 

### 15 to increase the replicates that we should first look at controls 

### 16 to increase the number of controls as a first step as that 

### 17 improves, but it would be nice to increase the actual replicates 

### 18 across the test and in doing so, it might be worth tradeoff in 

### 19 terms of increased replicates in a given level with fewer 

### 20 levels. 

### 21 **DR. MARY CHRISTMAN:**Correct. There are many 

### 22 ways you could proceed with this, but the two you just mentioned 

### 23 would be one way to consider keeping the same number of tanks, 

### 24 but varying the experimental setup itself. How many different 

### 25 levels and things of that nature? The second thing is I don't 

### 26 know enough about the actual physical lab setup. I don't know

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1 if your constraint is how many tanks you can fit in a water 

### 2 table or how many tanks could be attached to a diluter system, 

### 3 et cetera, et cetera. But if Dr. Johnson's recommendation about 

### 4 maybe more tanks and few animals per tank or not too fewer but 

### 5 some fewer, certainly the increasing in the breeding pairs, et 

### 6 cetera, et cetera, things of that nature could also be 

### 7 considered. 

### 8 **DR. LESLIE TOUART:**That's helpful. What also 

### 9 would be helpful for those that are, I guess, experienced with 

### 10 mysids and the test and the physical designs, any suggestions on 

### 11 how one may go about that increase without pseudo-replication, 

### 12 because that's the other concern that we have with the designs 

### 13 that are practical within typical lab setups and diluter 

### 14 systems. So, suggestions would be appreciated on that. 

### 15 **DR. MARY CHRISTMAN:**Just to respond -- you 

### 16 picked up a word that it a favorite word -- pseudo-replication. 

### 17 In fact, the lack of recognition of a tank effect leads to that 

### 18 problem with pseudo-replication. So if the statistical model is 

### 19 set up appropriately, that's not too much of an issue. The 

### 20 problem is the lack of recognition of exactly that, which 

### 21 doesn't seem to be too serious for the mysids, but some of the 

### 22 other assays certainly. 

### 23 **DR. MARTHA SANDY:**Alright, I think we are 

### 24 ready to move onto question five, Dr. Touart? 

### 25 **DR. LESLIE TOUART:**This is the Charge 

### 26 question 5. Considering the variability inherent in biological

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1 and chemical test methods, a test method needs to be repeatable 

### 2 and reproducible. A test is robust and reliable if the results 

### 3 are repeatable and reproducible within a laboratory and between 

### 4 different laboratories respectively. A test protocol should 

### 5 provide sufficient guidance to ensure proper and consistent 

### 6 performance across labs and chemicals. 

### 7 Please comment on the test method robustness 

### 8 and reliability and the repeatability and reproducibility of the 

### 9 results obtained with the mysid assay. 

### 10 **DR. MARTHA SANDY:**The lead discussant is Dr. 

### 11 Tuberty. 

### 12 **DR. SHEA TUBERTY:**So validation is a 

### 13 scientific process by which the reliability and relevance of an 

### 14 assay method are evaluated for purpose of supporting a specific 

### 15 use. This is from the ICCVAM 1996 statement. Reliability is 

### 16 the reproducibility of results from an assay within and between 

### 17 laboratories and robustness has been defined by other Panelists, 

### 18 addressing other species as a system that can handle variability 

### 19 but maintain status quo and consistency. 

### 20 The pre-validation process included 

### 21 demonstration of relevance or development of standard optimized 

### 22 protocols and determination of readiness for validation. 

### 23 Validation in multiple laboratories and demonstration of 

### 24 reliability across labs is the final process in this overall 

### 25 process. Earlier inter-laboratory trials assessing 

### 26 reproducibility of acute toxicity for certain aquatic test

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1 species demonstrated that LC50s may vary by a factor of four to 

### 2 five among different labs conducting these acute toxicity tests 

### 3 with the exact same species. You want to insert a reference to 

### 4 Rayan and Petricelli (phonetic) from 1985. 

### 5 Our current testing of these more complex 

### 6 multigenerational testing assays and protocols do not have 

### 7 similar metrics to compare to, but appear to fall within a 

### 8 similar range, more or less six, a factor of six based on the 

### 9 Lindane metrics reported here by EPA despite the increased 

### 10 exposure duration and increased complexities of the assays. So 

### 11 I think the overall outcome there is that we feel that there is 

### 12 fairly good reproducibility. Although the exact responses 

### 13 between labs don't seem to match up very well, there are 

### 14 responses between labs. 

### 15 We go on to another Panelist observed that one 

### 16 laboratory was unable to provide sufficient data for the F1 

### 17 generation and was therefore excluded from their analysis. 

### 18 Further, two laboratories that did provide data have results 

### 19 that led to different conclusions. Overall, this indicates that 

### 20 either one of the following occurred. The sample sizes are 

### 21 simply too small for reproducible analyses. Can you guess who 

### 22 put that in there? There are no treatment effects on the 

### 23 measured endpoints and all significant results were type 1 

### 24 errors. The appropriate concentrations of chemicals were not 

### 25 used to show effects or the laboratories failed to observe the 

### 26 protocols as directed. It is unclear whether the statistical

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1 methods are at fault since there is not sufficient information 

### 2 to determine whether the tests for checking assumptions were 

### 3 appropriate or adequate and how the decision for three 

### 4 replicates was made. 

### 5 The description given for power determination 

### 6 on page 43 of the EPA report is insufficient to determine how 

### 7 the final sample size was decided since it does not discuss 

### 8 whether the measures of time to first brood or length, 

### 9 presumably body length, were for individual breeding pairs as 

### 10 the measure of time or mysid shrimp length, or the average of 

### 11 the tank from which they were taken. 

### 12 Since much of the analyses are for 

### 13 experimental units as tank or basket within tank, the 

### 14 determination of power at the individual animal level is 

### 15 non-informative. 

### 16 As indicated in the ICR, an evolution of the 

### 17 some of the endpoints of the protocol occurred over the course 

### 18 of the studies, which allowed for more precise measurement, but 

### 19 disallowed for direct comparisons between tests, the beginning 

### 20 and the end of the demonstration process. For example on page 

### 21 42 of the mysid ICR, "During the optimization study, total young 

### 22 was evaluated as number per female, but the total was always 

### 23 calculated in two ways. For the first calculation, reproduction 

### 24 data for all females were treated equally. Therefore, 

### 25 reproduction data for female that die before producing a second 

### 26 brood or females no producing a second brood were being omitted.

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1 For the second, only the reproduction data for females that 

### 2 produced a second brood were included. Both sets of 

### 3 calculations were evaluated statistically and the results 

### 4 occasionally differed. The guidelines should clarify which 

### 5 approach meets the goals of the testing so that endpoint will be 

### 6 evaluated consistently by various testing laboratories." 

### 7 So this is one example that I picked up on, 

### 8 and I understand why they wanted to exclude a zero from their 

### 9 data. At the same time, that female that doesn't reproduce the 

### 10 second brood when the controls are all reducing second broods is 

### 11 a significant endpoint. Therefore, another endpoint was 

### 12 generated, which was percent-reproducing females, which never 

### 13 had a significant endpoint throughout the test. So they took 

### 14 the data away from where it could've been helpful and put it 

### 15 somewhere else or more informative and put it somewhere else 

### 16 where it was less informative -- something to be considered. 

### 17 In creating a simple table of reproductive 

### 18 versus growth and development effects and survival, so three 

### 19 different endpoints rather than 20 different ones that were 

### 20 chosen for the mysid test, and looking over the nine or 10 

### 21 different compounds tested, and then looking also which of the 

### 22 generations, F0 or F1 were more sensitive. We've already talked 

### 23 about the F0 being more sensitive a majority of the time, yet F1 

### 24 still being important at least 20 percent of the time. This was 

### 25 in attempt to look at the different known activities of some of 

### 26 these A and T compounds on invertebrates. There is some

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1 consistency in the results in our tests, and that indicates that 

### 2 100 percent of the time, we get growth and developmental effects 

### 3 in the invertebrates in the mysid. In about 70 percent of the 

### 4 time, we're getting effects in the reproduction. 

### 5 So again, these are really important apical 

### 6 endpoints for risk assessment and they're both very responsive, 

### 7 even to the E, A and T active compounds. So I don't want that 

### 8 to be lost on EPA that this is still, and I'm sure you're aware 

### 9 of this, this is still a relevant assay for looking at toxicity 

### 10 whether or not it's specially an EDC compound. We know that 

### 11 molting and growth, incremental growth in crustaceans and 

### 12 arthropods specifically is under ecdysteroid control. That is 

### 13 one of the most well understood hormonal pathways within 

### 14 invertebrates. 

### 15 Looking at the time we talked about the 

### 16 endpoints, day seven, day 14 or at the termination, in looking 

### 17 at the lengths, they are not direct measures of hormonal 

### 18 control, but they do indicate the incremental growth under the 

### 19 control of ecdysteroids. So if you have a significant effect on 

### 20 that growth rate, you're probably having an effect on the 

### 21 ecdysteroid growth. Not to say nutrition and other stressors 

### 22 can have an effect as well. But when we're looking at 

### 23 population level effects, you never see mechanistic effects 

### 24 inherited in those endpoints. We are not capable of doing that. 

### 25 26 Dr. Denver made this point with some of the

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1 other assays. It's what we teach our undergraduates. You can't 

### 2 do everything in one assay. So having that Tier 1 assay 

### 3 available or some other type of a screening test available or 

### 4 including an endpoint in that Tier 2 that is capable of doing 

### 5 that would be the smoking gun, I think that some of this think 

### 6 is important. That's all the comments I have ready. 

### 7 **DR. MARTHA SANDY:**Thank you. And the 

### 8 associate discussants, Dr. Klaine? 

### 9 **DR. STEPHEN KLAINE:**I have nothing to add. 

### 10 It is pretty well covered. 

### 11 **DR. MARTHA SANDY:**Dr. Perkins? 

### 12 **DR. EDWARD PERKINS:**I've got nothing more to 

### 13 add. 

### 14 **DR. MARTHA SANDY:**Dr. Scott? 

### 15 **DR. GEOFFREY SCOTT:**Just one point -- Shea 

### 16 really covered it all there. I think in looking at those 

### 17 inter-lab calibration exercises of acute toxicity that we 

### 18 referenced here, and in looking at the range, we saw in these 

### 19 assays using the most sensitive endpoints. There was a question 

### 20 raised early in this panel what constituted dose response. We 

### 21 said LOEC may be that. I know some people were looking at that 

### 22 and asking questions. Well, does that really define dose 

### 23 response? 

### 24 I think if you look at those acute assays, 

### 25 which were based on LC50s, you're in the middle of a dose 

### 26 response group, most sensitive part. These assays were at the

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1 LOEC, at the bottom of the dose response curve. I think in some 

### 2 ways the fact that it's pretty close tells me that my concerns 

### 3 on dose response are somewhat better satisfied. So I don't 

### 4 think anybody had pointed that out, but I would mention that 

### 5 because I think that's pretty important. Anyway, I just wanted 

### 6 to clarify that point. Thank you. 

### 7 **DR. MARTHA SANDY:**Dr. Christman? 

### 8 **DR. MARY CHRISTMAN:**I concur with Dr. 

### 9 Tuberty's comments. 

### 10 **DR. MARTHA SANDY:**Any additional comments 

### 11 from the Panel? Then I will turn to Dr. Touart to see if 

### 12 there're any issues that need clarification. 

### 13 **DR. LESLIE TOUART:**No clarification 

### 14 questions, but I do appreciate the comments, especially yours 

### 15 Dr. Scott. I think those are very helpful. I appreciate that. 

### 16 17 **DR. MARTHA SANDY:**I think we will take a 

### 18 break now. It is 10:10 by this clock. Let's take a 15 minute 

### 19 break and come back at 10:25. 

### 20 (Whereas a break was taken) 

### 21 **DR. MARTHA SANDY:**I think the Panel members 

### 22 have returned. Thank you. We're ready now if Dr. Touart, would 

### 23 you please read charge question 6.1? 

### 24 **DR. LESLIE TOUART:**The test protocol should 

### 25 be descriptive enough to be fully transferable to a functional 

### 26 laboratory. The protocol should describe the methodology of the

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1 assay in a clear and concise manner so that a laboratory could 

### 2 comprehend the objective, conduct the assay, observe and measure 

### 3 prescribed endpoints, compile and prepare data for statistical 

### 4 analyses, and report the results. Section 5 of the mysid ISR 

### 5 outlines the process of and challenges experience in the 

### 6 inter-laboratory studies. 

### 7 Please comment on the transferability across 

### 8 labs and provide any suggestions or recommendations or 

### 9 improvement of the mysid assay. 

### 10 **DR. MARTHA SANDY:**Dr. Tuberty is the lead 

### 11 discussant on this. 

### 12 **DR. SHEA TUBERTY:**Two major issues need to be 

### 13 addressed, which will improve these current mysid bioassays. 

### 14 The first, use of positive controls, which we've talked about in 

### 15 depth already, hit again on it here. Future inter-lab 

### 16 calibration exercises should include positive controls with 

### 17 known invertebrate EDC effects run at concentrations that should 

### 18 always elicit these responses. Positive controls will add cost, 

### 19 but are needed to assure the contract labs are adequately 

### 20 performing the assays. 

### 21 Positive controls should be added, such as 

### 22 methoprene or other known invertebrate EDCs, which would 

### 23 initially run at multiple doses, but with increased confidence, 

### 24 should be narrowed to a single dose with each new compound 

### 25 tested. Some thoughts should be given to this idea of positive 

### 26 controls, which may target different aspects of invertebrate

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1 endocrine pathways. As more and more data is generated for 

### 2 specific invertebrate EDC pathways represented by these positive 

### 3 controls, these QAQC data may become more important in 

### 4 establishing other benchmark matrices or metrics for use and 

### 5 comparisons on future EDC screening. 

### 6 This is in effect, doing QAQC with a purpose 

### 7 as one is not only assuring the quality of these data, but also 

### 8 enhancing our ability to better differentiate invertebrate EDC 

### 9 effects with specific invertebrate mode of action effects. This 

### 10 is particularly important in helping better distinguish what the 

### 11 different combinations of test endpoint effects actually mean, 

### 12 given the more indirect nature of discerning endocrine effects 

### 13 with these Tier 2 assays. Since mysid tests have been conducted 

### 14 on IGRs which have known AOPs for invertebrate-mediated 

### 15 endocrine effects, it would be important for EPA to 

### 16 statistically summarize those test endpoint responses for mode 

### 17 of actions and then use those as a point of comparison with test 

### 18 endpoints for future compounds tested. 

### 19 Using appropriate statistical comparisons, 

### 20 similarity analyses for example, to given probability based 

### 21 comparisons. This would provide a statistical-based approach to 

### 22 the toolbox for future weight of evidence analysis. 

### 23 The second point is importance of defining 

### 24 major or minor deviations in the labs performing the assay. The 

### 25 EPA EDC screening program would greatly benefit from a hazard 

### 26 analysis critical control point based approach used by other

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1 agencies for other regulatory purposes in reviewing the current 

### 2 crustacean EDC bioassays. 

### 3 The EPA in writing the conformance criteria 

### 4 should identify those test endpoints deviations, which represent 

### 5 major and minor nonconformities. Identification of major and 

### 6 minor nonconformities should focus on the conformance with 

### 7 identified control test endpoint criteria in terms of the 

### 8 survival, growth, development and reproduction endpoints. 

### 9 Defining these major and minor nonconformities can either be 

### 10 statistically based, for example, plus or minus one standard 

### 11 deviation of the mean or percentage based, less than 10 percent 

### 12 of mean or minor, greater than 10 percent of mean for a given 

### 13 test endpoint, which seems reasonable based upon best 

### 14 professional judgement, including statistical analysis for the 

### 15 endpoint. 

### 16 Major nonconformities should be significant 

### 17 test deviations of the protocols that may affect bioassay 

### 18 outcome or performance failures on positive and/or negative 

### 19 controls requiring the assay to be rerun. Minor nonconformities 

### 20 should be evaluated to determine if these deficiencies are 

### 21 personnel, facility's equipment or test support, suppliers in 

### 22 related issues. 

### 23 Review of these minor deficiencies should 

### 24 address how these deficiencies will be corrected and should be 

### 25 reported in subsequent bioassays to demonstrate that the 

### 26 inadequacies have been addressed and corrected.

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1 **DR. MARTHA SANDY:**Dr. Klaine? 

### 2 **DR. STEPHEN KLAINE:**Nothing further to add. 

### 3 **DR. MARTHA SANDY:**Dr. Perkins? 

### 4 **DR. EDWARD PERKINS:**Nothing further to add. 

### 5 **DR. MARTHA SANDY:**Dr. Scott? 

### 6 **DR. GEOFFREY SCOTT:**Nothing further to add. 

### 7 **DR. MARTHA SANDY:**Dr. Christman? 

### 8 **DR. MARY CHRISTMAN:**I concur with everything 

### 9 Dr. Tuberty said and anything specific to the statistical 

### 10 approaches have been handled in questions three, four and five. 

### 11 12 **DR. MARTHA SANDY:**Thank you. Other comments 

### 13 on this question from the Panel? We'll go to Dr. Touart of the 

### 14 U.S. EPA. 

### 15 **DR. LESLIE TOUART:**No clarifying question. 

### 16 **DR. MARTHA SANDY:**Would you please ready 

### 17 charge question 6.2? 

### 18 **DR. LESLIE TOUART:**This is the second part of 

### 19 the charge question number six, 6.2. Based on the validation 

### 20 results using the two different invertebrate species, the mysid 

### 21 and the copepod, the EPA is proposing the mysid protocol as the 

### 22 preferred Tier 2 assay. 

### 23 Please comment on the rationale to recommend 

### 24 the mysid protocol as the preferred Tier 2 invertebrate assay, 

### 25 as described in section 6.4 of the mysid ISR. 

### 26 **DR. MARTHA SANDY:**Dr. Tuberty, you are the

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1 lead discussant. 

### 2 **DR. SHEA TUBERTY:**Some Panelists felt the 

### 3 inclusion of both the mysid and copepod bioassays was warranted 

### 4 rather than selecting one bioassay at this time. Both assays 

### 5 have their strengths and weaknesses and are generally equally 

### 6 sensitive in comparisons presented by EPA in their presentation. 

### 7 The use of both bioassays has been recommended by the OECD at 

### 8 level five tests and the use of both bioassays provides enhanced 

### 9 protection. 

### 10 In addition, the OECD has used a broader 

### 11 toolbox approach for testing for invertebrates, which recognizes 

### 12 the broad and diverse areas of biological species the 

### 13 invertebrates represent, especially the crustacean, mollusk and 

### 14 annelids in aquatic habitats. 

### 15 The approach taken by EPA to use crustaceans 

### 16 as their Tier 2 testing is wise given the overall greater 

### 17 sensitivity generally seen in conventional, acute and chronic 

### 18 toxicity testing of pesticides and other chemicals using 

### 19 crustacean models. The choice of the mysid over copepod at this 

### 20 point seems premature based on the results presented to date as 

### 21 both assays have similar sensitivities and are responsive to 

### 22 most chemical exposures. 

### 23 The major short comings of the copepod assays 

### 24 seem to be more an issue of defining chemical exposures for 

### 25 low-volume dosage, lack of stage specific morphological 

### 26 character recognition and lack of familiarity with the testing

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1 protocol in general. Aside from determining the exposure 

### 2 chamber chemical concentration issues, these are generally 

### 3 issues than can be addressed through training and additional 

### 4 experience with running the bioassay. Similarly, some of the 

### 5 strengths of the mysid test are the familiarity and experience 

### 6 of labs running the bioassay in large database EPA has developed 

### 7 using the conventional mysid data mysid exposure testing. 

### 8 The present improvements to the mysid 

### 9 multigenerational bioassay do not include taking advantage of 

### 10 the current extensive mysid database and methods for exploiting 

### 11 this database through population modeling and other uses of 

### 12 existing data. 

### 13 If EPA is to select the mysid test over the 

### 14 copepod test, the following additional inputs of data would help 

### 15 the Panel in making this decision, including 1) Additional 

### 16 testing of known invertebrate EDC compounds such as methoprene 

### 17 and other known invertebrate hormone affecting chemicals in 

### 18 mysids and copepods to better compare sensitivity and 

### 19 effectiveness of multigenerational effects. These do not need 

### 20 to be testing between multiple labs, but should be testing only 

### 21 between the most competent with the goal of defining the 

### 22 broadest and greatest sensitivity to full range of known 

### 23 invertebrate EDC chemicals. 

### 24 2) EPA should select these chemicals for this 

### 25 testing where existing mysid 1 generational data exists, and 

### 26 these results should be modeled for multigenerational effect and

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1 compared with the bioassay results from the additional 

### 2 multigenerational mysid and copepod testing. This will allow 

### 3 two things to be defined, definitively determined, the value of 

### 4 the mysid and copepod multigenerational test in assessing 

### 5 invertebrate EDC effects, and secondly the value of the current 

### 6 one-generational mysid test in predicting known invertebrate 

### 7 endocrine disrupting effects. 

### 8 We are currently being asked to make a 

### 9 decision with results from chemical exposures to a large variety 

### 10 of chemicals, many of which have uncertain invertebrate EDC 

### 11 effects and modes of action. The concern we have is that we are 

### 12 not certain of which bioassays ability to currently discern 

### 13 known invertebrate EDC effects across a broader spectrum of 

### 14 invertebrate hormonal effects. Once this is established, we are 

### 15 certain the assays can discern this and it will be easy to 

### 16 decide which assay will be best. 

### 17 Inclusion of the evaluation of the additional 

### 18 analysis of the current one generation mysid bioassay results is 

### 19 a clear indication of the Panel's recognition of the extensive 

### 20 information that exists with the mysid bioassay, and its proven 

### 21 performances in protecting the environment and regulating 

### 22 current known invertebrate EDC chemicals or IGRs. 

### 23 As to the mysid protocol, the Panel has no 

### 24 issues with the performance of the test. However, the large 

### 25 number of endpoints seems to create duplication of effects. 

### 26 Therefore, consideration of a reduction in the number of

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1 endpoints assessed could streamline the protocol without losing 

### 2 power to assess adverse impacts. 

### 3 In Dr. Touart's slide show a couple of days 

### 4 ago, there is an acknowledgement that there were few endpoints 

### 5 that had signal to noise ratios greater than two, and many 

### 6 endpoints never demonstrated significant effects. Possibly an 

### 7 assessment of more powerful endpoints could be made of the more 

### 8 powerful endpoints and the resulting list of endpoints used as 

### 9 guidelines to ensure inclusion of data to bolster these 

### 10 endpoints when possible, even if this means reduction loss of 

### 11 other endpoints. For example, the example I gave just a few 

### 12 minutes ago regarding the second brood production in females in 

### 13 the F1 generation. 

### 14 There were two consistent comments from the 

### 15 EPA Industry regarding negative issues with the harpacticoid 

### 16 copepod assay that Dr. Scott's comments just a minute ago 

### 17 identified, and that was the lack of ability to discern the 

### 18 exposure concentrations and identify copepodite stages. The 

### 19 Panel suggests that this issue is overcome simply by the 

### 20 experience that will be gained once this test is required, and 

### 21 that this shouldn't be the main reason why we don't move forward 

### 22 with this second assay should it be accepted. 

### 23 **DR. MARTHA SANDY:**Dr. Klaine? 

### 24 **DR. STEPHEN KLAINE:**I have nothing further to 

### 25 add. 

### 26 **DR. MARTHA SANDY:**Dr. Perkins?

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1 **DR. EDWARD PERKINS:**I just want to reiterate 

### 2 that there was just a higher level of reproducibility seen in 

### 3 the copepod assay despite all the difficulties and inexperience 

### 4 of the labs in the assays. It was remarkably reproducible in 

### 5 comparison to the mysid assays, which people supposedly had a 

### 6 lot more experience doing, which supports suggesting that we 

### 7 keep both assays despite the cost. 

### 8 **DR. MARTHA SANDY:**Dr. Scott? 

### 9 **DR. GEOFFREY SCOTT:**As my fellow Panelists 

### 10 have said, I think both assays have merit. We provided, I 

### 11 think, some next steps and hopefully these are helpful next 

### 12 steps and certainly feel optimizing for a wide range of known 

### 13 invertebrate pathways may help us better define which assay may 

### 14 be most effective. 

### 15 **DR. MARTHA SANDY:**Dr. Christman? 

### 16 **DR. MARY CHRISTMAN:**I have no additional 

### 17 comments. 

### 18 **DR. SHEA TUBERTY:**I'd like to follow up on 

### 19 Dr. Perkins comment, and that is that an assessment of the mysid 

### 20 studies looking through 25 inter-lab comparisons of just two 

### 21 labs for Lindane, for example, really only one good 

### 22 collaborative result came out of all of those 25 comparisons, 

### 23 whereas if you look at the copepod assay, there were only given 

### 24 maybe the same number of comparisons, only two that didn't 

### 25 correlate as far as the direction and response, maybe the 

### 26 concentration of responses didn't match up between labs, but a

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1 lot more consistent responses. That is just a cursory review. 

### 2 I think that development of that assay needs 

### 3 to continue forward even given that the labs had less experience 

### 4 with it, they still were able to create a lot more consistent 

### 5 result. I think that's got to be one of the endpoints of 

### 6 concern here. So although the mysid assay seems to be ready to 

### 7 go right now and is definitely sensitive to the A, E and T 

### 8 compounds, I think the harpacticoid copepods are going to be as 

### 9 well. As far as getting false results, you're going to get a 

### 10 lot few of them with the copepod test. 

### 11 **DR. MARTHA SANDY:**Other comments from the 

### 12 Panel? Seeing none, Dr. Touart, any questions? 

### 13 **DR. LESLIE TOUART:**No further clarifying 

### 14 questions from me. 

### 15 **DR. MARTHA SANDY:**Shall we move onto charge 

### 16 question seven? 

### 17 **DR. LESLIE TOUART:**The purpose of the 

### 18 validation process is to determine the readiness of a test for 

### 19 inclusion in a testing program. A component of readiness of a 

### 20 test is the evaluation of the usefulness and limitations of the 

### 21 test, including the classes and types of test substances that 

### 22 can and cannot be tested. 

### 23 Please comment on the strengths and/or 

### 24 limitations of the assay, as described in section six of the 

### 25 mysid ISR. 

### 26 **DR. MARTHA SANDY:**The lead for this is Dr.

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1 Klaine. 

### 2 **DR. STEPHEN KLAINE:**Okay. You've probably 

### 3 heard some of these already. We'll go through -- the MTTT ISR 

### 4 describes the perceived strengths and limitations of the assay, 

### 5 pages 67 and 69, based on presentations, answers to questions 

### 6 and a lot of Panel discussions, we believe the strengths of the 

### 7 assay to be the follow. 

### 8 Historical use of the organism in bioassays 

### 9 and extensive experience of testing laboratories with animal 

### 10 husbandry associated with this organism. Test endpoints are 

### 11 generally simple and easy to measure. The assay includes 

### 12 endpoints that are relevant to population level effects and the 

### 13 results are amenable to common population modeling approaches. 

### 14 Males and females are distinguishable by conventional microscopy 

### 15 based on anatomical differences, and this assay is recommended 

### 16 as part of the OECD testing protocol at level five. 

### 17 We believe the limitations of that assay to be 

### 18 the following. Large number of endpoints that are included make 

### 19 the assay tedious and the apparent redundancy among the 

### 20 endpoints suggest that they may not have been strategically 

### 21 selected. Variability among laboratories in the ring test is 

### 22 troublesome. This variability includes inability of some 

### 23 laboratories to achieve control performance metrics suggesting 

### 24 that testing laboratories are not proficient in conducting this 

### 25 test, and/or that the test endpoints are highly variable. 

### 26 Earlier studies with mysids and other

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1 invertebrate species have indicated it is not possible to 

### 2 predict life sensitivity in the F1 generation. Similarly in the 

### 3 MTTT, it is not possible to predict generational sensitivity 

### 4 among life stage endpoints. This variability also includes 

### 5 variability in endpoints between replicates, treatments and 

### 6 laboratories. Whether this variability is intrinsic, for 

### 7 example animal husbandry, feeding, food quality, et cetera, or 

### 8 as due to test design or lack of laboratory proficiency is 

### 9 uncertain. However, it makes interpretation of the existing 

### 10 data speculative at best. Further, given this variability, it 

### 11 is uncertain if laboratories, if they were asked to repeat these 

### 12 bioassays would even get the same results the second time. 

### 13 Given the variability discussed above, there 

### 14 is insufficient sample size to adequately observe significant 

### 15 differences among treatments. The addition of a second 

### 16 generation significantly adds to the time and resources required 

### 17 to perform the assay. Effects in the first generation may 

### 18 result in an inability to complete the second generation due to 

### 19 insufficient young. 

### 20 Some methods were poorly described and may 

### 21 have led to poor test laboratory performance. There's concern 

### 22 that toxic effects in the F0 generation will result in less 

### 23 sensitive F1 organisms leading to less sensitivity in the F2 

### 24 metrics. We base that on the fact that some pesticides are 

### 25 known to cause pesticide resistance with increased generational 

### 26 exposure in insects, although we're not suggesting resistance

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1 but certainly selection of a less sensitive part of the 

### 2 population could occur. 

### 3 The flow-through nature of the test generates 

### 4 large volume of waste. We estimate 6000 to 8000 gallons of 

### 5 waste per test. It's a concern in terms of the test cost 

### 6 because waste disposal of 120 to 160 55-gallon drums at $1000 a 

### 7 drum -- so you can do the math -- substantial. 

### 8 Finally, there is no obvious mechanism from 

### 9 Tier 1 for trigging the need for this assay. Hence, in the 

### 10 absence of data that allows for any discrimination is likely 

### 11 that this assay would always be requested or alternatively, 

### 12 never be requested. So either of these will be problematic. So 

### 13 EPA in the document has proposed further refinements to the MTTT 

### 14 assay in light of the limitations. These include better methods 

### 15 for selecting doses based on results of a 21-day screening test, 

### 16 more judicious selection of test chemicals for the validation 

### 17 study based on known arthropod endocrine activity, and refining 

### 18 endpoints to potentially reduce animal use or improve 

### 19 statistical power. The Panel agrees with these additions. But 

### 20 while they may provide improvement, it is not assured that they 

### 21 would sufficient address all limitations. Hence, we recommend a 

### 22 few additional changes in the protocol. 

### 23 First would be increasing the number of 

### 24 organisms used to initiate the test. This would increase the 

### 25 probability of survival and adequate reproduction in the later 

### 26 generations, conduct a second validation study after allowing

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1 stakeholder comments on the test methods, to increase likelihood 

### 2 that test laboratories understand the protocol and are confident 

### 3 they can perform it under GOP. 

### 4 Finally, the addition as you have heard many 

### 5 times, the addition of a positive control that is a known 

### 6 invertebrate EDC -- for example methoprene -- with a known 

### 7 invertebrate hormonal pathway effect for inclusion in future 

### 8 testing, which will help establish growth, development and 

### 9 reproductive fingerprints that may be useful in better 

### 10 discerning invertebrate EDC effects in screening new compounds. 

### 11 12 **DR. MARTHA SANDY:**Thank you. And the 

### 13 associates on this, Dr. Scott? 

### 14 **DR. GEOFFREY SCOTT:**Nothing further to add. 

### 15 **DR. MARTHA SANDY:**Dr. Tuberty? 

### 16 **DR. SHEA TUBERTY:**My comments are included. 

### 17 **DR. MARTHA SANDY:**Dr. Christman? 

### 18 **DR. MARY CHRISTMAN:**No additional comments. 

### 19 **DR. MARTHA SANDY:**Dr. Perkins? 

### 20 **DR. EDWARD PERKINS:**No further comments. 

### 21 **DR. MARTHA SANDY:**Comments from others on the 

### 22 Panel? Mary Manibusan? 

### 23 **MS. MARY MANIBUSAN:**Just a clarification to 

### 24 Dr. Klaine's comment with regards to the Tier 1 and our 

### 25 selection process of Tier 2. Just for the record, the Agency 

### 26 has not declared its methodology for triggering Tier 2 assays

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1 and so there is no assumption that we will always require the 

### 2 invertebrate nor the assumption that we will never require the 

### 3 invertebrate assay. That's just for the record, thank you. 

### 4 **DR. STEPHEN KLAINE:**I appreciate that. And 

### 5 we did too, we understand that. It was just a concern that was 

### 6 raised. 

### 7 **DR. MARTHA SANDY:**Dr. Touart, any additional 

### 8 questions? 

### 9 **DR. LESLIE TOUART:**No additional questions. 

### 10 I think we understand the strengths and limitations that the 

### 11 Panel outlined, thank you. 

### 12 **DR. MARTHA SANDY:**I guess we are ready for 

### 13 question eight. 

### 14 **DR. LESLIE TOUART:**There is sufficient 

### 15 evidence to indicate that EDCs can disrupt normal development 

### 16 and reproductive success. However, the sensitivity of the F1 

### 17 generation compared to the F0 is less clearly defined. 

### 18 Please comment from a scientific and risk 

### 19 assessment perspective on the value added of multiple 

### 20 generations in the mysid assay. 

### 21 **DR. MARTHA SANDY:**Dr. Perkins is the lead. 

### 22 **DR. EDWARD PERKINS:**So the question at hand 

### 23 is the utility of multiple generations. The use of these 

### 24 multiple generations is intended to capture effects that may not 

### 25 be apparent through exposure of a single generation for a number 

### 26 that may occurred for a number of different reasons. Based on

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1 the tables that were provided by EPA in the presentation, slide 

### 2 172 by Les, the additional multiple generations of mysid tests 

### 3 provides information pertinent to risk assessment. 

### 4 In that comparison of mysid sensitivity by 

### 5 generation, the LOEC for the F1 appears to be more sensitive 

### 6 than LOEC observed in the F0 or number of chemicals. In that 

### 7 comparison, I'm not showing here, is the 10 chemicals available 

### 8 for comparison where there was both data in the F0 and F1 

### 9 populations. The F0 mysids were more sensitive than F1 to two 

### 10 chemicals. Mysids were more sensitive to three chemicals in the 

### 11 F1 generation than the F0 generation, while the F0 and F1 were 

### 12 equally sensitive to five chemicals. 

### 13 We also went through and looked at the tables 

### 14 ourselves in a cursory manner because we wanted to look at that 

### 15 ourselves in a little bit more detail. So as looking at many of 

### 16 the chemicals used, and this is dichlorophenol, if you look at 

### 17 the F0 generation, it looks like the -- look at total offspring 

### 18 per female. Some of the two most sensitive endpoints in that 

### 19 assay, the mysids, the most sensitive generation was the F1 

### 20 where it was at 7.21 and there was a direction going down, 

### 21 whereas in the F0, was around 200. So it's quite a bit 

### 22 different in the F1. And then time to molt, it was fairly 

### 23 sensitive in the F0, but I would argue that total number of 

### 24 offspring is more directly related to reproduction perhaps than 

### 25 time to molt. So this may have a heavier weight in a weight of 

### 26 evidence type of approach that we mentioned earlier and may be

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1 more biologically relevant to some of the mechanisms that you're 

### 2 looking at. 

### 3 Fenoxycarb, which is a molt inhibitor or 

### 4 juvenile hormone inhibitor I believe, 14 day length was the most 

### 5 sensitive. However, that wasn't done for some reason in the F1 

### 6 population, so we can't really compare it. Total offspring per 

### 7 female was actually more sensitive in the F1. 

### 8 However, it went up, which would rephrase some 

### 9 of the issues that the industry brought up that sometimes you 

### 10 get confusing data. However, recent molecular efforts using 

### 11 genomics to look at and examine these low-dose effects where it 

### 12 might be a nonmonotonic response shows that some of the 

### 13 inhibition pathways initiated by chemicals can cause changes in 

### 14 lipids that cause shifts in signalling ecdysterone metabolites 

### 15 and precursors that shift hormones in invertebrates. 

### 16 So you can get effects there that seem 

### 17 apparently confusing, but they're actually low-level toxic 

### 18 effects. So we can also suggest exploratory methods to 

### 19 understand why these are happening to reduce the confusion on 

### 20 that or reduce the uncertainty. 

### 21 In fipronil, the F0 was definitely more 

### 22 sensitive. Time to molt, seven-day length, ketoconazole as was 

### 23 pointed out, had to be redone, but the seven-day weight was most 

### 24 sensitive in the F0, whereas the time to second brood or the 

### 25 number in the second brood was much more sensitive and down in 

### 26 the F1. It kind of goes on like that.

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1 One of the big points I wanted to show out 

### 2 that much as Les pointed out earlier, the F1 does have some 

### 3 important information on several chemicals there. Just from 

### 4 this cursory one, at least from my view point, it seems like a 

### 5 lot of the endpoints being more sensitive in the F1 appears to 

### 6 more be directly related to reproduction, which is more directly 

### 7 related to the endocrine question that you're trying to look at. 

### 8 9 So in my eyes, this really supports the case 

### 10 that you do really want keep the F1. Then Shea Tuberty put 

### 11 together a second chart, looking at different factors. He can 

### 12 explain that one since he put that together late last night or 

### 13 early today. 

### 14 **DR. SHEA TUBERTY:**This table is a little 

### 15 small, I apologize for that. I didn't know it was going to be 

### 16 projected. So what I've done here is taken significant effects. 

### 17 These were indicated by either Dunn's or Dunnett's tests from 

### 18 the ISR, and broke things down into all things reproductive, 

### 19 growth or development. So these would have been including 

### 20 duration to reproduction or amount of time to reproduction or 

### 21 full development, or survival, which on occasion had a hit. 

### 22 Then as Ed has indicated those compounds that had more sensitive 

### 23 endpoints in the F0 versus the F1 or if they were equal. So 

### 24 this is similar to what Les gave us a few days ago. 

### 25 These are organized alphabetically, the 

### 26 compounds that were tested. I should have put them together by

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1 active ingredient. But if we look at just a couple of things 

### 2 that how similar modes of action like ketoconazole here or 

### 3 prochloraz, the prochloraz study had to be redone. So if we 

### 4 take the one that wasn't problematic here and compare it to 

### 5 ketoconazole, you see that the F1 generation for both of those 

### 6 compounds actually, as Ed indicated, had reproductive effects 

### 7 whereas only one of the compounds had reproductive effects in 

### 8 the F0. 

### 9 Growth and development is always affected in 

### 10 the F0 population, rarely so in the F1 generation. Survival 

### 11 only for fipronil, survivorship in these adults -- we want to 

### 12 have exposures where we're not getting lethalities. So that's 

### 13 not to be expected for an EDC to begin with. Of course, 

### 14 fipronil is not an EDC for endocrine compounds in invertebrates 

### 15 related to reproduction and development, but rather a 

### 16 neurotoxin. So I think this again, adds weight to inclusion of 

### 17 the F1 especially because of the effects on reproduction. 

### 18 **DR. EDWARD PERKINS:**So to summarize, 

### 19 basically these different analyses really support the use and 

### 20 maintenance in maintaining the second generation in the MTTT 

### 21 test which clearly captures effects at lower concentrations or 

### 22 certain chemicals more than just a one generation case. So a 

### 23 significant amount up to 30 percent of the chemicals that were 

### 24 looked at, so that is not a small amount. 

### 25 One other comment that would affect this and 

### 26 was brought up a few other times is that sample size in the F1

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1 generation, especially at the termination point. It's often too 

### 2 small to observe effects if they exist. Therefore, we again 

### 3 recommend that you look at the sample sizes in the F1 generation 

### 4 study so that there is enough samples in the F0 population so 

### 5 you have enough samples in the F1 population to look at 

### 6 subsequent effects. That was definitely a problem in some of 

### 7 the studies. There weren't enough to see effects, the N of zero 

### 8 and the N of one and two. That summarizes our comment. 

### 9 **DR. MARTHA SANDY:**Thank you. I will ask each 

### 10 of the associates if they have anything to add, so Dr. Tuberty? 

### 11 **DR. SHEA TUBERTY:**I've got nothing further. 

### 12 13 **DR. MARTHA SANDY:**Dr. Scott? 

### 14 **DR. GEOFFREY SCOTT:**Just a couple of points. 

### 15 I think the types of data that Shea and Ed have pointed out here 

### 16 are kind of moving towards what I think what we were trying to 

### 17 refer to as fingerprints or pattern recognition of effects. If 

### 18 you take a look at that table with fenoxycarb, which we know is 

### 19 an example of what we're talking about with positive controls, I 

### 20 think you can begin to see certain compounds follow some of that 

### 21 pattern recognition. If you take a look at a compound like 

### 22 fipronil for example, using both assays we know in the copepod 

### 23 assay it is male specific in its effect. 

### 24 So if you take a look in our long-term studies 

### 25 of monitoring Ag runoff sites, we see reductions in males 

### 26 because they don't have the lipid to be able to store the

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1 contaminant and they're more sensitive if you run tests between 

### 2 males and females, particularly for that class of compound. 

### 3 So probably what you're doing if you look at 

### 4 that fipronil where the F0 is most sensitive, you may be 

### 5 removing the males and that's why that's critical. To me, that 

### 6 kind of ability in a testing protocol to be able to have those 

### 7 kinds of insight are going to be critical in my opinion, to be 

### 8 able to do what the Agency wants to do. I think that's sort of 

### 9 where we are with looking at these data and proving you our best 

### 10 guidance as a panel here back to how we see you use it. Thank 

### 11 you. 

### 12 **DR. MARTHA SANDY:**Dr. Klaine? 

### 13 **DR. STEPHEN KLAINE:**I concur with my 

### 14 colleagues. 

### 15 **DR. MARTHA SANDY:**Dr. Christman? 

### 16 **DR. MARY CHRISTMAN:**I concur with my 

### 17 colleagues as well. 

### 18 **DR. MARTHA SANDY:**Any additional comments 

### 19 from the Panel? I will turn to EPA and Dr. Touart. 

### 20 **DR. LES TOUART:**No further clarifying 

### 21 questions. 

### 22 **DR. MARTHA SANDY:**So that concludes the last 

### 23 of the charge questions. I'd like to now go back and we need to 

### 24 revisit a few questions on earlier assays. My notes say that in 

### 25 the Medaka question four, I believe that Dr. Portier had made 

### 26 some suggestions, and we were going to confer back with Dr.

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1 Johnson and Dr. Christman what they thought about that. 

### 2 **DR. DALLAS JOHNSON:**Yes, thank you. I think 

### 3 that the way we've phrased our remarks so far about letting the 

### 4 nature of the variables drive the analysis rather than letting 

### 5 the data drive the analysis, I think that that is essentially 

### 6 what Dr. Portier was also getting at. He was just suggesting 

### 7 some particular routines that might be available in the 

### 8 statistical software that might help do some of those analyses. 

### 9 10 In that regard, I might also mention that I 

### 11 think it's our intention for Dr. Christman, Dr. Portier and 

### 12 myself to suggest some additional analyses or programs that 

### 13 writes some SAS code for you that might be included in the 

### 14 statCHARRMS module or macro, in hopes that we can increase the 

### 15 amount of charm that have with that macro. In other words, 

### 16 eventually showing how I mentioned confidence intervals this 

### 17 morning, we can show you the SAS code that would be used to get 

### 18 the confidence intervals. We talked about analyzing data that 

### 19 had unequal variances and we can show you the SAS code that 

### 20 would be necessary to do that. So it's our intention, I think, 

### 21 as well to perhaps put some of that kind of detail in the report 

### 22 that we come up with. 

### 23 **DR. MARTHA SANDY:**Dr. Christman, did you have 

### 24 anything to add? 

### 25 **DR. MARY CHRISTMAN:**No I do not. Dr. Johnson 

### 26 covered it all.

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1 **DR. MARTHA SANDY:**So before we leave Medaka, 

### 2 were there any other issues that anyone wanted to discuss? 

### 3 Seeing none, thank you. The next thing I have on my list was 

### 4 the amphibians. It was question 3.3 and had to do with the RT 

### 5 qPCR assay. I see Dr. Denver. 

### 6 **DR. ROBERT DENVER:**Yes, we discussed the 

### 7 issues whether or not to include vitellogenin messenger RNA as a 

### 8 means for detecting estrogenic compounds. The possibility is 

### 9 that it could represent a more sensitive biomarker for 

### 10 disruption of estrogenic signalling, but this would need to be 

### 11 evaluated. To our knowledge, there is no current validated 

### 12 assay for this. RT qPCR assay presented this vitellogenin 

### 13 messenger RNA. There are technical challenges that are 

### 14 different in character, but perhaps similar in complexity to the 

### 15 vitellogenin ELISA. For example RNA isolation, evaluation of 

### 16 RNA quality, reverse transcription, qPCR, they're all 

### 17 technically challenging and they could be beyond the expertise 

### 18 of some contract labs. 

### 19 Also a panel of appropriate reference that is 

### 20 housekeeping genes would need to be identified, assays developed 

### 21 and then run in parallel with the vitellogenin assay. 

### 22 Lastly, the concordance between plasma 

### 23 vitellogenin and liver vitellogenin messenger RNA and the 

### 24 relative sensitivities of those measures would need to be 

### 25 evaluated. 

### 26 So at this point, we recommend that the EPA

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1 consider evaluating the feasibility of including it, but it's 

### 2 something that would need to be investigated to determine each 

### 3 of those issues that I laid out. 

### 4 **DR. MARTHA SANDY:**Dr. Degitz? 

### 5 **DR. SIGMUND DEGITZ:**Thank you for your 

### 6 comments. 

### 7 **DR. MARTHA SANDY:**Thank you. Any other 

### 8 comments on the LAGDA assay? Okay. So there have been a number 

### 9 of crosscutting issues that have been brought up and common 

### 10 themes across all four assays. I think allowing each assay 

### 11 discussant group to hear the others has helped with that. Our 

### 12 report may pull some of these common issues together if that's a 

### 13 plan. 

### 14 Now, I'd like to give every Panel member a 

### 15 chance as we go around to say any final comments, other issues 

### 16 they'd like to discuss. We'll start here on my left with Dr. 

### 17 Delclos. 

### 18 **DR. K. BARRY DELCLOS:**I'll maintain 

### 19 consistency across days and pass on this. 

### 20 **DR. JAMES MCMANAMAN:**After listening to all 

### 21 of this, I really appreciate all the hard work that went into 

### 22 trying to develop the assays by the EPA and to deal with the 

### 23 complexities. But I'm beginning to think that we're in kind of 

### 24 a Rumsfeldian dilemma in the sense that we have known unknowns 

### 25 and unknown unknowns. The known unknowns include the problems 

### 26 with experimental design, validation, experimental execution by

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1 some of the contract labs, which have suggest that maybe we're 

### 2 not ready -- some of these assays are not quite ready for 

### 3 primetime and there should be some further evaluations of the 

### 4 assays. 

### 5 Regarding the unknown unknowns, the whole 

### 6 invertebrate section seems like that there seems to be little 

### 7 rationale for including it in a Tier 2 assay since we didn't 

### 8 even consider these kinds or compounds or consider doing this in 

### 9 the Tier 1 or in the pre-assay evaluations. Had we known that 

### 10 we were going to consider that and had we known that, we might 

### 11 have come up with some better suggestions or some better 

### 12 observations help in trying to integrate the Tier 1 and the Tier 

### 13 2. 

### 14 So these concerns have me a little leery that 

### 15 we really not ready for Tier 2. There may be other factors that 

### 16 are pushing us ahead, but I don't hear a lot of -- not really is 

### 17 very sanguine about it at all. 

### 18 **DR. DANIEL SCHLENK:**I've been thinking a lot 

### 19 about this over the week. It has been very interesting to try 

### 20 to put an academic mind on a regulatory issue perspective, which 

### 21 I think if I were the Agency, I think that's what you'd probably 

### 22 unfortunately are getting is a severe academically challenged 

### 23 Panel here that is real easy to criticize things, particularly 

### 24 when not all the data is there, when not all the methods are 

### 25 laid out clear and properly, and maybe you're missing a little 

### 26 bit here, little bit there. It kind of reminds me of a grant

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1 review panel when a red flat goes up, the sharks just feast. It 

### 2 may just be a little, you know like a cut on your leg kind of 

### 3 thing, but everybody just kind of falls in on that. 

### 4 My heart is heavy because I know where the 

### 5 Agency wants to move in this regard, but again I think there are 

### 6 some issues, particularly I think on the positive control side 

### 7 as you keep hearing over and over again. I think again, we just 

### 8 want to be sure. Everybody wants to be sure. I know you guys 

### 9 want to be sure too. 

### 10 The good news is I think people want these 

### 11 assays to move forward. I think that's definitely what I've 

### 12 heard at least in conversations is that we do want them to move 

### 13 forward, but we also want them to be correct in terms of linking 

### 14 the responses to an endocrine-based response. At least the 

### 15 consistent theme I hear, at least through most of the assays is 

### 16 that it's just not there. I mean, we see the great apical 

### 17 endpoints, but honestly, you already have those endpoints if the 

### 18 compound is going through FIFRA or being used for some other 

### 19 component, maybe not for the SDWA compounds, but at least for 

### 20 FIFRA you've got those data. 

### 21 The point here is that you want to link it to 

### 22 an endocrine response to an AOP and EAT particularly. I think 

### 23 that's where, at least as I hear this, this is the common sort 

### 24 of negative component that I hear throughout this whether it's 

### 25 the inverts not even having a Tier 1 or even some of the bird 

### 26 and amphibian things. It's really difficult to link those

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1 apical endpoints to that EAT AOP, and I think that's at least 

### 2 the challenge that I see most people sort of presenting in this 

### 3 particular Panel. 

### 4 So I'd like to say to we encourage the EPA to 

### 5 keep moving forward because I think this is obviously something 

### 6 that needs to be done. It's just a difficult challenge to do 

### 7 and may take a bit. I can't define what a bit means, but I 

### 8 think we're trying at least to provide you at least some of 

### 9 that. That's all I have to say. 

### 10 **DR. STEPHEN KLAINE:**I want to thank the EPA 

### 11 again for all the work as this was a lot of work to put 

### 12 together. Also, I want to thank the public commenters because 

### 13 it was also a lot of work to put together. I'm not going to go 

### 14 and reiterate what Jim and Dan have said, but I think the length 

### 15 of their comments reflects the work and discussions we've had 

### 16 this week and how arduous this task has been. Knowing where you 

### 17 want to go and our concern with getting there in a fashion that 

### 18 allows you to have data that you have confidence in that you can 

### 19 make conclusions and decisions upon has really driven a lot of 

### 20 our comments. 

### 21 So yeah, we might have jumped on a few wounds, 

### 22 but I think that we really tried to be objective in how to 

### 23 improve these tests. We do want to see this move forward. It 

### 24 just has to move forward in a fashion that everyone had 

### 25 confidence in. So thanks again. 

### 26 **DR. GEOFFREY SCOTT:**Thank you for the

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1 opportunity to serve with my distinguished colleagues on this 

### 2 Panel. I have learned from every one of you and thank you for 

### 3 your collective wisdom in sharing in across the assays because I 

### 4 think we all learned something along the way. I want to 

### 5 particularly thank my colleagues on the invertebrate panel who 

### 6 have come together to put our collective wisdom forward to 

### 7 provide EPA with the best guidance we can. I really want to 

### 8 thank EPA because I know all the hard work that has gone into 

### 9 this. 

### 10 Dan's comment about the grant and the cut on 

### 11 the leg and the sharks descended reminded me of something. I'll 

### 12 share it as an anecdote to close my comments. My favorite movie 

### 13 of all time is Jaws. Recently, it was just 38 years ago since 

### 14 we had Jaws come out. The reason it's my favorite movie is 

### 15 where industry, the fishing industry, academia and government 

### 16 come together to solve a problem. You ever thought about that? 

### 17 Now if you start at the beginning of the 

### 18 movie, everybody's trying to do it their way and they get 

### 19 nowhere. But you know when they finally come together and they 

### 20 start picking up their legs and comparing scars, that's when 

### 21 things started to happen. In the end, everybody got to try it 

### 22 their way. The fisherman tried it his way. The academic 

### 23 mission tried it his way. It didn't work and he got away. At 

### 24 the end, government had to make the decision. 

### 25 So I think the process -- that's why it's my 

### 26 favorite movie because in many ways, we're not trying to kill a

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1 shark, but we are trying to regulate an issue. To me, this 

### 2 Panel emulates in many ways what we try to do when we all come 

### 3 together. Thank you for letting me be a part of it. 

### 4 **DR. REYNALDO PATINO:**I would like to thank 

### 5 Sharlene for the opportunity to serve on this Panel. I also 

### 6 enjoyed -- I was here a month ago and even though I'm supposed 

### 7 to come here and share scientific advice, I feel that I learned 

### 8 more than I shared. So I appreciate the opportunity for the 

### 9 discussions and also the EPA for listening. 

### 10 As far as overall thoughts, I think that I'm a 

### 11 little bit -- I don't know if confused is the right word, but I 

### 12 heard a number of times what matters really, the outcome that 

### 13 matters from the Tier 2 assays are the applicable endpoints of 

### 14 growth, development and reproduction. So I'm wondering why are 

### 15 we looking at AOPs when the concern is in regards the Tier 2 

### 16 assays. If that really in some ways does not matter in terms of 

### 17 the outcome that is going to be useful for the agency to make 

### 18 risk assessment and regulation. 

### 19 So I guess I'm leaving here with a little bit 

### 20 uncertainness to what the purpose is of many discussions that 

### 21 we've had concerning the AOPs, there being no modes of action if 

### 22 in some ways that may have been addressed already in the Tier 1 

### 23 and it's not going to affect the purpose of the Tier 2. But 

### 24 it's probably just me. I may have gotten things wrong, but I 

### 25 just wanted to share that with you, my parting thought. Thank 

### 26 you very much everybody.

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1 **DR. CATHERINE PROPPER:**I also want to thank 

### 2 the EPA for all their amazing hard work. The amount of data 

### 3 presented through these different assays is stunning. I truly 

### 4 appreciate the complexity of trying to evaluate both within 

### 5 assay variation across laboratories and even within 

### 6 laboratories, and certainly now trying to pull together what it 

### 7 means across assays is going to be a huge task. 

### 8 So in fact, one of my questions is how are you 

### 9 going to really do that, and is there a plan to move forward 

### 10 once these data really do start coming in and a regulatory 

### 11 mechanism for -- how are you going to make the decision -- 

### 12 what's the next step beyond just running the assays to get to 

### 13 that regulatory standpoint? I'd be very curious, but I do 

### 14 appreciate just how much work's even gone into getting this far. 

### 15 16 One of the other issues I just wanted to 

### 17 reiterate was for the statistics, I really appreciated Dr. 

### 18 Christman's comment about using some multi-variant kinds of 

### 19 analysis might be a way to try to pull in those data in a way 

### 20 that can give you a broader picture. So that was just my last 

### 21 kind of suggestion on the stat side of things. Then I just want 

### 22 to say that also this is my second Panel and I learned so much 

### 23 from being here that I take it as an opportunity to serve for my 

### 24 own selfish gain, but also because part of my personality is I 

### 25 want to fix things. This gives me an opportunity to try to give 

### 26 input to help better the environment in a way that I find very

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1 personally satisfying. So I really appreciate the opportunity. 

### 2 3 **DR. EDWARD PERKINS:**I wanted to thank EPA and 

### 4 my Panel members for all the hard work they've done in 

### 5 presenting the tests and developing over so many years and try 

### 6 to get something out of it after all that work. I was a little 

### 7 bit overwhelmed by all the different panels or subpanels I was 

### 8 on. There was a lot of commonality to them. So I was happy to 

### 9 learn a lot and be able to contribute. 

### 10 One think probably might help and might have 

### 11 helped would be little better overall conceptual presentation of 

### 12 AOP context in relation to the Tier 2. AOP concept really only 

### 13 took hold over the last two years throughout EPA. So I think it 

### 14 was sort of slapped on to the Tier 2 after the fact, and it's 

### 15 really unclear how AOPs will be used in risk assessment within 

### 16 EPA and out. That's really an active area of research. 

### 17 Nevertheless, I think AOPs what they can in 

### 18 the risk assessment context is help use a weight of evidence 

### 19 approach and ensure that you're very sure about how and why it's 

### 20 doing it. So ultimately, you can extrapolate to other species 

### 21 and other things like that. It may not be directly the species 

### 22 that you're doing the test on, but gives you more confidence in 

### 23 extrapolation. 

### 24 It would help to have more of the molecular 

### 25 endpoints to be able to do that, but I don't think it's 

### 26 necessary to do a final assessment of whether or not this causes

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1 reproductive effects and extrapolate potential populational 

### 2 effects. So that is going to be a big thing to struggle with is 

### 3 how do you really integrate this AOP concept into a risk 

### 4 assessment and use that to drive these assays, which were 

### 5 developed and started some 10 or 15 years ago back when they 

### 6 used to call it mode of action or mechanism of action. 

### 7 Anyway, I've enjoyed serving on the Panel and 

### 8 anxious to see what you actually do with this. 

### 9 **DR. ROBERT DENVER:**I'm also appreciative of 

### 10 the hard work of the EPA scientists and I hope that our comments 

### 11 will help to move this program forward. I actually sat on the 

### 12 SAP that evaluated the Tier 1 assays back in 2008. At the time, 

### 13 the Tier 2 assays were just a concept that was being developed. 

### 14 So I'm glad to see that the Tier 2 assays have come along. I 

### 15 remain uncertain still how the decisions will be made to move 

### 16 from the Tier 1 to the Tier 2. So I hope that that can be 

### 17 sorted out in the near future. 

### 18 **DR. DALLAS JOHNSON:**I also want to thank the 

### 19 EPA for their hard work and for the opportunity to serve on the 

### 20 Panel. I do appreciate the amount of statistical thinking that 

### 21 went into these reports. While I think the stats can be 

### 22 improved, I do know that they're being used to a much higher 

### 23 degree than they were being used in some of the earlier SAPs and 

### 24 human study review board panels that I've served on over the 

### 25 last about eight years now that I've been serving on SAPs and 

### 26 HSRVs. So the level of statistics has been going up. I'm glad

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1 to see that and hoping to see it rise to even more. I hope that 

### 2 I've provided some insight that can maybe help the stats to be 

### 3 done better. 

### 4 It's also good to get out of the house and 

### 5 clear some of the cobwebs out of my mind. Being retired, I 

### 6 don't get opportunities like this, so it's good to know I've got 

### 7 something up there that I can use once in a while. Even though 

### 8 I was trying to write some SAS code the other day and I had to 

### 9 keep going to the help manual to figure out how to do it. Some 

### 10 of the things that I used to just do automatically, now I have 

### 11 to stop and think about and they don't come as automatically as 

### 12 they used to. Again, thanks for the opportunity to serve. 

### 13 **DR. COLIN SCANES:**This is going to one of 

### 14 those on the one hand, on the other, the yes but. First of all, 

### 15 it's been a privilege to be on this Panel. I think great 

### 16 colleagues, but also very well organized by Sharlene and her 

### 17 staff, and very attentive listening from EPA. Obviously, you 

### 18 are here to listen and to think and to show passion on 

### 19 occasions. 

### 20 Now we're going to move onto some observations 

### 21 and the buts. One of the nicest things coming out of this 

### 22 meeting is that there are a series of common themes from 

### 23 virtually every group. Positive control, statistics, number of 

### 24 replicates and we'll come out with more of these. What I found 

### 25 very useful was that in many ways the fish, the amphibians, the 

### 26 invertebrates inform me on information that could be very useful

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1 on the quail act. I hope that this will be used by EPA that 

### 2 some of the information coming from one species test or one test 

### 3 be useful to inform you on different species. 

### 4 One of the things, we've all been on grant 

### 5 review panels and the very good proposals are often easy to see. 

### 6 The blood in the water is easy to see, but sometimes what is 

### 7 not as easy to see is the fatal flaw. Sometimes, one can have 

### 8 the first time you go through a proposal, you say, wow this is 

### 9 from an outstanding group, outstanding proposal and then you go 

### 10 back again and move from an excellent down to a poor because of 

### 11 a fatal flaw. I think in some ways, despite looking at the 

### 12 common features is something which is also looking for things 

### 13 that need to be corrected, that must be corrected. I think that 

### 14 that's something which having this final wrap up and looking at 

### 15 common themes is extremely useful. 

### 16 One minor suggestion or two minor suggestions 

### 17 -- one is Les and his colleagues gave excellent presentations. 

### 18 I would have appreciated the chance to have those before the 

### 19 meeting as the power point before the meeting because it would 

### 20 give an impression of where EPA was thinking. A second area 

### 21 that I think might be useful -- I said two, there's going to be 

### 22 three, I can't count -- the second one is it might be useful to 

### 23 EPA given that the questioning is in EPA language to have one 

### 24 question at the end, and that is are there any concerns that 

### 25 have not been addressed by these questions. 

### 26 Now I think basically as academics, we always

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1 give those anyway and we fit them in under other questions. But 

### 2 if there was that, it could be helpful to EPA. It's not 

### 3 addressing the question, but it could be points that could be 

### 4 valuable to you. 

### 5 The final area that I want to encourage EPA to 

### 6 think about, and that is a lot of these supporting work was in 

### 7 the form of EPA reports. If the work could be put in the form 

### 8 of published work perhaps with appendices as EPA reports, it 

### 9 would give a degree of confidence in the work that it's already 

### 10 gone through some form of peer review. I say that because I 

### 11 think obviously a regulatory agency wants to be on as firm a 

### 12 footing as possible. 

### 13 I do want to go back to what Geoff said about 

### 14 the example of Jaws. I'm not going to use another movie 

### 15 example. But I was very encouraged by the process, not only of 

### 16 the Panel but the process EPA is using and the quality of the 

### 17 EPA staff. 

### 18 **DR. SHEA TUBERTY:**I also would like to parrot 

### 19 my colleagues in saying thank you for inviting me to be part of 

### 20 the Panel. It is really an honor. I thank my other subpanel 

### 21 members for their input and guidance and levity at times. 

### 22 Geoff, good job. It was needed. 

### 23 In talking with Bob and others that had some 

### 24 history with the EDSTAC program -- I teach it every year to my 

### 25 toxicology class, a little bit of the history and how it got 

### 26 started. I really do emphasize endocrine disruption in my class

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1 as what my students need to be worried about in their future. I 

### 2 just want to applaud EPA's attention to endocrine disruption as 

### 3 an issue that needs attention and continue pushing forward, even 

### 4 though it seems to be in jumps and spurts given that Tier 1s 

### 5 were in 2008 or 2009. I understand the amount of time that goes 

### 6 into the EPA's development of these assays having been part of 

### 7 it myself during my post doc. 

### 8 I sit here and watch you guys kind of cringe 

### 9 and wince as we pick your assays apart. I think that's like Dan 

### 10 put, you find one little think wrong with it and you make a big 

### 11 deal out of it. It's really not. I think we appreciate the 

### 12 amount of effort that goes into this work, both at the bench and 

### 13 at the office. But I just want to mention again that this is 

### 14 really important work. This toxicology is where we need to be 

### 15 going. Having a screening program that will catch these 

### 16 compounds before they're released in the environment is 

### 17 critical. Whatever the amount of work it takes and how many 

### 18 generations it takes to get the program finalized is well worth 

### 19 it. I applaud your attention to it and I hope you continue to 

### 20 move forward with it. 

### 21 Having said that, there seems to be no letter 

### 22 of process in the EDSTAC program as it is to go back and change 

### 23 things that were overlooked. I say overlooked because it's not 

### 24 there and I wonder why and I can't get good answers from anyone 

### 25 except for there's not enough public outcry for one thing or 

### 26 another. Why is there no bird Tier 1? Maybe because there is

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1 just no good assay. I don't think Colin would agree with that. 

### 2 Why is there no invertebrate assay? I know there are good ones 

### 3 out there and we use them all the time. 

### 4 So, it just seems like it was overlooked in 

### 5 the first -- maybe it was all the way to the wingspread 

### 6 conference. Maybe there wasn't good invertebrate 

### 7 representation. It's hard to believe that that would be true. 

### 8 It's something I think we need to take a step back and correct. 

### 9 I just wanted to use the platform as an opportunity to say that 

### 10 out loud and hope that it get corrected at some point in the 

### 11 future and we don't just keep moving forward regardless of how 

### 12 important we all know it is. So thanks once again. 

### 13 **DR. JENNIFER FREEMAN:**Well, what's left to 

### 14 say? I think going near the end here, my other Panel members 

### 15 have really highlighted the all last comments that I had jotted 

### 16 down that I wanted to share. So I'll just keep it short and say 

### 17 thank you to the EPA for giving me the opportunity to serve on 

### 18 the panel, and that I really appreciate all the hard work that 

### 19 has gone into developing these Tier 2 assays. Thank you. 

### 20 **DR. MARY CHRISTMAN:**I'm just going to say 

### 21 ditto. Thank you -- hard work -- can't believe you did all this 

### 22 so quickly. That's a lot of information. 

### 23 The only comment that I wanted to make 

### 24 separately was I would like to encourage EPA. There's an 

### 25 enormous amount of data that's been collected and analyzed. I 

### 26 would like to encourage the EPA to maybe take a step back and

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1 consider reanalyzing it in a more complete and correct manner 

### 2 and allow you to sort of think in terms of where it is you want 

### 3 to go with this information based on slightly different -- 

### 4 because I'm trying to take this step back to this idea of what 

### 5 is the optimal experimental design for these problems and how 

### 6 might they be approached. Taking a step back and thinking about 

### 7 that more generally, and using data from different assays to get 

### 8 to that to answer that question, I would encourage you to think 

### 9 about it if you have the resources. Thank you once again. 

### 10 **DR. MARTHA SANDY:**We haven't been as vocal 

### 11 during this panel until now. I apologize for that, but we do 

### 12 all recognize all the hard work that EPA staff has put into 

### 13 preparing the ISRs that we reviewed and all the immense amount 

### 14 of documentation that supplemented that. I am very appreciative 

### 15 of that. 

### 16 I found the ISRs to be written very clearly. 

### 17 It was very, very helpful. It was really worthwhile to have all 

### 18 that other documentation so that the panel members could read 

### 19 and look at that and give you detail, hopefully very helpful 

### 20 informative comments on how to improve these assays. We are 

### 21 very appreciative of that. 

### 22 I appreciate all the hard work that this panel 

### 23 has done. Lots of thoughtful comments were made, a lot of work, 

### 24 a lot of material read. Thank you very much. 

### 25 Now we'll turn it over to Mary Manibusan. 

### 26 **MS. MARY MANIBUSAN:**So thank you everybody.

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1 I'm just going to take just a few short minutes to make my 

### 2 appreciation known as well as giving you a brief glimpse into 

### 3 the next SAP, which is our final SAP for 2013. 

### 4 I too want to acknowledge the work of the Tier 

### 5 2 team that broadly includes experts from across the Agency, our 

### 6 Office of Research and Development as well as our programatic 

### 7 office. 

### 8 Although there have been experts coming in and 

### 9 out of that program in that project, there has been one mainstay 

### 10 expert, and that is Dr. Les Touart, who I want to recognize 

### 11 specifically for his leadership and expertise as well as his 

### 12 eloquence and persistence in ensuring that we do this correctly 

### 13 and we ensure sound science is maintained. That's why we're 

### 14 here today. So again, I just wanted to acknowledge Dr. Les 

### 15 Touart for all his work. 

### 16 I want to acknowledge Dr. Bradbury, Dr. Dix 

### 17 and Mr. Knott for their management support without which we 

### 18 cannot move forward with the endocrine disruptor screening 

### 19 program. 

### 20 Dr. Matten, who has been organizational leader 

### 21 in ensuring that logistics happen and that we're here today and 

### 22 we can have a timely discussion as well as the SAP Executive 

### 23 Secretary, Laura Bailey and her staff, ensuring that all of your 

### 24 travels are completed on time to make sure you're here and that 

### 25 you have the necessary information loaded onto the public 

### 26 docket, which seems to increase by the day.

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1 I definitely want to acknowledge Dr. Sandy for 

### 2 stepping up and serving as chair for this meeting, and Dr. 

### 3 Schlenk for stepping down to serve as an expert on the Panel. 

### 4 We definitely appreciate everyone's participation because we too 

### 5 want to ensure confidence as we move forward with these Tier 2 

### 6 very expensive, very complex designs. 

### 7 We hear you all loud and clear in terms of 

### 8 making sure that are test guidelines are specific and where we 

### 9 are requiring certain parameters to be met that we state so. 

### 10 But not only that in terms of just making a test guideline 

### 11 available, but ensure that we do the right thing and make sure 

### 12 that laboratories demonstrate proficiency, they have the 

### 13 necessary training within YouTube videos or different medias 

### 14 that we have in the 21st century to deploy all those to ensure 

### 15 that the message gets out, the training is available and that 

### 16 we're here to provide consultation. It is no different than how 

### 17 we pursued our other test guidelines. We will continue to do 

### 18 that as we move the science forward. 

### 19 I just want to reiterate that this is the 

### 20 close of the EDSTAC conceptual framework. We've completed our 

### 21 Tier 1 test method validation. We've issues our test 

### 22 requirements for the initial list. We'll be pursuing the second 

### 23 list shortly. This is to validate the Tier 2 test methods. 

### 24 So that concept now becomes a reality. We 

### 25 understand that the validation process includes several steps, 

### 26 one of which is held here, the Scientific Peer Review. The next

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1 step of course, is a lot of the work in terms of getting the 

### 2 test guidance regulatory accepted for use in our program as we 

### 3 seek to address the mission that's stated before us, which is 

### 4 quite large. We recognize that as well. 

### 5 So a couple of next steps will be finalizing 

### 6 test guidelines. We look to your specific comments to help us 

### 7 do so. We will be developing scientific evaluation procedures, 

### 8 SEPs, as we've done for the Tier 1 assays. We've heard also 

### 9 recommendations and we'll be taking into serious consideration 

### 10 development of workshops specifically focused on statistical 

### 11 methods as well as histopathology. So look forward for those 

### 12 additional activities. 

### 13 Needless to say before the Agency deploys Tier 

### 14 2 test orders and these methods, we will have to complete the 

### 15 work of the Tier 1 weight of evidence evaluations. That is the 

### 16 subject of the next SAP come the end of July. There, I just 

### 17 want to calibrate expectations we will be bringing before the 

### 18 Panel some specific case studies. Not all 52, not even all 21, 

### 19 but specific examples that are quite challenging to how we 

### 20 consider melding together the Tier 1 battery of information 

### 21 along side with other science equally relevant information. 

### 22 That definitely will include the pesticide submission 158 data 

### 23 as well as mode of action information where we have that 

### 24 information, dose response to a setting, so toxicokinetics, 

### 25 toxicodynamics, and we will be utilizing the AOP concept to kind 

### 26 of use as a framework to fill out the information that we have

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1 before us to make that weight of evidence determination. 

### 2 Certainly weight of evidence approaches are 

### 3 not new to the Agency. We do that every day, in fact, with the 

### 4 158 information. What is unique in this circumstance is the 

### 5 data and the decision that we'll be making post that evaluation. 

### 6 7 So we look forward to seeing some of the 

### 8 permanent members back in July, and we welcome some of the 

### 9 remaining members who will be re-invited to that meeting. That 

### 10 will close out our 2013 year of SAPs. Going back to the analogy 

### 11 of building our house, we've built our first floor, we're here 

### 12 to discuss a second floor and the last meeting will close out 

### 13 our house. Thank you very much. 

### 14 **DR. MARTHA SANDY:**Thank you Ms. Manibusan. 

### 15 Thank you Dr. Bradbury and thank you Dr. Touart for all your 

### 16 hard work during this last week and prior to that. 

### 17 I've been negligent. I need to thank Sharlene 

### 18 Matten for all her hard work in planning this meeting and 

### 19 organizing it so well and putting together such a great panel 

### 20 and keeping my on track. And to the rest of the SAP staff, 

### 21 thank you. 

### 22 **DR. SHARLENE MATTEN:**I have a few comments, 

### 23 perhaps not quite as eloquent as Mary's, but I would like to 

### 24 thank a number of individuals. I'll start with of course 

### 25 thanking the Panel and Martha Sandy for sitting next to me and 

### 26 indulging me in a few nudges and comments about keeping the

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1 discussions on track with the scientific issues. This meeting 

### 2 was somewhat challenging in that regard and I'm hoping that in 

### 3 the report, there is a clear distinction with the reference to 

### 4 the charge questions and then other scientific comments. That's 

### 5 always important to capture everything. It's just a challenge 

### 6 sometimes to write it. 

### 7 So what you hear here might translate into 100 

### 8 plus pages, which is great and hopefully the agency will find 

### 9 that very informative. I am glad that everyone has enjoyed 

### 10 their time here and have made their way through some of the 

### 11 hiring process for the newest people. That was a bit more 

### 12 challenging than usual. So I appreciate that and everyone's 

### 13 travel here and being so gung-ho in doing the homework and 

### 14 coming prepared to the meeting. I'm glad everyone appreciated 

### 15 the fact that in organizing this meeting that I made sure that 

### 16 everyone was the lead to write up something. That's always 

### 17 important to me to be very inclusive. 

### 18 So in that theme, I wanted to thank the EPA 

### 19 team of folks. Of course there are the folks that sit in the 

### 20 table and of course, we have in this case two program lead 

### 21 offices, Office of Science and Coordination Policy, which 

### 22 encompasses the group and the Office of Pesticide Programs that 

### 23 encompasses the group. There is also the Office of Research and 

### 24 Development. I can't count either -- that's three offices and 

### 25 we all are part of the big ship of the EPA, but you only see a 

### 26 portion that give the presentations. So the only people

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1 remaining in the back are all important people. So Sig, 

### 2 Patience, Pat, Amy, Ed, Catherine, they're from -- we have the 

### 3 Environmental Fate and Effects Division. I was in that division 

### 4 in 1993 to 1997. The Office of Research Development in Duluth, 

### 5 they have a longer name but that's what I call them, Office of 

### 6 Research and Development Duluth. I appreciate those folks for 

### 7 coming down and being warmed up. 

### 8 So there is Pat and Sig and Rodney who 

### 9 escaped, and Kevin, and there was a young man over here with the 

### 10 glasses, John. Now in the Office of Science and Coordination 

### 11 Policy, we had Patience. You know I could give the last names, 

### 12 but this is a personal remark. And then Les because Les was in 

### 13 the Environmental Fate and Effects Division. I'm never quite 

### 14 sure how he responded and went back then, but he's been a very, 

### 15 very, very important scientist in the development of this 

### 16 program on the environmental side. He deserves the credit that 

### 17 Mary has given him from 1997 to present. That's really 

### 18 important that he's been here that long and has endured. 

### 19 Mary Manibusan -- she is the boomer. She is 

### 20 one of the best question readers ever. Les had a hard task to 

### 21 follow, but he came around. Then Steve Bradbury, who is always 

### 22 very poignant, and I always appreciate learning various comments 

### 23 on -- because I sat on that side of the table and I'm one of the 

### 24 few that has been DFO and technical expert. So I go the whole 

### 25 ball of wax. I know how people are thinking and so I try to 

### 26 make sure that there is something for everyone for all

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1 audiences. 

### 2 There are other people back across the river, 

### 3 as we say. There are other people that deserve some thanks, 

### 4 Bill and Scott, so then I would also like to thank my 

### 5 colleagues, Don Wood, Laura Bailey, Fred Jenkins, Joe Bailey, 

### 6 Joyce, Shirley Percival, Carlos and Linda for all of their hard 

### 7 work as well as the contractors. I got the same note that my 

### 8 husband always gives me. 

### 9 With that, I'll wrap things up. I really 

### 10 appreciate everyone's time for being here. I will meet with the 

### 11 Panel in the back room. 

### 12 (Whereas the meeting was adjourned) 

### 13 ***** 

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87:11 93:17 99:19,20 101:22 103:1 117:16,19 141:2 161:16,18 207:22 218:6,7 256:17 257:7 265:8 266:25 267:18,24 268:5 280:25 289:15 306:9 352:6 378:1 405:15 412:9 479:2 506:22 559:19,20 577:19 579:26 604:11 605:18 671:22 680:16 694:1 case-by-case 34:20 case-specific 32:26 33:18 cast 14:11 catch 154:20 662:6 739:15 categorical 578:10,16,17 category 53:25 Catherine 6:12 66:23 67:8,17 68:12 180:24 181:20

182:3,7,17 183:4,9,13,26 198:11,20,25 242:20 460:8 545:25 549:16 554:9 563:8 565:2 569:1 573:25 574:16 575:8,23 598:15 600:26 606:14 607:19 609:3 611:10 654:16 655:10 660:25 665:10 733:1 747:2 Cathy 6:12 242:20 460:8 665:10 cattling 394:8 caudal463:5 469:19 caught327:15,16 647:15 causal 329:26 330:8 449:16 525:8 causative 329:23 cause 25:24 92:6 230:8 236:4 282:2 328:22 447:23

448:17 455:14 495:10 499:11 537:2 630:23 638:3 638:6 669:10 715:25 720:13,14 caused 95:4 97:2,3,4 126:22 495:7,22 553:18

571:22 572:8 667:3,7

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causes 92:9 168:19 371:19 405:5 581:25 671:4

734:26 causing 298:25 586:6 caution449:26 cautions 220:6 cautiously 689:14 cavalier 633:1 caveat274:13 431:23 caveated 636:21 caveats430:15 433:26 631:14 648:18 649:21 cavity 169:13,15 CC494:21 cDNA 512:23,24 cell 85:13 92:13,21 274:16 562:6 cells 111:23,24 158:13,18 233:20 378:24 379:4

571:18 cellular 6:20 557:2,21 censored483:23,25 center 1:14 5:9 6:1,5 151:15 160:6 242:5 459:18

664:20 centered479:19 centimeters 578:7 centrifugation 571:20,22 centrifuged 571:18 cents 686:13 century 11:15 743:14 certain 58:24 63:14 80:13 120:10 122:10 123:20 129:20 146:9 159:16 172:4 194:21,23 202:2 247:10 298:24 341:22 361:11,12 388:9 391:1,26 396:24 401:11,13 415:23 417:14 419:22 421:14 448:17 449:23 465:8 533:9 600:13,21 621:21,22 625:3 654:12 658:8,26 659:10 697:26 710:12,15 722:22 723:20 743:9 certainly 13:9,23 19:12,17 26:20 32:8 78:9 86:7

87:16 118:8 121:19 124:16 133:4,20 134:26 136:11 140:4,17 187:3 231:25 259:3 305:22 335:26 336:6 364:24 367:21 369:7,10 374:16 377:19 382:13 392:1 396:15 409:12 423:16 428:24,25 435:24 456:22 510:10 514:9 516:1,2 518:4 523:4,9,14,17,22 524:22 525:24,26 526:26 527:6 537:8 542:20 565:3 633:1 634:16 635:2 636:3,4 642:11 646:6 656:15 674:2,6 696:5,22 712:12 716:1 733:6 745:2 certainty 23:1 540:19 553:15 634:10 certificate 657:12 certification428:19,20,22 651:11 652:10 654:23

655:4,15,24 656:9,11,26 657:8 659:2,14 660:6,11 661:3 certification-like 650:14 certified 655:5 certify 657:20 658:5 cetera 37:5 272:21 348:5 445:1 446:21 479:1 513:1

671:6,6 676:25,25 677:10 696:3,3,6,6 715:7 cetra410:13 chain424:15 chair2:11 5:3,16 12:20 205:26 459:11 743:2 chairing 241:25 chairperson 305:18 challenge 27:26 70:2 174:12 276:19 280:4 327:15

634:21 730:2,6 746:5 challenged 59:1 382:2 728:22 challenges 57:14 325:21 422:9 507:24 568:19

607:14 704:5 726:13 challenging4:23 11:14 60:4 113:15 313:25 329:14

559:18 726:17 744:19 746:2,12 chamber211:10,10,10 221:24,25 228:3,3 425:18

471:26 709:2 chambers 120:21 221:24 225:4 228:8 472:1 chance70:5,6 134:6 267:12 270:6 303:18 309:19

333:5,12 363:25 386:3 390:22 519:18 520:1 629:4 648:5 652:14 661:18 727:15 737:18 chances 622:19 Chandler248:18 change 30:4 55:3 79:3 93:25 102:12 115:24 118:14

118:17 163:2,26 182:15 185:22 187:8 235:24 247:1 262:16,16 298:22,24 379:18 408:3,3,3 416:12 437:2 487:21 498:12 505:12 531:4 542:13 551:17 557:4 585:25 595:15 633:2 688:17 739:22 changed75:5 93:21,24,26 98:3 162:14 174:1 198:24 299:6 312:15 557:5 600:6 changes 24:7 94:12,12 99:3 101:7 110:22 161:14 164:12 197:19 204:6 237:14 262:14,15 274:14 292:8 335:1 361:17 366:18 377:19 410:16 417:7 464:14 498:13 533:26 534:3 594:10 612:13 667:20 716:22 720:13 changing 99:2 119:7 695:7 character 84:9 92:1 708:26 726:14 characteristic 120:26 318:18 503:7 characteristics 55:23 57:3 90:20 95:5 103:4 140:21 194:23 223:6 298:1 303:3 357:16 366:4,5 366:21 371:24 409:26 425:6 431:10 448:7 451:5 451:5 452:18 453:4,10,22 454:16 462:14,17,23 462:25 464:4,5,17 466:7 469:25 479:6 494:21 509:4 514:13 516:1 524:5 543:16 550:6 574:8 597:10 637:5 characterization 278:24 279:1 448:6 characterize 26:12 38:1 39:23 58:26 69:19 76:12 155:11 212:19 222:9 278:9 282:12 316:25 329:3 347:7,11 349:15 350:24 436:9 443:8,12 447:10 448:10 534:21 545:12,16 613:6 616:9,23 624:10

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699:2 checkpoints 143:1 chemical 8:22 11:7,22,26 17:6,13,22 19:10,17 20:8

20:12 21:9 22:12 23:1 25:7,24 32:13 33:5 34:16 34:17 36:22 58:6 75:21 76:14,23 84:17 95:26 96:1,2,18 98:18 101:20 112:19 113:26 114:26 126:21 132:13,23 133:4,6,10,16 142:11 148:15 161:24,25 167:1,1 214:7 225:1 230:13 233:16 238:11 271:5 275:10,15,15 279:11 280:1,15,18 296:1 317:19 319:18 326:7,25 329:20 330:13 368:25 370:3 386:9 390:2 407:20 409:17,25 412:10,13 413:13 414:5 438:4 446:9 470:17 492:10,11 493:6 494:14 497:1,10 500:7 505:9 524:18 529:13 531:24,25 550:24 551:12,26 552:6 553:18 556:19 559:6 568:14 576:20 601:16 602:10 617:12 621:22,23 631:11 647:2,8 648:1 652:7 669:7 684:18,18 685:10 697:1 708:22,24 709:2 710:9 chemicals 11:17 12:3 14:7 15:5,6,7,11,19,24 16:1,4 16:19 17:7,21,24 18:5,8,14,24,25 19:22 22:5 24:5 26:25 29:2 41:19 59:23 72:23 93:19 100:21,22 113:18 123:20,22 155:22 159:15 161:21 163:17 197:19 217:22 234:21 275:19 279:23 283:5 293:2 299:3 313:8 348:14 351:12,14 356:2 383:8 387:2 407:11,14 414:10 416:16 419:13 423:25 424:5,13 424:17,22,26 425:2,20 426:21 427:3,16,21 435:7 438:26 446:18 447:2,5,17 469:22,23,25 470:14 485:6,11 487:16 490:10 492:11,11 493:12 494:8 524:21 525:18 528:19 534:10 537:26 539:17,22 540:18,24 542:11 543:22 544:12 550:17 556:26 557:6 560:4,8 564:24 565:24 568:17 576:17 601:22 602:6 616:18 617:15 618:6 621:7 626:15 634:2,18,19,19 638:9,14,20 646:15 662:9 667:10 674:18 697:6 698:24 708:18 709:17,23,24 710:10 710:22 716:16 719:6,7,10,10,12,16 720:13 721:3 722:22,23 chemical's 278:9 623:2 chemical-induced 175:14 chemist 399:21 chemistry 113:16 133:6 147:24 289:2 374:13

378:3 410:17 511:9 656:9,10,14 chest425:9 chick45:6 339:12 368:4 chickens 366:13 569:20 chicks 41:2 43:24 56:16 64:15 73:22 74:9,11

288:21 289:21 290:9,12 338:23 339:2,2,13 340:19 392:10,13,14,23 424:6 Chief5:5 children 663:9 chitchat 343:14 chlorpyrifos 669:16 choice42:12 143:25 349:23 352:11 373:5,6 409:6

418:18,21 425:18 427:12 439:18,21 469:18,26 487:25 501:23 549:4,5 562:1 563:23 564:2,3 585:9,13 597:13 627:11 708:19 choices492:16 563:24 688:4 choose 253:15 292:15 325:6 369:19 576:1 choosing 324:24 325:2 349:26 628:25 chose 59:12 76:16 77:2 156:24 158:18 167:1 170:24 373:25 420:19 512:5 548:6 chosen49:8 59:13 145:1 258:26 344:13 383:19

389:3 409:9,20 470:26 487:16 488:15 559:25 563:20 577:2 596:11 628:23 644:15 675:4 685:11 689:8,10,12,23 700:20 Christman7:18,19 8:7 35:24,25 36:13 137:10,11

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571:6 circumspect 153:2 circumstance 312:12 745:4 circumstances 401:11 417:14 547:17 citation494:1 citations 482:6 cited 301:1 claiming 672:3 clarification 30:2 35:26 59:8 61:6 63:19 70:18 114:24 140:11 152:25 179:4 198:19,20 199:13 201:18 202:21 218:10 229:18 245:7 249:22 253:19 261:24 267:10 270:7 276:2,15 300:16,18 301:3 334:1 337:18 353:14,16 357:6 358:10 362:12 375:17 382:10 411:14 429:24 434:19 442:2 452:8 468:21 470:7 491:16 492:19 507:8 521:10,11,15 539:10,24 540:7,9,20 541:5,6,10,24 543:13 555:23 570:2,4 645:23 649:12 650:9 703:12,13 717:23 clarifications 205:11,18 361:26 441:3,6 452:10

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274:23 333:13 343:15 411:13 468:24 484:19,21 528:13 533:18 549:22 555:24 564:11 575:5 595:25,26 596:1,4 601:11 607:3 610:22 615:15 636:7,8 660:4 685:4 695:3 707:15 713:13 724:20 clarity 64:5 195:5 203:9 331:22 516:19 652:13

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611:5 619:8 621:22 713:21 classical 116:14 classifies 478:10 clean 113:20 114:1 190:14 565:7 620:26 cleaning 113:21 clear 34:15 53:21 55:13,24 68:18 73:13 122:23 140:7 141:21 149:18 157:12 194:25 205:22 243:19 253:19 263:10 264:11 274:15,26 276:23 278:10,25 306:11 307:10 318:12 328:3 330:26 347:5 348:6,12 358:4,20,21 359:3 367:19 380:19 388:16 389:8,10 392:20 395:5 407:8 409:13 413:1 421:21 422:4,23 424:6 426:5,23 432:7 440:11 443:4 444:10,13,22 445:5 452:10 477:18 479:23 487:18 495:24 500:8 506:23 507:19 517:16 518:15 532:26 542:9 545:8 553:7 556:24 556:26 561:19 566:1 571:26 586:14,17 597:14 599:4 607:9 609:22 614:19,26 616:6 621:15,17 622:11 634:9 654:16 655:6,25 659:9 660:14,16 684:5 704:1 710:19 728:25 736:5 743:7 746:3 clearance416:24 clearcut 173:21 cleared 236:5 309:26 clearer 518:18 clearly 11:13 38:18 53:15 68:20 69:9 103:22 118:10 194:15 209:19 229:6,15 231:1 280:6 284:26 288:5 315:20 346:26 350:1 352:12 357:26 365:4 367:8 426:15 431:21 435:10 438:10 446:6 481:26 496:4 504:13 508:7 513:3 521:16 528:21 537:21 547:18 551:2 558:4 586:16 617:13 618:2 618:15 619:17 631:21,24 652:22,24,26 654:2 658:16 660:23 671:7 673:25 677:17,17 718:17 722:21 741:16 clear-cut 69:23 122:9 Clemson 5:20 242:10 459:23 664:25 clichI 660:19 clicking 22:19 climax 551:6 560:24 561:14 567:1 568:1,4,6 clinical 600:16 clip 80:21,22 cloacal45:19 46:4 366:23,26 clock 153:8 507:11 703:18

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239:4 246:3 248:12 283:22 346:26 475:21 509:25 633:20 661:16,21 663:10 703:2 731:12 743:20 745:10,12 closed 8:9 closely 25:13 52:6 156:19 398:13 closer 8:1 316:19 567:10 closes 345:20 closest503:15 closing4:18 10:16 14:26 304:9 305:9 cloud 304:4 clouds 630:26 clustering 153:20 clutches 223:15,19 CMP96:22 479:16 505:8 506:9 Coady 295:25,26 326:24 coastal 6:2 620:10,16,19 coauthor 130:17 cobwebs736:5 Cochran-Armitage481:19 code 129:6 154:6 183:20 725:13,17,19 736:8 coded483:21 662:10,13 coefficient218:18,20,22 235:5,13 236:1 237:18

366:1 477:12,18 572:19,21 coefficients 181:13 218:26 680:8 cognisant 693:8 cognition 361:6 cohort 686:23 cold 278:7 393:15,17,20 colder 393:18 Colin7:3,3 36:15 37:1 38:7 63:15,16 64:2 145:19

146:4 147:2,15,21 177:15,18,25 178:10,14,19,24 203:15 204:19 205:2 243:4,4 263:23 264:5,16 267:15 268:3,15 269:7 338:12 339:5,14,18,22,25 340:3,7,21 347:22 352:25 354:13 356:7 357:24 359:20,24 360:2,13 362:20 363:6 364:16 372:21 373:26 374:15 375:23 376:21 405:26 407:26 408:12 411:2 413:6,7,16 418:8 419:5 428:16 430:13 440:3 460:18,18 569:17 646:23 648:3,12 649:18 650:26 651:22 652:11 654:6,26 655:17 659:7 660:8 661:8 665:18,18 672:24 736:13 740:1 Colin's 356:12 collaborate 25:13 collaborated 27:2 collaboration 26:4,15 43:12 161:24 collaborations 152:9 198:7 collaborative 9:24 712:22 collaborator 193:22 collaborators 168:8 colleague 359:17 colleagues 3:23 4:21 13:24 93:8 133:21 151:23 184:24 343:4,13 356:8 359:13 577:7 617:7 632:4 635:13 642:15 653:21 679:10 724:14,17 731:1,5 736:16 737:17 738:19 748:5 collect58:21 62:12 109:5 268:12 288:20 359:7

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374:25 638:19 645:11 652:9 combinations 705:11 combine 688:11 combined 99:22 424:26 542:24 556:23 557:5

558:26 634:18 come 11:21 24:14 29:11 33:4,14 36:18 38:13 54:7 104:25 105:2 150:10 151:25 153:3 154:16 162:15 171:10 187:14 193:13 194:19 204:14 254:23 257:4 264:1 276:4,10 277:1 285:20 286:5 305:15 306:6 307:11 311:3 312:26 322:19 324:4 332:1 333:12 350:20 356:7 358:7 359:13,23 363:25 364:17 378:8 386:21 389:20,23 393:11 400:14 401:18 403:16 441:22 480:21 484:13 500:26 511:24 539:22 544:17,19 564:19 575:25 576:7 577:20 593:13,18,24,25 596:16 617:9 629:24 632:12 633:2 645:26 648:4,6,23 650:19 652:4 654:11 659:11,19 671:14 672:21 693:17 694:12 703:19 725:22 728:11 731:6,14,16,19 732:2,7 735:14 736:11,24 744:16 comes 24:17 33:6 37:6 116:6 146:22 165:21 178:20 184:21 194:22 195:15 245:14 246:8,11 322:7 373:5 396:2 399:4 471:25 542:18 662:24 comfortable 329:9 384:4,16 coming 11:23 15:1 16:23 38:15 55:5 108:21 111:6 150:26 174:5,24 296:12 303:16 319:8 327:8

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462:26 463:3,6,16,24 495:3 504:11 514:8,9 517:24 536:16 550:23 683:13 735:23 738:9 degrees491:22 492:9 DEJ 592:17 delay 166:3,5 307:9 309:13 604:5 delayed424:16 596:25 603:18 delays 170:16 604:10 Delclos 5:8,8 242:4,4 374:9,10 459:17,17 664:19

664:19 727:17,18 deliberated 16:10 delineate 30:26 deliver 303:20,25 649:25 delivered 121:3 delivering 237:22 delivery 114:4,5 213:26 214:7 223:16 301:12

641:17 delta 594:13,19,22,25,25 595:4,6,13 delve 32:6 demand404:25 demonstrate 27:23 30:8 60:26 61:24 122:13 200:1

214:12 230:21 280:18,22 318:21 364:10 375:21 461:18 517:22,23 529:25 537:18 542:11 556:9 559:26 562:22 574:9 650:7 652:6 653:14 656:25 660:15 661:11,12,12 666:10 706:25 743:12 demonstrated 26:7 28:6,19 42:6 72:11 122:18

230:13 258:13 281:19 283:1 313:11 357:14 364:22 436:7 438:10 449:14 452:16 454:12 462:3 500:17 535:13 536:15 550:3 571:8 605:15,23 606:6 613:15 614:25 637:3 660:12 662:9 698:1 711:6 demonstrates 111:25 185:21 216:6 demonstrating 39:10 43:7 283:18 436:3 643:8

650:13,18 652:10 654:23 demonstration 39:18 209:14 297:14 298:11

364:14 383:7,19 384:11 431:15,21 461:22 464:11 470:12,26 556:13,17 560:4 576:16 577:2 613:13 666:14 667:23 668:21 669:1 674:17 675:4 681:19 681:20 683:21 689:5 697:21,23 699:20 denominator 254:14 584:10 588:17 denoted 594:15,21 densities 558:21 density 558:24 582:26 608:5 Denver 6:19,20 34:8 35:7 68:13,14 184:5 190:3,4 190:17,26 191:3,9,14,17,22 192:20 193:2,5 242:24,24 460:12,12 548:17,18 550:13,15 556:15 556:16 564:18,26 565:21,22 566:24,25 570:17,18 575:16 576:8 598:11,12 606:8,9 609:10,11 610:9 615:2,3 665:14,14 701:26 726:5,6 735:9 Denver's 36:16 denying 144:20 depart483:5 departments 7:24 departure 35:21 74:6 185:25 199:22 482:26 departures 403:2 depend 263:4 548:14 dependence 31:26 476:15 525:8 582:26 dependency 129:8 dependent73:9 118:25 173:3,3 247:17 351:8

387:1,1 399:7 402:7 449:15 454:1 597:2 602:8 624:15 depending 66:19 87:26 89:9 211:8 212:9 254:17

257:5 262:1 265:22 397:23 489:18 535:16 581:22 594:10 685:12 depends 199:3 383:13 470:20 473:20 532:25

576:23 671:17 674:24 694:22 depiction 17:17 deplete 220:12 deploy743:14 deploys 744:13 deposited 58:7 515:26 deposition47:11 depth496:11 704:15 deputy 3:10 8:14,20 635:12

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582:19 637:22 651:21 description 138:26 222:24 324:13 346:5 364:5

461:13 465:1 511:4 515:14 555:1 556:5 586:11 666:6 676:13 699:5 descriptive422:2 428:9 507:17 600:20 607:7

703:25 descriptively 582:6 deserve748:3 deserves 369:4 747:16 design 17:11 24:16 39:13 109:2,23 110:4,17 111:19,20 120:11 140:7 148:7,14,22 149:9 153:25 168:10 191:23 211:9 219:17 227:19 271:4 286:7 287:11,15 290:1,25 292:7,23 293:5 294:4 297:6,11,15 299:8,11,13 302:1,4 304:10 370:8 383:14 384:20,21 386:7 392:5 393:7 403:26 420:22 446:14 456:9,11,14 470:21 480:6 481:24 511:6 524:25 531:22 532:13 576:24 583:14,24 584:22 590:16,16,17,18,20,23,24 591:1 641:8 649:14 650:23 670:18,20,22 671:2,5,14,15,23 672:1,6 673:20 674:3,25 675:14,21 676:6,8,16,16 678:14 687:20 688:26 695:7 715:8 727:26 741:5 designated 2:2 3:2 9:11 241:2 459:2 517:21 664:2

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297:9 347:10 349:14 365:11 443:11 529:11 545:15 616:22 618:10 632:25 635:20 639:8 681:2 682:1 designing 354:19 590:11 designs 32:10 220:4 290:4 293:1 294:1,23 385:25

582:16 671:26 673:12,15 696:10,12 743:6 desired449:8 despite 290:14 698:9 712:3,7 737:11 destroy 113:26 detail 20:2 21:2,24 25:15 148:4 173:10 179:13 187:3 309:20 348:25 417:8 446:2 466:20 469:6 485:8 500:26,26 510:18 511:26 512:1,14 514:9 514:10,18 521:16,25 522:5,10 599:21 666:22 676:21 682:11 719:15 725:21 741:19 detailed 27:20 42:13 209:8 210:10 318:4 349:24

364:4 369:16 377:4 412:11 425:24 426:2 461:13 466:1 510:13,20,22,25 514:21 515:10,13 516:13 535:11 537:10 556:4 575:6 666:5 details 89:17 154:24 158:23 196:26 346:11 349:20

418:8 442:12 465:9,13 466:25 467:1,9,19 469:5,7 666:23,26 detect25:7 37:23 55:7 56:6 67:20 110:15,26 111:16 112:8 136:2 148:2 190:24 214:13 220:2 221:4 247:13 281:13 293:11 302:22 417:7 438:7 439:11 517:18 550:16 552:24 568:2 570:25 571:4 659:25 detectable 129:10 421:1 572:23 573:4 detected 338:18 396:24 427:4 437:6 448:14,18

505:25 552:26 588:8 detectible 190:22 detecting 54:26 110:18 111:2 280:20 553:17 559:1

573:11 622:23 667:8 726:8 detection 176:1,21 191:12,13 370:1 396:23 397:1

397:20 419:24,26 421:9,16 535:14 560:20 571:7 572:26 detections 291:18 determinable 611:26 determinance451:14 determinates 275:12 determination 20:14,18 35:16 103:23 128:8 139:17 140:16 160:2,3 169:24 199:21 231:22 232:20 253:17 348:15 376:9 433:14 450:20 463:22 464:3 466:26 531:5 536:13 697:22 699:5 699:14 745:1 determinations 35:3 141:11 195:16 263:3 394:17

449:4 determinative 308:18 determine 20:8 25:9 26:1 28:4 37:22,25 40:25

42:23 55:9 58:8 64:25 66:3 72:16 73:1 76:6 80:20 112:12 125:2 128:1 130:21 185:13,18,24 195:24 219:7 232:24 236:2 275:2,4 289:2 298:17 300:6 300:11 317:11 319:8,24 322:3 324:2,10 326:7,23 329:26 358:22 430:4 438:18 445:8 454:20 455:24 469:20 482:26 490:18 515:14 516:19 522:15 536:22 548:9 555:9 563:1 566:26 567:17 581:17 582:4 588:10 599:5 611:2,20 612:6,11 613:13 622:20 625:14 634:17 659:17 684:1 699:2,6 706:20 713:18 727:2 determined 37:26 48:23 58:23 80:19 81:12 282:3

287:22 307:14 326:13 394:8,16 411:25 463:4

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483:17 506:21 706:11 deviations 52:10 122:7 254:2,15 474:20 502:24 558:4 705:24 706:4,17 device 391:10 devil 196:26 devising 690:26 de-gased 117:16 DFO 2:7 9:1 10:23 13:7 657:17 747:24 diagnose 261:5 diagnoses 68:18 83:24 84:5 85:2 diagnostic 69:6 84:8 85:1,16 174:13 187:7 212:22

260:1 273:11 299:21 302:25 378:11 551:20 552:17,23 557:13 570:19 654:10,14,18,24 diagnostics 404:16 523:22 diagrammatic46:10 diagrammatically 46:11 diagrams 108:9 diameter 178:18 dichloroacetic 84:17 dichlorophenol 209:18 719:16 dichotomously463:2,25 464:1 dictated 171:8 die 690:9 699:25 died 587:21 591:9 diet47:2 170:21 171:24 172:1,2,6,7,9,22 173:6 196:22,24 237:8 390:8 653:25 dietary48:25 56:17 61:14 diets 172:5 differ 57:8 207:17 416:24 526:17,18 differed 321:6 700:4 difference 52:17 54:26 55:3 63:8,9 67:9 70:19

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249:16,18 266:20 317:11 354:5 462:18 497:13 567:23 605:17 712:3 difficulty 144:26 236:4 237:5 238:24 diffuse 274:7 dig 175:24 dilation 290:8 dilemma 334:2,3 727:24 diligence 114:18 diligent239:16 dilute 397:20 diluter 114:5 267:3 696:2,13 diluters 266:16 dilution 114:5 182:4 264:9 265:1,9,21 300:24

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243:17 260:23 308:26 323:12 423:17 455:17 488:6 552:15 553:24 559:8 616:13 635:20 639:1 641:8 656:20 667:11 719:24 721:6,6 734:21 director 5:26 6:24 8:14,16,20,23 10:9 635:12 disadvantage 515:3 641:12 disagree 143:24 disagreement524:19 668:23 disagreements 348:5 disallowed 699:19 disappointed 330:3 disappointing 333:6 disaster417:21 discarded469:17 531:9 discern 32:9 56:10 66:6 74:15 281:10 564:16 641:6

641:23 667:24 677:12 679:4 688:19 710:12,15 711:17 discerning 645:12 705:12 717:10

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594:26 division 10:10,12 75:13 747:3,3,13 Dix 8:23 742:16 DMY 80:23,25,26 122:22 123:11,13,19,22 128:8 128:25 129:3,4 156:13 462:18 515:24 dNTPs 513:16 doable 113:13 192:17 457:23 655:21 660:17 doc739:7 docket 1:8,10 4:2 239:26 241:10 277:23 346:15

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31:18,19,20,22,23,24,26 32:2,19 33:2,15,15,20 71:19 102:7,8 105:23 106:3,5 112:17 113:19 127:21,22 149:1,3,21,22 182:2,4 192:3 212:19 219:5 222:9 237:18,22 272:8,11,12,14,18,19,21 274:23 275:2,4,5 278:10 290:6 292:24 302:18 312:6,7 316:25 329:4 345:9,11,14,15 347:8,13 349:17 358:14,21,22,22 362:13,14,18 363:4,14 363:15 371:10 374:12 375:11 386:9 388:19 389:4 389:5,10 438:17 439:18 447:11,26 448:21,22,25 448:26,26 449:6,24 451:9,12,18 476:15,19 480:3 482:7,14,16 525:8 545:18 554:10 561:13,14,19 574:10 585:9,10,12 589:16 613:6,14 614:26 616:10,25 617:26 621:15 622:21 624:11,26 625:15 641:17 646:21,25,26 647:6,7,20 668:24 680:1 688:10 690:20 702:20,22,25 703:1,3 704:24 744:24 doses 22:13,16 23:3 25:25 31:11,24 32:8,22 33:6

33:24 47:17 71:17 92:4 192:1 312:22 362:21 374:12 438:18 448:24 482:19 489:21 561:9 564:24 594:16 648:1 680:3 690:17,18 704:23 716:15 dose-dependence 33:23,25 128:1 dose-dependents 127:16 dose-related49:21 dose-response 11:26 17:14 30:13,14,23,26 31:1,3

31:18 32:1,20 33:12,21 35:26 48:4 63:21,25 76:12 127:19 443:9,13 545:13 dosing42:18 43:22,24 203:4 420:23,23,25 444:20

599:6 dot594:21 dots 505:7 double48:3 doubling 171:7 202:26 Dow 174:25 296:1 downright580:1 dozen247:5 593:24 Dr2:1,10 4:25 5:2,8,12,15,19,23,26 6:4,7,12,16,19

### FIFRA SCIENTIFIC ADVISORY PANEL DOCKET #EPA-HQ-OPP-2012-0818

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600:10,10 725:4,5 735:4 driven436:11 730:19 driver 61:11 186:2 231:14 drives 526:6 driving 392:7 drop 236:17 264:21 673:7 dropped 61:5,19 101:6 214:20 237:12 389:7

609:21 dropping 350:22 drops 344:20 drove 621:1 Drs 347:26 Drug 14:6 drum716:7 drums 716:6 dry 214:1 220:14 221:2 236:9,10,14,17 261:18

262:8 duck 64:8 286:21 340:13 432:25 ducks 338:16 426:25 duct 164:10,12 167:17,18,19,24 169:19 177:22

546:16 561:4 603:7,12,15 604:26 606:16,20 611:22 ductal 552:11 559:5 560:17 ducts 166:20 170:18 175:15,16,17,19 561:8 565:5

### FIFRA SCIENTIFIC ADVISORY PANEL DOCKET #EPA-HQ-OPP-2012-0818

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217:14 221:6,19 222:12 235:8 247:20 255:8 262:5 278:2 301:24 640:6 698:10 721:20 durations 214:3 dust408:20,22 duty 629:17 dwell 641:25 676:26 dwelling 621:19 dynamics 33:1 132:10 133:6 D.C 154:16,18 E E 20:8,16 36:23 233:17 300:13 315:8 316:26 337:3

447:12 448:13 449:21 455:18 457:7 525:25 561:12 595:3,3,4 618:19,22 619:4 623:7,12 630:17 634:13 637:23,25 683:4 701:7 713:7 earlier 21:7 29:13 35:26 82:26 85:20 93:11 171:4 176:10 201:11 204:4 220:8 332:21 351:8 389:22 404:9 417:1 418:3 426:12 439:8,17 450:11 458:5 475:15 515:18 546:9 557:13 583:12 612:14 620:25 641:4 667:16 680:12 697:25 714:26 719:26 721:2 724:24 735:23 earliest 620:2 early 24:14 37:6 61:9 83:2,2 85:7 88:10 99:25 100:25 122:17 142:15 159:9 171:3 185:19 189:25 233:18 321:12 365:12 523:16 702:20 721:13 Early-life 657:25 earnest46:15 earth 628:20 629:1 ease444:26 523:7 546:10,11,11 easier 25:17 204:22 226:5 236:15 249:14 257:26

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288:23 289:9 313:21 361:7 370:23 373:15 403:7 408:23 410:26 425:5,7,14 427:26 429:7 513:23 581:4 666:25 Easton481:21 easy 13:10 48:23 69:23 173:8 199:6 210:19 274:8

350:3,17,17 355:25,25 360:25 392:2 425:23 427:9,9 446:21 574:8 583:6 584:14 640:2 678:4 710:15 714:11 728:23 737:5,6,7 eat 390:22 448:1,6,11 449:20,22 516:23 524:14

537:3 631:12 632:19 667:10 682:6 685:14 729:22 730:1 ecdysal 259:20 ecdysis 259:10 ecdysone 318:1,2 618:17 623:4 625:26 626:9 644:7

683:7 ecdysteroid 207:24,26 230:24 231:2 259:22,25

643:24,26 667:15,21 685:12 701:12,21 ecdysteroids 207:13 259:9,17 261:6 318:1 701:19 ecdysterone720:14 ECD-mediated 638:7 echo 10:23 eco 186:2 337:25 ecological 7:21 12:1 18:14 20:1 21:22 24:25 26:20

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50:1 53:10 54:3 58:24 59:2 63:4 68:26 69:5,9,11 69:20 70:3 78:8,8 79:8,22 80:4,18 81:5,7 82:3,8 82:24,25 83:1,11,12 84:26 85:1 87:2 90:19 91:4 95:3,16 99:7 103:23 107:1,9 108:4,7,13,14 109:16,18,24 124:1 130:20,22 140:1 152:5 157:10 158:26 159:12,24 161:8 163:23 164:1 166:19 167:8 169:21 175:6,8,18 184:20 186:13 196:13 197:3 199:5 204:20 212:21,23 214:1,26 216:9,16 217:1,5 218:22,25 219:16,20 222:11,12 225:23 226:6,24 235:8,20,26 236:6 247:20 255:1

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66:25 72:7 77:15 85:16 99:7 131:15 133:5 144:11 198:15 208:22 222:20 236:12 269:14 275:23 289:16 292:22,25 303:19 323:1 334:14 344:8 354:5 360:25 368:23 462:18 465:19 495:23 513:24 546:19 573:6 598:17 603:8 605:12 611:13 614:12 636:19 675:19 681:22 685:7 703:14 708:13 722:17 723:1 essence 141:10 168:4 490:17 518:8 essential 98:22 104:18 113:24 280:9 528:1 essentially 101:25 103:10 122:5 123:6 124:14 144:18 148:21 490:25 510:8 511:4,16 522:23 532:11 542:26 554:26 587:19 628:4 725:5 establish44:8 47:17 61:15 139:24 274:13 326:26

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716:4 estimated 676:1 estimates 90:16,17 380:14 estimating 503:16 estimation483:23 estimator 587:18 estradiol42:19 47:23 48:8 51:26 68:20 94:3,15,22

99:10 105:18 106:5,17 107:10 177:5,9 370:6,8 374:5,5 413:10 553:1 559:24 estrogen 11:23 17:7 19:3 47:23 59:20 69:17 76:9

80:13 96:1,13 112:15 135:8 164:5,7 173:23 177:4 189:7 230:23 299:14 302:22 303:2 317:13 329:5 347:8 368:4 374:4 426:13 443:9 488:10 490:24 538:9 545:13 546:4 547:6 552:12 560:22 561:10 613:7 616:10,14 618:1 622:17 625:15 626:2 682:2,3 estrogenic 16:19 49:14 166:22 168:3 186:20 408:8

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247:2 296:6 316:5 336:13 400:2 404:22 546:1 684:8 729:3,8 731:21 732:26 741:26 everybody's 334:11 731:18 everyone's 743:4 746:12 748:10 evidence 17:9,10 18:3 20:5,18 34:2 136:11 187:11

225:16 319:7 328:24 330:11 333:21 336:4,7 337:6 351:10 355:3 358:1 366:25 371:25 376:24 377:12 379:18 411:9 423:17 435:6 436:1 445:13 482:8 483:4 485:24 486:3,11,25,26,26 487:4 528:18 552:11 581:9 582:10,10 609:7 630:10 668:3 684:2,8 705:22 718:15 719:26 734:18 744:15 745:1,2 evident 16:25 39:20 50:16 176:13 568:10 evidently 397:3 evolution 195:11 699:16 evolutionary 351:26 432:11 exacerbation 136:12 exact 316:17 581:21 611:25 698:3,12 exactly46:11 77:12 79:2 87:26 141:8 146:17

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245:20 246:9 247:10,18 306:2 509:22 531:2 543:26 629:8 642:9 709:12,25 715:9 exists 289:2 551:19 709:25 710:20 exit253:12 expand 15:21 16:14 19:16 369:15 373:18 470:4

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214:8 280:5 304:23 305:7 402:15 411:23 436:6 451:19 467:4 490:18 494:18 495:1,3,5,11,19,20 495:25 496:3,8 499:18 511:19 531:22 536:14 548:5 563:26 600:7 602:11 603:22 650:7,20 651:6,19 652:5 656:4 722:13 expecting 202:16 374:6 482:6 expedience75:12 expended 680:14 expense 262:18 375:5 expensive 24:19 313:15 583:8 634:20 688:3 743:6 experience 28:12 77:19 83:2 91:17 103:18 104:18 118:2 151:22 179:12 188:18 219:1 225:11 236:6 263:10 284:25 293:25 294:11 303:17 311:1 314:2 314:3 372:8 381:19,20 399:6,12,15 424:12 497:3 500:19 539:16 540:11 573:7 607:14 643:4 704:5 709:4,5 711:20 712:6 713:3 714:9 experienced 104:8 197:12 200:8 422:9 497:2 500:7

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586:4 590:12 671:14 experiment-wise478:6 expert 11:16 19:4,7,16 250:26 251:8,15 385:20

579:16 583:4 742:10 743:3 747:24 expertise 7:5,8,12,20 189:23 282:23 285:10 293:25

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647:16 explanations 562:15

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139:11 141:12,25 148:2 156:10 161:14 225:17 319:22 346:23 348:8 349:22 356:26 370:16 382:11 403:1 424:7 432:8 436:2 439:9 441:9 453:17 456:15 462:8 465:3 478:18,20 482:2 494:1,12 496:9,19 505:17 511:5 516:6,7 522:6 523:6 525:22 561:22 569:9,15 573:4 598:2 602:8 610:4 611:13 666:20 667:2,6 668:23 709:1 generalization 81:18 generalized 148:1 303:11 480:19 515:21 525:22

684:3 generally45:18 49:25 52:24 57:26 76:20 128:17 141:14 149:11 213:8 224:11 225:17 226:22 246:19 269:1 291:6 293:3 311:14 312:11 317:24 432:10 465:20 494:18 495:25 497:26 503:20 505:24 506:8 516:12 568:6 602:14 603:20 605:14 611:12 620:23 625:10 671:24 708:5,17 709:2 714:11 741:7 generate 278:16 443:11 462:23 545:16 616:23

624:9 640:17 generated 161:3 307:6 313:6 463:23,24 478:11

544:1 571:20 676:18 691:26 700:12 705:1 generates 347:11 349:15 641:11 716:3 generating 160:26 572:6 generation 23:21,22 24:1,2,6 41:12 42:21 44:1,9

46:24,26 47:6 50:16,19,19,21 51:6,24,25 53:7 54:4 58:4,14 64:13 67:24 70:19,23 72:9,10,14,15 72:17 73:1,2,14 75:6 78:18,19,20,21 79:14,15,24 80:4,6 93:15 94:16,19 95:1,2,5,6,12,12,15,20,21 97:11,19 98:2,5,20 104:5 107:19 143:9,13 206:15 211:20 212:14 213:23 215:10,11 217:4,11,13 218:6,7 219:20 222:7 245:11,15,19,23,26 246:12 247:6,24 250:3 256:2,5,6,10,13,16 257:13,22,23 258:1,6,16,21,23 260:25 261:19 264:6,13,20 273:6,8,9 277:26 286:24,24 290:9,10,17 291:1 293:21 294:7,17 295:1 316:14,15 319:12 320:8 320:10,25 323:9,9 325:10 339:6 340:6,15 341:1,5 341:6,7 344:25 350:4 352:10 366:7,7 392:19 419:7 431:16,16 432:22,24 435:9,12,21,23 436:17 437:1,11 438:15,23,24 439:16 451:2 474:17 504:6 505:1 523:10 524:9,12 528:21 529:2,9,11,21 530:17,19,20 533:5 535:3 536:13 536:19 624:4 626:13 683:18 689:3,16 690:9,11 690:21 692:9,17,23 698:17 710:18 711:13 715:2 715:16,17,18,22 718:17,25 719:5,11,11,17,19 722:5,10,20,22 723:1,3 generational 72:20,23,26 73:6 217:8 530:11,12,18

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54:6 71:7 76:15 78:5,6,17,17 79:9,9,11,19,23 104:4,22 107:24 108:24 109:3 217:3,6,16 226:10 249:8 250:10 254:24 291:15 294:19 318:26 322:25 332:2 435:9,17 436:19 437:17,24,26 439:1,8 440:18 483:13 504:7 529:23 530:13 533:3 535:20 624:23 630:26 668:12 677:9 690:19 690:23 700:22 716:26 718:20,23,24 719:2 739:18 generic 578:2 generically 194:18 genes486:10 513:8 522:11 726:20 genetic 26:1 77:5 80:10,18,20,25,26 81:2,13 102:9

103:12,13,23 104:17 113:3 122:18 128:8 130:18 131:10,15 140:12,18,22 156:2 160:2 165:4 169:24 417:20,23 420:24 429:3,7 445:8,17,23 448:8 453:11 454:21 455:4 466:26 468:25 469:6 487:6 495:14,14,17 498:21 515:11,14 523:23 530:23 531:3 552:26 603:17 605:8 genetically 26:11 109:12 123:17 155:24 156:12

389:26 423:5 546:25 659:18,25 genetics 429:6 691:23 genital463:18,19,20 genome 352:14 359:11 427:11 445:1 523:18,20

544:7 genomes434:14 genomic 304:12 genomics 11:16 356:14,17 720:11 genotype 80:15 86:14,15 130:21 141:5 445:24

450:19,23 455:5 470:2 499:8 genotype/phenotype 129:12 genotypic 123:18 281:2 301:10 451:7,8 455:2

469:20 487:6 524:1 530:26 557:15 604:11 genotypically 130:26 genotypic-phenotypic 302:7 genotyping 298:12 653:9,26 Geoff5:26 242:14 243:26 334:4 460:1 665:3

672:24 691:21 738:13,22 GEOFFREY 5:26 150:24 152:1,4,25 153:4 187:13

187:18,20 188:1 189:5,9 190:1 202:20 235:2 236:19 237:17 238:5,10 242:14 243:25 248:2 249:21 250:2,5,9,16 253:1,18 266:13 333:16 335:13 336:16 337:14 460:1 569:9 617:6 627:16 632:17 635:1 636:2 637:13 645:2 656:8 665:3 670:1 672:10 682:15 684:5 685:19 690:6 702:15 707:6 712:9 717:14 723:14 730:26 Geological 6:5,8 242:16,18 460:4,6 665:6,8 geometric 362:18 George7:22,23 65:7 66:13 180:14 204:2 243:14,14

347:21 352:22 355:11,23 359:17,19,20,21,25 360:6 361:4 362:5 368:9 372:5 373:3 375:9 380:17 405:23 406:18 407:10,14,17,20 408:1,9 408:10 410:6 411:9 413:7 414:16 422:15 429:3 434:3 439:26 461:2,2 650:3,15 655:2,18 657:3 662:1 George's 360:13 germinal 164:19 germs 158:18 getting 11:20 12:7 79:20 85:8 88:1 94:1 113:8

119:18 126:10 131:18 136:16 181:8,22 195:3 219:13 250:23 271:3 304:15 306:5 336:18 346:2 367:14 379:18 388:7 396:19 404:26 470:3 489:22 520:1 541:24 584:11 589:17 646:17 652:17,22 701:4 713:9 722:12 725:6 728:22 730:17 733:14 744:1 ginning 166:15 girls 584:7 give 11:21 13:15 21:11 32:20 39:12 47:21 55:6

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121:25 136:3,16 155:15 156:3,23 157:14,25 175:10,19 190:17 191:19 223:18 235:23 237:21 239:9 241:13 246:24 258:2 259:15 325:13 333:4 347:14 353:11,20 355:2 365:10 366:11 368:23 369:3,4,4,5 375:6 378:2 387:24 403:12 406:26 411:21,25 412:10 417:8 418:20 433:17 435:13 436:5 437:7 442:11 445:5,7 448:4 449:1 450:1 453:4 454:19 462:18 486:24 487:4 495:26 505:10 519:12 525:7 531:5 536:5,9 537:24 555:10 562:26 568:19 571:19 574:25 576:10 582:16 588:8 594:11 595:17 619:7,10 624:1,17 627:2 629:19 631:14,15 637:25 639:13 642:18 668:1 669:9 687:4 695:19 699:5 704:25 705:12,20 706:12 708:16 712:23 713:3 715:10,13 737:23 739:4 747:17 gives 32:21 60:18 84:8 158:22 176:19 236:14

247:20 369:21 420:21 630:25 648:5 652:14 684:19 691:1 733:25 734:22 748:8 giving 11:2 22:20 33:16 43:18 257:13 451:2 600:12

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746:9,14 gland45:19 46:4,4 167:9 169:11 178:1 180:20

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39:8 60:5 61:16 64:23 67:14,15 68:1 70:1 75:19 76:1 84:20 85:17 86:4 89:17 90:7 92:1 93:14 96:8 99:8,11 102:11 105:11,25 110:20 112:22 113:20 114:24,24 115:4 116:8 137:18 140:13 142:13 143:6 148:10 150:7 151:6 153:14 154:26 155:9 157:8,22,26 162:11,23 163:2,3 168:7 181:11 183:23 184:1 188:10,13 189:17 194:17 196:19 203:11 206:10 222:2 227:16 229:21 232:12 235:11,14 241:22,26 244:20 247:2,7 251:17 254:7,13 255:18 256:11 260:2,14 263:15 267:16 271:20,25 276:9 283:20 284:18 285:14 296:21 301:6 307:8 309:19 322:4 325:26 327:18 331:3 333:17,21 336:22 337:2,22 343:2 345:21 346:21 346:23 353:21 364:16 377:2,3,3 378:4,19 379:3 380:1 381:6 383:5,23 392:18 395:23 401:15 404:5 407:11 410:3 422:22 428:17,20 444:6 452:7,12 459:13 469:6 473:24 475:23 476:7 484:18 487:14 491:18,19 492:8 510:18 514:9 521:17 538:3,4 539:21 543:24 544:5 575:13 576:1 579:6 580:18,24 581:1 588:25 595:19 599:6 601:9 602:16 606:2 617:10 624:6 628:1 632:4 633:6 635:3 639:4 649:3 653:5,6,6,8,14 654:22 655:14 656:10,12 658:19 661:20 664:14 676:14 689:18 696:11 698:15 707:13 713:7 714:3 724:23 727:15 730:13,17 737:8,9 738:13 739:22 741:3 747:24 goal 200:4 287:25 290:12 348:18 349:21 352:3

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143:16,22 145:4,8 146:17 158:4 159:10 160:1 164:8 167:16 170:18 309:4 378:17 536:11,16 556:20 560:17 gonadal78:11 82:16,17,19 131:1,5,13 142:7

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298:26 305:11 329:10 332:10 351:20 426:5,7 533:3 552:19 683:14 684:2 729:21,26 linkage 618:3,21,23 619:19,23 626:16 639:6 linkages 308:3 linked 156:14 262:6 291:25 453:11 linking 629:7 632:19 638:22 729:13 links 329:26 330:8 369:5 lipid 723:26 lipids 720:14 list 17:22,24 18:21,23 45:26 254:23 283:4 284:23

285:5 291:23 321:19 422:22 441:13 444:22 465:8 466:18,19 522:24,25 571:16 580:17 597:2,8,13 597:22 634:2 639:23 643:22 711:8 726:3 743:22 743:23 listed 97:16,22 195:22,25 248:12 259:5 287:26

305:2 330:4 404:1 445:2 485:22 486:2 487:10 596:19 602:17 603:3 640:14,24 listen736:18 listening 205:14 276:13 440:5 459:7 648:12 659:8

664:6 727:20 732:9 736:17 listing 600:13 liter 94:4,9,15,22,26 95:1,4,6,8,10,11,15,16,23

96:11,26 97:1,7,12,19,19,23 98:1,12,19,21 101:11,13,17,18 102:6,6 105:20,21 106:3,4,7,10 106:12,13,14,21,22,23,24 107:1,6 115:3,4,6 168:18 177:9 217:19,25 535:15,17 551:15,16 560:16 561:5,15 literally 83:22 100:18 116:7,9 142:14 181:21

580:13 literature 27:18 49:13 91:15 94:19 95:13,22 96:12

97:9,16,23 98:20 115:20 136:11 184:8,14 230:20 248:17 275:12 297:24 374:16 375:12,13 404:11 494:15 496:3 567:6,11 569:11,14 575:9 602:20 603:23 613:1 little 8:3 15:22 21:1,23 28:24 39:9 43:2 47:7 50:8

50:13 52:3,18 55:11 56:3 63:19 64:5 69:5 88:21 100:20 105:4 109:1 120:19 126:10 134:7 140:11 141:8 150:4,5 151:19 157:18 165:16 181:4 182:20 186:9,16 187:10,15,24 189:19 193:11,26 194:9 196:8 206:7,10,12,18 210:20 211:8 212:25 213:25 215:21 216:12 217:15 218:26 220:11 222:24 224:24 225:10,19 226:13 229:15 234:26 238:21 245:25 247:12 248:15,16 249:4,19 251:2 253:13 254:20 259:26 262:14 266:23 279:13 281:7 283:26 296:21,23 302:25 303:12 305:24 306:1,13 307:19 309:20 314:10 315:7,23 323:22 328:26 329:8 333:6 335:7 342:20 343:14 351:6 355:18 358:9 362:17 366:25 369:3 373:24 374:1 385:4 386:10 390:3 392:16 396:19 398:6 418:19 422:16 436:1 437:15 440:4 445:14 446:12 456:24 464:10 473:7 479:12 480:25 481:2 485:7 486:6 487:14 488:16,23 494:19 496:3,11 501:26 509:19 511:25 518:18 537:14 542:9 547:8 555:2 582:1

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85:15 88:13,15,15 92:26 96:10 106:20 107:1,4,6 125:20 139:19,19 143:22 144:7,7,23 145:8,21 146:23 147:6 159:22,23 163:13 167:7 168:26 169:1 170:11 204:7 274:1 281:24 308:5 368:14 376:10 377:14,23 378:20,21 380:12 489:8 512:11 512:11 537:2 553:7 554:3,16 556:24 557:1 558:26 562:9 563:1 565:3 570:19 573:26 591:12 609:18 726:23 livers 172:19 lives 620:9 loaded 241:10 663:2,3 742:25 loading 183:20 loathe 374:1 LOBBY 1:14 local 558:9 located4:1 387:9,26 location 388:4 417:21 locations 2:16 227:26 387:10 388:5 391:15 393:20

417:24 429:5,6 472:14 LOEC 23:5 30:13 31:21 32:2 36:1,2 93:22 94:3,8

94:14,17,26 95:10,14 96:26 97:10,18 98:19 101:4 101:5,8,10,21 102:1 105:19,24 106:2,4,6,10,11 106:14,18,19,20,21,24 107:4,5,10,11,14 112:3,5 138:20,25 151:13 161:17 164:11 215:2 216:2 218:12 239:3 304:19 311:24,26 345:13 449:4,15 449:19,21 482:1 496:25 509:3,16 529:26 535:9 535:14,18 551:15 560:15 561:5 589:14 605:2,4 613:2,14 646:25 647:6 702:21 703:1 719:5,6 LOECs 94:14 101:24,25,25 106:26 107:8 161:15 198:15,16,21 199:1 200:4 215:25 304:23 312:26 319:15 449:26 558:18 560:7,15 561:2 581:7 614:5,12,26 679:25 LOEL 23:5 log 358:18,18,18,20 362:22,23,24 363:7 399:3

400:26 479:9 564:3 logged 63:22,23 logic 337:7 597:13 613:9 686:6,10,11 logical 621:4 logically 13:10 73:19 logistical 227:5 logistically 210:19 222:14 350:8 logistics 742:21 Logit578:19 log-base 63:24 log-based 64:22 log-bases 65:4 long 51:13 98:21 108:16 123:10 145:16 156:8 178:1 206:1 225:21 278:2 293:8 303:17 312:5,21 316:10 322:9 343:25 354:5 355:22 386:4 401:8 406:24 410:22 415:20 444:24,25 446:21 512:12 547:13,14,17 589:22 600:4,7 611:11 636:21 747:18 longed-lived424:10 longer20:24 23:3 25:9 73:26 77:11 94:6 165:12

211:8 262:7 303:18 398:15 424:12 438:19 456:12 456:13 505:14 622:22 747:5 longer-term 634:20 long-lived 57:23 long-term40:25 41:19 57:14 64:7 308:16 376:6

581:20 606:18 723:24 long-winded 114:21 look 10:17 14:12 20:9,11 21:17 24:24 27:23 28:18

32:8,16,19 36:16,18 37:10,20 39:1 40:10 50:14 51:3 53:24 54:25 61:10 63:20 64:24 71:14 77:6 78:5 85:15 86:2 89:25 96:17 100:20 101:10,26 105:15 115:4 118:10 122:22 127:21,22 134:21 142:11 143:8,10 147:5 148:1 149:24 151:2 152:8 153:16 157:8 159:19,19,22 160:3 162:23 166:23 168:6 170:5 177:7,26 178:20 184:8 188:13 189:19 195:23 196:3,23 204:16 209:9,25 210:25 217:6 230:26 235:10,11 239:2 244:6 250:18 255:18 256:21,22 258:10,26 260:19 273:23,26,26 274:1 285:12 295:5 296:14 297:2,10 308:21 309:19 311:9,11 312:1 316:12,14 317:15 320:16 321:5 322:19 324:21,25 325:4,4,13 327:2,25 328:10 329:10 331:11,21 334:18,20 337:10 340:2 349:13 356:14 365:14 368:23 370:22,23 371:12 373:13 380:6,13 386:17 395:1,2 397:1 403:13 409:15 410:12,16 421:23 434:26 437:17 451:1 468:5,11,12 473:4 488:2 489:1 509:10 512:22 517:11 535:18 569:14 575:9 578:24 582:20 587:18 590:19 594:21,23 596:1 632:6 636:13,17 639:13,18,21 642:3,8 643:22 644:3 645:10 646:1 648:15 649:14 657:22 658:1,2 673:8,16 678:3 685:25 686:24 691:10 693:26 695:5,15 700:25 702:24 712:23 719:14,16,17 720:11 721:7 722:1 723:3,5,18,21,24 724:3 741:19 744:6,11 745:7 looked 37:23 45:23 59:20,25 64:19 128:24 132:2 163:17 192:7 217:2 224:25 255:13 290:5,7 309:10 316:12 321:17 345:2,10 354:3 366:5 367:1 379:9 400:22 411:16,26 474:16 493:25 498:11 540:3 631:18 719:13 722:24

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24:8 27:18,24 28:4,11 32:15,23 34:23,23 35:13 37:18 46:2,6 49:22 50:4 52:13 63:2 71:9 74:10,16 82:19 94:13 98:23 104:6 129:12 131:19 133:7 140:20 142:12,14,22,25 143:3 145:21 146:17 151:8,12,17 152:5,8 163:19 165:24 167:8 178:3 182:4,15 184:12 189:24 192:13 194:26 201:12 204:6,10,13 214:10,14 216:14 217:17 232:7 233:22,23 235:5,6 244:14,26 250:12 252:16 253:2 254:23 255:2,25 258:24 259:16,18 261:17 269:7 275:18 285:12,16 291:18 297:12 300:9 302:11 304:19 306:14 307:1,2 308:22,24 309:16 310:3 311:21 312:4,8 316:2,18,19 319:14 326:2 330:11 332:26 334:13 336:7 337:3,3,18 338:21 343:4,15 344:17 345:6 356:20 359:15,16 360:18 361:17 362:26 368:3 371:14 374:2 378:20 379:16 384:19 394:1 395:16,18 399:13 400:10,21,25 402:11 403:24 405:15 407:1 411:10 419:16 430:16,17 436:17 444:21 457:6 468:7 469:12 503:15 506:7 508:8 512:6 515:6 523:20 525:22 565:12 582:6,7 587:16 599:5 631:12 632:23 634:14 642:4 643:6 644:12 646:6 647:8 650:8,17 662:7 670:11 672:13 690:11,15,17 692:24 693:5 700:20,21 701:9,15,16,22 702:16,18,21 712:20 719:15 720:2 721:11 724:9 732:15 737:11,12,14 looks 16:12 68:2 70:20,22 71:16,18,20 85:26 89:24 115:1,5 189:8,8 224:1 253:25 276:5 306:6 308:23 339:5 365:3 395:25 396:6 409:10 474:19 482:9 504:9 640:7 650:12 652:23 657:26 658:19 678:2 719:17 loops 554:13 lose 111:10,11,13,16 191:19,26,26 192:3,4 312:25

515:4 691:5 losing 182:24 711:1 loss 111:5,9 182:20 183:12 258:20 304:18,21 359:5

527:26 528:1 531:7 532:12 579:15 643:15 669:4 689:2 693:2 711:10 losses 690:3 lost 162:24 172:16 192:5 220:14,16 221:22,24

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33:20 38:25,26 55:6 57:15 59:22 66:24 69:1,26 86:19 91:16 99:25 100:21,22 102:26 110:18 112:23 114:18 151:6,7 154:23 163:16 171:26 185:3 190:9,14,15,21 192:9 195:1 196:13,13 202:25 204:22 220:9 248:17,19,19 249:6,11 254:2,15 267:24 268:16 276:26 285:9,10 286:20 287:12 288:7,11,12,25 289:5,15,17 290:16 292:25 293:15 295:3 297:23 298:20 301:23 302:14 305:21 306:15,16 309:7 310:24 311:1 315:10 316:17,20 320:17 323:16 326:14,17 327:6 334:10 344:3 345:6 354:23 359:7 364:26 366:3 367:21 370:18 379:15 385:25 386:5 392:1 396:7 396:18 398:20 400:24 404:13 405:5,20 409:5,11 410:19 420:17 427:11 440:12 454:22 471:9 472:4 472:15,22 473:4 474:4 484:17 498:1 502:18 521:18 564:24 576:3 585:20 590:12 607:19,25 608:16 613:22 623:14,21 625:4 629:5 630:1,10 631:26 633:25 647:15 649:16 671:8 679:20 680:2 683:19 687:7 691:19 692:15 694:5,6,13 712:6 713:1,4,10 714:6 721:5 728:16,18 730:11,13,19 734:8,9 738:6 740:22 741:23,24 744:1 lotions 168:2 lots49:10 79:22,22 82:23 102:25 114:10 148:2

369:10 399:15,15 400:9 491:18 572:5 630:4 694:11 741:23 loud740:10 743:7 low 86:15 88:13 100:10 102:25 119:21 120:13 121:11 128:21,26 129:13 177:9 191:13 217:24 231:13 295:6 303:24 367:11 374:26 416:8 417:7 436:11 437:24 503:22 505:25 506:11 538:7 540:4 541:1 552:21 557:8 571:5 625:3 644:9 689:25 692:18 693:14 lower22:4,6 31:9 51:16 52:10 70:22 87:5 99:14 101:14 102:2 105:25 106:2,22,24 107:10,11,14 107:15 165:13 169:14 185:23,26 215:11 216:3,25 217:20,25 231:13 246:13 268:5 272:21 275:23,23 297:26 298:1,1,23 304:22 319:23 396:25 415:17 415:21 438:18 448:26 464:5 505:16 525:6 535:14 535:21 556:25 567:9 622:18 669:7 690:20 692:16 722:21 lowered 257:23 lowest36:2,5,10 75:4 99:13 105:15 106:19,20 127:22 217:20,26 219:8 224:5 226:23 255:23 320:26 561:13,19 605:5 688:10 low-dose720:11 low-level 720:17 low-observed70:11 low-volume708:25 LSE 215:4 LSI 161:10 162:24 163:8 165:18 168:25 luckily 327:15 lumen 170:2 lumped44:26 45:10 lunch 104:24 343:10 345:21,24 544:17,19,21

591:26 luxury 157:6 lyophilization 572:4 M

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macro 592:2,2 725:14,15 macrophages 164:20 magnified 136:8 magnitude71:18,26 87:19 177:10 216:11 226:18

237:21 256:21 363:5 520:18 main48:17 122:1 166:19 235:25 278:21 290:12

317:24 362:7 406:22 688:21 711:21 mainstay 742:9 maintain 120:25 195:7 222:19 226:2 241:7 391:11

446:21 635:2 697:19 727:18 maintained 62:15 358:5 742:13 maintaining 58:16 214:8 223:17 237:25 497:18

722:20 maintains 22:23 62:15 maintenance 518:16 666:24 722:20 major72:7 117:9 129:24 275:21 288:18 294:21

296:6 321:3,14 343:24 344:8 358:7 366:18 401:9 465:12 497:12 498:3 533:26 587:8 673:25 675:11 679:21 704:12 705:24 706:5,5,9,16 708:23 majority 129:22 155:7 351:7,23,25 445:4 446:26

534:25 628:20 700:23 making 13:9 24:6 38:20 125:21,22 140:15 186:26 199:4 234:5 268:13 269:2 296:6,19 311:13 334:23 372:23 382:17 392:8 402:4 424:18 439:10 481:26 505:11 574:8 614:17 654:1 691:19 709:15 743:8,10 745:5 male40:13 53:7 65:15,17 66:15,16,19,20 82:10,11

87:8,20 92:25 95:5 97:3 99:23 103:8 109:14 123:16,19,21 124:9 132:4 142:9 158:11,11 163:13 167:7,16 169:18,26 170:1 221:24,25 267:26 270:23 303:3 365:19 373:8 378:15 387:6 389:14 413:10 418:14,17 425:10,21 426:14 464:1 464:5 469:23,26 519:16,17,23 561:8 571:24 572:14 583:23 659:17 723:23 maleness 659:25 males 68:20 80:26 87:13,17 88:5,7 91:20 97:4,14

98:5 99:21 109:13,20 124:8 128:11,19 129:6,8,9 129:14 140:17,22 146:2 163:7 164:4,10,11 167:17 168:24 173:16 223:6 273:13,14 289:7 290:8 303:1 339:3 348:15 372:23 373:8 389:16 413:12,14 425:9,19 451:3 453:25 469:24 480:7 494:21 495:2,14,24 496:22 498:12,24 557:7 560:18 584:6 603:13,15,17 604:6 605:13 640:2 669:5 714:14 723:25 724:2,5 males/females 130:21 male's 92:6 Male-biased 553:2 male-specific495:8 male/females 53:17 mallard 64:8 340:13 432:24 mammalian 21:18,19 23:19 24:13 33:3 37:12 294:2,4,6 329:7 407:1,2,3,22 408:5 614:7 mammals 40:12 137:3 162:6 348:13 man747:9 management241:11 251:12,13 252:23 433:21

742:17 managers 351:6 mandate 14:5 324:14 mandated 2:19 15:14 mandates 15:14 Manibusan 10:9 12:23,24 30:1,18 34:14 36:20 199:12,13 201:15 205:16 233:3 239:21 276:14 277:3 283:19,25 346:4 363:10,11 382:11 441:7,8 461:7 539:9,10 540:7,15 633:16,17 646:2,3 649:11,12 650:9 651:23 660:9 717:22,23 741:25 741:26 745:14 747:19 manifest40:4 41:9 43:6 48:11 53:9 61:23 125:16 126:15 213:12 552:1 manifestations 69:4 manifests 73:11 manipulate 196:24 manipulated 517:7 manipulation 517:6,12 523:7 manner269:16 281:11 356:26 374:20 384:6

387:19 422:5 488:4 507:19 603:19 605:2 607:10 651:13 667:25 704:1 719:14 741:1 manual 672:20 736:9 margin 146:21 marine 6:3 222:19,20 619:20 621:23 Marion 5:23,23 61:4 242:12,12 337:16 459:25,25

654:9 657:7 665:1,1 Marion's 338:21 mark 38:9 218:13 marked 50:6 264:6 339:7 408:24 marker 156:14 186:20 markers 123:17 448:8 453:12 market55:19 573:19 marks 218:15 Martha2:10 4:26 5:2,4 7:26 8:13 12:21 29:26 34:6

35:24 36:14 38:10,15 59:6 61:3,26 63:15 65:6 66:22 68:13 70:15 75:7 104:23 105:2,6,9 114:22 115:10,13 126:9,13 128:6 130:14 133:15 134:4 137:9 139:8 145:15 148:10 150:23 153:5,10 154:7 176:7 177:3,13 179:1,20 180:13,23 184:4 187:12 189:11 190:3 195:17 196:9 198:9,18 199:12 200:13 201:17 202:19 203:13,22 205:3,23 227:15 229:20 239:8 241:22,24,24 242:1 243:16 253:22 254:21 263:22 266:12 267:9 270:1 273:20 274:21 276:1,12 277:6 283:19 284:1,2 285:22 295:22 305:14 314:21 323:19 333:8,10 337:15

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34:14 35:25 36:13,20 137:11,17 138:8,11,17,24 139:7 153:13,13 176:7,8,12,15,20,25 199:12,13 201:15 205:9,16 227:17 228:5,15,23 229:8,12,16 229:19 233:3 239:21 243:12,12 276:12,14,25,25 277:3,6 283:19,25 346:4,12 363:10,11 382:11 397:15 404:4,6 405:12 441:7,8 442:1,8 460:26,26 461:7 484:6 539:9,10 540:7,15,23 548:25 554:21 563:12 569:5 574:5 577:5 591:17,23,23,25 596:8 606:3,4 610:8 615:9 630:19 633:13,16,17 643:22 644:24 646:2,3 649:11,12 650:9 651:23 655:26 656:1 660:9 665:24,24 670:8 671:12,16,21 673:22 675:9 680:10 681:9 687:25 690:7,16 691:14 695:11,21 696:15 703:8 707:8 712:16 717:18,22,23 724:16 725:25 740:20 741:25,26 747:17,19 Maryland43:10 Mary's 229:25 661:12 691:13 745:23 masculinization495:10 masked 281:15 mass48:15 55:18 166:11 418:5 match48:14 133:18 249:10 300:14 302:7 498:22

499:7 698:13 712:26 matched 248:26 604:12 605:8 matches 128:10 370:1 633:24 matching 129:12 421:7 499:7 mate 373:6 mated 387:11 material 38:26 40:17,19 43:5 70:14 73:9 208:14

209:17 231:23 238:15 246:15 260:4,24 266:3 299:18 301:12 409:1,2 644:14,14 741:24 materials 37:22 69:26 207:23 219:26 220:23

230:22 248:1 266:3,22 300:13 358:17 494:19 515:24 516:21 522:2,7 602:15 652:2 670:18 maternal 25:10 40:20 47:10 58:5 72:1 125:10,16 126:4,15,21,21 446:11,13,18 447:5 448:9 524:20 524:22 529:12,14,15 531:12 536:24 639:11 math 310:10 543:21 716:7 mating 23:24 54:7,8,11,15 55:5 65:8,12,13,22 66:1

66:14 373:6 529:17 matrices 705:4 matrix 182:11,14 Matten 2:1,2 8:4 9:10 13:8,11 29:17 205:9,23

239:16,18 241:1,2 283:16 284:2 342:26 343:2 346:2,3 441:25 442:21 458:12,14 459:1,2,12 544:22 545:2 593:6,10,15 615:21 626:21 633:18 635:11 649:1 662:22,23 664:1,2 742:20 745:18 745:22 Matten's 629:26 632:13 matter 196:7 389:21 580:3,13 732:16 mattered 239:5 matters 343:6,6 732:12,13 maturation41:3,12 42:10 44:16 45:19 47:4,6

55:23 73:23,26 74:12 155:13 157:23 159:11 214:2 431:7 433:5 536:11 mature 58:9 84:14 213:16 447:6 571:8 620:4

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354:15 360:11,12 368:16,17 373:18,19 374:21,22 381:8,9 406:1,2 409:4,5 411:3,4 412:8 413:5 418:26 419:2 426:12 428:6,7 434:5,6 435:18,19 460:14,14 647:11,12 656:17,18 660:2,3 661:21 661:23 McManaman 5:12,13 70:15,16,26 71:12 73:12

74:13 134:10,14 135:13,19 136:6,14 137:2,6 242:6,6 459:19,19 664:21,21 727:20 MD481:21 mean 36:4,10,17 61:9 68:21 71:20 81:18 82:23 102:18 117:13,26 118:5 119:5,18 122:5,10,23 123:5,13,15 124:17 127:1,2 131:9 132:7,19 137:6 137:17 138:2 140:6 141:9,22 143:2,24 144:22 148:1,6,21,21 160:26 171:7,11 172:16,16,18 176:20 183:2,17 184:23 185:11 187:8 190:20 191:11 192:18,18,20 201:19 204:10 218:24 230:19 232:6,7,11,12 238:19 245:3 246:9,10 247:4 252:19 253:3 254:11 266:16 267:22 269:11 270:3 312:16 325:2,6,16,24 326:6 328:4,21,24 329:9 334:5,7,25 336:13 350:22 374:6,10 379:11 393:11 395:3,3,15 396:26 407:22 411:22 477:20 477:21,22 482:21 483:16,18,19 492:24 493:21 499:12 505:19 506:24 521:16 541:14 549:4 564:19 574:12 579:13 580:12 584:14 587:21,24 587:24 594:10,21,22 606:17 612:14 622:23 649:13 656:2 658:14,25 660:23 671:25 672:4 679:26 680:7 691:8 705:11 706:11,12,12 729:16 meaning 34:20 58:20 65:13 68:6 69:6 87:4 219:20

228:3 298:20 448:23 meaningful 27:14 204:20 334:10 453:3 637:15 means 23:2 48:1,3 90:18 127:13 137:19 153:25 161:3,6 162:14,21 163:25 166:5 183:21 254:11 311:23 312:13 320:25 324:11,13 326:11 327:9 329:12 379:6 390:4 393:10 395:12 404:22 468:16 474:19 494:2 497:14 505:8 551:11 565:23 581:15 582:6 583:4 584:2 587:20 588:21 589:3 592:16 600:19 610:14 690:10 711:10 726:8 730:7 733:7 meant 162:19 233:11,15 234:23,23 386:15 389:8

394:19,20 492:23 555:4 653:4 measurable 102:8 173:16 176:1,4 373:16 555:10

630:14 measure 54:17,22 56:2 63:4 66:5,15 67:3,4 82:14 84:19 87:9 108:19,22 124:17 126:26 132:16 157:3 174:25 180:17,18,19 181:3 188:12 190:5,7 190:19 191:6 204:21 221:5 222:4 228:21 236:11 236:15,18 259:17 260:6,14 273:10,11 288:7,8,9 364:11 365:1 366:26 368:12,13 373:1 379:5 413:4 418:9 422:6 423:2,23 425:16,17 426:1 428:1 431:20 447:21 461:19 473:13 475:4 496:9 496:9 507:20 514:18 520:7 533:7 543:1,2,4 555:14 556:10 567:15 574:18 578:6 607:11 609:19 612:1 626:14 638:25,26 640:2,15 666:11 667:19 699:10 704:2 714:11 measured45:4 64:16 65:9,15 79:8 80:5 81:1 86:1

87:10,12 89:6,15 96:18 105:16 108:4,5,13,21 120:20 124:20 132:18 158:26 187:16 188:6 219:26 254:26 255:1 260:9 275:5 279:5 289:21 291:15 308:14 310:14 339:17 364:5,6 367:20,20 411:26 416:18,21 418:14 423:10,16,18,20,20 425:5,7,13 426:19 431:11 461:13,14 474:13 478:12,13 497:15 506:17 542:25 551:6 552:15 556:5,5 567:8 574:7 602:22,23 666:6,7 668:12 694:23,24 698:23 measurement55:25,26 119:11 137:24 180:16 187:8,26 236:22 286:13 295:16 314:9 397:23 408:16 426:21 463:5 465:3 498:6 504:25 566:21 569:21 586:18,19 699:18 measurements 30:14 80:1 119:10 124:15 137:24 159:1,26 161:11,11,12 174:21 175:9 188:13 226:7 308:17 313:21,25 314:8 382:18,26 400:25 418:5,17 441:10,13 449:15,22 456:3 462:19 483:21 504:5,10 514:16 566:23 568:21 569:12 570:16,23 600:14 641:23 645:9 666:20 668:17 673:4 measures 45:20 46:5 56:8,12 58:12 66:20 67:5

69:7 79:22 88:1 89:14 137:23 138:3,12,22,25 153:25 157:8 159:13 173:18 177:21 186:11 221:2 226:4 236:7 260:6 308:25 313:4 338:17 342:17 365:19 412:23 420:24 425:4,15 426:10 428:24 433:4 456:25 475:3 478:20 479:6 480:9 532:17 547:6 551:9 552:8 553:19 555:7 560:26 561:20 567:4 568:23 609:5,14 639:2 643:14 645:10 699:8 701:17 726:24 measuring 65:9 100:15 108:16 142:5,14 154:3 166:9 174:20 184:18 203:5 204:21 259:22 308:19 372:18 416:20,21 498:15 513:24 543:14 566:17 567:24 568:20 570:13 573:10 586:26 609:18 689:3 690:2 meat-market 55:17 mechanical 303:19 mechanism 33:1 69:13 286:16 295:20 348:14

381:11 386:19 426:6 429:8 435:25 439:23 445:7

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328:22 667:17,25 701:23 MED 100:10 116:25 449:3 medaka 25:16,20,21 29:22 75:18,20,24,25 76:16

77:2,3 78:2 80:24 84:3 94:21 103:19,21 107:17 107:22 115:23 116:18 117:8 128:20 151:17 156:13 157:6 270:8 277:26 296:3,3,7,9,10,15,16 296:24 297:6,15 299:8 302:10,13,21 304:26 305:2 308:23,25 314:12 392:16 395:7 441:21 442:3,23 443:3 444:22,23 445:6,11,14 446:20,25 448:5,10 450:12,20 452:6,14 453:5,23 454:4,15 454:24 456:23 457:14 458:19 459:9 461:6,11 462:4 471:15 486:26 487:5 488:18 495:13,16,25 507:25 509:22 511:21 513:7 519:10,10,11,14,16 519:17,23 520:25 526:13 528:22 531:6 533:22,24 536:9 543:18 544:16 546:26 548:4 559:12 592:13 595:8 596:13 602:3 605:24 610:3 612:16 653:22 662:5 724:25 726:1 Medakas 192:15 media 220:21 225:5 238:18 518:17 medial 146:21 median 54:4 68:3,3,5,10 146:25 211:19 221:7,13

221:15 587:22 588:1 medias 743:13 mediated 81:24 159:9 184:10 304:3 338:23 440:11

453:26 455:17 537:3 626:11 638:3,4,6 681:25 mediator 186:21 Medicine 277:19 283:14 medium485:15 MEDs488:16 meet 16:3 58:21 122:14 202:4 239:18 327:20

328:14 342:2 364:21 365:23 367:8 397:22 405:1 478:5 588:11 613:10 748:10 meeting 1:2 2:3,4,7,11,13,14,17 3:3,16,26 4:2,5,7,7

4:10,13,15,15,17,20,22,23 5:3 8:9,11 9:1,9,11,14 9:16 11:14,19,24 12:15 13:2,3,5,9,9 29:11 89:12 240:7 241:3,4,26 251:1 252:21,23,24 311:25 348:11 459:3,11 484:7 545:3 626:22,23 633:21 634:9 649:2,20 661:21 663:10 672:15 736:22 737:19,19 743:2 745:9,12,18 746:1,14,15 748:12 meetings 9:3,12 10:24 meets 20:14 122:12 195:8 199:20 365:2 444:17

700:5 melding744:20 melted 20:6 member 5:9,14,18,21,25 6:15,18 7:8,13,16 251:21 446:2,5,12,17,24 447:15 619:7 668:14 727:14 members 2:12 3:5,15 4:19,19 5:1 8:1 12:10 38:16

38:20 105:7 239:15,19 243:17 270:2 345:26 349:19 353:8 354:16 382:8 404:1 406:13 410:2 411:11 421:20 428:17,19 429:22 434:19 435:2 436:8 438:7 441:1,18 452:6 458:2 468:8 484:14 521:8 528:8 539:5 549:18 555:20 567:21 569:24 569:24 573:9 593:24 596:7 598:9 599:25 606:25 610:19 615:11 617:9 637:16 644:26 649:25 660:24 661:17 662:21 671:10 672:9 686:3 693:22 703:21 734:4 738:21 740:14 741:18 745:8,9 memory 365:4 mention44:19 80:8 92:14 98:25,25 131:26 145:4 188:18 206:25 310:6 318:5 404:21 406:19 424:6 425:3 431:8 480:18 514:8,24 517:14 575:5 590:11 609:18 641:26 643:19 645:15 671:22 678:10 680:11,12 703:4 725:10 739:13 mentioned 61:4 63:17 65:7 73:25 82:26 84:15

85:20 87:3 90:9 93:11 99:2,6 100:10 113:14 120:18 133:23 152:9 159:25 161:23 189:2 227:18 236:25 237:17,19 264:2 286:6 289:11 290:3 298:6 307:11 321:15 336:26 343:11,21 368:21 372:17 379:7 384:25 414:26 417:2 418:3 420:25 423:12 425:14 426:10,12 427:10,15 428:13 430:16,16 432:17 433:20,26 434:23 437:15 453:5 458:5 465:12 467:11,12 468:17 469:14 471:18 473:21 474:5 492:22 514:12 519:7 523:23 525:18 526:3 527:25 583:3 586:10 587:14 592:15 596:21 602:18 608:21 610:3 627:12 631:3 643:19,23 651:4 667:16 678:25 691:21 695:22 719:26 725:16 mentioning71:3 397:12 mentions 419:20 684:1 mere 23:22 26:22 merit426:20 712:10 message 28:8 311:5 349:8 381:25 435:20 743:15 messages 170:4 messenger 184:9 368:13 494:22 570:19 726:7,13

726:23 met2:9 3:14 89:12 194:21 237:1 241:9 320:7

341:23,24 347:26 349:21 443:23 444:12 445:5 488:9 614:20 654:19 743:9 metaanalysis 396:12 metabolic 60:5,9 metabolically 318:8 metabolism 60:8 260:22 486:13 metabolites 60:5 416:21 510:11 720:14 metabolomic432:16 metabolomics 371:18 372:3

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568:5 metamorphosed 213:19 metamorphoses 604:22 metamorphosis 155:19 158:5,8,8 159:5,11 166:4 167:14 175:9 191:12 314:13 550:20 551:24 557:4 561:18 568:21 591:11 596:25 603:18 methimazole 559:22 method 19:2 25:1,15,16,22 26:7,8,22,23,24 27:9,19

39:26 40:1 44:26 54:10,20 55:26 60:18,24 87:14 122:22,24,25,26 123:5 136:2 145:1,2 148:2 155:17 166:18 191:2 195:11,12,13 211:4 222:7 223:12 225:24 227:10 249:17 252:5,25 260:6 347:4,6 348:23,24 366:14,16 383:7,9,12 412:7 414:5,11 421:6 443:4,6 444:9 470:13,15,18 493:6 493:13 503:6,19 504:4,12,14 512:4,5 515:2,8 517:9 534:11 545:10 562:16,21 566:16,16,21 567:15,19 571:12 573:12 576:16,18,21 581:18 587:17 601:16,23 616:5,7 636:13 643:2 662:8 674:17,20,22 681:1 697:1,7,14 743:21 methodologies 42:14 194:7 209:10 211:3 219:6

222:18 232:16 266:1 269:3 521:23 539:21 577:14 580:20 584:13 methodology 11:24 14:16 29:14 39:5,11 41:23

42:3 49:4 54:19 187:24 210:6,21 252:9 353:19 404:14 422:4 435:1 464:24 465:5,6 507:18 547:10 578:2,26 581:21,22 584:24 587:6 589:20 591:26 596:10,14 607:9 676:21 703:26 717:26 methods 11:17,21,21 15:18 16:23 20:1 21:18 23:20

24:9 25:20 27:5 28:21 29:10,12 78:9 113:3,13,22 113:23 114:3 147:7,11 166:16 174:26 188:15,16 195:20 208:26 209:1 282:19 290:21 291:5 300:4 344:2 354:8 357:12,25 369:10 382:18,21 383:15 383:18 384:10,17 398:3,20 403:9,26 404:15 410:5,8 411:5 414:5 434:23 437:15 441:14 452:14 457:24 469:5 470:22,25 474:9 476:3 484:26,26 485:6 491:12 493:6 511:1 512:20,24 514:25 518:15,15 534:8 545:8 550:1 557:25 564:16 569:13 576:25 577:1,7 578:22 579:21 580:9 587:11 590:2 601:16 633:22 637:1 674:26 675:3 677:3 679:10,11,14,20 680:19 681:6,11 688:20 697:1 699:1 709:10 715:20 716:14 717:1 720:18 728:24 743:23 744:11,14 method's 281:12 methoprene 318:18,18 335:16 621:11 704:22

709:16 717:6 methoxychlor42:5 methylation 372:2 methylene 515:7 metric 115:19 125:1 188:6 203:12 236:23 239:7 metrics 202:24 203:5,9 235:5 237:1 423:7 571:13 623:3 656:16 698:7,9 705:4 714:23 715:24 mg 169:14 mic 10:19 mice 381:21 Michigan 6:21 242:25 460:13 665:15 microarray 514:4 microgram 95:11,23 96:10 97:26 98:8 221:4 micrograms 94:26 95:1,4,6,8,10,15,16 96:26 97:1

97:6,18,19,23 98:19,21 101:11,13,17,18 102:5,6 106:3,4,7,10,12,13,23 115:3,3,6 217:19,25 221:3 224:6,6 535:15,16 551:15,16 560:16 561:4 572:17 microliter 223:13 572:10 micronnutrients 117:2 microns 146:10,24 micronutrients 501:9 microphone4:25 8:2 38:18 241:21 243:18 272:5

283:18,22,23 346:26,26 347:2 458:16 459:10 488:7 490:22 544:26 615:24 664:9 microphones 8:1 Microplate 224:13 microscopy 640:3 714:14 microwell 211:22 225:2 238:14 mid 151:14 423:15 middle 101:2 192:3 243:23 403:22 461:5 687:5

702:25 midline 146:24 midst 173:26 Mid-Continent75:13 567:12 Mihaich 284:7,8 323:20,21,24 333:9 334:4 337:24

340:8 342:8,13,19,24 343:1 mild 378:23 mile 620:12 miles 653:5,8,9 milestone 9:22 Miller 115:22 116:2 455:9 milligrams 76:20,20 88:5 98:12 106:24 107:1 168:18 177:9 370:9 506:11,12 561:14 milliliter 506:12 milliliters 506:11 million 52:9 310:10 374:17 693:7,7 millions 285:8,8 Milwaukee7:5 243:5 460:19 665:19 mind 14:1 15:2 232:1,3 267:13 271:13 299:15

305:13 322:14 328:23 329:22 330:17 342:9 385:16 386:21 387:3 392:20 393:4 397:4 399:4 401:14 405:2 471:23 508:17 514:7 542:18 652:8 666:3 678:22 682:1 683:13,22 728:20 736:5 minds 156:5

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527:8,13 minnows 115:22 116:16 157:6 450:12 455:10 minor 161:14 569:17 577:15 705:24 706:5,6,9,12

706:19,23 737:16,16 minus474:18,18 694:3 706:10 minute 162:9 441:22 444:16 577:20 703:18 711:16 minutes 3:17,21 4:13 10:7 13:14 18:22 24:10 205:5

239:19 276:8,10 285:24 323:3 544:23 593:20 687:1 711:12 742:1 MIQE 514:1 misidentification 224:22 misidentifications 224:19 misidentifying 224:18 mislead 183:7 misleading436:13 mismatches 128:18 129:17 130:1 487:6 498:24

499:12 mismatching 281:2 misnomer475:6 misreading 115:6 missed 294:21 321:15 339:8 367:2 429:12 477:11

527:25 missing 105:8 289:15 383:1 478:16 555:1 618:14

659:9 671:8 728:25 mission 14:4 15:3 29:13 731:23 744:3 mistake 133:9 mistaken 65:26 misunderstand 540:6 misunderstanding 160:22 597:19 misunderstood 627:16 misuse 588:3 mitochondria 260:21 mix 150:14 627:25 mixed 226:13 271:11 475:7 604:19 605:10 mixes 626:24 mixing 150:3 392:14,23 393:3 mix-sexed 170:3 MMP494:17 MMRT445:21 MMT 78:15,15,22 82:17 85:2,18 90:18 91:17 97:9

97:24 99:26 100:21 103:18 104:22 107:25 108:1 110:8 114:25 116:7 129:11 134:19 135:5 142:13 142:21 279:17 281:5 297:6 299:4,4 300:15 301:22,22,23 304:10 305:9 333:1 444:8,13,18 445:16,21 447:21 449:5,16,20 451:19 452:24 454:18 455:1,3 456:6 464:12,13,21,26 471:1 478:11 479:5 486:8 493:15 495:21 496:15,20 497:10 498:19,20 501:2 505:2,25,26 506:14 507:23 508:3,23 509:23 510:26 511:13 516:20 517:17,21 522:21 524:10,15 525:6,10,16 526:20 527:8,13 528:26 529:7,19 530:3,25 531:15 533:23,26 534:8,26 535:1,15,18,24 537:9,13 541:16 589:25 610:12 MMTs 85:19 93:15 304:20 474:13,14 MMT/MRT462:1 465:1 MOA 517:19 mode 68:18 83:9,10 133:8 135:4 148:4,8 296:20

297:22 298:25 299:19,22,24 300:3 302:5 303:2 304:6 305:12 307:14,14,18 308:8,18,21 314:8 325:1,16 328:9 329:13 333:22 334:17,22,24 336:11 357:17 376:8 452:20 464:18 486:9 489:9 548:11 550:8 552:6 553:3,14 554:2,4 559:26 565:13,18 566:1 611:21,25 629:9 630:15 637:6 638:4 705:9,16 735:6 744:23 model 33:13 51:26 56:16 57:22 59:20 75:1 115:18 115:21,22,22 116:10,11 164:24 257:15 352:8 359:12 404:18 413:17 418:21 425:5 445:6,9,11 445:20 446:11 447:16 448:5,10 450:21 454:17,21 454:25 455:4,8 456:23 457:1,7 475:7 478:3,5,6 478:19,20 480:20,24 484:3 523:2,3,5,24,26 546:26 553:1 583:26,26 594:7 595:11,20 611:15 612:18 696:18 modeled 594:10 709:26 modeling 226:9 245:12 322:16,18 535:11 642:1,5

642:10 676:3 677:19 680:21 709:11 714:13 models 116:2,4,5,8,9,14,15,17 359:10 370:17

413:17 439:4,8 445:12 454:26 456:26 465:20 480:19 523:6 578:19,19,20 586:7 598:8 611:13 708:19 moderate 177:5 378:24 463:8 490:24 508:24,25

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531:3 535:2,25 536:4 modified 58:6 72:18 464:14 480:15 622:4 623:25

641:10 modify 152:11,12 194:6 237:7 modulated 136:3 modulation 307:13 529:14 modulator 318:19 module 89:5,13 90:10,12 137:22,23,25 138:1,3,22 161:7 725:14 modules 89:4 90:10 molecular 6:18,20 7:8,9 21:8 113:3,6 233:18

642:23 667:14 670:3 720:10 734:24 mollusk 619:8 708:13 molt260:15,15 585:26 719:22,25 720:3,22 molting 207:13 259:8,10,16 260:8 638:26 639:1,5

667:5 701:11 molt-inhibiting 261:6 moment 184:1 295:26 575:12 Monday 251:1 money 224:15 309:7 336:19 375:5 692:10 monitor 225:5 287:20 monitored454:7 monitoring 51:23 546:10 723:25 monitors 552:10 monodeiodinases 568:16 monopolize 180:25 monotone482:7 monotonic 30:12 31:1,5 127:19 300:9 449:24

481:17 482:11,15 483:7 585:11 600:4,7 688:6 monotonicity483:1,4 Monte 110:6 139:4 month 12:12 15:1 19:20 284:10,24 325:20 539:19

575:17 630:12 732:6 months 12:11 56:21 118:3 447:3,7 543:23 593:16

643:25 morass 660:5 morning 2:1 5:3,12 6:19 7:6,22 10:22 12:24 13:13

38:23 154:10 205:19 239:10,11 241:1,24 243:26 243:26 284:7 314:23 347:26 390:21 450:11 451:14 458:19 459:1 468:6 475:26 592:15 646:19 661:18,26 662:16 663:11 664:1,10 725:17 morphological455:21 546:10 708:25 morphology45:14 143:16 378:14 433:14 536:16

552:11,16,20 557:12 560:17 561:4 morphology/histopathology 45:13 morphometric 147:4,7,16 377:23 380:13 462:22 462:25 464:4,7 561:24 562:6 mortalities 159:19 364:20 mortality 168:19,20 171:3 307:9 309:23 453:18

462:9 591:9 600:15 601:2 689:2,25 690:18 motility433:14 motivated 354:18 675:22 motivation 355:8 mount 65:17 146:10 365:18 400:25 mounts 65:15,16 mouth 283:23 move 10:2,7,14,26 12:8,14 13:4 18:4 24:15,18,25

25:22 28:2,9 33:17 34:18 35:11 75:8,8 88:20 91:13 104:21 145:17 153:6 154:11 156:24 157:1 157:7 174:16 182:13 251:23 263:14 277:12 283:23,23 285:2 302:14 311:6 315:21,22 329:24 342:2 363:26 368:10 376:2 382:12 414:2 430:1 435:3 470:10 518:7 533:19 540:17,19 546:22 548:10 549:25 566:7 610:24 620:5 624:25 629:23 633:10 634:3,19 636:23 642:11 660:10,19 666:2 679:6 681:4 687:15,16 696:24 711:21 713:15 729:5,11,12 730:23,24 733:9 735:11,15 736:20 737:10 739:20 742:18 743:5,18 moved 18:16,23 24:3 93:12 98:6 157:19 349:11

578:26 630:19 movement 117:2 228:24 266:8 moves 20:12 movie731:12,14,18,26 738:14 moving 19:25 33:10 40:17 47:7 57:19 119:8 152:11 161:1 195:4 250:24 253:7 269:20 308:19 310:4 335:1 344:8 347:2 408:21 629:11 652:21 676:24 681:1 695:14 723:16 730:5 740:11 mRNA 82:15 87:12,17,20 96:10 100:14 185:4,4

410:11 453:25 494:22,26 495:9 536:16 609:19 MRT 25:21 98:17 107:24 112:11 116:6 124:13 126:17 129:11 134:19 135:5 141:10 142:13,21 143:7 144:11 275:8 297:9 299:11 300:3,14 301:24 302:8 305:9 333:1 444:13 445:16 447:21 450:4,6,8 454:18 455:1,3,13 456:9 464:16,21 471:1 476:24 490:8 493:15 501:2 508:3 511:1,13 516:20 517:17,21 522:21 524:16,24 525:1,4,6,12 525:13,15 526:20 527:8,13,26 529:9,9 530:3,4,26 531:15,22 534:8,11,17,21 535:1,1,20,23 536:1 537:7,11,13,20 538:11,13 541:16 MRT-test528:24 MSMS 174:25 MTT 273:21 323:14 MTTT 315:15 316:1,18 637:19 666:21 714:3

715:3 716:13 722:20 muddied 234:26 muddled 359:22

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209:6 278:1 296:3 305:2 318:14 338:14 431:11 442:4,23 443:3 458:19 459:9 461:6,11 471:15 529:7 530:5 624:4 multigenerational 18:9 430:14 444:19 446:1

456:13 511:10 529:10 535:25 547:13 577:15 624:25 639:7 641:1 669:3 698:6 709:9,19,26 710:2,4 multigenerations 136:7 multiple 20:24 23:12 41:20 43:25 59:22 70:1 89:16 100:23 107:23 150:26 153:26 167:2 192:15 199:5 199:23 212:21 219:14 245:5 251:19 287:13 291:7 293:1 298:11 307:24 331:23 332:2 370:19 371:14 377:4 382:3 405:11 435:16 437:1 449:6 478:7,13 524:9 619:16 637:20 660:24 668:2 680:5 685:24 692:15 697:23 704:23 709:20 718:19,23,24 719:2 multiples 148:25,26 multiple-generation 318:15 multivariant 679:2 multi-gen 93:6 multi-generation 23:22 25:20 31:6 39:21 40:5

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217:19,24 218:1 219:12 220:10,10,13,26 225:9 225:11,17,20,20,25 226:4,25 227:8 228:4 231:10 244:22 246:17,18 247:4,14 248:22 249:5,8 255:1 258:25 259:5,18 260:13 261:26 263:13 266:21 318:12 322:1 341:20 343:25 344:25 442:10 582:3 585:24 643:26 644:5 691:26 696:10,21 709:18 714:26 719:9,10,19 mysis 643:26 M1 510:11 M2 510:11 N n254:17 589:4 691:16,20 723:7,8 nail 199:18,26 303:13 nailed 181:22 naive 54:12,13 141:24 name 2:1 5:4,15 6:26 8:20 133:15 213:17 241:1

305:16 314:24 459:1 621:12 660:18 664:2,16 747:5 names 590:1 747:11 name's 284:8 naming 104:3 660:19 nanograms 94:4,9,15,22 97:10,12 105:20,20 106:14,21,21 107:6 nap 577:20 narrate 158:23 narration 154:25 narrow 68:11 narrowed704:24 NAS 31:3 33:11 national 5:9 6:1 242:5 251:18 381:18 459:18 460:1

664:20 665:3 nations 251:19 native446:25 447:1,2,13 448:3 549:9,10 natural417:21 479:9 520:7 naturally 15:19 234:11 476:25 nature 94:12 168:3 215:23 258:3 269:3,16 307:20

329:3 347:7 350:2 371:10 383:14 443:8 447:10 447:26 448:3,6,11 470:20 524:25 545:12 554:12 576:23 585:11 610:13 613:6,10 616:9 617:25 624:18 640:16 641:8,18 674:24 695:25 696:6 705:12 716:3 725:4 nauplii 223:1 224:12 nauplius 223:1 nauseam443:24 NBP-2 162:3 near2:12 96:17 156:8 205:19 479:3 735:17 740:14 nearly 111:6,9 181:7 Nebraska 6:25 243:1 460:15 necessarily 30:23 55:25 64:19 71:25 81:17 85:26

92:7 100:17 169:4 173:3 175:7 179:8 221:12 298:8 313:13 325:25 328:21 330:8,16 331:2,9 336:14 489:8 510:1 521:25 524:15 526:23 527:2

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322:15 331:8 349:20 351:2 352:18 406:11 411:16 455:26 456:12 457:25 469:5 483:4,24 490:9 492:6,19 509:23 516:14 537:15,18 538:5 562:16 562:18 563:6 577:23 609:25 612:16 632:14 661:24 668:15,18 704:19 738:22 needing 185:14 309:24 432:18 589:13 Needless 744:13 needs 34:17 36:7 113:2 144:22 164:23 203:18

287:4 291:11,24 292:14 300:15,18 301:3 309:26 313:22 314:7 326:12 343:26 350:13 358:19,21 365:7,25 376:23 377:18 388:24,24 414:5 416:4 416:18 422:23 464:22 468:14 472:7 480:13 483:1 493:6 501:3 502:18 522:5 542:25 565:15 571:8 574:20,25 581:23 584:25 586:13 599:19 601:16 612:8,26 614:17 647:1 658:17,22 672:5 697:1 713:2 730:6 739:3 negative 96:12 261:21 292:15 300:20,24 326:25

331:17,21 375:1 399:22 408:6 412:14 413:22,25 483:18 489:6,24,24,25 499:21 547:26 580:12 598:19,20 604:9,13 605:7 606:15 706:18 711:15 729:24 negatives 22:6 317:8 375:2,2,4 547:26 613:25

622:19 negligent745:17 negligible 322:24 neighborhood 219:2 neither 183:4 282:20 315:17 530:25 neomysis 643:26 nerdy 594:5 nesting 137:20 587:12 589:17 neural 370:7 neuro 373:12 neurobiology427:20 neuroendocrine 638:15 Neuroscience7:24 neurotoxin722:16 never 117:22 120:2,3 154:17,18 155:9 166:23 171:23 227:18 282:2 345:2 359:7 399:21 400:22 402:23 421:13 629:13,21,23 700:12 701:23 711:6 716:12 718:2 747:13 nevertheless 611:22,26 734:17 new 143:7 151:25 152:9 180:26 197:13 201:25

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481:10 484:9 non-crustacean 667:7 non-definitive 321:23 non-EDC 624:17 638:21 non-endocrine 81:23 125:2 304:3 326:25 455:14

489:5,7 531:12 536:23 537:5 538:10 551:3 552:6 553:5,8 554:1 557:3,20 645:13 non-endocrine-active 299:17 non-endocrine-mediated 293:22 non-expert 669:2 non-informative 699:15 non-invertebrate 630:7 638:4,7 non-linear48:4 non-mating483:12 non-monatomic 161:19 non-monotonic 30:26 31:3,6 482:9,15 564:4 non-normal 398:22 400:1 non-normality 677:5 non-parametric474:8 579:7 580:25 675:20 non-parametrics 581:1,24 non-pesticide 19:8 non-reproductive 535:26 non-reproductively 367:5 non-solvent517:15 non-statistical 383:15 470:22 576:25 674:26 non-statisticians 579:25 non-steroidal 644:7 non-zero479:19 481:5 noon276:22 normal 26:13 83:26 84:2 147:19 398:9 399:11,19

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484:1,4 579:6 676:24 677:5 normalization467:6 normalize 121:12 517:8 normalized402:14 478:24 479:24 normalizing 121:16 normally 398:13,20,25 399:5 483:16 579:3 580:26 North7:7 243:6 460:20 northern 6:13 242:21 460:9 526:14 665:11 notable487:5 note 8:4 51:12 87:13 444:1,3 480:5,17 481:10

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600:8 607:11 698:25 704:2 715:14 723:2 observed 36:10 46:21 47:3 48:4 53:4,9 54:8,11

60:25 65:18 75:4 95:21 103:4,6 105:15 159:18 161:17,18 163:5 164:7 167:6,8 170:6 226:23 269:17 281:8 316:21 318:23 345:9,16 418:25 451:20 482:19 490:18 529:18,22 538:2 578:9 600:5 610:12 698:15 719:6 observing 153:26 269:6 obtainable 173:8 obtained 226:8 319:11 414:13 438:1 493:15

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74:13 115:13,15 116:21 119:8 120:18 121:6,9,21 123:9 124:5,12 126:13,24 128:5 129:15 133:11 137:16 138:11,17 139:7,9 140:10 141:6 143:5,7 149:8,13,15,19,24 150:15,22 153:10 162:9,18 166:25 176:2,9,15 179:19 180:22 181:20 182:3,7 182:17 183:18,26 187:20 188:1 189:11 192:12 193:4 198:9,20,22 199:5 201:6 203:14,26 204:9 205:2,26 229:17,19 230:15 231:25 233:2 234:3 243:16 245:22 246:2 250:5,16 261:17 271:1,14 271:23,25 272:3,10,15 273:3 276:13 277:24 285:26 290:11 297:17 300:5 326:2 327:5 328:24 329:14 330:2,20 334:9,21 335:12 336:25 339:20 340:11,14,19 341:9,16 342:5,13,19 345:17,18 346:21 347:1,20 355:23 357:2 359:21,25 360:17 361:23 363:26 376:3 383:4 393:4 399:24 401:2 404:4 405:10 406:12,14 407:13,24,25 409:4 414:16 422:15 427:22 428:4 429:23 452:6 453:1 457:5 468:7,13,16,19 470:6 476:8,11 478:1 484:13,17 485:10 493:1 508:6 511:11 512:14 519:6 540:15,22 542:10 544:13,15 593:23 595:18 596:3 601:6 606:8 624:7 662:18 714:2 727:8 old 87:4 181:22 212:7 552:22 older 87:9 Olmstead 157:10 167:5 181:17 Omaha 6:25 243:1 460:15 omitted 699:26 once 34:16 85:6 87:25 88:10,12,15 89:6 92:12,16

92:26 95:3,7,12 97:14,15,22 98:17 100:13 101:16 102:1,11 106:4,18 114:6 129:10 168:7 195:4 213:20 221:8,14 234:1 314:25 318:23 319:9 321:2 390:8,9,12,13 415:22 478:12 489:15 518:7 533:17 539:21 614:19 644:14 649:2 670:13 678:12 710:14 711:20 733:10 736:7 740:12 741:9 ones45:11 72:11 99:3,3 194:2 209:19 220:1 234:4

235:11 255:6 280:12 285:2 320:23 324:4,26 325:1 328:10 387:17 420:19 578:5,14 623:17 630:14 653:26 700:19 740:2 one's 248:25 268:13 one-by-one 602:16 one-gen 288:13 292:26 294:4,10 335:6,6 337:5

344:23 one-generation 21:20 24:4,23 234:12 286:19,23

291:4,20 292:23 294:12,23,25,26 320:13 338:19 339:17 621:13 639:9 one-generational 710:6 one-gen/two-gen 294:8 one-one 689:11 one-third 689:11 one-tier 339:9 one-way 90:10,12 137:14 153:18 401:22,23 589:5 one/type403:7 ongoing 649:5 652:19 online 16:23 83:15,16 84:12 onset54:12 64:9 oocyte 85:11 157:8 167:20,25 oocytes 84:4 85:5 147:5 158:10 169:6 170:2 546:14

557:10 oogonia 378:16 OP 165:1 494:21 opaqueness 515:6 OPDDT479:15 open 1:2 4:11 11:12 29:24 89:5 270:12 295:24

362:16 521:7 533:12 592:21,23 663:5 opening 12:26 opens 15:20 operate 15:13 32:11 operating 69:16 472:19 591:3 operationally 18:17 operations 11:10 opinion 174:6 329:15 333:22 334:16 342:7,15

356:1 373:14 375:7 385:20 398:26 413:14 431:1

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464:11,12,24 466:15,18,22 507:25 556:12,17 557:23 558:8 563:19 666:13 667:23 668:21 669:1 682:1 689:5 699:21 optimize 27:24 78:10 99:6 166:8 574:25 674:3 optimized 296:11 299:7 305:4 557:25 697:21 optimizes 579:4 optimizing467:11 579:2 712:12 option44:16 73:22 74:11 90:23 180:1 425:23

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744:14 ordinal 89:19 398:3 478:14 578:10,16 organ 274:7,8 380:1 531:10 562:3 organigenesis 171:4 organism46:8,9 76:7 77:8 112:13 158:2 305:26

307:22,22 312:9 352:8 526:8 551:5 596:24 641:10 684:16 714:8,10 organismal 170:22 organisms 165:9,15 171:5,9 186:1 207:2 305:26

310:7,11 313:3 621:18,19 639:2 687:11 689:18 715:23 716:24 organization 11:11 22:25 32:23 142:20 210:4

634:15 662:25 organizational 141:7,9,13,15,23 142:1,26 143:4

213:7,10 446:4 453:21 462:12 529:16 530:14 742:20 organize 83:23 184:2 organized 81:6 485:9 577:22 635:26 721:25

736:16 organizing9:14 745:19 746:15 organogenesis 165:10 552:2 553:2,26 556:21 organs45:15,16 274:1,2 376:10 562:4 organ/gland45:13 oriented 162:26 origin 392:10 463:13 original 72:10 78:22 161:15 252:7 originally 222:7 397:6 597:3 602:14 OSCP29:18 151:23 Oshima 359:13 ostensibly 143:3 ought 87:15 396:9 399:17 400:5,15 401:1 577:22

595:19 673:26 674:9 688:21 outcome 21:7 22:24 25:24 32:18 37:25 41:24 42:6

42:23 45:6 70:6 84:7 90:3 91:2,2 92:4 96:5,25 101:3 104:6 133:18 141:4,18 143:3 185:18 188:7 188:10 198:15 252:21 327:7 330:1,4 409:20 420:15 436:2 541:22 553:1 565:12,14,16,20 598:18,19 603:6 606:19 608:12 611:26 612:2,6,9 614:4 618:26 619:3 621:10 628:4 656:5 689:15 698:11 706:18 732:12,17 outcomes 47:14 58:5 61:25 63:12 69:15 75:21,22

81:1 89:9 90:25 91:17 94:23 103:10 107:8,17 117:23 148:2 210:16 251:8 254:26 255:3 308:3 447:11,22 499:18 533:22 548:2,5,13,14 553:13 555:8 597:15 606:18,22,23 607:25 609:5,7 611:17 612:15,17 614:8,23,26 638:1 658:25 661:5 684:1 686:17 outcry739:25 outlier 680:14 outliers 68:6 479:4 680:20,21 681:1,2,3 outline 39:6 42:16 155:5 206:9 351:1 354:24

607:13 outlined 252:1 302:14 348:26 364:14 461:22

540:23 556:13 614:20 666:14 718:11 outlines 422:8 507:23 704:5 output 86:6 105:14 139:20 140:20 143:21 144:15 144:16 505:18 outputs 505:4 642:5 outside 62:16 193:19 353:3 355:5 366:15 449:3,4

488:6 636:15 688:24

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553:21 557:11,12 562:5 604:6,11 605:13 oviductal 552:16 Oviparity40:14 oviparous 73:8 oviposit552:18 oviposition47:11 ovo 25:10 40:26 368:2 ovulation423:21 oxidative 260:20 oxymoron 119:5 o'clock 38:12 458:18 P P432:22 package 205:13 464:8 592:7 627:13 Padilla 515:12 page 88:19 288:15 421:4 442:4,6 479:4,14,15,16

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82:6 91:10 109:5,6,21 110:2,3,5,16,19,19,22,23 110:23 111:1,8,15,22,23 112:1,1,4 140:13 149:13 221:22 267:23 268:11 271:7,10,10,11,21,22 272:4,6,6,11,12,16,17,19,20 273:1,1,15,16 358:12 387:4,5,11,12 388:18 389:9,14,15,16,26 392:23 437:5 445:23 454:12 455:5 469:21 470:1 470:3 479:12,18,20 480:5,9,11,14,22 481:11 483:12 484:10 505:22 519:13,19,23 520:22 521:1 521:5 535:3 696:5 699:9 pair-wise 138:18 482:5 pale 179:8 panel 1:1 2:3,8,12,18 3:5,24 4:8,19,21 5:1,10,14,18

5:22,25 6:15,18 7:8,17 8:1,5,24,26 9:7,16,26 10:25 11:5,9 12:9,19,26 13:1 19:20 21:2 30:1 34:7 38:11,16,19,24 45:26 54:21 59:5,8 70:21 84:16 105:7 114:23 134:5 153:10 186:13 188:8 195:23 203:23 204:1 239:15,19 241:3,4,18,20,22 243:17 246:24 251:4,6 267:10,11 270:1,6 283:24 295:24 332:20,24 333:12,26 343:3,7 345:26 346:9 353:8 375:16 382:9,12 394:6 404:1,3 406:12 410:2 411:11 419:23 420:1 421:15,20,24 428:19 429:22,26 434:19 435:2 441:1 442:2,11 442:14 443:25 446:2,5,11,17,20,24 447:14 449:25 452:6,10 458:3,16 459:3 468:8 480:18 484:14 486:20 490:10,15 491:11,14 493:20 496:17 497:14 499:15,25 500:15,24 507:2 509:7 509:19 510:4,15,26 515:19 516:18 517:17 521:8 521:20 524:20 525:12 528:8 533:13 539:6,12

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542:5 paradigms 76:19 paragraph 381:25 parallel 182:5 250:20 619:1 726:21 parallelism 181:12 parameter 365:17 367:6 433:7 440:14 parameters 116:26 163:6 165:21,26 168:24 298:8

341:23 373:1 415:9 416:2,24 425:6 431:13 465:17 467:10 474:12 478:26 508:11,26 509:9 515:17,20 516:14,15 517:2 520:16 535:7,26 537:15 558:2,13 573:5 614:20 650:23 653:17 658:13 659:10 743:9 parametric 397:21 398:5 403:3 parametrics 474:6 paraphrase 554:23,25 paraphrasing408:10 parasagittal 274:12 Pardon 593:8 parent 132:11,15,17 211:18 510:10 parental41:12 42:21 44:1 50:16 51:6,24 52:15 53:7 54:4 57:15 71:7 76:14 112:19 351:9,16 354:26 360:14 389:15,16 425:12 427:12 435:9 455:24,25 456:7 524:18,20,22 691:23 parentheses 47:26 48:1,3 675:25 parenthesis 91:26 parents41:1 351:6 393:11,21 424:24 436:26 parings 691:15 parochial 634:8 parrot738:18 parsed 148:22 part4:11 5:7 39:10,15,16 40:22 43:25 44:19 49:4

51:15 61:20 67:25 80:8 81:4 98:16 111:11,12 113:1,17 119:11 120:12,12 130:8 132:7 136:21 137:22 138:5 142:6 144:7 146:17,24 163:6 180:21 181:25 186:8 189:22 193:26 196:5 201:20 206:11 210:23 225:4 228:19 241:8 252:7 257:4,4 264:14 271:24 276:17 292:6 330:21 333:3 334:11 347:15 349:7 350:26 364:3,3 377:17 382:13 383:14 384:9,14,15,16,17 393:26 394:6 410:4 439:7 443:16 470:20 475:24 485:5 497:24,26 510:22,25 516:26 519:9 522:8 541:8,16 543:13 545:21 556:3 559:11 563:13 566:9 576:23 578:21 579:23 584:7 587:2,8 595:4 617:2 622:12 640:10 640:12 642:12 650:14 651:25 652:7 654:17 656:15,21,24 658:15 664:18 674:16,24 678:14 681:5 682:6 686:7 687:19 702:26 707:18 714:16 716:1 732:3 733:24 738:19 739:6 746:25 partial 64:13 456:8 partially 135:13 participant 3:8 participants 641:20 participate 2:13 9:3,17 160:16 284:14 652:4 participated4:20 170:8,25 214:18 215:23 557:24 participating 9:8 162:2 282:25 562:20 participation 743:4 particular 14:17 15:15,18 18:8 23:7 25:13,18,19

25:25 26:2,3,6,9,21,24 27:3,15,26 29:4 30:13 33:23 35:2,18,19 38:5 39:5 43:4 46:8 48:7,10 49:17 50:1 52:18 56:19 59:13 60:24 61:18 62:5 62:11,19 81:1 83:9 87:20 89:24 90:3 98:7 110:20 117:7 119:10 121:24 123:12 124:1 137:25 138:1 147:26 148:4 184:13 186:4 208:14 209:15 210:6 221:11 223:10 232:20 237:24 246:6 251:22 252:17 253:16,24 254:8,19 255:12 256:21 262:1 262:26 263:21 264:3,15 265:24 268:12 271:17 274:8 297:15 311:19 324:26 325:15 328:9 349:13 349:22 355:5 375:22 384:4 410:11 412:1 431:13 444:26 445:20 453:4 455:11,22 466:4 482:25 488:12,15,24 498:8 501:6 511:17 523:5 526:1

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437:13,25 482:11 574:14 685:15,16 723:17,21 patterns 162:11 234:9 436:17 482:16 pay 63:12 73:4 178:22 201:9,25 paying 336:26 PBDE488:22 PCR 80:22 82:16 87:14 136:17 139:16 457:13

514:2 573:26 575:10 pdf442:17 593:7,9,11,17,20 594:2 peek 187:25 peer2:20,25 10:1 11:12 492:17 591:24 592:5,6,8

664:4 738:10 743:26 peers 237:21 pen 389:16 392:10 393:23 394:25,26 penaeid 620:4 pens 290:7 389:14 393:23,24 433:11 people 112:24 113:23 114:10 134:1 136:18 145:16 184:5 198:1,5 206:1 270:3 277:17 283:13 284:23 310:16 311:24 345:21 360:4 370:20 371:1,4 372:13 391:25 399:15 428:21 458:17 473:26 488:6 494:15 518:18 527:1 579:9,10 590:22 591:2,4 620:11 702:21 712:5 729:10 730:2 746:11,26 747:1,25 748:2,3 peptide410:18 perceived 262:12 714:4 perceiving 202:12 percent54:1,2,4,5,26 55:3 67:21,22,23 68:2,3,4

86:11,11,16,17,24 87:2,24,25 88:11 110:11,12,13 110:21,25 111:2,3 123:25 170:25 171:6,6 196:20 197:15 214:4,14,15 219:2 230:11 235:7,12,21 238:1 258:8,9,9 264:19 284:22 291:17 307:23

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431:12 445:8,17 451:4,7,8 454:15,20 455:2 469:24 523:25 524:1,5 526:23 530:23 531:1,3 552:26 557:14 phenotypically 546:25 phenyl446:11

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628:16 630:22 677:6 716:12 722:4 problems 102:9,10 103:12,25 104:1 129:22,24 130:26 131:9 136:16 196:12 219:11 224:20 237:20,22 249:18 279:16 301:10 316:20 319:3 331:13 370:18 498:5 499:7,19 560:24 571:16 581:2,26 586:15 604:24 608:19 610:17 629:9,20 641:17,18 670:23 677:8 727:25 741:5 probs 123:8 PROC473:5 procedure 89:24 127:9,13,20 138:20,22,23 139:3 166:14 502:25,26 589:11 591:3 680:4 procedures 166:8 417:11 467:7 501:6 521:23

582:12 605:18 673:20 675:10 744:7 proceed 249:15 695:22 proceeded 50:9 proceedings 9:18 proceeds 158:24 process4:11 9:16 11:12 12:6 14:15 27:8,16 28:3,9

32:7 33:17 41:14 83:18 93:13 120:21 141:18,21 142:18 143:4 159:11 160:18 190:10 206:13 231:2 241:8 248:5 251:8 252:1 253:20 256:1,11 261:1 283:4 310:13 321:13 326:16 337:2 369:1 387:19 388:15 389:24,25 392:25 393:26 394:11 401:7 422:8 430:4 454:14 471:21 502:15 507:23 522:15 539:14 573:15 592:5 607:13 611:1 617:21 621:16 623:11,18,19,26 626:11 628:5 633:22 634:23 635:9 642:12 649:1,7 650:1 652:10,25 656:11 663:4 670:26 697:13,20,24,25 699:20 704:5 713:18 717:25 731:25 738:15,16 739:22 743:25 746:11 processed45:24 processes 14:18 30:9 82:10 98:5 139:26 259:8,23 261:5 357:13,26 364:8 451:17 452:16 461:15 463:2,14,15 504:24,26 512:19 550:3 556:7 625:16 626:1 637:2 666:8 processing 188:23 499:2 604:15 prochloraz 97:18 99:13 119:9 134:15 135:24 161:22 162:1,5 163:5,14,16 170:9,10,26 171:1 173:18,26 209:21 281:23 311:22 485:21 487:10 488:26 489:26 492:23,24 495:12,21 505:15 551:13 555:13 560:11,15 561:16 562:10 596:19 596:23 602:12,17,23 603:26 604:2 625:11 682:2 722:3,3 produce 23:25 53:2 88:8 91:22 143:15 172:10 177:11 181:24 201:5 273:13 290:12 348:2 389:6 482:14 489:17 502:23 513:23 519:19 650:20 produced 50:5 55:16 64:12 81:11 149:25 172:11

223:18 237:6 265:16 271:22 280:25 290:9 319:5 320:8 338:24 339:2,2,23 340:1,4 505:10 521:3 568:7 616:12 691:26 700:2 produces46:25 162:6 171:16 172:8 478:22 479:24 producing46:18,19 51:19 197:20,22 203:3 214:15

489:16 505:22 699:25,26 product 64:7 201:5 587:18 620:12 production 18:26 41:1 45:3 50:3,18,24 51:11,14

51:16 52:16,21,23 55:19 63:6 70:19 71:15 79:25 96:19,19 135:3 141:18 143:22 208:1 214:10,23 215:25 237:11 247:12,20 255:11,22 263:17,20 339:13 350:5 364:19 368:3 375:1 431:11 438:10 486:13 494:26 519:26 529:12 551:7 552:12 561:18 568:18 711:12 productive 51:19 68:7 products 29:7 168:2 professional 706:14 professor 5:13,16,20 6:20,24 7:1,4,11,16,20,23

243:2 401:9 679:22 proficiency 199:17 375:22 650:13 652:6,10 660:12

661:12 715:8 743:12 proficient456:23,24 714:24 profiles 683:3 Progesterone423:20,21 program 1:5 2:6 9:20 11:3,13 12:3 13:4,15,15,19 13:26 14:4,10,12 15:12,16 17:2 18:20 20:13,15 27:6 29:14 41:26 88:23,26 89:7 90:5 179:23 181:2 195:5 204:26 251:19 253:13 268:21 269:22 282:6 306:14,17 315:13 316:8 324:14 326:18 349:7 354:10 355:3,6 365:25,26 381:19 428:19 428:22 430:5 472:23 473:2,6,8,17 475:7 483:6,18 522:16 543:19 611:3 614:2 633:25 639:20 650:14 652:15 659:1,6,15 661:14 664:5 705:25 713:19 735:11 738:24 739:15,18,22 742:9,19 744:2 746:20 747:16

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265:20 459:5 464:6 592:3 649:22 651:2 659:23 725:12 746:22 Program's 241:6 progress 182:8 330:2 progressed 505:19 659:14 progressing 86:3 progression46:20 142:22 362:18 551:5 prohibitive 547:16 project239:5 742:9 projected 53:20 545:6 721:16 prominent56:25 185:2 promise 305:1 404:7 471:10 537:21 promote 319:25 prompt549:13 promptly 105:3 prone427:21 pronounce 595:9 pronounced 217:9 425:5 proof597:15 proper 379:7 384:12 414:9 419:12 478:2 493:11

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728:25 properties 19:10 275:15 383:11 470:17 479:25

551:12 576:20 621:10 674:22 prophylactic 113:21 proportion 87:1 481:8,13 591:12 Proportional-Odds 578:20 proportionately 378:20 proportions400:10,10 proposal 210:5 308:7 484:8 737:8,9 proposals 737:5 propose 122:12 153:5 386:23 proposed 1:4 2:22 9:21 21:17 25:2 112:11 152:9 187:13 195:20 202:8 210:6 222:7 241:6 251:26 253:9 299:9 300:2 301:21,24 302:4,8 307:14 349:12 352:2 377:1 383:12 444:10 454:3,18 456:6,9 459:5 470:18 475:20 507:25 528:25 533:26 534:3,17 576:21 664:4 674:23 687:19,22 716:13 proposing73:21 75:23 87:9 107:22 108:2 121:13 122:24 124:1,14 136:15 209:15 475:8 528:22 649:14 707:21 Propper 6:12,12 66:22,23 67:8,17 68:12 180:23,24 181:20 182:3,7,17 183:4,9,13,26 198:10,11,20,25 242:20,20 460:8,8 545:23,25 549:15,16 554:8,9 563:7,8 565:1,2 568:26 569:1 573:24,25 574:15 574:16 575:8,23 598:14,15 600:25,26 606:13,14 607:18,19 608:22 609:3,12 610:9 611:9,10 654:15,16 655:9,10 660:24,25 665:10,10 733:1 proprietary 335:25 336:3 protect 15:4 323:10 619:9 642:20 687:12 protected 335:10 617:15 619:7 protecting 18:13 315:14 621:6 686:8 710:21 protection 1:13 16:14 18:13 244:19 285:6 323:10

459:16 619:18,24 620:8,15 621:4 708:9 protective 15:11 24:8,21 30:24 31:9,14 254:25

255:3 256:2,3,7,14,17,24,25,26 307:18,19 308:1 314:12 334:19 686:1 692:22 protects 686:11 protein 190:9 410:12 416:15 proteins 184:9 567:14 protocol 27:21 28:1,2 39:12 40:6 41:11 42:4,16

43:7,19 56:9 57:19 58:19 63:18 64:3 75:19 76:2 76:24 77:6 78:15,15,16,26 79:3,21,22 80:9,10,11 89:22 98:23 100:25 103:18 104:16 107:20,26,26 108:8 112:11 139:10,17 143:7 160:14,15 173:11 193:6,14 197:5 209:15 210:7,7 273:25 275:26 278:20 292:18,18 296:7,11 299:6 300:14 301:21 302:8,14 305:4,8 313:17 347:21 348:1 349:6,20 350:8,26 352:17 354:19 356:5 358:8,19 364:4,10 364:23 367:11 368:20 370:16 384:20,21 386:24 403:26 408:14,17 409:17 412:15,17 413:11 414:9 417:9 418:2 419:11 420:13 422:2,3 428:14 433:13 436:12 437:4,5,7 438:25 439:10 461:12 461:18 465:14,21 466:14 469:14 470:2 471:15,16 472:19 480:6,13 483:1 491:24 493:10 502:14,16 502:17,24 503:2,4,5 504:11 505:12 506:6,18 507:16,18,26 510:20,20,26 511:1,22 512:15 513:21 515:11 516:19 520:13,17 528:25 529:3 546:9 555:16 556:4,9 558:8 567:13 570:13 571:15,17 573:20 574:9 579:14 586:11,17 598:25 599:8,12 601:20 607:7,8 608:4 610:15,17 614:3 622:14 623:14 638:11 640:5,24 642:12 649:22 650:23 651:1,12,13 652:1,7,14,14 654:11,23 655:6,7,20,21,25 659:10,22 660:13,16 661:13 666:5,10,21 667:1 670:12,15 671:7 680:13 682:21 697:4 699:17 703:24,26 707:21,24 709:1 710:23 711:1 714:16 716:22 717:2 724:6 protocols 27:25 28:21 40:21 77:22 78:23 79:1

89:10 93:12,12,14 114:13 160:17 194:13 195:14 198:1 206:14,17 208:24 282:22 325:25 330:19,20 330:20 331:14 335:2 349:1,3 426:2 428:9 433:17 465:4,10 466:13 471:17 497:3 509:22 514:21 517:22 526:3,26 558:4,5 585:3,6 599:22 607:26 614:19 621:1 622:26 639:21 645:5 668:25 697:22 698:6,26 706:17 protracted 197:1

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43:3 57:21 59:11 63:19 64:5 70:7 104:14 107:19 114:7,9 175:24 195:7 196:4 205:18 228:14 229:18 251:9 274:17 277:4 301:13 325:11 347:12 348:5 349:16 355:3 356:21 363:12,19 364:11 366:17 371:19,21 374:18 382:25 384:5 407:4 414:9 419:11,21 422:11 434:16 443:13 444:13 453:13,15 457:4,14,24 461:19 462:6 467:19 483:6 486:14 487:3 493:11 494:19 508:2 516:9 521:16 530:2 545:17 556:10 558:25 561:26 564:15,17 566:5,21 568:4 570:14 572:24 575:6 596:25 597:14 601:21 603:1 607:16 608:13 609:14,20 613:20 614:10 616:24 624:11 626:11 631:15 638:13 642:24 645:11 650:7,24 657:20 662:26 666:11,26 667:17 674:8 679:25 680:2 695:6 697:5 698:16,18 704:8 705:21 716:20 730:8 731:7 743:16 provided 10:8 90:24,25 122:2 155:1 160:14,17 173:11 179:11,17 244:19 250:19 282:7 296:24 300:13 302:9 309:21 346:5,10 347:23 366:25 367:18 376:13 418:8,13 434:24 437:12 442:10 446:3 448:22 449:11,14 465:4,13,17 469:16 493:25 510:21 511:13 528:25 533:23 561:26 571:14 578:18 580:21 627:6 636:11 666:24 670:17,24 676:4,22 678:8 712:10 719:1 736:2 provides 2:19 10:3 41:11 58:14 83:17,18,21 84:9 172:8 313:3 314:10,15 322:15 323:17 365:4 369:16,26 376:23 421:4,14 426:7 445:26 464:26 520:7 621:17,26 622:2 680:6 708:8 719:3 providing 9:9 10:1,25 25:15 62:17 172:22 251:7

277:17 296:2 346:17 350:15 372:25 377:12 455:16 459:4 492:17 518:15 534:26 566:1 572:7 620:14 686:24 proving 247:11 724:9 provisions 2:9 3:6 proxy 551:7 552:14 prudent 124:17 343:11 438:21 451:11 pseudo-replication 696:11,16,18 PTP529:18 PTU457:13 pubertal 233:6 pubertals 233:20 puberty424:11,16 public 2:14,14 3:15,19,23 4:1,4,5,19 9:8,17 10:26 12:17 13:25 15:4,11 20:19 43:9 61:23 154:10 239:26,26 240:3,4 241:13,15,19 277:8,12 284:3 314:24 325:23,23 330:23 343:5 345:20,20 346:14 428:3 442:18 451:13 510:21 514:2 623:16 624:20 642:7 648:21 663:5 669:11 730:12 739:25 742:25 publically 514:6 publication 352:14 462:22 515:12 publicly 10:1 31:1 publish489:23 published 27:17 94:19,22 95:13,22 97:6,9 98:10,20 157:10 163:16 174:22 181:16,16 225:15 248:17 431:24 547:11 560:13 567:24 738:8 pull 8:1 13:10 17:10 29:4 109:6 114:18 187:11

230:8 270:11 364:18 508:22 587:22 653:24 658:15,21 727:12 733:6,19 pulled 27:7 109:9 164:3 165:8 311:20 pulling 217:7 653:10,13 punctuated 634:22 Purdue7:15 243:11 665:23 purely 228:9 purification 513:5 purity 214:7 purple 179:14 purpose 25:6,19 27:10,11,15 39:7,22 42:19 58:25

76:4 112:11 155:11 206:10 208:19 212:16 222:8 260:2 278:8 285:16 306:14 324:11 329:3 332:7,7 334:12 346:23 347:5,7,14 348:7,8 357:21 426:17 430:4 443:5,8,15 444:7,14 445:22 452:23 453:4 503:23 522:15 545:9,12,20 550:11,16 551:22 597:16 611:1 613:5,15 614:22 616:6,9 617:1,11 626:13 627:17,21,26 631:11 635:3 637:10,16 685:9,14 697:14 705:6 713:17 732:20,23 purposes 29:7 375:4 409:24 436:14 500:15 598:3

605:22 662:7 681:21 706:1 pursue 148:3 206:20 252:18 266:25 356:22 455:8 pursued 116:1 252:19 743:17 pursuing743:22 purview 655:13 push 13:25 22:14 480:20 492:2 630:10 646:5 pushed 643:3 pushing 643:17 728:16 739:3 put 11:10 13:8 14:10,20,22 15:16 18:18 31:2 67:12

86:20 87:1 100:18 116:14 117:14 121:2 139:24 140:3 150:11 169:5 178:7 202:25 239:25 244:11 248:4 298:5 299:25 306:17 311:20 314:14 330:24 336:17 342:4 346:14,15 361:9 369:26 372:20 387:12 391:19,23 395:19 397:10 425:18 429:13 431:23 442:17 484:9 490:15,16 491:17 494:1 502:12 513:17 514:6 521:1 584:16 585:5,17 586:13 590:7 592:8,9 593:8,11,11 594:4 614:17 617:7 623:22 630:1 635:8 648:5,13,24 649:9 650:3 662:11 673:19,26 687:3,13 698:22 700:14

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423:12,26 512:7,8 513:2,2,10,19,20 575:18 726:5 726:12,16 QSAR 11:16 19:3,4 quadratic483:9 quail 25:3 29:21 39:3 42:5,10 43:20 45:19 46:17

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31:17 34:15 36:1,16,22,25 37:3 54:17 64:1,2,20 67:22 68:23 71:13,21,22,26 73:13,17 74:2 75:2 115:1 122:21 124:7,13 126:11 130:3,8,11 131:3 132:23,25 133:12 134:18 135:14,20 136:15 138:12 139:23 143:8 153:11,23,24 164:3 173:20 175:6,7,7,11 177:12 179:21 184:19 187:14 188:17,21 191:17,22,26 192:18 193:3,9 196:16 198:17,26 203:16 208:15 218:13,15 227:17 231:5 232:22,25 233:16,24 234:1,14,15,20 239:1 241:12 243:24 245:14 246:8,9,11 247:6,24 250:16,17,26 256:16 257:2,21 258:24 259:25 265:9 268:15 272:1 274:23,26 275:7,15 281:12 286:17 287:10 297:18 298:9,9 304:17 306:15,18 308:9 316:4 323:13 325:16 326:2,14 330:25 332:1 335:17 336:21 338:14 344:22 346:11 347:3 353:13 354:14 355:14,19,21 356:2,8 357:3,10,11 359:14 360:4,5 361:23,24 362:13 363:3,23,26 364:2,3 370:25 373:9,23 375:16 376:2,3 382:9 383:5,6 385:6,10 389:2,19 391:7,12 395:4 403:22 404:2 406:14,20,22 409:9 410:4 411:6 412:5,21 414:2,4 421:20,26 422:1,16 429:14,23 430:1,3 434:9 435:4,5 436:15,16 437:18 441:2,19 442:9 443:3,22,24 452:6,12,14 458:10 459:9,14 461:9 461:11 466:20 467:12,19 468:9,22,24 469:8 470:10,11 471:4,7 475:25 476:1,15 484:25 485:5 485:7,8 487:15 490:16,25 491:11 492:20 493:2,4 493:5 496:10 501:1,22 502:1,2,12 507:2,14 508:8 510:4,13 511:17,25,25 514:15 518:5 521:8 522:13,14 523:4 526:7 528:16,16,18 529:9 532:25 533:20,21 535:21 539:6 541:6 545:4,6,7 550:1 556:1,3,3 559:11 561:16 564:7,12 565:9 566:7,9 567:20 569:18 570:7,8 575:5 576:14,16 585:22 592:4 593:26 595:26 596:8 597:5 601:13 601:15 602:3,4,5 603:24 607:4,6 608:26 609:10 610:19,25,26 612:8 613:4 615:12,22 616:1,2,4 618:5 623:18,19 627:8,17,17,25 628:8,11 632:18 633:3 635:3,18,20,23,23 636:7,8,24,26 639:16,17 642:24 645:1,14 646:18,19 647:20 649:12 656:2 656:20 660:2,4 662:4 666:1,2,3,4 667:16 668:14 669:22 670:9 671:10,11,17 674:15,17 675:7,11

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303:19 387:19 388:15 389:25 393:19,26 394:2 402:15 429:3,9 472:9 475:8 512:25 578:15 592:14 594:19,20 595:11 randomization 62:7 386:11,14,16,19 387:10 388:8

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493:14 503:2,9 504:4,4,14 517:25 570:15 601:24 697:8 repeatable414:6,7,24 493:7,9 504:10 563:2

601:16,19 697:1,3 repeated 89:14 137:23 138:3,6,12,14,22,24 153:25

180:1 202:16 215:10,18 219:9 475:3,4,6 478:20 479:6 480:9 502:26 503:3 581:19 repeatedly 229:1 578:22 582:23 585:3 586:10 repeating 278:17 repetition414:20 422:16 repetitions 581:20 repetitive 66:14 rephrase720:8 replace 316:2 386:18 replaced 221:23,25 238:20 replacement210:26 316:1 replacing 680:24 replicate 91:12 109:5,13,16,19,21 110:2,3,5 111:1

111:5,9,15,22,23 112:1,1,2,4 138:4 149:13 160:24 191:18 211:11 221:26 228:2 271:21 273:12 433:11 471:26 481:23 506:7 524:25 586:26 587:3 659:13 688:25 694:17,18,21 695:5 replicated 35:5 replicates 106:9 111:10,11,13,21 148:24,25 160:25

168:11,12,13,13 192:8,17,25 211:24 254:17 267:17 271:26 359:1,3 367:11 377:26 378:7 476:24 503:11,11,26 504:2 505:6 511:7 513:16 534:1 535:11 583:7,8 585:17 687:20,21 688:13 691:14 693:14,16,25 694:14,20 695:4,7,8,15,17 695:19 699:4 715:5 736:24 replication 90:13 98:16 101:5 106:9 108:6 109:4

112:7 137:25 152:19 191:18 271:17 297:7 299:13 300:2 301:25 302:19 464:15 526:20 535:2,8 583:25 584:6,8 687:23 688:3,8 report 3:9 4:8,11,13,15 8:12 9:3 12:16 13:5 16:11

33:11 47:9 63:23 93:23 129:16 130:2,25 150:20 154:23 160:15 161:16 165:9 172:20 200:15 268:16 289:15 300:15,22 301:2,17 329:1 348:10 349:25 363:19 365:3 368:7 369:15 379:10,15 382:14 384:26 385:1,8,9 388:20 389:4 396:4,20 397:13,15 398:26,26 399:25 401:5 422:7 425:4 429:15 430:18 431:25 433:21,24 438:2,11 447:9 455:11 470:5 471:13,24 472:4 474:14 477:11,19 477:24 480:26 485:22 486:2,14,24 487:18,19 491:17 498:11,13 502:25 503:14,20 504:16,18 505:2,3 506:14 507:22 520:5 522:26 547:19 575:7 590:10 591:5 595:16 596:2,26 597:3 603:2 603:25 604:3 607:12 627:13 635:20 636:3 660:7 662:24 679:19 691:26 699:6 704:4 725:21 727:12 746:3 reported 93:23 95:7 100:13 150:16 161:15 164:9

164:11 165:1 171:14 184:14 254:5,15 279:24 280:2 281:23,25 395:15 455:9 482:16 487:6,13 496:20 498:23 509:2 560:23 561:12 567:3,6,10 572:23 575:17 602:19 603:5 605:5 641:19 647:14 676:5 683:22 698:9 706:25 reporting 94:23 100:14,16 112:26 163:18 282:15

570:14 reports 3:13 38:25 65:26 76:6 346:24 361:11 365:5

379:15 392:9 394:24 399:1 402:11,19 504:15 735:21 738:7,8 represent46:12 110:8 383:10 390:1 443:25 470:16

531:16 553:8 576:19 628:25,26 674:21 706:4 708:13 726:9 representation 16:7 271:16 629:15 740:7 representative 86:2 145:22 379:26 380:3 381:1,5

383:8 431:3 432:8 446:25 447:14 456:14 470:14 525:17,17 538:7 562:2 576:17 625:14 674:19 representatives 29:18 represented 21:23 37:14 271:6 553:9 559:17 630:6

630:22 705:2 representing 3:20 9:23 110:6 223:24 241:15 553:6

577:24 693:7 represents 48:4 110:10,25 420:26 506:20,21

594:13 repro 291:20 reproduce 58:5,9 116:12 157:4 165:5 297:25

472:17 533:8 700:9 reproduced 320:9 503:4 603:22 669:2 reproduces467:8 reproducibility 27:12 53:1 100:21 114:25 196:11

198:2 293:21 301:15 304:9 311:7 412:4 414:12 420:11 488:3 491:1 493:14,23 496:9,10,11 497:4 499:22 500:12 503:3 504:12 509:13,15,20 526:19 537:16,19 560:10 566:20,22 567:18 570:16 582:18 585:5 601:25 603:25 697:8,16,26 698:12 712:2 reproducible 60:18 190:11 196:14 197:6 198:4,5

282:24 414:6,7,24 493:7,9 500:6 502:18 550:22 601:17,19 652:21 697:2,3 698:21 712:4 reproducing 117:8 221:22 223:3 582:11

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27:1 40:22 42:11 45:3 47:17 49:23,26 50:18 51:7 52:8 54:3 55:2,4 64:8,14 66:25 67:1,3,4,17 68:3 72:8 74:1,26 75:25 78:23 79:23 81:7 88:4 91:5 92:17 93:17,18 97:11 99:9,18 102:25 103:1 107:22 108:7,11,16,17,20,23 109:21,25 110:2,5 115:24 118:5 142:3 144:18 155:14,25 175:21 185:20 186:20,23 206:6 207:14 212:15,24 213:24 214:12,26 215:15 222:3 236:7 237:4 255:4,14 260:12 263:6,7 273:16 279:21 280:12 281:4 288:13 296:3,8,11,16 297:8,11,15 298:5,19 299:8 302:13,20 304:22 305:1 309:1 316:13 318:3 325:15 329:16 334:15 338:8,17 339:7,8 342:17 344:17,19,20 351:17 354:6 430:24 431:20 433:5 438:24,25 440:7 444:26 448:5 479:15,16,16 480:7 481:6 496:21 505:24 507:26 524:11,12 528:20,22 529:8 530:3 532:15 533:7,24 534:2,6 535:3 537:3,6 541:9,13,21 542:26 543:1 552:17 557:14 565:25 612:2 616:15,20 618:8,11,18 624:19 625:23 631:5 638:20,25 639:3 640:8 643:5 645:9 664:13 667:6 689:1,7 699:23,25 700:1 701:4 706:8 716:25 719:24 721:6,20,20 722:15,17 732:14 reproductive 5:5,10,14 7:12 33:13 40:24 42:9 46:3

55:23 69:10 73:15 89:14 91:9 98:16 105:14,21,23 107:9 110:7 141:18 142:5,23 144:15,16 145:7 162:7 164:1 166:19 167:9 169:21 170:13,18 175:18 185:22 187:9 211:10 212:17 213:12 214:13 221:20,24 222:12 225:21 228:3,18 255:11 261:9 273:15,16 275:8 279:4 290:11 335:7 340:24 426:25 431:6 435:8 437:24 438:19,23 447:22 448:7,9 455:15 456:1 465:26 466:3,9 495:20 497:20 503:10 505:18,18 520:25 521:2 526:4 530:15 532:17 533:9,10 535:7,10,11,14 544:5 552:9,13 561:1,20 603:12 605:13 628:17 637:21 638:13 669:7 683:3,6 700:17 717:9 718:16 721:18 722:6,7 735:1 reproductively 214:4 request283:5,12 309:2 332:23,25 363:12 382:12

382:12 658:12 requested441:21 677:22 716:11,12 requests 615:10 require 166:11 194:24 225:9 366:16 530:4 533:10

576:9 626:5 641:16 649:9 687:21 718:1,2 required 9:2 15:23 35:19 56:9 152:20 220:6 227:2

287:5 294:10 313:13,16 314:10 332:17 341:17 342:3 391:11 449:9 464:25 625:20 675:15,20 711:20 715:16 requirement4:12 197:8 222:13 328:1,5 341:13

342:12 405:1 534:22 660:18 requirements 3:13 104:8 127:18 194:18 241:9 331:23 341:21 342:15,16,21 465:14 508:9 525:10 624:14 625:10 626:2 666:22 743:22 requires 15:16 55:26 60:4 221:3 367:17 572:3

573:11 requiring 282:23 706:19 743:9 reran 137:22 rerun 138:10 194:24 706:19 research 5:9 6:2,9 10:13 13:24 16:9 27:18 29:19

49:3 75:18 78:15,15,26 79:21 107:17 108:1 148:8 160:7 204:9,11 242:5,18 322:3 323:6 354:9 356:9 365:15 366:17 367:17,24 368:2 377:22 380:13 382:22 383:2 419:6 430:22 434:16 436:5 441:11 459:18 465:14 639:13 664:20 665:8 734:16 742:6 746:23 747:4,6 researched 377:18 researcher471:20 472:17 researchers 370:4 473:4,10,11 480:14 resembled463:10 resembles 683:5 reserve 298:3 460:6 reside 169:14 residential-only 234:10 residuals 484:3 resistance 335:21,23 447:18 669:11,15,19 715:25

715:26 resistant 690:11 resolution 178:9 resolve 313:8 resolved 314:7 resonating 363:16 resource 534:12 resources 173:26 259:12 307:5 309:8 334:11 523:9

643:22 687:21 715:16 741:9 respect76:9 96:11 99:9 109:16 112:14 151:24

271:11 275:18 279:7 281:16 313:11 385:19 387:4 403:2 492:21 503:5 504:4,12 534:17 539:21 586:21 587:1 594:14 612:9 671:3 676:17,20,23 respectable 219:3 respectful 308:2 respective 602:11 respectively414:8 493:10 530:1 601:20 697:4 respond 62:9 64:20 184:15 227:14 299:22 322:1

351:13 368:4 409:17 411:19 416:22 520:21 610:16 617:17 696:15 responded 520:4 747:14 responding 178:6 666:1 response 23:4 30:8,12 39:24 48:9 51:2,21 60:21

68:10 69:13 72:17 85:14 89:5,15,19,25 95:22 96:3 112:17 125:21 126:2 132:5 133:2 134:26

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269:4,15,16 276:21 277:4 280:2,23 288:2 290:22 293:2,3 297:20 303:10 308:1 318:10 322:26 325:9 329:17 351:20 357:8 377:6 383:11 412:3 413:12 416:10 432:17 448:14 449:21 453:8,9 454:22 455:13,14,23 467:4 470:17 482:14 484:1 499:14 500:5 516:22 520:13 526:18 529:18 530:15 531:17 537:3 554:10 564:1,1,5 574:24 576:20 608:16 627:8 668:9,11 674:21 675:26 691:1 698:12,14 704:18 705:16 712:26 713:1 729:14 responsibility 3:3 responsible 2:8 49:11 277:19 283:14 318:2 433:22 responsive 39:26 219:16 255:19 357:14 397:20

452:17 550:4 637:3 701:6 708:21 responsiveness 416:18 423:3 643:8 689:22 rest4:26 33:24 180:19,20 256:7 383:24 442:16

484:25 511:26 542:2 633:7 635:25 658:15 672:17 672:21 745:20 restate 311:6 restricted 23:6 365:19 result 68:22 88:3 96:16 125:15 132:21 141:23

166:10 174:20 175:20 183:3 185:19 197:25 278:17 280:6 292:9,10 375:1 401:25 407:15 415:1,2 416:11,24 424:12,14 436:24 489:25,25 489:26 494:23 495:1,14,17 496:4 524:14 528:1 565:7 581:10 582:20 601:25 602:25 604:19 605:3 605:8 611:20 619:15 620:18,20 630:25 656:4 669:6 712:22 713:5 715:18,22 resulted 171:6 219:18 494:20 495:6 603:11 resulting 27:1 125:15 260:25 402:2 415:8 420:20

530:10 616:20 669:7 711:8 results 27:13 39:18 43:22 47:20 51:4 60:12,16,25

81:24 88:9 118:22,22 133:12 151:3 167:23 190:12 194:4 206:15,17 214:16 226:21,25 239:3 246:2 248:10 249:1 255:26 269:6 278:11 279:16 280:25 281:1,15,20,21 282:4 291:2,2 292:7 301:14 311:12 318:21 320:5,6,9,16 326:4,20 344:16 348:18 351:3 371:1,4 375:1,6,25 386:17 387:1 391:21 405:7 409:14 412:14 414:7,13 420:11 422:8 436:3 437:12,17 438:4,9,13 440:10 487:12,12 489:16,24,26 490:18 493:8,15,23 494:5 496:18,19 497:11 498:15 499:21 500:8 502:23,26 503:3 507:22 511:15 515:22 519:20 531:22 547:26 551:12 552:6 557:23 558:5 561:16 562:9,13,25 563:14,15 572:5 581:2 582:11 597:5 601:18 602:11,20 603:25 604:9,23 605:7,11,12 605:17 607:13 609:1 614:14 620:11 647:2 649:25 650:7,20 651:5,19 652:5 659:18 667:26 670:21 678:8 683:26 685:15 686:25 690:17,19 693:17 697:2,9,16 698:18,23 700:3 701:1 704:4 707:20 708:20 709:26 710:1,9,18 713:9 714:13 715:12 716:15 retained 109:17 retains 57:2 retention40:16 rethink 584:20 639:4 rethinking 633:24 retinkering 655:7 retinoid 643:24 retired 736:5 retreat28:24 retroactively 322:4 retrospective 24:4 269:20 return 213:5 345:21 441:26 492:4 returned 61:21 703:22 returning 80:22 reverify 225:3 reversal 80:14 81:13 85:3,10 86:13 92:12,16 165:1

364:19 377:13 495:10,14,17 496:23 499:11 541:14,21,22 553:22 603:17 605:9 reversals 91:22 reverse726:16 reversed 80:15 85:9 547:2 reversible 213:1,4 review 1:4 2:4,20,25 8:10 10:1,6 11:12 14:20 20:17

21:4 27:20 28:17 31:4 42:13 75:19 76:3 81:5 83:11 91:14 93:5 109:1 147:11 209:8 210:10 241:4 248:17 254:7 284:14 306:5 318:4 330:24

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512:20,22,25 570:19 726:7,13,15,16,23 RNAs 184:9 road 183:24 312:20 343:3 684:7 roadmap 14:22,24 20:11 ROBERT 6:19 34:8 35:7 68:14 190:4,17,26 191:3 191:9,14,17,22 192:20 193:2,5 242:24 460:12 548:18 550:15 556:16 564:18 565:22 566:25 570:18 575:16 576:8 598:12 606:9 609:11 615:3 665:14 726:6 735:9 robin409:24 robust25:18 40:24 63:18 64:3 123:14 156:1,9

291:21 330:13 398:22,22 414:6 493:8 494:2,6 502:18 524:7 549:8 559:1,5 563:2 573:11 601:18 610:13 680:20 681:1 697:2 robustly 157:14 robustness 25:4 175:10 414:11 419:7 445:14

493:13,24 500:16,18,20,24 502:21 503:6 582:5 601:24 605:22,24 606:6 653:3,4 697:7,17 rocks 155:3 rodent 36:17 Rodney 30:5 75:9,10,10 104:26 105:12 115:8,12 115:26 117:4 118:8,19 119:12,15,24 120:2,9 121:1,8,15 122:6,26 123:3,7,15 124:3,11,16,21 124:26 125:5,11,25 126:7,20 127:4,8,12,17 128:2 128:4,11,14,21 129:19 130:6,11,16,23 131:2,6 133:1,19 134:12,24 135:17,21 136:10,19 137:5,8 137:16,21 138:9,15,21 139:5,9 140:2,25 141:12 143:2,23 144:25 145:24 146:7 147:7,20,25 148:19 149:2,12,17,22 150:6,9,13,19 151:21 152:3,14 153:2,13 154:19 161:22 174:10 189:2

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182:5 199:23 285:10 291:7,8 292:6,8 310:8 312:20 313:23 328:12 412:17 423:12 503:4 511:20 516:16,17 527:1 573:7 614:21 653:5 656:13 688:18 709:4,6 733:12 runoff723:25 runs 293:8 351:23 513:13 517:1 rush 327:19 rushed 327:14 RXR644:1 667:21 S S 216:20 SAB 539:11 sack 288:23 289:1 sacrifice 144:8 sacrificed 221:10 228:21 safe 14:6 15:25 17:20 329:21 342:25 safeguard446:15 safeguards483:6 safely 335:15 621:8 639:16 safety 8:22 11:7 319:25 323:18 sagittal 145:7 146:19 274:8 sake 178:5 255:25 651:8 659:16 salt 129:25 sample 103:25 104:1,11 120:6 131:19 145:20

254:13,13 289:5,12 293:15,16,17 359:5 366:12 370:9 371:3 394:12 396:23 399:7,8,9 405:6 417:14 418:23 421:2,8 437:4,8 439:7,10,13,16 469:20 473:23 499:1 555:10 568:1 571:22 572:13 582:24 588:10 589:8 594:17,26 595:2 604:15 654:10 694:1 698:20 699:7 715:14 722:26 723:3 sampled 82:7 100:2,3,3 109:15,22 158:26 380:20

534:5 samples 26:1 104:4 113:8 145:12,22,26 146:26 166:17 181:24,26 182:5 188:23,23 254:17 268:5 268:8 287:17 288:16,25 289:14,24,25 293:17,18 295:6 366:5,12 367:14 376:9 379:22 382:3 402:7 566:17 569:22 571:14 572:9,17,21 573:1,3,15 656:12,13 723:4,5 sampling 85:22 91:13 93:16 99:4 100:9,11 145:23

254:18 268:9 289:3,23,26 418:4 424:2 571:18 sandpaper515:1,2 Sandy 2:10 4:26 5:2,4 7:26 8:13,18 12:21 29:26

34:6 35:24 36:14 38:10,15 59:6 61:3,26 63:15 65:6 66:22 68:13 70:15 75:7 104:23 105:2,6,9 114:22 115:10,13 126:9,13 128:6 130:14 133:15 134:4 137:9 139:8 145:15 148:10 150:23 153:5 153:10 154:7 176:7 177:3,13 179:1,20 180:13,23 184:4 187:12 189:11 190:3 195:17 196:9 198:9 198:18 199:12 200:13 201:17 202:19 203:13,22 205:3,23 227:15 229:20 239:8 241:22,24,25 242:1 243:16,25 253:22 254:21 263:22 266:12 267:9 270:1 273:20 274:21 276:1,12 277:6 284:2 285:22 295:22 305:14 314:21 323:19 333:8,10 337:15 338:11 341:10 344:21 345:18,25 346:17

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19:1 20:4 21:3 33:23 35:2 171:19 198:4 283:10 284:24 325:20 419:22 421:15 482:13 539:11 549:7 567:21 568:22 573:9,16 608:8 620:25 625:25 632:26 634:23 735:12 742:3,3,22 744:16 745:20 SAPs 14:22,26 633:20 735:23,25 745:10 SAP's 2:25 SAS 154:6 183:20 592:2,2,3 725:13,17,19 736:8 SAS-based 88:26 sat 639:16 735:11 747:23 satisfactory 221:26 236:8 374:20 651:13 satisfied 3:5 703:3 satisfying 530:16 734:1 Saturday 600:19 Saulnier 89:20 481:21 save 31:2 61:14 585:16 602:5 627:12 saw 39:19 52:17 123:12 132:2 138:26 139:15 161:18 163:13,23,24 166:4,5,20 167:16 169:1,2 169:17,22 170:10,12,13 172:18 197:13 227:18 238:24 249:1 253:22 254:19 284:19 294:14,15,16 297:5 311:2 320:5 321:3 323:1 330:20 341:5,6 365:5 377:16,20 433:17 438:9 509:26 586:15 587:2 588:13 613:16 651:9 662:12 672:12 702:18 saying 5:4 30:17 86:21 104:10 112:7 126:16,18 137:13 152:1,4 155:5 162:24 172:13 178:9 179:11 186:18 194:11 197:25 228:16,25 233:26 235:16 248:13 249:12 263:9 327:22 334:21 335:20 337:26 339:10 340:16 341:5 342:14 379:23 380:3 407:8,13 408:9 411:9 413:1 421:7 489:12 579:19 580:4 582:20 584:14,15 630:3 642:22 646:12 651:16 654:21 655:14 656:22 660:8 738:19 says 162:13 229:1 236:24 239:23 319:11 380:25

390:7 392:9 408:16 420:13 469:14 474:14 479:10 479:26 503:6,19,20,25 504:13,18 505:3,5,6 506:22 507:11 520:5 542:26 548:26 555:12 563:18 569:19 571:17 574:7 599:12 670:18 675:14,25 694:10,10 scale46:11 350:10 463:7,17 537:14 564:3 scales 405:16 Scanes 7:3,3 36:14,15 37:1 38:7 63:15,16 64:2

145:18,19 146:4 147:2,15,21 177:14,15,18,25 178:10,14,19,24 180:15 203:14,15 204:19 205:2 243:4,4 263:22,23 264:5,16 267:14,15 268:3,15 269:7 338:11,12 339:5,14,18,22,25 340:3,7,21 347:22,26 352:24,25 354:12,13 356:6,7 357:23 357:24 359:20,24,26 360:2 362:13,20 363:6 364:16 368:10 372:16,21 373:26 374:15 375:23 376:20,21 380:18 381:4,7 405:25,26 407:25,26 408:12 411:1,2 413:15,16 419:4,5 428:15,16 430:12,13 440:2,3 460:18,18 569:16,17 646:21 646:23 648:3,11,12 649:18 650:4,26 651:22 652:11 654:6,21,26 655:17 659:7 661:5,8 665:18 665:18 672:23,24 736:13 scarified 195:2

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scars 731:20 SCC483:20 scenario 306:3 scenarios 508:21 scenes 9:15 scheduled 3:21 252:23 276:8 507:9 schema 253:3 schematic46:10 212:25 213:14 223:8 scheme 253:6 256:12 632:6 691:1 schemes 228:13 Schlenk 5:15,15 30:3 31:16 33:22 34:1,5 115:14,15

116:21 117:25 118:12 119:8,14,20,26 120:7,18 121:6,9,21 122:21 123:1,4,9,26 124:5,12,19,24 125:1,9,14 126:1,9,12,14,24 127:6,10,15,26 128:3,5 242:8,8 443:18,20 457:20 459:21,21 467:26 468:1 476:13,14 488:7,8 492:21,25 501:19,20 508:14 519:1,2 528:3,4 532:21,22 538:16,17 540:22,23 541:26 542:6 664:23,23 728:18 743:3 School7:15 science 2:25 3:11 6:5 7:4 8:15,21 10:3,3 13:19 16:22 31:2 155:15 332:24 348:12 366:16 382:20 400:20 473:12,25 474:22 475:15,19 492:17 591:14 600:10 631:1 632:8 634:26 636:19 694:16 742:13 743:18 744:21 746:21 747:10 Sciences 7:15 scientific 1:1,4 2:3,4,18,20,26 3:24 4:8,21 5:1 9:26 10:4,8 11:5,9,12 20:10 27:8 241:3,4,5,18,20 284:9 306:12,16 323:25 326:5,10 343:4 347:5 348:7 400:5 435:15 443:5 444:9 459:3 534:16 545:9 616:6 635:14 664:7 697:13 718:18 732:7 743:26 744:7 746:1,4 scientifically 15:18 18:4 20:7 202:3 330:12 357:13

357:26 452:15 550:2 637:1 scientist 6:17 10:12,12 694:8 747:15 scientists 16:9 28:26 77:15 327:16,20 390:4 393:12

735:10 scissors 514:26 scoliosis 171:14,16,23,25,26 172:8,10,11 scope 63:21 162:1 198:26 439:14 631:23 636:15 score 84:5,6,7 89:18 90:1 91:22 177:20 378:9

379:1 462:24 463:22,23 464:3,5 478:15 562:25 scored483:22 556:25 scores 85:5 379:1,9,9,11,12,20 402:8,15,16 scoring 147:4 269:8 scorning 84:10 Scott5:26,26 148:11 150:23,24 152:1,4,25 153:4

184:5 187:12,13,18,20 188:1 189:5,9,14 190:1 198:10 202:19,20 203:13,16 235:2 236:19 237:17 238:5,10 239:8 242:14,14 243:25,26 248:2 249:21 250:2,5,9,16 253:1,18 266:12,13 333:15 333:16 335:13 336:16 337:14 460:1,1 569:8,9 617:5,6 627:16 632:16,17 633:17 635:1 636:2 637:12,13 643:19 645:1,2 646:11 656:7,8 659:2 665:3,3 669:26 670:1 672:9,10 681:14,16 682:14 682:15 683:26 684:5 685:19 686:15 690:5,6 702:14,15 703:15 707:5,6 712:8,9 717:13,14 723:13,14 730:26 748:4 Scott's 203:16 243:23 628:15 672:25 711:16 scratch 547:11 screaming 169:7 screen 9:20 14:8,10 15:23 16:4 17:3 22:5,9 121:2,5 148:13 187:15 234:17,23 299:12 349:14 368:3 470:12 594:1 622:16 623:10 626:8,13 634:16,17 screened 220:11 644:2,4 screening 1:5 2:6 11:3 12:3 13:15,19 14:13 15:4 17:2 18:1,2 21:3 22:3,15 41:26 96:4 241:6 282:6 336:7 355:3,6 407:11 459:4 622:14 628:5 630:12 630:20 631:12 637:23 638:1 644:2 664:5 682:6,7 682:21 686:18 702:3 705:5,25 716:15 717:10 739:15 742:18 screenings 623:13,24 624:7 screens 284:26 screw586:13 SDWA 634:18 729:19 se 126:2 135:6 226:7 259:18 353:24 411:23 412:6

553:25 652:25 655:7 667:25 seamless 9:16 search 115:20 270:25 seated 276:12 seats 38:16 105:7 153:11 345:26 441:26 seawater211:13,26 222:21 266:21 second 20:16 24:1,6 27:21 64:20 153:24 162:20

210:14 211:19,20 212:13,14 213:22,23 214:12 215:11 217:17 221:7,10 241:4 250:8 255:22 256:2,4,4,9 258:21 263:24 290:9 291:1 293:21 294:7,17 295:1 296:10 316:15 319:12 320:7,10 320:25,26 323:8 325:10 335:17,21 340:5 354:2 356:12 369:23 381:25 384:9 404:21 417:17 423:2 428:23 432:7 451:9 457:8 465:25 497:6,7,19 510:22 524:12 566:9 597:7 598:23 604:21 681:5 687:19 692:23,25 695:25 699:25,26 700:1,2,10 700:10 705:23 707:18 711:12,22 715:12,15,18 716:26 720:24,25 721:11 722:20 733:22 737:20 737:22 743:22 745:12 secondary 69:13 80:5 82:8,11 87:2 90:20 91:22

92:2,7 94:5 95:5 97:5,13,20 98:3 100:8,11 101:13 102:5 103:3,26 106:25 108:15 109:23,25,26 110:2,3 120:6 131:1,4,12 132:3 140:21 142:8 298:1 303:3 371:24 425:6 448:7 451:5 453:9,22

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348:10 361:10 368:21 369:16,18,26 370:2,6,11 376:14 379:26 380:2 381:2,5 397:16 410:16 420:21 421:4 422:8 430:10 444:17,20 445:3,26 452:24 453:20 461:22 462:3,11 464:26 469:14,19 503:13 504:16 506:14 507:23,25 509:10,14 528:26 534:26 545:22 566:16 607:13 637:11 660:6 704:4 707:25 713:24 728:6 sectioning 145:3,7 146:19 178:20 188:22 sections 146:5,10,11,18,24 158:4 159:6 174:13 179:12 274:10,11,12 347:16 357:22 364:14 380:25 422:26 443:17 511:1 523:4 550:12 556:14 562:2,20 617:3 666:15 secure 21:8 security 257:16 sediment 344:14,18 447:5 621:21,24 622:4 sediment-dwelling 641:10 see 22:15 24:9 30:10 32:1 38:11 40:18,19 47:19

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214:22 215:14,19 216:9,24 217:24 252:8 267:6 267:11 276:3 330:3 365:6 379:20 412:24 431:17 444:25 473:7 491:14 507:5 528:11 533:15 539:8 555:22 564:9 581:6 599:9 601:9 615:14 618:24 689:25 693:2 713:12 726:3 745:7 seek 28:20 35:17 246:23 744:3 seeking 25:23 28:7 363:12 seen47:5 48:8,11 49:19 51:10,21 52:18 53:8 56:6

60:14,19 63:6 72:4 74:4,21 87:5 108:8 130:12 198:9 203:9 206:17 207:22 214:17 215:23,24 216:11,19,26 217:18 218:12 223:7,25 228:11,14 229:5 239:3 248:22 262:5 264:20 269:4,16 280:17 294:16 315:2 318:10 319:13 326:19 331:17 334:25,25 340:23 358:17 364:25 413:3 419:13 420:16 432:14 435:26 436:4 479:13 524:11,14 546:26 551:14 552:3 553:6 558:19 560:23 562:10 563:26 564:2 582:4 588:6,7 590:4 598:17 651:10 667:18 668:23 669:8 676:25 678:20 685:15,15,16 693:13,15 708:17 712:2 sees48:20 671:18 segways 124:7 seldom 595:6 select 35:3 268:8 280:14 387:11,14 501:23 519:25

709:13,24 selected 25:3,17 26:2,8,9,24 27:25 34:9 55:18,18

60:15 62:5 232:10 287:19 289:24,24,25 380:19 380:21,22 387:10,12,13,18 394:1 407:14,21 422:24 488:26 629:2 667:8 714:21 selecting 62:11 228:8 287:2 392:18 559:18 671:13

685:24 708:4 716:15 selection 33:2,15,21 219:5 292:14 293:6 294:22

298:10 321:10 364:7,13 368:17 372:23 383:13,17 384:1,8 406:15,22 410:3 425:9 461:14,21,26 462:13 470:19,24 478:3 484:26 491:12 556:6,12 556:17,18 576:22,26 596:10,14,17 623:13 646:22 666:7,13 674:23 675:2 679:13 681:5 682:8 686:10,12 716:1,16 717:25 selective 23:17 280:20 selfish 733:24 self-starting 220:9,12,15 semantics 660:5 semen 366:12,12,15 semi-domesticated 55:15 semi-static 211:21 223:12 225:2 send 117:4 406:7 569:25 585:4 657:8 680:15

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76:11 84:25 107:9 112:16 175:17 197:21 207:2 208:10 215:3,24 216:2 218:1,5,6,7 221:3 223:25 225:17 226:14,18 231:6 244:12,23 245:3 246:6 246:19 247:5 248:23 249:6 254:24,25 255:6 257:9 258:2,6 259:5 263:19 280:13 282:11 302:15 306:8 317:20 318:13 324:20 342:17 351:12 376:5,8 413:10 424:17,21 433:4 450:24 454:2 526:4,9 534:13 546:13,17 549:8 558:1 571:7 576:3 609:19 620:23 622:9 626:14 643:13 669:4,6 680:20 684:16 687:11 690:10,12 691:22 692:9,9 700:22,23 702:19,26 708:6 713:7 715:23 716:1 719:5,9,10,12,18,19,23 720:5,7,22,24,25 721:5,22 724:1,4 726:9 sensitivities 226:20 518:9 633:18 708:21 726:24 sensitivity 48:10 97:8 197:19 224:23,26 226:11

231:12,26 245:25 246:18 255:15 258:20 278:24 279:1,4 286:13 295:17 306:7 309:4 324:17 325:4 326:12,23 328:14,15 332:3 364:10 390:2 413:9 435:8 436:18 437:16 438:15 439:1 446:19 461:17 483:11,24 503:22 518:3,8 531:7 534:18 535:14 535:22 537:22 556:9 568:15 612:23 620:22 622:11 634:22 635:8 666:10 684:23 686:7 690:23 708:17 709:18,22 715:2,3,23 718:16 719:4 sensitization71:21 340:22 440:17 sent 8:7 137:13 152:14 188:24 239:21 277:23

326:13 392:12 442:4,13 654:11 657:10 separate 65:23 109:7,10 223:6 255:10 289:6

385:18 590:25,25 635:24 686:19 separated 100:26 228:24 677:7,9 separately 284:18 285:20 325:26 583:23 674:7

677:5,10 740:24 separation 362:14 385:19 SEPs744:8 September 20:20 sequence 370:10 sequenced 129:3 sequences 523:20 sequential 31:24 sequentially43:8 serial451:23 572:6 series 93:18 137:24 158:7 325:19 346:18 356:25

376:26 403:11 592:2 599:13 648:18,25 662:9 736:22 serious 111:5 125:6 131:24 279:15 498:23 574:24

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332:22 374:7 583:20 surprising 169:1 249:4,5,8 406:8 506:4 surrogate 157:8 surrogates 548:13 surrounding448:24 Survey 6:5,8 242:16,18 460:4,6 665:6,8 survival44:14,23 49:25 64:15 82:26 83:1,3 88:10 108:14,15,20 109:25 110:1 186:6 202:6 212:23 214:1,14,26 221:21 222:1 223:14,17,17 263:16 297:12 313:5 316:13,15 325:14 329:16 334:15 338:8,9 481:16 515:7 542:20 587:19,20,20 618:11 624:19 637:21 638:25 691:2 700:18 706:8 716:25 721:21 722:10 survived 72:12 257:11 268:9 survives 691:8 surviving 116:11 469:15,17 survivorship 690:1 722:11 susceptible45:11 58:2 63:14 257:12 suspect49:1,14 57:10 278:13 436:19 suspected 37:21 282:12 suspended 121:3 suspicion41:18 suspicious 83:8 sustained 71:3 Sweden210:5 251:26 swiftly 277:4 swimming 169:4 switched 183:16 switching 222:6 symmetry 373:8 425:9 sympathetic 374:23 syncs 317:7 syntheses 231:3 synthesis 168:5 207:26 348:3 512:23,24 synthesize 678:26 679:2 synthesized 8:8 syringe 267:3 system 11:23 12:4 17:26 18:15 19:4,7,12,16 21:10

21:13,18 22:12 23:7 57:6 114:1,5 120:19,23,24 121:5 132:11 147:4 148:6 175:18 178:6,8 180:16 192:14 211:6,14 219:25 222:19 226:1 241:11 266:15,21,24 267:2 269:8 283:16 286:9 294:6,7 317:13,17 323:7 340:24 371:8,13 373:12 378:20 407:22,23 427:18 430:18 432:3 456:5 471:26 472:1,3 487:1 494:2,6 534:10 552:13 557:19 617:16 622:20 623:2 626:17,23 641:23 667:12 670:19 696:2 697:18 systematic 88:25 146:5,12 178:19 381:4,6 478:10 systematically 88:24 380:1 systemic 69:13 327:5 systemically 178:2 systems 10:6 11:16 15:17 76:9 79:18 112:15 113:18,20,21,25,26 114:5,6 168:9 207:7,12,16 220:10 223:13 230:14 266:1,4,10,15 267:3 294:2 317:6,10,23 337:11 370:7 379:7 407:1 426:26 430:19 432:26 471:25 486:23 617:17,19 618:16 619:25 622:17 623:6 631:16 637:26 640:16 681:25 696:14 T T 20:8,16 36:23 138:18 139:1 233:17 315:8 316:26

337:4 449:22 455:18 457:7 618:19,22 619:4 623:7,12 630:17 634:14 637:23,25 678:13 680:24 683:4 700:26 701:7 713:7 table 30:4 49:22 66:26 145:18 161:26 170:5,19

216:6,12 241:20 311:19,21,23 319:13 395:11,12 395:18 396:18,21 435:13 436:20 437:13,18 471:14,16,19 474:11,11 504:20 520:8 548:3,5,7 600:12 633:4 648:14,24 691:16 696:2 700:17 721:14 723:18 746:20 747:23

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719:13 tackle441:22 544:10 tackling492:15 tactful 635:13 tadpole 567:4 586:1 594:9 tadpoles 190:18 567:1,9,15,24 654:20 tail 151:13 680:25 tails 680:23 take 3:6 9:6 38:11,16 49:4 58:6 59:4 104:24 108:10 108:11 114:18 118:5 120:20 127:21,23 129:24 131:23 134:4,20 145:20 146:9,23 149:25 152:23 152:24 153:6 154:6,26 162:9 166:23 176:5 178:10 182:14 183:21 191:25 205:5 210:25 212:12 236:21 258:7 273:12,13 276:7 284:17 285:19 291:13 295:26 296:14 311:5 316:15 318:15 321:4 330:5 332:18 344:19 345:11,26 354:14 364:3 380:2 381:25 384:24 391:4 396:11 397:2 403:16 404:13 412:9 419:16 441:21 468:11 468:16 484:24 494:4 507:10 520:26 533:4 542:10 544:16 547:14 576:3 577:20 578:15 593:19 595:10 615:18 628:24,24 629:12 635:25 646:19 677:12 678:23 680:4 681:12,14 703:17,18 722:4 723:18,21,24 730:7 733:23 740:8,26 741:4 742:1 taken 24:12 38:14 46:5 78:22 82:4 89:6 105:5 110:2,3 144:9 146:26 153:9 169:15 182:24 203:10 205:8 276:11 288:24 345:24 387:11 388:6 403:19 441:24 458:21 507:12 544:21 591:12 593:22 594:8 607:26 615:20 618:12 663:12 668:18 674:9 699:11 703:20 708:15 721:16 takes 9:14 73:21 86:19 138:4 152:21 153:11 159:2 173:9 195:5,12 197:1 210:19 285:7 323:2 424:10 739:17,18 take-home 170:4 172:24 174:17 196:17 talk 8:2 14:13,15,23,23 15:22 20:1 21:23 33:4 39:6

39:14,19 43:2 72:24 87:22 88:21 100:7 122:18 130:17 133:21 134:1 141:7 148:14 151:25 155:6 162:9 163:10 165:20 177:19 196:16 206:4,7,13 206:16,18 210:20,21 213:26 224:24 271:18 318:5 323:22 327:6 333:1 334:6 343:9 346:12,24 363:23 385:8 386:4,4 393:2 397:15 398:6 421:5 464:10 484:12 508:7 543:7 546:24 555:14 628:3 640:23 672:22 686:12 687:2,8,8 692:7 talked 29:1 179:22 203:16 209:20 259:13 270:13

271:4,9 290:24 324:22 338:15,16 359:9 373:26 378:14 394:23 414:24 416:26 418:7,18 423:26 424:1,20 432:4 487:26 490:8 526:22,24 532:11 584:22 588:7 591:26 592:12 635:11 643:10 658:6 659:2 682:22 700:22 701:15 704:14 725:18 talking 11:15 21:1 35:26 71:5 141:16 154:13 155:8 157:20 183:24 192:26,26 200:19,22,25,25 206:22 216:23 307:19 311:25 379:2 432:26 543:23 591:16,17 623:15 646:25 656:21 692:15,26 693:1 693:6 723:19 738:23 talks 212:25 323:24 337:17 346:22 401:6 402:11

432:21 433:11 479:12 481:1 632:18 Tamoxifen 96:25 107:4 485:19 494:24,26 495:2

506:11 tank 80:22 90:15 91:13 109:7,9,10,14,14 121:5 132:16 137:18 153:19,21,21 160:26 161:3,4,4,6 171:8,9 173:3 182:21,23 183:14,17,21 192:4,15 211:9,11 228:3,17,26 229:9 273:13,14,14 395:8,8 472:3,8,10,11,13 475:7,10 477:21 519:14,15,18 519:24 584:8 586:20,23 587:3,24 592:14,16 594:12,14,18,19,21 673:26 694:24 696:4,17 699:11,13,13 tanks 108:26 109:10,14 191:18,19 192:26 273:12

471:12 472:13 502:19,20 583:1,15,15 584:8 594:17 595:11 674:1 695:6,23 696:1,2,4 tank-to-tank477:14 target 336:1 355:7 410:11 488:22 522:11 691:15

704:26 targeted 34:19 232:16 356:22 376:7 513:7 targets 47:14 125:20 457:10,11 489:8 task 39:1 520:6 584:16 730:16 733:7 747:20 tasked 308:20 taxa20:25 21:23 23:12 34:25 35:18 36:18,26 37:2

37:5,7,17 63:10 72:5 189:23 206:22 348:17,20 550:26 teach 391:24 399:20 590:11 702:1 738:24 Teacher5:24 team 2:15 205:17 428:17 742:5 746:19 tech 5:24 6:9 113:1,12 136:26 137:1 242:13 459:26

665:2 technical 224:17 227:1 301:9 497:12,15 499:19

500:21 511:6 513:16 566:19 567:18,23 568:19 604:14,24 605:17 726:13 747:24 technicality 662:5 technically 313:25 726:17 technique 54:10 174:19 269:24 289:3 370:18

402:18 583:24 techniques 11:15 100:16 103:17 123:6 174:22 189:19 266:6 269:21 372:10 379:8 509:22 546:5 578:17 582:15 630:5 644:11 technologies 13:25 181:6 technology 19:16 26:12 181:1 382:20 646:6,14 tedious 714:19 teleconferenced 251:2 Telephone 1:10 tell 35:9 69:11 87:15,16 88:16 104:5 117:24 123:16

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594:25 622:18,22 650:21 660:22 661:12 690:12 691:7 termed 22:4 481:19 terminated44:14 108:12 221:6,16 terminates 528:24 terminating 304:24 534:2 termination 23:24 159:21,22 433:12 532:18 535:4

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32:7 34:3 39:12,16 40:10,14,17 41:2,3 42:9,11,20 43:4,7 44:23,25 45:5,6,7,9,23,26 46:13,20,26 47:20,24 48:24 49:14,22,24 50:22,23 51:1,10,11 51:13 52:7,12,23,26 53:4,15,21,23 54:2,17 55:9 55:14 56:1 57:19 58:8,16,23 59:16,26 60:1,13,19 60:23 61:11,19,20 62:11 63:3,4,5,5 64:19,21 65:1 65:4,12,21,22 67:2,3,23 68:10 69:3,12,15 70:13 71:26 72:8 73:5,20 74:3,6,22,25,26 101:3,24 121:26 123:5 147:8 150:26 157:20 159:7 170:15 170:17 171:19 173:17 174:18 175:18 176:18 182:9 185:13 186:6,13 187:6,11 189:19,22 191:6 192:1 193:24 194:6,7,21 196:1,1,4,18,19 200:23 202:11 206:14,19,21 209:3,11,11,18 213:12,26 214:22 215:15 216:23 217:7,20 218:2 219:6 220:3,6 221:19 222:1,2 223:20 224:8,17 226:25 227:23 229:4 230:10 231:2,7,11,14,26 232:8,19 232:26 233:22 236:2 237:7 238:8,18,23 239:1,3 245:12 246:2,5,8,23 247:11,26 249:19 250:21,21 251:4,9 254:9 256:16 257:5,18 260:2,8 262:10,17 264:10,14,24,26 265:5,19 267:21 269:19 309:9 311:7 339:7 340:23 348:18 353:18,26 354:8 356:3 358:8 359:15 362:1,3,9,9 372:23 378:11 382:17 386:26 400:2 408:5 409:1 410:20 411:10 411:15,24,26 412:24 413:3 423:12 426:18,20 427:24 431:10,19 434:22,22,24,24,26 441:6 444:1 453:1 472:5 491:19,23 492:4,5 493:19,21 500:21 501:22 503:15 506:23 508:25 519:26 542:4 566:4 586:25 588:24 590:11 598:4 602:4 605:9 617:19 620:24 632:10 636:13,19,19 645:12 647:4,20,22,26 651:5 673:3,5,15,17,18,19,20 674:11 685:8 695:19 706:7 716:5 729:13 732:16 741:2 743:7,10 744:1 terrestrial 619:20 terribly 167:10 670:15 test2:6 9:22 10:6 14:8,16 15:17 16:1 17:11 18:24

20:1,17 21:17 23:17,20 24:9,15,25 25:1,13,15,16 25:20,21,22 26:6,8,22,22,23,24 27:5,14 28:13,21 29:9,12,14 30:7,20 31:7,11 32:3 35:19 36:2,16,17 37:6 39:3,5,7,16,21,22 40:5,5,19,22 41:7,9,10,23 42:5,19 43:10,22,26 44:8,14 45:3 47:18 49:4,5,18 50:9,11 54:26 56:20,21 58:10,14,17,25 59:7,13 60:3,11,13 61:5,16,22 62:22 64:7,8,10 74:2,25 75:14,18,20,22,23,23,24,25 76:2,4,6,7,17,23,23 77:6,10 78:2,3,3,16,24 79:13 80:8,17,18,19 81:1 81:3,3,4,4,6 83:1,2 86:3,4 88:9,17,25 89:20,22 91:16 93:6,18 98:23 99:7 104:15,17,19 105:16 107:18,20,21,22,23,26,26 110:16 112:12,12,13 113:7,21 114:2,8 121:2 122:19 127:13,18,26 132:15,23 133:24 134:1 136:2 139:1 142:5 148:6 151:7 152:9,11,12,21 155:6,6,7,7,11,17,21,23 156:1,26 157:2,14,25,26 159:21,22 160:1 161:21 164:15 165:13,16 167:3,4 168:17,21 169:18,19 175:2 177:8,10 188:3 194:6,7,18,18,19,20 195:19 195:20,20 196:7 199:26 200:10,12,16,18 202:16 202:17 206:5,6,11,15 208:17,19,24 209:6,7,12,21 210:6 211:7,22 212:3,5,5,10,12,13 213:14,15,22 214:6,7,18 217:26 219:2,20,24,25 220:4,5 221:1 221:16 222:18 223:16,20 224:12 225:4,17 226:5 227:9,24 230:8 232:2,4,5,9 234:14 237:16 245:17 245:20 246:10 247:3,11,16,18 248:5 249:14,26 251:9,14,18,20 252:4,25 253:8,10 258:16 260:5

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23:16 25:3 27:23 29:7 31:12,12 34:19 42:15 43:16 76:19 77:22 90:25 103:18 114:1 132:11 138:25 150:26 151:18 156:17 182:14 184:19 208:22 224:13 225:13 226:16 230:2,3,4,5 232:1 234:3 244:21 245:1 249:20 251:13 253:6 283:6 284:24 285:8,9 286:8,8,12,22 287:5,9,25 290:18 302:4 306:1 310:4 316:10 322:10 324:16 325:10 332:17 334:9 341:12,14,17 347:15 349:7 352:15 354:11 356:26 369:11 374:8 377:15 378:14 383:8 406:25 407:5 428:26 430:5 433:22 437:20 439:7 442:12 443:16 444:19 462:6 470:13 478:5 500:13 522:16 529:8 534:15 538:6,12 539:15 545:21 546:8 558:16 570:11 576:17 582:17 584:9 589:1 589:12 599:7 611:2 614:2 617:2 618:20,23 621:5 622:10 623:7,13 624:23 625:9 629:15 634:20 638:9,10,16 641:3,14 642:17 648:22,26 649:4,13 649:15,15,17 650:10,12,22 651:25 652:4 655:19 655:20 659:22,23 660:11 661:2 666:25 668:24 669:3 674:18 680:5,5 681:20 682:8,9 693:13,15 698:5,6 700:5,6 708:11,16,18,26 709:7,16,20,20 709:25 710:2 713:19 714:9,16,24 717:8 724:6 testis 84:2,3 85:3 158:12 169:26 170:1 testosterone 207:24 626:3 tests 1:6 17:12 40:26 47:10 56:13,24 57:11 58:19

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478:18 481:18 592:3 687:20 709:11 USGS 25:14 43:12 50:12 51:10,22 60:26 74:14,18 usual45:1 746:12 usually44:3 199:23 220:11 236:12 238:17 270:26

306:4 309:11 344:14 345:10 350:4 391:23 489:14 586:23 uterotropic 233:19 utility 39:11 48:25 181:25 246:22 433:6 447:15

457:7 547:12 718:23 utilization 15:17 29:12 445:10 454:24 utilize 54:20 56:23 57:24 58:22 59:24 201:19 221:5

222:5 225:12 265:19 620:8 utilized 34:2 43:25 44:3 57:12 58:19 62:19,23

64:22 175:23 189:20,21 211:11 214:6 220:7 221:11 223:18 224:4 228:8 235:24 237:13 247:26 268:22 353:20 445:16 455:1 526:23 529:12 utilizing 160:6 548:1 744:25 UV 168:2 U-shaped 215:9 482:14 U.C 7:23 461:2 U.S 6:5,8,17 25:14 101:4,5 152:22 160:6 242:15,17

242:22 252:17 253:13 310:20 429:4 431:2 460:3 460:5,10 474:13,17 509:1,12 549:9,10 620:9,11 622:10 640:13 650:23 665:5,12 707:14 V vacuoles 147:6 vague 369:2 423:1 valcuolization 290:8 valid 32:3 282:9 285:16 289:18 293:2 324:14 490:3

577:14 669:11 validate 182:1 542:14 596:18 597:17,26 743:23 validated 15:17 16:23 23:20 27:7 155:18 286:9

288:2 289:11 290:21 293:12 295:12,13 314:6 315:17 320:15 332:7 457:12,12 518:7 539:21 541:11 542:21,25 546:5 551:22 580:8 657:25

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25:12 26:3,17 27:3,8,16 28:3,5,9 37:20 39:9 44:20 59:11 73:25 132:24 181:12 189:22 194:1 206:13 208:9 209:20 251:12,13 252:22 253:2 278:11 280:4 281:16 282:26,26 287:9 292:8 295:3 296:9,12 299:6,18 302:2 312:19 319:26 320:3 324:10 326:3 376:12 383:15,19 384:11 430:4 433:21 444:8 454:13 464:21 470:21,26 487:26 491:1 494:9,9 508:9 510:19 522:15 537:6 537:12,17 538:1 539:13,14 540:14,17 541:8 543:4,8,8 547:9 548:6 551:13 560:5 566:10 576:3 576:24 577:2 597:22,25 598:7 602:7 611:1 614:25 633:22 650:11 652:19,25 656:12 662:7 670:12 674:25 675:4 697:12,22,23 707:19 713:18 716:16,26 727:26 743:21,25 validations 130:9 validity 214:6 223:15 280:9 306:12,16 324:2,13

326:6,10 367:24 562:23 598:22 625:17 648:18 validly496:18 valuable 12:19 16:16 176:5 245:11 423:11 438:1

547:3,5 614:10 738:4 value 23:5 39:20 66:26 67:3 68:9 74:24 87:15

88:11,12 107:19 138:7 151:16 157:18 162:22 245:14 246:23 247:24,24 257:17,20,20 258:14,14 291:17 294:7,18 307:6 314:10,15,16 319:17 321:5,21 322:25 328:12 338:13 376:15 405:7,17 435:16,21 436:16 479:19 528:20 529:2 572:23 574:17,20 605:5,5 629:5,6 669:20 690:2 695:8 710:3,5 718:19 valued420:14 values 23:6 85:18 86:10,11 99:1 100:12 101:10 110:10 115:3 118:13 120:20 138:6 190:10 275:20 295:4 304:19,23 345:2 365:6 420:3 496:25 509:3 529:26 543:2 567:5,9 680:23 variabilities 218:14 417:10 418:4 variability 55:24 66:5,24 67:6,11,13,23,26 139:14 157:14 190:18 196:13 198:12,14 199:10 200:3 278:14 281:14,17 282:15 292:16,21 293:14 294:12 309:22 322:25 385:9 390:23 391:4 394:25 394:26,26 395:1 398:1 414:4 416:5 417:9,10 418:25 420:15,20 421:10,12 422:17 427:24 436:25 449:1,11 454:12 465:15 466:11 476:25 480:3 493:5 494:3 497:23 498:1 503:14,21,25 504:17 505:10 506:7 508:11 511:15 520:14,15,17 520:19 530:9 536:8 537:25 557:23 560:14 572:2 584:8 594:13,14 600:23 601:15 604:17 605:16 607:25 610:15 668:23 671:20 692:5 693:13,15 696:26 697:18 714:21,22 715:4,5,6,10,13 variable 54:9,16 78:25 157:11 289:20,21 297:3 390:15 399:26 400:3 404:18 417:3 451:3 455:22 472:26 474:24 475:18 478:23 479:8 481:10 500:19 510:5 558:24 561:1 567:5 571:25 578:12 587:23,25 714:25 variables 55:24 291:13,19 292:21,22 361:14

395:17 397:3 400:13 405:21 437:21,23 473:9,13 473:26 474:1,16 475:16,20,20 479:2 501:8 520:8 578:8,15 725:4 variance 85:25 90:17,17,17 98:26 146:16 229:2,5

235:5 237:18 394:18,20 395:2 396:3,6 401:6 417:4 571:21 578:25 583:13,15,17 584:5 594:24 594:24,25 595:3 677:24 678:19 variances 89:9 398:10,14,17 402:20,24 403:6,9

404:26 473:19,21 474:7,8 574:11 584:2,4 725:19 variants 676:25 variation 89:21 151:4,8 181:14 199:2 218:18,21,26

235:12,13 279:14 311:2 366:1,6,19 372:12 394:23 395:2,7,7,8,10 396:5 415:5 417:14 436:22 437:14,21 438:2 440:15,16 477:12,13,14,14,15 477:16,18 503:8,16,18 520:8 546:7 558:7,8,11,16 558:18 560:8 567:26 570:22,25 571:25 572:20,21 573:4 585:23 608:3,4,14 613:16,23,24 614:11,24 670:21 673:26 677:25 733:5 variations 52:3 65:8 218:22 236:1 579:13 680:8 varied 62:3 145:24 164:12 561:2,4 573:1 varies 580:20 684:23 variety 8:8,9 36:18 37:21,24 200:8 209:10,16

216:16 217:22 235:26 287:20 494:3 619:6 638:12 658:11 710:9 various 85:5 217:1 299:12 302:6 446:19 504:14

533:23 564:19 580:20 582:8 597:16 700:6 747:22 vary 57:17 390:2 446:19 466:7 508:11 698:1 varying 581:22 695:24 vast 351:25 vastly 281:24 vector 513:25 vehicle 249:26 250:2,8 263:26 264:4,6,11,19,26

265:5,7,8,10 358:24 671:3 vehicles 201:19 vehicle's 264:26 Velcha 306:6 ventral463:3,16,17,19,20,26 verbal 382:13,16 verbatim4:15 verbose482:10 verification 238:16 322:10,12 344:6,7 verify 229:11 versa 361:21 version 83:16 89:1 91:12

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212:17 217:23 230:14 246:16,18 252:12 277:25 282:8 318:6,9 322:1,2 337:11 598:8 615:23 616:19 617:18 618:23 619:18 620:7 623:13 628:5 631:20 637:26 640:16 641:22 667:10 vertebrates 40:12 207:17 246:20 317:24,26 611:13

616:14 618:4,22 619:5,20 625:12,24 628:18 630:16 687:12 vertebrate-active 209:17 258:4 260:4 verts 337:13 vessel481:23 vetted 315:12 332:15 viability44:12 45:5 74:11 213:13 214:4 394:7

466:25 515:4,5,9 521:2 viable 141:19 481:8 576:2 vice 361:20 vice-versa 143:17 Vicki 6:4,4 179:1,3,7,15,19 242:15,15 273:21

450:10 456:19 460:3,3 467:15 477:6 491:3 502:4 518:26 527:24 532:4 534:25 539:26 540:13 548:23 554:15 563:10 569:3 574:2 599:24 606:12 610:2 615:7 665:5,5 Vicky 147:12 videos 370:20 372:13 514:19 518:17 522:3 743:13 view247:23 323:15 335:26 362:20 363:8 371:8

380:8 413:24 495:1,3 555:15 582:9 654:26 655:3 676:3 678:13 721:4 viewed440:8 viewing 359:3 viewpoint44:22 380:4,5 414:19 views 258:19 vigor533:7 vinclozolin43:17 52:12,17,19 53:4,6,14,16 59:13

59:16,18,25,26 60:4,20 63:20 67:9 97:25,25 98:13 101:20 102:15 103:1,6,11 121:10 129:16 130:5 131:24 132:12,24 151:1 210:1,24 226:19 238:25 255:10,13,21 266:3 269:14 279:13,18,26 280:15,17 281:11 292:1,25 295:7 301:7,11,18 395:13 409:9 411:15,17 412:6,14 414:25 436:1 438:8 486:2,8,12,16,19,21 487:5,8,10,18,25 492:23,25 494:17 496:7 497:9,16 498:19 499:9 499:14,16 500:8,10 501:21,24 503:14,17 504:15 510:1,4,8,9,16 517:6 violated402:25 violation402:26 Virginia 1:17 5:24 242:12 459:25 664:8 665:2 virtually 51:7 319:9 350:16 736:23 visibility 483:1 visible463:8,15 vision 14:4 18:17,18 29:13 visit597:4 visual 17:17 482:25 577:8 visualization 122:25 514:21 visually 582:6 vital 620:6 621:14 vitellogenic 143:14 169:6 vitellogenin 80:4 82:15 83:7 84:20 87:12,17 90:20

91:21,26 92:11,21,24 94:10,18 95:8,17,18,19,22 96:9,10,18,19 97:2,2,3,14,16 98:9,21 100:13,14 100:15,17 101:16 102:11,12,15 103:9,26 106:20 107:1,3,4,6 108:15,21,21 109:25 110:1 124:9,10 125:22,22 133:14 135:3 139:18 140:7,8,8,9 143:11,12,16,19,20,22 144:1,6,9,19 160:3 161:10 164:4 169:5,6,8,17 170:17 173:15,21 175:26 176:3 181:11,15 185:2,4 186:19 187:1,11 298:1 298:23 299:25 303:1 308:11,13 309:23 314:8 338:6,6 367:18,25 368:3,12,12,13 433:16 453:9 453:24 454:2 466:23 468:25 479:1 482:21 494:22 494:26 495:2,23 496:22,24 512:2,6,18 522:11 523:19 532:15,17 536:2,9,13,16 541:23 543:14 546:23 552:11,20,21,23 553:21 557:7,9 559:10 560:19 570:9,18,19,23,26 571:3,4,6,11,23 572:15 573:10,18,19,26 575:10,18 603:5,14 609:15,19 726:7,12,15,21,23,23 vitro 17:4 21:4,6,14 22:19 37:9 95:26 96:3 368:2 vivo 17:5 21:5,11 22:19 23:7,11 29:8 96:23 233:20

376:6 462:5 vocal427:14 741:10 vocalizations 373:9,11 volume 18:26 180:19 181:4 204:11 716:4 volumes 132:16 238:17 Vtg 185:19,22 467:1 483:26 513:6,23,25,26 515:25

524:4 570:9,12,14,16 574:7 VTG1 504:24 513:7 VTG2 513:7 vulnerability 56:10 309:16 vulnerable 57:26 351:19 354:25 W W 156:12 wait539:22 589:9 594:1 waiting 663:3 waiver589:21

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339:1,18 378:8 380:19 388:16,16 389:8,10 409:17 416:6 442:11 488:17 490:1 527:25 547:11 586:17 587:4 598:25 599:4 628:7 633:21 655:12 655:17 662:6 687:5 720:5 722:4 740:6 waste 360:20 640:17 641:12 716:4,5,6 wasted 334:11 375:4 585:20 watch739:8 water 13:23 14:7 15:25 16:1 17:20 116:22,25 117:5,9,17,20 118:1,2 121:3 214:13 220:12 222:20 223:16 265:1,9,21 275:4 303:24 322:13 344:15,18 358:13 387:20,20,23 390:20,24 391:3 447:6 465:11,11,12,14,17,21 501:8 511:9 515:17 515:19 554:18 558:8,9,15,18,20 608:2 620:26 696:1 737:6 waterborne 303:20 waters 116:24 117:11,15,16,17,24 325:8 water-only 344:19 Watts462:21 wax747:25 way23:12 24:15,20 56:2 61:13 64:24 70:6 73:20

74:17 78:23 88:25 103:22 108:10 110:12,23 111:6 116:6 134:21 140:13 145:21 146:5,5,8 152:24 153:3 154:18 160:21 162:26 175:24 180:7 180:9 182:23 183:19,23 186:22 190:2 193:16 195:25 197:21 200:8 201:3,24 256:4,9,13 257:26 270:26 279:13 292:12 307:17 311:8 312:11,16 321:20 322:2,12 325:7 326:16 331:10 340:17 342:4 345:3 348:13 355:24 356:13 358:15 366:21 367:19 385:18 391:6,26 392:1,6,7,25 393:5 396:16,17 397:6 398:12 400:13,14 401:7 410:11 414:25 420:10 424:23 425:20 431:18 436:23 440:19 442:3,3,18,18 473:10,11,24 474:26 475:9 477:19 478:1 481:4 485:6,9 489:6 494:5,5 502:22 544:10 550:23 559:15 579:5,25 580:13,19 584:14 584:18 586:4,13 587:17 590:13,26 591:15 592:14 608:1 609:4 610:4 618:12 623:5 635:13,13,19 636:4 640:19 642:6,10,11 645:8 651:4,20 654:3 656:10 657:5 658:19 662:25,26 676:13 679:4 682:7 685:16,16 686:21 689:19 690:22 691:18 695:23 725:3 731:4,18,22,22,23 733:19,19,26 740:5 746:10 ways46:1 93:13 100:14 137:18,19 147:18 178:3 179:24 207:18 258:13 322:18 331:26 362:26 392:1 440:15 475:16 477:17 513:20 544:1 564:21

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490:24 496:14 549:1 560:21 597:8 603:3 673:18 682:3 weaker451:22 weakness432:7,15 506:17 weaknesses430:17 432:3 448:4 639:24 640:14

708:5 weanling23:23 weary206:2 web 1:9 365:26 366:20,21 428:12 514:20 webcast243:18 283:17 664:16 webcasts 514:22 website 20:19 331:9 web-based 189:3,4,18 week 10:6 12:16,19 13:3 44:15 46:20,22 49:13

50:5 79:17 80:1,2 90:22 100:5 108:14 109:12,12 159:2 251:1 273:10,11,12 301:26 390:9,13 469:16 505:16,20,22,26 506:16 521:2 536:3 641:5 728:19 730:16 745:16 weekly 51:20 505:4 weeks4:17 44:5,13 46:13,16,18 51:15,16 64:11

78:24 79:11,17 80:1 85:23,23,24 87:4,8,10 100:3 100:4 108:4,16 143:18 158:7,8,18,25 159:3,5,5,5 175:12 181:21 289:6 333:26 340:14 389:5,12 393:15,24,24 424:11 456:7,10 497:10 530:22 532:17 536:3,8 571:1,6 week's 9:19 11:24 13:2 Weibull401:1 weigh 328:7 335:11 391:1,1 654:21 weighed 259:3 389:13 390:21 408:18 686:16 weighs 257:24 weight 17:9,10 18:3 20:4,18 34:2 99:23,23,24 121:11,12,16,17 161:10 171:7 172:16 187:11 214:1 221:2 236:10,10,14,17,20 262:8,11 263:16 263:18 319:7 328:24 330:11 333:21 336:4 337:6 361:19 390:19,23,26 395:16,19 432:5 433:6 480:11 555:13 578:6 591:12 592:8 595:2 599:4 668:3,17 673:4 684:2,7 705:22 719:25,25 720:23 722:16 734:18 744:15 745:1,2 weighted 161:4 180:5,6 192:16 396:11 480:10

594:7 595:1,5,7 weights45:13 100:6,17 172:18 180:2,3 261:18

291:14 533:5 592:17,20 595:13 weight-of-evidence 356:22 371:21 411:10 434:16

608:8,10,24,25 623:20 624:15 welcome 8:23 241:25 323:20 421:23 434:25 545:3

590:5 664:10 745:8 wells 641:18 well-balanced 691:7 well-characterized 597:21 well-constructed 570:4 well-defined445:7 500:7 well-documented 575:10 well-established 226:6 523:6,25 524:3 well-known 184:14 559:22 well-sequenced 523:18 well-studied 302:21 well-validated 294:6 went66:26 90:22 98:4,6 155:20 163:4 166:10,13 187:23 190:13,15 198:26 249:15 269:14 337:17 341:7 379:8 547:23 589:13 599:18 616:18 675:18 676:9 677:2 719:13 720:8 727:21 735:21 747:14 weren't269:15 288:18 325:22 404:26 409:18

723:7 we'll 276:21 277:11,12 284:3 286:5 295:24 311:6

329:25,26 333:2,12,25,26 345:22 346:1 347:23 358:7 364:3 378:8 399:24 404:3 410:3 416:2 421:25 428:5 434:2 435:2 441:22 444:15 452:7 455:10 458:10,18,19 459:14 468:11 476:8 484:13 484:18 485:4,4,25 487:14 494:7 501:12 544:20 546:20,24 547:7 549:16 576:8 583:6 592:11 596:16 597:4 601:9 609:9,19 610:24 615:26 622:4 624:5 625:4 626:18 628:1,12 629:24 633:5 633:6,10 638:17 643:23 645:19 646:18 653:2 675:7 692:7 707:13 714:3 727:16 736:24 741:25 743:22 744:9 745:5 we're 195:2 234:20 241:26 250:18 253:23 260:10

267:6,22 268:9,24 276:7,19 277:2,7 284:22 285:2 285:5 286:3 290:13 295:11 296:11 302:10 303:15 305:3 306:14 307:1,2,13 308:4,6,14,18,20,22,23 311:10,21 312:20,21 313:16 316:2,18,19 319:14 320:10,11 324:3 326:17 327:10,12,22,24 329:19 330:6,9,10 331:18 333:21 334:7,13 335:10 336:2 336:3 338:4,5,21 340:21 343:3,14 346:18 350:21 350:22 353:13 355:19 357:9 359:3,5 363:15 366:22 371:14,14 373:12 376:1 379:21 380:5 382:10 386:1 390:19 392:17 403:21 412:5,13,24 417:8 419:14 424:18 437:16,25 442:1 469:6 470:9 489:11 493:1 507:9 511:16 515:8 542:18 542:19 543:12 544:22,23 549:21 576:13 580:23 590:19 615:22 623:6,17 627:18 629:23 630:9 631:25 632:11,24 633:2 634:3,11,12,14,21,25 635:6 636:23 640:5,19 645:15 646:18 650:26 652:17 656:22 657:26 659:21 660:11 662:21 672:18 674:14 684:16 690:10 692:19,26,26 693:4 693:6 701:4,22 703:22 715:26 722:12 723:19 727:23 728:1 730:8 731:26 736:20 742:13,21

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