· the use of all available existing information; · the development of a relational database to efficiently access and utilize information; · an initial sorting of the universe of chemicals into categories based on an operationalized Conceptual Framework; · the development of high throughput pre screening data and its incorporation into the database; · the use of the database to summarize empirical data and estimate fate and effect parameters where possible; · the use of the database to establish criteria for sorting chemicals where appropriate; and · the use of a compartment based concept to accommodate subjective weighting where appropriate.

The EDSTAC viewed its role within EPA's broader mandate to protect human health and the environment and the broader testing authorities available to EPA. As such, the EDSTAC did not limit itself solely to requirements of the Food Quality Protection Act and the Safe Drinking Water Act Amendments of 1996. The Committee believes it is important to have priorities driven by scientific considerations and explicit value judgments, rather than by existing regulatory requirements.

B. The Universe of Chemicals and Initial Sorting 1. The EDSTAC recommends that pesticides, commodity chemicals, environmental contaminants, naturally occurring non steroidal estrogens ( e. g., phytoestrogens, mycotoxins), food additives, cosmetics, nutritional supplements, and a set of representative mixtures be prioritized for endocrine disruptor screening and testing.

2. The EDSTAC recommends that scientific considerations be used as the primary basis for prioritizing chemicals for endocrine disruptor screening and testing. Statutory authority to protect human health and the environment is embedded in long standing federal legislation, as EDSTAC Final Report Chapter Four August 1998 4  79 well as the Food Quality Protection Act and the Safe Drinking Water Act.

3. The EDSTAC recommends that the chemicals under consideration ( approximately 87,000 compounds) be sorted into the following four primary categories based on the operationalized Conceptual Framework:

· Polymers are to be placed on hold ( with some exceptions) pending review of their monomers, oligomers, other components, additives, and degradation products ( approximately 20,000 to 25,000 compounds). · Chemicals to be considered for endocrine disruptor screening ( approximately 62,000 compounds) which lack sufficient data to be placed on hold or to proceed to definitive testing or hazard assessment will be subjected to the priority setting process for T1S. · Chemicals with sufficient data are to bypass screening and proceed directly to testing or hazard assessment ( approximately 500 to 600 compounds). · Chemicals with sufficient data are to go to hazard assessment ( expected to number approximately 50 to 100 compounds)

C. Polymers 4. With some exceptions, the EDSTAC concluded that, due to molecular weight, polymers are less cause for concern than other classes of chemicals with regard to endocrine disruption. However, there is some concern regarding the intestinal absorption capacity of neonates. Because of the lack of information on polymers produced prior to 1979 ( the date of the initial TSCA Inventory), coupled with the low likelihood that polymers themselves are a concern for endocrine disruption, the EDSTAC offers the following recommendations.

· All new polymers with a number average molecular weight ( NAMW) greater than 1,000 daltons and all previously manufactured ( or " existing") polymers ( regardless of NAMW) are to be held from priority setting for endocrine disruptor screening and testing pending the outcome of the screening and testing of their monomer, oligomer, and other components. · The monomers, oligomers, and other components of polymers, as well as " new" polymers ( i. e., those that went into production after 1979) with a NAMW less than 1,000 daltons are to undergo priority setting, screening, and testing as appropriate. · Chemicals on the EPA SDWA Contaminant Candidate List ( CCL) should be used to identify the potential degradates of polymers which are most likely to present environmental exposure and which should, therefore, be subjected to priority setting, screening, and testing, as appropriate. EDSTAC Final Report Chapter Four August 1998 4  80 · If monomers, oligomers, or other components of a polymer are determined to have endocrine disrupting properties, an exposure assessment should be performed. At this stage, all potential exposure routes for a component would be determined, including the potential for the component to be available from the polymer. · As the Agency gains experience with endocrine disruptor screening and testing of monomers, oligomers, and " new" polymers ( i. e., those that went into production after 1979) with NAMW less than 1,000 daltons, it should apply that experience toward development of an approach to address " existing" polymers ( i. e., those that went into production before 1979).

D. Priority Setting Information Categories and Criteria 5. The EDSTAC recommends using existing exposure related and effects related data and information to establish criteria for accomplishing initial sorting. The Committee identified the following subcategories of information that could be used as the basis for sorting and priority setting and developed principles regarding their use.

Exposure Related Information and Criteria a) Biological sampling data b) Environmental, occupational, consumer product, and food related data c) Environmental releases d) Production volume e) Fate and transport data and models Effects Related Information and Criteria a) Toxicological laboratory studies and databases b) Epidemiological and field studies and databases c) Predictive biological activity or effects models ( e. g., SARs, QSARs) d) Results of high throughput pre screening E. High Throughput Pre Screening 6. The EDSTAC found there was a general lack of endocrine effects data for the vast majority of chemicals. To address this problem, the EDSTAC recommends that, if demonstrated to be feasible, eight in vitro transcriptional activation assays should be conducted in a high throughput pre screening mode ( i. e., with the use of robotics and other automated processes). The objectives for conducting these assays in a high throughput mode is to:

· provide some information about the affinity of chemicals to bind to the estrogen, EDSTAC Final Report Chapter Four August 1998 4  81 androgen, and/ or thyroid hormone receptors; · use this information in conjunction with other exposure and effects related information to determine the priority by which chemicals should be advanced to T1S; · improve QSAR models; · provide a source of information to help focus the selection of Tier 2 tests for those chemicals that bypass T1S; and · generate data that can be used to identify chemicals that may be of concern at low doses.

7. The EDSTAC recommends that the high throughput pre screening ( HTPS) transcriptional activation assays be conducted on:

· the estimated 15,000 chemicals that are currently produced in an amount equal to or greater than 10,000 pounds per year; · chemicals that are permitted to bypass T1S and go directly to T2T; · chemicals that are permitted to bypass both T1S and T2T and go directly to hazard assessment; and · all pesticides ( both active ingredients and formulation inerts).

8. The EDSTAC recommends that HTPS results for the " bypass" chemicals not be used to set priorities for T1S, but to improve QSARs and inform dosing considerations, particularly during the interim period when research on low dose is being conducted, and to inform decisions regarding the types of tests that would need to be conducted in T2T.

9. The EDSTAC recommends that existing QSAR models be derived and supplemented with data from the HTPS assays, thereby expanding the predictive ability of these models.

10. The EDSTAC recommends that EPA explore the feasibility of creating an archive of a subset of HTPS project chemicals which can be accessed by researchers interested in studying endocrine mediated toxicity or in validating new screens for endocrine disruptors.

F. Mixtures 11. The EDSTAC recommends that EPA include a limited set of mixtures that span a range of physical and chemical properties in both the feasibility demonstration project for the HTPS assays, as well as the validation effort for the T1S assays.

12. If the screens are shown to be capable of handling a diverse set of mixtures in the HTPS feasibility demonstration project and the T1S validation steps, EPA should use expert judgment, guided by a set of prioritization criteria, to evaluate the literature and to decide on a limited set of mixtures to enter HTPS.

13. The battery of screens validated for use in the screening program should be used to evaluate the mixtures examined in HTPS. If appropriate, screening should be followed by testing. EDSTAC Final Report Chapter Four August 1998 4  82 14. The EDSTAC recommends that a comprehensive literature evaluation be undertaken to identify exposure and effects data on mixtures that do not undergo HTPS. This information would be used to inform the prioritization for Phase II and subsequent phases of the screening and testing program which would use the same prioritization criteria as those used for single chemicals.

15. The EDSTAC recommends that representative sample mixtures be selected from the following categories and be subjected to HTPS ( if feasible) and to T1S:

· contaminants in human breast milk; · phytoestrogens in soy based infant formulas; · mixtures of chemicals most commonly found at hazardous waste sites; · pesticide/ Fertilizer mixtures; · disinfection byproducts; and · gasoline.

G. Naturally Occurring Non Steroidal Estrogens ( NONEs)

16. Naturally occurring non steroidal estrogens include natural products derived by plants ( phytoestrogens) and fungi ( mycotoxins). Due to the ubiquitous presence of these compounds in foods, and due to the potential additive and antagonist effects of NONEs with other endogenous and exogenous hormonally active chemical substances, the EDSTAC recommends that:

· NONEs be included in the endocrine disruptor screening and testing program singly and in complex mixtures; and · the following NONEs be screened and, if necessary, tested.

Representative NONEs:

· Isoflavones: genistein, daidzein, miroestrol, biochanin A, formononetin, equol · Flavones: kaemferol, naringenin · Coumestans: coumesterol · Dihydrochalcones: phoretin · Triterpenes: betulafolienetriol ( ginseng) · Lignans: enterolactone Representative estrogenic mycotoxins:

· Beta resorcyclic lactones: zearalenone, zearalenol, zearanol EDSTAC Final Report Chapter Four August 1998 4  83 H. Nominations 17. The core priority setting process recommended by the EDSTAC focuses on giving high priority to chemicals with widespread exposure at the national level. The EDSTAC recognizes such a process could result in a low priority for chemicals where exposures are disproportionately experienced by identifiable groups, communities, or ecosystems. Therefore, the EDSTAC recommends that EPA establish a nominations process that:

· runs parallel to, but is separate and distinct from, the core priority setting process; · is designed to allow chemical substances and mixtures for which there may not be widespread exposures on a national scale, but for which there are exposures on a smaller scale, to be eligible to receive a priority for T1S; · allows for an early opportunity to submit nominations during each phase of the Endocrine Disruptor Screening and Testing Program; and · draws no less than 5% of the total number of chemical substances or mixtures subjected to T1S from substances receiving nominations but not selected through the main priority setting process.

18. The EDSTAC recommends that any nominated chemical substances and/ or mixtures that becomes a priority for T1S through the core priority setting process be removed from consideration within the list of nominated chemicals in order to ensure that the priorities drawn from the nominations process will compete only against other nominated chemicals.

19. In keeping with the overall purpose of the nominations process, the EDSTAC recommends that a different set of exposure related criteria be used to evaluate the priority for nominated chemicals compared to the exposure related criteria that will be used for the core priority setting process. Specifically, the nominations process should focus on exposures that are disproportionately experienced by identifiable groups, communities, or ecosystems rather than focusing on chemicals for which there is widespread exposure in the aggregate.

20. The EDSTAC recommends that if there are effects data for the nominated chemical, or if the chemical is similar to another chemical substance or mixture for which effects data are available, EPA should utilize those data as a secondary source of information to help set priorities among nominees.

21. The EDSTAC recommends that when the relative priorities of nominated chemical substances or mixtures are evaluated, EPA should consider those that meet the following criteria to be a higher priority than those that do not:

· chemical substances or mixtures where there is a likelihood of regular exposure, in contrast to those for which exposure occurs only rarely or occasionally; EDSTAC Final Report Chapter Four August 1998 4  84 · chemical substances or mixtures that affect a high proportion of people within a given community or workplace; and · chemical substances or mixtures for which there may be empirical or estimated ( i. e., model derived) effects related data regarding endocrine disrupting potential.

22. The EDSTAC recommends that EPA make use of all available information when evaluating nominations, including anecdotes, and other information gathered as part of the core priority setting process ( e. g., information contained within the Endocrine Disruptor Priority Setting Database).

23. To assist EPA in evaluating nominated chemicals, the EDSTAC recommends that EPA request the following types of information from the public regarding nominations:

· how exposure to the nominated chemical substances or mixtures may be disproportionately experienced by identifiable groups, communities, or ecosystems; · the reasons for the nomination ( which may include both exposure and effects related concerns) and any information that provides a basis for those concerns; and · the degree of support for the nomination from the potentially affected communities and/ or workplaces.

I. Endocrine Disruptor Priority Setting Database ( EDPSD)

24. The EDSTAC identified and evaluated numerous data sources associated with the exposure and effects information categories and criteria ( Appendix G). The Committee endorsed the integration of relevant and useful data sources into a prototype relational database, referred to as the Endocrine Disruptor Priority Setting Database. Although promising, the EDPSD could not be completed within the EDSTAC's time and resource constraints. Consequently, EDSTAC made a number of recommendations regarding continued development and use of the EDPSD.

· EPA should continue to develop and maintain the EDPSD as a tool that can be used to expeditiously sort and prioritize chemicals for endocrine disruption screening and testing. · The process used by EPA in developing the EDPSD, as well as the process by which it is used, should be open and transparent. · EPA should convene a multi stakeholder group prior to the completion of the EDPSD tool to ensure effectiveness, openness, and transparency. · After completion of the HTPS assays, this group should make use of the tool, along with the " compartment based" approach to priority setting described below, in assisting EPA as it develops the final priorities for T1S. · The EDPSD should not be limited to effects data that can be easily placed into a database format, but should also include data from peer reviewed literature. EDSTAC Final Report Chapter Four August 1998 4  85 · EPA should update the EDPSD at least every six months, and more frequently if time and resources permit.

25. The EDSTAC recommends that EPA provide resources to complete the Quality Assurance/ Quality Control investigations of files that are currently in the EDPSD. The EDSTAC further recommends that EPA provide resources to add new files to the EDPSD in stages. These files and stages for their addition could include:

1st stage: EPA's and others' databases that provide data on use for industrial chemicals and pesticides; information from pesticide ecotoxicity, fate, and toxicity one liners; chemicals that are non food use pesticide active ingredients and non food use other pesticide ingredients; chemicals on the Generally Regarded As Safe ( GRAS) list; and chemicals in the FDA Priority Assessment of Food Additives ( PAFA) database. 2nd stage: Data on chemical use that were not readily available in databases; chemicals and concentrations of chemicals in National Health and Nutrition Examination Survey ( NHANES), Total Exposure Assessment Methodology ( TEAM), and Agency for Toxic Substances Disease Registry's ( ATSDR) Hazardous Substances Emergency Events Surveillance ( HSEES) files; measured chemical fate data; and additional QSARs for endocrine disruptors. 3rd stage: Inclusion of HTPS data and improved QSARs.

The EDSTAC recognizes that the time and resources required to add new files will depend upon a number of factors, including: when pending files are received, the format of received files, the determination of whether to use files as sources of numerical or logical data, conversion of logical files to numerical files, completion of QA/ QC investigations of the files and data, and expediency of the input process.

J. Recommended Approach to Priority Setting 26. The EDSTAC identified a number of obstacles to the development of an " ideal" priority setting system, including the uneven quality and quantity of both exposure and, even more so, effects related data sources. Major characteristics of this unevenness include:

· Many more data are available on the effects of the relatively small number of currently registered active ingredients in pesticides ( approximately 900) than on the thousands of industrial chemicals produced in much larger quantities. · Biological monitoring data for humans are scarce. A relatively small number of chemicals ( on the order of 100 or less) have been routinely sampled in human blood and EDSTAC Final Report Chapter Four August 1998 4  86 urine in the United States, and the major U. S. national program for sampling concentrations in human tissues was discontinued in 1990. · Monitoring data for other organisms, while more numerous than human data, still focus on a relatively small number of chemicals. · Data on routine chemical releases to the environment, while markedly better than they were prior to the creation of the Toxic Release Inventory about 10 years ago, still encompass only 528 industrial chemicals and pesticides and frequently rely on engineering estimates rather than actual releases.

27. The EDSTAC recommended several principles to guide the development of a strategy for setting priorities for the large number of chemicals for which there are insufficient data to go to T2T or hazard assessment. The selected system should be transparent, should make use of the guiding principles for exposure and effects related data sources, and should be driven by empirical data, but not be held captive by them.

28. The EDSTAC recommends a " compartment based priority setting strategy" for prioritizing chemicals for T1S.

· The strategy builds upon the identification and evaluation of the different exposure and effects related information categories and criteria. · The term " compartment" refers to the consideration of these information categories either singly or in combination. · Illustrative examples of the four different categories of compartments include: · the integration of exposure and effects information; · the consideration of exposure information; · the consideration of effects information; and · specially targeted priorities ( mixtures, nominations, and naturally occurring nonsteroidal estrogens).

The specific compartments and the weights and/ or order in which they should be utilized have not yet been agreed upon. A target number of chemicals to go through T1S in the first phase of the program or during the life of the program has not been determined. Possible targets and how these targets might be affected by the compartmentalized approach to priority setting have not been agreed upon.

29. The EDSTAC recommends a number of next steps to further develop and refine the compartment based approach to priority setting, including:

· use of the EDPSD by a multi stakeholder group to further characterize and define what will be contained in each compartment; · whether, and if so, how to prioritize the compartments; and · how to address the possibility of overlaps between compartments. EDSTAC Final Report Chapter Four August 1998 4  87 30. The EDSTAC recommends using the schedule EPA has established for tolerance reassessments and pesticide re registration under the FQPA for setting priorities for those food use pesticides that meet the criteria for bypassing T1S and going directly to T2T. When planning for the registration renewal process begins, the FQPA requirement for endocrine disruptor screening and testing should be designated as a criterion for priority setting.

31. The EDSTAC recommends that priorities for T2T for all other chemicals ( i. e., non food use pesticides and other chemicals where the owner either wishes to voluntarily bypass T1S, or where the owner has met the criteria for completing the alternative, functionally equivalent, T1S assays) should be established on a case specific basis. However, the EDSTAC recommends that chemicals which receive a high priority ranking for T1S should retain that high priority ranking for T2T, even when the owner wishes to voluntarily bypass T1S.

XIII. Literature Cited Ankley, G., S. Bradbury, J. Hermens, O. Mekenyan, and K. E. Tollefsen, " Current Approaches to the Use of Structure Activity Relationships ( SARs) in Identifying the Hazards of Endocrine Modulating Chemicals to Wildlife," in Tattersfield, L., P. Matthiessen, P. Campbell, N. Grandy, and R. Länge ( Eds.), 19 40 SETAC Europe/ OECD/ EC Expert Workshop on Endocrine Modulators and Wildlife: Assesment and Testing, SETACEurope Brussels, 126, 1997. Adams, N. R., " Phytoestrogens: Lessons from Their Effects on Animals," Proceedings of the Nutrition Society of Australia, 1996. Baintner, K., " Intestinal Absorption of Macromolecules and Immune Transmission from Mother to Young," CRC Press, 1986. Cassidy, A., " Physiological Effects of Phytoestrogens in Relation to Cancer and Other Human Health Risks," Proceedings of the Nutrition Society, 55, 1996, pp. 399 417. Clarke, R. et al., " Estrogens, Phytoestrogens and Breast Cancer," Advances in Experimental Medicine and Biology, 40, 1996, pp. 63 85. Cowan, C. E., D. Mackay, T. C. J. Feitel, D. van de Mount, A. DiGuardio, J. Davies, and N. Mackay, " The Multi Media Fate Model," 1995, pp. 78. Gavaler, J. S., E. R. Rosenblum, and S. R. Deal, " Hidden Hormones in Alcoholic Beverages," R. R. Watson ( Ed.) Drug and Alcohol Abuse Reviews, 6, Alcohol and Hormones, Humana Press Inc., Totowa, N. J., 1995. Heindel, Jerrold J. et al., " Assessment of the Reproductive and Developmental Toxicity of Pesticide/ Fertilizer Mixtures Based on Confirmed Pesticide Contamination in California and Iowa Groundwater," Environmental and Applied Toxicology, 22, 1994, pp. 605 621. Jensen, A. A., and S. A. Slorach, ( Eds.), " Chemical Contaminants in Breast Milk," Boca Raton, FL, CRC Press, 1990. Johnson, B. L., and C. T. De Rosa, " Chemical Mixtures Released from Hazardous Waste Sites: Implications for Health Risk Assessment," Toxicology 105, 1995, pp. 145 156. Kleinman, R. E., and W. A. Walker, " Antigen processing and uptake from the intestinal tract," EDSTAC Final Report Chapter Four August 1998 4  88 Clin. Rev. Allergy, 2( 1), 1984, pp. 25 37. Lecce, J. G., and C. W. Broughton, " Cessation of uptake of macromolecules by neonatal guinea pig, hamster and rabbit intestinal epithelium ( closure) and transport into blood," J. Nutr. 103( 5), 1973, pp. 744 750. Lien, L. L., and E. J. Lien, " Hormone Therapy and Phytoestrogens," Journal of Clinical Pharmacy and Therapeutics, 21, 1996, pp. 101  111. Markovits, J., " Inhibitory Effects Of The Tyrosine Kinase Inhibitor Genistein on Mammalian DNA Topoisomerase II," Cancer Research, 49, 1989, pp. 5111 5117. Richard, S. E., and L. U. Thompson, " Phytoestrogens and Lignans: Effect on Reproduction and Chronic Disease," Antinutrients and Phytochemicals in Food, American Chemical Society, Chapter 16, 1997, pp. 273 293. Safe, S. H., and K. Gaido, " Estrogens and Anthropogenic Estrogenic Compounds," Environmental Toxicology and Chemistry, 17, 1998, pp. 119  126. Sheehan, D. M., and K. Medlock, " Current Issues Regarding Phytoestrogens," Polyphenols Actualites., 13, 1995, pp. 22 23. Swanson, M. B., and A. G. Socha, " Chemical Ranking and Scoring: Guidelines for Relative Assessments of Chemicals," SETAC Press, Pensacola, FL, 1997, pp. 186. Thomas, J., " Phytoestrogens and Hormonal Modulation: A Mini Review," Environmental Interactions, 1, 1997, pp. 5 12. U. S. EPA, Guidelines for Developmental Toxicity Risk Assessment, Fed. Reg. 56: 63798 63826A, 1991. U. S. EPA, Reproductive Toxicity Risk Assessment Guidelines, Fed. Reg. 61: 56273 56322, 1996. U. S. EPA, Guidelines for Neurotoxicity Risk Assessment, Fed. Reg. 63: 41845, 1998. Verdeal, K., and D. S. Ryan, " Naturally Occurring Estrogens in Plant Foodstuffs   A Review," Journal of Food Protection, 42, 1979, pp. 577 583. Walker, W. A., " Antigen Handling By the Gut," Arch Dis Child, 53( 7), 1978, pp. 527 531. Weaver, L. T., M. F. Laker, et al., " Milk Feeding Changes in Intestinal Permeability and Morphology in the Newborn," Journal of Pediatr Gastroenterol Nutr., 6( 3), 1987, pp. 351 358. Weise, T., and W. R. Kelce, " An Introduction to Environmental Estrogens," Chemistry and Industry, 1997, pp. 648 653. Westrom, B., J. Svendsen, and C. Tagesson, " Intestinal permeability to polyethyleneglycol 600 in relation to macromolecular " closure" in the neonatal pig," Gut., 25, 1984, pp. 520 525. Westrom, B. R., C. Tagesson, et al., " Decrease In Intestinal Permeability to Polyethylene Glycol 1000 During Development in the Pig," J Dev Physiol., 11( 2), 1989, pp. 83 87. Wiseman, H., " Role of Dietary Phyto Oestrogen in the Protections Against Cancer and Heart Disease," Bioactive Components of Food, 658th Meeting of the Biochemical Society, Liverpool England ( UK) April 16 19, 1996 Biochemical Society Transactions 24 ( 3), 1996, pp. 795 800. EDSTAC Final Report Chapter Five August 1998 Chapter Five Screening and Testing EDSTAC Final Report Chapter Five August 1998 Table of Contents I. Chapter Overview ................................................................................................................. 1 II. Tier 1 Screening Concepts and Design Parameters ............................................................ 2 A. Introduction to T1S ........................................................................................................... 3 B. Criteria for T1S.................................................................................................................. 3 III. Recommended Tier 1 Screening Battery ........................................................................... 4 A. Outline of Recommended T1S Battery and Possible Alternatives........................................ 4 1. Recommended T1S Battery............................................................................................ 4 2. Alternative Assays for Possible Inclusion........................................................................ 5 3. Validation of the Battery ................................................................................................ 8 4. Assays not Included in T1S ............................................................................................ 8 5. Developmental Exposure Screening Assay ..................................................................... 9 6. Methods to Select the Appropriate Dose Level( s) for In Vivo Assays ........................... 10 7. Routes of Administration ............................................................................................. 11 B. Scientific Basis for In Vitro Screening for Estrogen, Androgen, and Thyroid Activities..... 11 C. In Vitro Assay Overviews................................................................................................. 13 1. Estrogen Receptor Assays............................................................................................ 14 2. Androgen Receptor Assays .......................................................................................... 17 3. Steroidogenesis............................................................................................................ 18 D. Scientific Basis for In Vivo Screening for Estrogen, Androgen, and Thyroid Activities ..... 19 1. Unique Thyroid Action Properties to be Considered in Design and Interpretation of T1S ................................................................................................... 20 2. In Vivo Assays Using Other Vertebrates....................................................................... 23 E. In Vivo Assay Overviews.................................................................................................. 24 1. Rodent 3 Day Uterotrophic Assay ( Subcutaneous)....................................................... 25 2. Rodent 20 Day Pubertal Female Assay With Thyroid ................................................... 26 3. Rodent 5 7 Day Hershberger Assay.............................................................................. 27 4. Frog Metamorphosis Assay .......................................................................................... 28 5. Fish Gonadal Recrudescence Assay .............................................................................. 28 F. Alternative Assays for Possible Inclusion .......................................................................... 29 1. Placental Aromatase Assay........................................................................................... 29 2. Modified Rodent 3 Day Uterotrophic Assay ( Intraperitoneal)....................................... 29 3. 14 Day Intact Adult Male Assay .................................................................................. 29 4. Rodent 20 Day Thyroid/ Pubertal Male Assay............................................................... 30 IV. General Principles in Evaluating Tier 1 and Tier 2 Results............................................ 31 A. Introduction..................................................................................................................... 31 B. False Negatives and False Positives Within the Context of T1S and T2T .......................... 34 C. Specific Principles for Evaluating T1S.............................................................................. 35 V. Tier 2 Testing Concepts and Design Parameters .............................................................. 40 A. Introduction to T2T ......................................................................................................... 40 B. Outline of Recommended T2T Battery ............................................................................. 41 EDSTAC Final Report Chapter Five August 1998 C. Guidance for Selecting Tier 2 Tests.................................................................................. 42 1. Determining Which Taxa Should be Included in Tier 2 Testing for a Specific Chemical......................................................................................................... 43 2. Determining When to Perform Alternative Tests........................................................... 45 3. Determining Selection of Endpoints ............................................................................. 47 4. Chemicals That Bypass T1S and go Directly to T2T..................................................... 47 5. Potential Need for Supplemental Information to Complete T2T.................................... 48 D. Low Dose Considerations for T2T................................................................................... 48 1. Introduction to the Issue .............................................................................................. 48 2. Recommended Project to Address Low Dose Issues..................................................... 50 E. Methods to Select the Target Doses for T2T.................................................................... 52 F. Testing Antithyroid Activities in T2T................................................................................ 53 VI. Recommended Tier 2 Testing Battery ............................................................................. 54 A. Outline of Recommended T2T Battery............................................................................. 54 B. Two Generation Mammalian Reproductive Toxicity Study............................................... 54 C. Alternative Approaches to Mammalian T2T ..................................................................... 55 1. Alternative Mammalian Reproduction Test................................................................... 58 2. One Generation Test.................................................................................................... 59 D. Description of the Tests for Other Animal Taxa ............................................................... 60 1. Avian Reproduction Test ............................................................................................. 61 2. Fish Life Cycle Test ..................................................................................................... 62 3. Mysid Life Cycle Test .................................................................................................. 64 4. Amphibian Development and Reproduction.................................................................. 66 VII. Validation, Standardization, Methods Development, and Research............................. 66 A. Concept of Assay Validation and Standardization............................................................. 67 B. Statutory Need for Validation .......................................................................................... 67 C. Addressing the Validation Issue........................................................................................ 67 D. Validation and Standardization Process............................................................................ 68 E. Levels of Effort Necessary to Validate the Recommended Screens and Tests.................... 70 F. Screens and Tests Recommended for Further Research..................................................... 71 G. Stakeholder Involvement in the Validation Program ......................................................... 73 H. Preliminary Cost Estimates for the T1S and T2T Batteries ............................................... 74 VIII. Compilation of Chapter Five Recommendations.......................................................... 77 A. Tier 1 Screening............................................................................................................... 77 B. Principles for Evaluating Tier 1 and Tier 2 Results ........................................................... 79 C. Tier 2 Testing .................................................................................................................. 79 D. Validation of the Screening and Testing Batteries............................................................. 80 IX. Literature Cited ................................................................................................................ 80 Figures EDSTAC Final Report Chapter Five August 1998 Figure 5.1 Potential Sources of False Results in Screening and Testing................................ 36 Figure 5.2 False Positives Possibility Graph......................................................................... 37 Figure 5.3 False Negative Possibility Graph......................................................................... 38 Tables Table 5.1 Assays Included in Recommended T1S Battery and Possible Alternatives............. 6 Table 5.2 T1S Assays Related to Biological Activities Detected........................................... 7 Table 5.3 Mammalian Tier 2 Test Endpoints...................................................................... 56 Table 5.4 Avian Reproduction Test Endpoints ................................................................... 63 Table 5.5 Fish Life Cycle Test Endpoints ........................................................................... 65 Table 5.6 Estimated Costs per Chemical for Tier 1 Screening ............................................ 75 Table 5.7 Estimated Costs per Chemical for Tier 2 Testing ................................................ 76 Appendices Appendix J: References and Sources for Chapter Five  Screening and Testing Appendix K: Brief Overview of Assays Considered for Tier 1 Screening Appendix L: Protocols for Tier 1 Screening Assays Appendix M: Assays not Included in Tier 1 Screening Appendix N Endocrine Disruption and Invertebrates Appendix O: Protocol for Possible In Utero Developmental Screening Assay Appendix P: Examples of " Weight of Evidence" Determinations Appendix Q: Tier 2 Testing Study Designs Appendix R: STWG Preliminary Categorization of Tier 1 Screens and Tier 2 Tests Appendix S: Survey of Cost Estimates for the EDSTAC's Proposed Endocrine Disruptor Screening and Testing Assays EDSTAC Final Report Chapter Five August 1998 5  1 I. Chapter Overview This chapter describes the EDSTAC recommendations regarding development of a screening and testing program for assessing the potential of pesticides and other chemicals to disrupt endocrine function in humans and wildlife. Where appropriate, strengths and limitations of options are discussed and possible future research projects are identified to develop needed procedures. The EDSTAC established the Screening and Testing Work Group ( STWG) ( see Appendix D for a list of work group members) to assist in their efforts to provide guidance to EPA regarding the development and implementation of its endocrine disruptor screening and testing program. The STWG work formed the basis for this chapter and recommendations. Literature cited for all sections are found at the end of the chapter. Additional sources can be found in Appendix J.

After this introduction, the chapter is comprised of seven main sections: ( 1) the concepts and design parameters involved with Tier 1 Screening ( T1S); ( 2) the Recommended T1S Battery; ( 3) the general principles in evaluating Tier 1 and Tier 2 results; ( 4) the concepts and design parameters involved with Tier 2 Testing ( T2T); ( 5) the recommended T2T Battery; ( 6) a discussion of validation, standardization, methods development, and research; and ( 7) a summary of the recommendations made throughout the chapter.

The T1S sections begin with an explanation of the purpose of screening and identification of five criteria used to design the screening battery. An outline of the recommended T1S battery follows with brief overviews of each recommended assay and discussions of the value of including both in vitro and in vivo assays. Four alternative assays for consideration are also discussed. Finally, a section on evaluating the battery includes a discussion of a " weight of evidence" approach to evaluating T1S results.

In developing the T1S battery, the EDSTAC considered screening endpoints for their utility in screening chemical substances or mixtures for their potential to interact with the endocrine system. The goal of T1S is to detect chemical substances or mixtures capable of interacting with estrogen, androgen, or thyroid ( EAT) hormone systems. Assessing these activities is relevant as changes in them may adversely affect the development, reproductive function, or chronic health status of humans or animals. The objective of T1S is not to determine dose response relationships, confirm the mechanism of action, or determine the adversity of the chemicals' effect on reproduction and/ or development; however, screening assays must be sensitive enough to detect all known xenobiotics that act via the mechanism of action each assay is designed to detect.

The screening battery presented here has been designed to ensure that interaction with hormone systems will be detected. There are instances in which a choice had to be made between an assay that was highly specific for a hormonal activity and one that may be less specific but more sensitive and apical ( i. e., a more comprehensive assessment of functions that are relevant to reproduction, development, or chronic health). In those instances, the EDSTAC opted for the latter since it better fulfills the first criterion for screens ( that they be sensitive), and is better aligned with the overall mission of detecting effects regardless of mechanism of action. These EDSTAC Final Report Chapter Five August 1998 5  2 assays require varying levels of additional development, standardization, and validation before they can be reliably and routinely implemented as part of T1S.

The T2T sections begin with discussions of the purpose of testing, guidance for selecting Tier 2 tests, and the issues of low dose considerations and selecting target doses for components of T2T. An outline of the recommended T2T battery is followed by overviews of the mammalian twogeneration reproductive toxicity study, alternative mammalian tests, and tests using other vertebrate and invertebrate taxa. Finally, recommendations regarding implementation of the standardization, validation, and research program are presented.

The goal of T2T is to determine whether a chemical substance or mixture causes endocrinemediated adverse effects for EAT and to determine the consequences to the organism of the activities observed in T1S and their dose response relationships. This is done in the larger context of testing for reproductive and developmental toxicity potential by any mechanism ( including EAT) using study designs that provide a comprehensive assessment of relevant functions.

II. Tier 1 Screening Concepts and Design Parameters Chemical substances or mixtures can alter endocrine function by affecting the availability of a hormone to the target tissue, and/ or affecting the cellular response to the hormone. Mechanisms regulating hormone availability to a responsive cell are complex and include hormone synthesis, serum binding, metabolism, cellular uptake ( e. g., thyroid), and neuroendocrine control of the overall function of an endocrine axis. Mechanisms regulating cellular response to hormones are likewise complex and are tissue specific. Because the role of receptors is often crucial to cellular responsiveness, specific nuclear receptor binding assays are included. In addition, tissue responses that are particularly sensitive and specific to a hormone are included as endpoints for Tier 1 screens.

The following definitions are utilized in this chapter. Estrogenic refers to compounds whose effects are mediated through the estrogen receptor ( ER), initiating a cascade of cell/ tissue specific effects similar to those initiated by estradiol, as opposed to estrogen like for those chemicals resembling estrogen which are not or have not been shown to be mediated through the ER. Similarly, androgenic effects are androgen receptor ( AR) mediated, as opposed to androgen like effects, which may not be mediated via the AR. In contrast, the terms antiandrogenic and antiestrogenic are not specifically limited to AR and ER mediated interactions. In this context, agonists bind to the receptor and act like the endogenous hormone; antagonists bind to the receptor and appear to act opposite to the endogenous hormone. Antihormones can act via: ( 1) the steroid hormone receptor; ( 2) steroid hormone synthesis inhibition; ( 3) reduction of bioavailability by reducing the amount of free hormone in the serum; ( 4) increased hormone metabolism leading to reduced serum hormone levels; and ( 5) other mechanisms. EDSTAC Final Report Chapter Five August 1998 5  3 A. Introduction to T1S The number of chemicals needing evaluation is huge. T1S is intended to make the evaluation process more efficient by distinguishing those chemical substances and mixtures which may interact with the endocrine system from those that may not. The EDSTAC considered all known endocrine disruptors of EAT in developing the T1S battery and believes that the recommended battery, if validated, will have the necessary breadth, and depth to detect all currently known disruptors of EAT. Therefore, following application of the T1S battery, a chemical substances or mixture will be designated as having either: ( 1) the potential for EAT activity, which will require further analysis in T2T to verify and evaluate that potential; or ( 2) low or no potential for EAT activity, which will allow assignment of chemical substances or mixtures to the " hold box" ( see Section IV in this chapter, on general principles in evaluating Tier 1 and Tier 2 results for further discussion of how this decision is made).

In developing the recommended T1S battery, many existing and potential assays were evaluated for their relative strengths and weaknesses ( overviews of evaluated assays can be found in Appendix K). The recommended T1S battery contains mammalian in vitro and in vivo assays and in vivo nonmammalian assays. The T1S battery is designed to be a sufficiently sensitive screening mechanism so that chemical substances and mixtures which ultimately prove to be endocrine active for EAT in humans and wildlife are not missed. In this regard, sensitivity of the battery has been prioritized above specificity. In addition, T1S results should inform T2T, in terms of providing guidance on which tests to perform, which endpoints to include, and to assist in determining the range of doses to be used. These goals include identifying those doses ( doseresponse life stages ( most sensitive), and organisms ( most appropriate, sensitive, and at risk) in which adverse effects are likely to occur.

B. Criteria for T1S The T1S battery recommended by the EDSTAC has been developed such that, at the completion of the selected assays, the EPA and other stakeholders will accept, both scientifically and as a matter of policy, the assignment of chemical substances or mixtures as either having: ( 1) low or no potential for estrogen, androgen, or thyroid endocrine activity; or ( 2) as having such potential. The ability to accept either outcome requires that the chosen T1S battery meets the five criteria identified below.

1. The T1S battery should maximize sensitivity to minimize false negatives while permitting an as of yet undetermined, but acceptable, level of false positives. This criterion expresses the need to " cast the screening net widely" in order not to miss potential EAT active materials.

2. The T1S battery should include a range of organisms representing known or anticipated differences in metabolic activity. The battery should include assays from representative vertebrate classes to reduce the likelihood that important pathways for metabolic activation or detoxification of parent chemical substances or mixtures are not overlooked. EDSTAC Final Report Chapter Five August 1998 5  4 3. The T1S battery should be designed to detect all known modes of action for the endocrine endpoints of concern. All chemicals known to affect the action of EAT hormones should be detected.

4. The T1S battery should include a sufficient range of taxonomic groups among the test organisms. There are known differences in endogenous ligands, receptors, and response elements among taxa that may affect endocrine activity of chemical substances or mixtures.

5. The T1S battery should incorporate sufficient diversity among the endpoints and assays to reach conclusions based on " weight of evidence" considerations. Decisions based on the battery results will require weighing the data from several assays.

The T1S must be relatively fast and efficient while meeting the criteria described above. The EDSTAC recommends that if changes are made to the recommended T1S battery, based upon development of new, validated assays, the " amended" battery also needs to meet these criteria.

III. Recommended Tier 1 Screening Battery A. Outline of Recommended T1S Battery and Possible Alternatives1 1. Recommended T1S Battery The T1S battery recommended by the EDSTAC includes three in vitro assays, three in vivo mammalian assays, and two in vivo nonmammalian assays. Those chemicals which go through the HTPS program, if it is technically feasible and validated, would not be required to do the first two in vitro assays at the bench. Based on existing data, the EDSTAC believes this battery will detect EAT activity, provided all of the component assays can be properly developed, standardized, and validated.

In Vitro 1. Estrogen Receptor ( ER) Binding/ Transcriptional Activation Assay; 2. Androgen Receptor ( AR) Binding/ Transcriptional Activation Assay; and 3. Steroidogenesis Assay with Minced Testis.

In Vivo 1. Rodent 3 Day Uterotrophic Assay ( Subcutaneous); 2. Rodent 20 Day Pubertal Female Assay with Thyroid; 3. Rodent 5 7 Day Hershberger Assay; 4. Frog Metamorphosis Assay; and 1 Protocols for all these assays can be found in Appendix L. EDSTAC Final Report Chapter Five August 1998 5  5 5. Fish Gonadal Recrudescence Assay.

2. Alternative Assays for Possible Inclusion In addition, the EDSTAC has identified one in vitro assay and three in vivo assays as possible substitutes, if properly developed, standardized, and validated, for some of the component assays in the recommended battery. These assays are:

In Vitro 1. Placental Aromatase Assay.

In Vivo 1. Modified Rodent 3 Day Uterotrophic Assay ( Intraperitoneal); 2. Rodent 14 Day Intact Adult Male Assay With Thyroid; and 3. Rodent 20 Day Thyroid/ Pubertal Male Assay.

Combinations of the alternative assays, if validated and found to be functionally equivalent, could potentially replace three of the component assays in the recommended T1S battery ( in vitro steroidogenesis assay with testis, 20 day pubertal female assay, and 5 7 day Hershberger assay), thereby possibly reducing the overall time, cost, and complexity while maintaining equivalent performance of the overall T1S battery. Table 5.1 shows the assays included in the proposed battery as well as two possible batteries that would include the alternative assays. In addition, Table 5.2 shows the assays in relation to which of the biological activities they are expected to detect, that may be affected by exogenous agents and lead to EAT related toxicity.

One alternative battery would include the ER binding or transcriptional activation assay, the AR binding or transcriptional activation assay, the modified rodent 3 day uterotrophic assay ( administered by intraperitoneal injection), the rodent 14 day intact adult male assay with thyroid, the frog metamorphosis assay, the fish gonadal recrudescence assay, and, possibly, the placental aromatase assay.

The other alternative battery would include the ER binding or transcriptional activation assay, the AR binding or transcriptional activation assay, placental aromatase assay, the rodent 3 day uterotrophic assay ( administered by subcutaneous injection), the rodent 20 day thyroid/ pubertal male assay, the frog metamorphosis assay, and the fish gonadal recrudescence assay. EDSTAC Final Report Chapter Five August 1998 5  6 Table 5.1 Assays Included in Recommended T1S Battery and Possible Alternatives Assays Recommended T1S Possible Possible Battery Alternative 1 Alternative 2 In Vitro Estrogen receptor binding x x x Androgen receptor binding x x x Steroidogenesis x Placental aromatase ? 1 x In Vivo 3 day uterotrophic x ( sc) 2 x ( ip) 3 x 20 day pubertal female x Hershberger x 14 day intact male x 20 day pubertal male x Frog metamorphosis x x x Fish gonadal recrudescence x x x 1 = may be needed in battery to meet criteria 2 = subcutaneous 3 = intraperitoneal EDSTAC Final Report Chapter Five August 1998 5  7 Table 5.2 T1S Assays Related to Biological Activities Detected Anticipated to Detect:

Assay in Option Estrogen Agonism Estrogen Antagonism Androgen Agonism Androgen Antagonism Thyroid Related Effects Steroid Synthesis Aromatase Inhibition 5 a  Reductase Inhibition HPG1 In Vitro Estrogen receptor binding 1,2,3 X X Androgen receptor binding 1,2,3 X X Steroidogenesis 1 X Placental aromatase 3, 2? X In vivo 3 day uterotrophic 1,2,3 X ( X) 2 20 day pubertal female 1 X X X X X X Hershberger 1 X LH3 Hershberger + T 1 ( X) 4 X X LH3 14 day intact male 2 X X X X ( X) 5 X X 20 day pubertal male 3 X X X X X X X Frog metamorphosis 1,2,3 X ? 6 ? 6 ? 6 X X ? 6 ? 6 X Fish gonadal recrudescence 1,2,3 X X X X ? 6 X X ? 6 X Notes: 1 HPG   indicates that the model has an intact hypothalamic pituitary gonadal axis ( except for the Hershberger assay which does not have an intact HPG axis), and that effects on hypothalamic pituitary control of gonadal endocrine function would be evaluated. 2 It is likely that aromatizable androgens would be detected in this assay; however, given that there are no examples of environmental androgens, this point cannot be empirically demonstrated. 3 Agents that affect LH level would be detected in the assay. 4 Empirical demonstration that the assay detects estrogens is limited. The biology of the system suggests that they will be detected. 5 Empirical demonstration that aromatase inhibitors are detected is limited. If sensitivity to aromatase inhibitors is lacking, a placental aromatase assay would be added to this option. 6 The biology of these organisms suggests that these effects may be detectable. However, there are no empirical data to support the sensitivity of the assay for these endpoints. EDSTAC Final Report Chapter Five August 1998 5  8 3. Validation of the Battery In order to provide sufficient data to allow informed decisions about the relative merits of the recommended T1S battery component assays and alternative assays ( based on sensitivity, specificity, technical complexity, inter and intra laboratory variability, time, and cost), EDSTAC recommends that validation studies be initiated on all of the assays in the recommended battery as well as the alternatives.

If the assays comprising either of these alternative combinations ( see Table 5.1) are validated, the EDSTAC recommends performance of the alternative battery containing these assays using the same standard test substances recommended for validation of the initial battery, which were selected on the basis of predetermined criteria ( see Chapter five, Section VII, G). Sufficient information could then be available to allow an informed choice between the recommended battery or a variation, including the alternative assays, as the preferred T1S battery. This approach would be most expedient in the event that one or more of the recommended battery assays cannot be properly standardized and validated, since information would be immediately available on the alternative assays. The EDSTAC believes this process provides a model for validation and incorporation of new assays, as they may be developed and proposed, into the T1S battery.

The EDSTAC believes it is critical to acknowledge that the state of the science, with respect to assay development and species selection, is rapidly evolving, and bioassays are currently being developed that may offer distinct advantages over those assays and species presently recommended for use. This is particularly the case for selection of non mammalian species currently recommended for use in in vivo assays. Specific bioassays and species should be selected on a performance based approach. As improved bioassays and/ or those utilizing more appropriate species are developed and validated, EDSTAC strongly encourages their use as assays for screening and/ or testing. Selected assays identified as research priorities by the EDSTAC are discussed in Section VII, F of this chapter. The EDSTAC recommends that EPA set up a specific mechanism for evaluating and incorporating these and other new developments, as appropriate, into the program.

Given the wide range of species that may be adversely affected by endocrine disruptors, continued development of screens and tests is particularly important to ensure that a representative range of species and potential endocrine related effects can be evaluated.

4. Assays not Included in T1S Currently, there are no data available to suggest that thyroid effects of chemical substances or mixtures are mediated through the receptor. Therefore, the recommended T1S battery does not currently include a thyroid receptor ( TR) binding and/ or transcriptional activation assay. Nevertheless, the EDSTAC is recommending that the HTPS program include evaluation of the TR. The Committee believes including the thyroid assays in the HTPS program will enable EPA, EDSTAC Final Report Chapter Five August 1998 5  9 and others, to obtain a better understanding, at relatively low costs, of whether effects could be mediated through the TR.

Brief overviews of all assays considered by the STWG can be found in Appendix K. In addition, Appendix M includes more thorough discussions of assays that the work group considered in detail, but decided not to include in the recommended T1S battery. The EDSTAC's thoughts on the role of invertebrates in T1S can be found in Appendix N.

5. Developmental Exposure Screening Assay The EDSTAC considered all known endocrine disruptors of EAT in developing the T1S battery and believes, to the best of its knowledge, that the recommended battery, if validated, will have the necessary breadth and depth to detect any currently known disruptors of EAT. There is a concern, however, that chemical substances or mixtures could produce effects from prenatal/ prehatch exposure that would not be detected from pubertal or adult exposure. Furthermore, there are differing views within the EDSTAC about whether there is scientific evidence of known endocrine disruptors or reproductive toxicants that can affect the prenatal stage of development without affecting the adult or prematuration stages, and whether effective doses and affected endpoints may differ among the three life stages.

Notwithstanding these differing views, the EDSTAC recommends that EPA take affirmative steps, in collaboration with industry and other interested parties, to attempt to develop a protocol for a full life cycle ( i. e., with embryonic exposure and evaluation of the adult offspring) developmental exposure screening assay that can be subjected to validation and standardization. In addition to the general principles and criteria, set forth in Chapter Three, that guide selection of all T1S assays, the EDSTAC believes such an assay or assays must involve prenatal or prehatch exposure and retention of offspring through puberty to adulthood and structural, functional, and reproductive assessment.

The EDSTAC recognizes it may be difficult to develop a developmental exposure screening assay that meets both the criteria specified above, and the more general criteria for selecting T1S assays set forth in Chapter Three. However, the EDSTAC believes it is worth the effort. Furthermore, in Section VII, F, the EDSTAC has summarized protocols for in utero and in ovo developmental screening assays that could be further evaluated for this purpose ( an expanded discussion of an in utero protocol is included in Appendix O). Inclusion of these protocols is not intended to limit the creative effort that will be necessary to achieve the EDSTAC's recommendation.

Finally, the EDSTAC recommends that if such an assay were identified, validated, and standardized, the decision on whether it should be included in the T1S battery should include an evaluation of its potential to replace one or more of the recommended T1S assays and its overall impact on the cost effectiveness of the T1S battery. It should be noted, however, full life cycle assessments are included in the recommended T2T battery for mammals, other vertebrates, and invertebrates. These tests will employ a full range of doses, embryonic exposures, rearing offspring to adulthood, and a full complement of reproductive and developmental endpoints. EDSTAC Final Report Chapter Five August 1998 5  10 6. Methods to Select the Appropriate Dose Level( s) for In Vivo Assays All T1S in vitro assays ( including the steroidogenesis assay) will involve multiple dose levels, whether performed by HTPS or bench level methods, so a dose response curve and assessment of relative potencies can be developed. Results from the HTPS ( or its equivalent) will provide potency information ( i. e., EC 50) relative to a positive control such as 17 beta estradiol ( E2), diethylstilbestrol ( DES), testosterone, or T4 for those chemical substances or mixtures which bind to the E, A, or T receptor. Information on the in vitro effective doses of E2, DES, testosterone, or T4 can be used to set the dose level( s), based on the validation process, for the remaining T1S assays for these chemical substances or mixtures. There are no current data which indicate that thyroid toxicants act via binding to the thyroid hormone receptor( s). Thus, the recommended in vitro receptor binding/ transcriptional activation assay may not inform dose selection for in vivo T1S assays for thyroid endpoints. For these chemical substances or mixtures, prior information and range finding studies will be critical.

Subject to the results of the validation process, the EDSTAC recommends using one or more dose levels in the performance of the in vivo assays. Information to assist in selecting the dose level( s) includes:

1. prior information, such as that available during the priority setting phase; 2. results from the HTPS ( or its equivalent bench level assays); and 3. results from range finding studies, utilized for T1S dose selection ( see below).

A range finding study can be performed at multiple dose levels ( at least five) with a few animals per dose level and a limited number of relevant endpoints. The range finding studies specifically performed for each in vivo TIS assay will include the following:

· use of the same species strain, sex( es), and age as in the T1S assay; · use of the same route of administration, vehicle, and duration of dosing as in the T1S assay; · use of multiple dose levels ( the number of dose levels will depend on the availability and extent of prior information); · use of multiple animals per dose level which may be fewer than the number used per group in the T1S assay; · use of relevant endpoints, which may be more limited than those in the T1S assay ( for example, the range finding study for the T1S uterotrophic assay may employ only body weights and uterine wet weight, while the assay may also evaluate uterine gland height, serum hormone levels, and/ or vaginal cornification, etc.); · use of comparable animals ( e. g., ovariectomized females for the uterotrophic range finding study or castrated males for the Hershberger range finding assay). However, there may be circumstances under which exceptions occur ( e. g., use of intact males in the range finding study for the Hershberger assay to define doses producing systemic toxicity and any effects on the reproductive system as a first pass approximation); and · use of more than one range finding study if the initial version does not identify the dose level( s) to be used in the specific T1S assay if necessary by extrapolation or interpolation. EDSTAC Final Report Chapter Five August 1998 5  11 The dose level( s) to be selected for the in vivo assays should not result in excessive systemic toxicity, but should result in effects useful for detection of potential EAT disruption. However, no dose level higher than one gram/ kilogram body weight/ day ( i. e., a " limit" dose) should be utilized. The rationale for selection of dose levels for each range finding study, all of the results for such studies, and the logic employed to select the dose level( s) for the T1S assay should be included in the submission of T1S results for evaluation by the Agency as to the appropriateness of the study design, conduct, and conclusions.

7. Routes of Administration The route of administration for the recommended uterotrophic assay is subcutaneous ( sc) injection while the route for the modified uterotrophic assay and 14 day intact adult male assay with thyroid is intraperitoneal ( ip) injection. The route for all other mammalian in vivo assays is gavage ( orogastric intubation). The parenteral ( non oral) routes avoid the first pass metabolic effect of the liver and will permit detection of potential EDCs that are active as parent compounds and which undergo significant first pass metabolism. Hepatic xenobiotic metabolism does occur eventually after parenteral administration ( substantially with ip), so the potential effects of metabolites will be evaluated as well by these routes. Compounds are occasionally metabolized by the gut microflora; this type of metabolism has been shown to be important for some plantderived estrogens. The oral route of exposure will allow for this type of metabolism.

The EDSTAC believes EPA should propose a policy for route of administration for each assay and test. Since T1S seeks activity, an ip route or other in vivo approach seems most realistic for detecting potential endocrine activity. Conversely, T2T should focus on developing a policy for route of administration based on exposure route( s) which approximates the ecologically relevant exposure pathway, dependent on the test species and fate of the chemical in the environment.

B. Scientific Basis for In Vitro Screening for Estrogen, Androgen, and Thyroid Activities General agreement has been reached on the strengths and limitations of most currently available in vitro, in vivo, and ex vivo methods for detection of toxicants that act via ER, AR, steroid hormone synthesis inhibition, and/ or altered hypothalamic pituitary gonadal ( HPG) mechanisms. With this in mind, several short term in vitro assays for AR and ER receptor binding or transcriptional activation and minced testis steroidogenesis inhibition ( SI) activity were identified as quite useful in screening. In vitro methods also include steroidogenic enzyme/ hormone synthesis, biochemical assays, and in vitro and testis steroid hormone synthesis.

Advantages of in vitro assays include:

a) sensitivity to low concentrations increases detectability; b) high specificity of response; c) low cost; d) small amount of chemical substance or mixture required; EDSTAC Final Report Chapter Five August 1998 5  12 e) in vitro assays can be automated, including use of robotics; f) high throughput assays ( thousands/ month) can be developed; g) results can be coupled with QSAR models and for database screening; h) can be used for complex mixtures ( sludge, water contaminants); and i) reduces or replaces animal use.

The EDSTAC recognizes two categories of in vitro assays that may be used in T1S to assess the binding of test substances to receptors, i. e., cell free assays for receptor binding and transfected cells designed to detect transcriptional activation. The specific assays chosen, whether done " at the bench" or through the high throughput pre screening process ( discussed in detail in Chapter Four, Section V), should have the following characteristics:

a) evaluate binding to estrogen, androgen, and perhaps thyroid nuclear receptors; b) evaluate binding to the receptor in the presence and absence of metabolic capability ( e. g., one or more of the P450 isozymes, CYP1A1, CYP3A4, etc.); c) distinguish between agonist and antagonist in functional assays; and d) yield dose responses for relative potency of chemical substances or mixtures with endocrine activity.

If high throughput procedures are used, receptor binding assays should be performed for EAT receptors. If the assays are done at the bench level, only estrogen and androgen receptor assays are recommended and/ or functional assays should be performed for estrogen, androgen, and perhaps thyroid receptors ( specifically recommended is a stably transfected cell line like the MVLN cell line, if available, to assess transcriptional activation). If stably transfected cell lines are not available, then transiently transfected reporter gene assays should be used. MCF 7 proliferation assays are also acceptable; however, yeast based assays are not recommended at this time. These assays can be performed either high throughput or at the bench level.

Receptor binding assays can use rat, mouse, or human ER or AR. These assays evaluate the ability of the xenobiotic chemical substances or mixtures to displace the radio labeled endogenous ligand from the binding site, in a cell free or whole cell system. Relative potency can be determined for positive chemical substances or mixtures. Assay limitations are solubility in the culture medium, inability to distinguish agonists from antagonists, lack of metabolic capability, and risk of degradation of the receptor.

The functional assay, specifically transcriptional activation, requires, for agonist or antagonist activity, that the chemical substance or mixture bind to the receptor. In addition, there is a consequence to the binding, i. e., transcription ( synthesis of mRNA) of a reporter gene and translation of the mRNA to an identifiable detectable protein such as firefly luciferase or betagalactosidase In the case of the firefly luciferase, with substrate and cofactors present in the culture, there is a light flash detected from formation of the product when the enzyme is synthesized in response to transcriptional activation and acts on the provided substrate. In the case of the beta galactosidase, with substrate and cofactors present in culture, the product is detected colorimetrically when the enzyme is synthesized in response to transcriptional activation and acts on the provided substrate. The assay uses intact cells and may use different cell lines for EDSTAC Final Report Chapter Five August 1998 5  13 assessment of effects on EAT binding domains with transfected ( transiently or permanently) receptors and reporter gene constructs. This assay can distinguish between agonists and antagonists. Assay limitations are solubility, toxicity, permeability of the cell membrane, and lack of or limited metabolic capability. If a chemical substance or mixture must be metabolized to an active moiety, it will not be detected unless the limited residual metabolic capacity of the cultured cells is sufficient to transform the chemical to its active form. Metabolic activity might be provided by either preincubating the chemical substance or mixture with an S9 fraction ( supernatant from 9000g x centrifugation of homogenized liver from a metabolically induced rat) or incorporating the S9 fraction into the treatment mixture. In addition, cell lines are being genetically engineered to incorporate genes for P450 enzymes as a method for extending their metabolic capacity and, perhaps, obviate the need for use of the S9 fraction.

For assessing receptor binding in vitro, EDSTAC recommends both the receptor binding assays and the transcriptional activation assays be incorporated into the T1S battery, and subjected to validation and standardization. There is agreement that the transcriptional activation assays can provide more information than the receptor binding assays, since they measure not just binding capacity but also the physiological and biochemical consequences of that binding. However, the limited database on the relative utilities of receptor binding and transcriptional activation assays do not allow the EDSTAC to recommend one category of assay over the other at this time. Including the receptor binding and transcriptional activation assays in the standardization and validation program is expected to provide the data needed to reach a decision on whether both assays should be required or, if not, whether the receptor binding or transcriptional activation is preferred. It is important to keep in mind that these assays evaluate just one of the possible mechanisms of endocrine disruption; if a chemical substance or mixture acts via another mechanism than the receptor, it will not be detected in these assays.

Large scale high throughput pre screening ( HTPS) programs for chemicals have been employed, using standardized in vitro functional assays ( i. e., transcriptional activation of a reporter gene), in the pharmaceutical industry. Several companies involved in drug design routinely screen chemicals for hormonal activity on a large scale ( thousands per month).

In vitro evaluations can provide both false positive and false negative results. In vitro false positives ( i. e., active in vitro but not in vivo) arise when a chemical is not absorbed or distributed to the target tissue, is rapidly metabolically inactivated and excreted, and/ or when some other form of toxicity predominates in vivo. False negatives are considered to be of greater concern if in vitro tests were used to the exclusion of in vivo methods. In vitro evaluations can result in false negatives due to their inability, or unknown capacity, to metabolically activate toxicants. As a result, the EDSTAC's recommended battery includes in vivo methods in conjunction with in vitro techniques. Nevertheless, some in vitro assays may offer distinct advantages over in vivo assays when investigating the activity of specific metabolites.

C. In Vitro Assay Overviews EDSTAC Final Report Chapter Five August 1998 5  14 The EDSTAC recommends a specific assay for each of the ER receptor binding, ER transcriptional activation, AR receptor binding, AR transcriptional activation, and steroidogenesis categories in order for standardization and validation to occur efficiently. The receptor binding and transcriptional activation assays would be performed only on those chemical substances or mixtures not going through HTPS, while the steroidogenesis assay would be performed on all chemical substances or mixtures going through T1S. Equivalent assays could replace these if they meet specific performance criteria and were similarly validated. Even if HTPS is implemented, standardization and validation of these additional in vitro assays would allow them to be conducted in individual labs on a more limited basis. The following assays are the specific ones recommended for inclusion in the standardization and validation program.

1. Estrogen Receptor Assays a) ER Binding: Cell Free ER Alpha Binding b) ER Transcriptional Activation: MVLN 2. AR Assays a) AR Binding: Cell Free AR Binding b) AR Transcriptional Activation: AR Transcriptional Activation 3. Steroidogenesis a) Minced testis 1. Estrogen Receptor Assays In vitro rat ER binding assays provide a rapid and fairly inexpensive method for quantifying the ability of chemicals to compete with DES or estradiol for ER. The assay can be used for measuring ER in cell free extracts obtained from various tissue homogenates following in vivo exposure to an environmental chemical. In addition, the assay may be used to determine the ability of a given compound to compete with radio labeled estradiol for binding to the ER. The technical aspects of the ER binding assay are well documented for receptors obtained from cytosolic or nuclear extracts of various mammalian and other vertebrate tissues ( Anderson et al., l972; Korach et al., l979). In brief, cytosolic or nuclear extracts containing ER are incubated with [ 3H] estradiol for 18 hours at 4o C in the presence or absence of increasing concentrations of radio inert DES or test chemicals. Nonspecific binding is assessed by the addition of 100 molar excesses of radio inert DES. Bound [ 3H] and free ligands are separated using hydroxyapatite extraction, or charcoal dextran adsorption, and are quantified by scintillation counting.

The ER binding assays are less sensitive than the functional assays, of short term duration, and can be standardized between laboratories. The assay is useful for evaluating effects of a test compound on ER distribution and number following in vivo exposure. In addition, the assay can be used to rapidly evaluate test compounds for their ability to bind to the ER in the absence of any of their metabolites. Comparison of IC50 and Ki values for the chemicals tested in vitro with that of endogenous and synthetic estrogens provide an indication of the potential of a given chemical to disrupt ER function in vivo. However, this assay does not distinguish between ER agonist and antagonists. The cytosolic rat ER binding assay may also yield false negative results if metabolic activation is required prior to binding EDSTAC Final Report Chapter Five August 1998 5  15 to the ER or if the test chemical is not completely solubilized in the assay buffer. In addition, the results may be artifactual if ER is altered by detergent/ denaturation effects of the test chemical, particularly if concentrations greater than 10 micromolar are used. At present, ER binding data are not entirely comparable from lab to lab because of methodological differences between labs in the conduct of this assay. However the rat cytosolic ER binding assay has been used for about 20 years; it is less complex than whole cell binding assays, and competent laboratories should be able to obtain similar results with minimal effort.

Cell free and whole cell binding assays using human ER ( hER) are rapidly being developed and offer both advantages and disadvantages over the above assay, one advantage being the use of the human rather than the rat ER. However, being relative new, they have not been standardized in their examination of xenoestrogens. Assays for ER beta binding and/ or transcriptional activation should be considered as they become more widely available, and included in screening if warranted ( i. e., if it is determined that some xenobiotics bind only to, or more avidly to, ER beta and would be missed in current assays with ER alpha).

a. ER Binding The cell free estrogen receptor alpha binding assay, a long standing and relatively simple in vitro assay that detects specific mechanisms of endocrine activity, is recommended. This is important because several xenobiotics display affinity for the estrogen and/ or androgen receptors. Binding assays identify, but do not discriminate between, agonists and antagonists. The apical nature of these assays is an advantage rather than a limitation because either activity can produce adverse reproductive effects. These assays typically lack metabolic activity, which is an advantage if one wishes to identify the specific compound with endocrine activity. However, the lack of metabolic activation is also a limitation because some xenobiotics require metabolic activation.

b. ER Transcriptional Activation Binding of estrogen to ER alpha in target cells results in the initiation of specific transcription activation events. Various estrogen regulated genes have been identified in MCF 7 cells ( pS2, Cath D, PgR, TPA), and their corresponding gene products can be measured as an endpoint for estrogen action ( VanderKuur et al., 1993a; Pilat et al., 1993; Davis et al., 1995). However, such endogenous genes are additionally regulated by other cellular mechanisms ( Nunez et al., 1989; Cavailles et al., 1989; Zacharewski et al., 1994), and the quantification of gene products ( mRNA) may be relatively laborious and difficult. Therefore, the introduction of artificial, ER regulated reporter gene constructs into MCF 7 cells has become a routine method of measuring ER transcriptional activation ( VanderKuur et al., 1993b; Meyer et al., 1994). These reporter assays utilize the human ER of MCF 7 cells for transcriptional regulation of a reporter gene that codes for an exogenous enzyme that can be easily measured in a cell lysate. Of the typical reporter gene products of chloramphenicol acetyl transferase ( CAT) and luciferase ( Luc), the more sensitive assays utilize luciferase. Reporter genes can be introduced into cells for the duration of the experiment only ( transient transfection) or permanently, creating a genetically altered subline ( stable transfection). EDSTAC Final Report Chapter Five August 1998 5  16 Transcriptional activation assays are a direct manifestation of receptor mediated responses on gene expression ( i. e., the presence of a functional estrogen receptor and a reporter gene are sufficient to express estrogen mediated induction). The MVLN assay ( stably transfected MCF 7 cell line with an artificial gene including ER alpha, a controller segment of vitellogenin, and promotor regulating expression of luciferase), which detects transcriptional activation after receptor binding using a luciferase reporter gene, is recommended. This rapid and sensitive assay ( IC50= 20 pM range) confirms ER binding and appropriate controls can distinguish agonists from antagonists. These assays should be conducted in a manner that allows them to detect receptor antagonists as well as agonists. Although these assays often provide information similar to the above binding assays, this is not always the case, and there are well founded biological reasons for a chemical to be positive in either the binding or the transcriptional activation assay but not both. However, due to a higher degree of difficulty, concern exists that proper execution of whole cell assays requires a level of skill and training that may not currently exist in the toxicology community. If so, these assays might be much more difficult to implement than the binding assays, some of which have been used for decades and are less complex.

In spite of the difficulty of establishing stably transfected cell lines, various MCF 7 cell derivatives have been created. As mentioned above, the MVLN cell line is an MCF 7 cell derivative containing an artificial gene consisting of the ER controlled segment of the vitellogenin promoter, regulating the expression of luciferase ( Pons et al., 1990; Gagne et al., 1994). These cells also contain a neomycin resistance gene that was used in the stable transfectant selection process. Therefore, since all MVLN cells contain the reporter gene, estrogen regulated transcription can be measured with a high sensitivity. However, the metabolic capability of the MVLN assay has not been studied in detail; it is assumed to be similar to that of MCF 7 cells from which they are derived. In principle, there are several advantages of this assay over other in vitro assays that assess estrogen action. The MVLN cell assay is easy to use because it is permanently transfected and it is a short term assay. In addition, the MVLN cell assay has been standardized to the degree that is has been employed in high throughput transcription assays involving robotic manipulation of large numbers of sample wells containing relatively few cells ( e. g., 96 well plates). A procedure that has been used to characterize estrogen agonists as well as antagonists can be characterized with the MVLN assay ( Gagne et al., 1994). In addition, a systematic comparison of more than 25 chemicals, including phthalates, alkylphenols, chlorinated pesticides, and steroids in the MVLN and the MCF 7 proliferation assay found that these assays were of equivalent sensitivity and responsiveness. Assays like the MVLN are deemed desirable because they are stably transfected and hence relatively easy to use and standardize, have high throughput potential, and are typically run to detect both agonists and antagonists.

The MVLN assay has been reported to have a disadvantage though, namely, that when the cells are briefly exposed to hydroxytamoxifen, their reporter gene cannot respond to estrogens. The mechanism underlying this effect is presently unknown. In principle, avoiding exposure to hydroxytamoxifen should prevent this from happening; however, this raises the issue of instability due to inadvertent exposure to chemicals during maintenance or propagation of the cells ( this requires a serumsupplemented medium). The MVLN cells, like all other cell culture models, requires monitoring in order to ascertain that the initial response is preserved through extensive propagation ( Badia et al., 1994). In addition to the MVLN, other stably transfected cell lines have been or are being used to detect for ER and AR action. EDSTAC Final Report Chapter Five August 1998 5  17 2. Androgen Receptor Assays a. AR Binding The cell free AR binding assay, used to determine the ability of environmental chemicals to compete with endogenous ligand for binding to AR, is recommended. This is an easy, time honored task, with decades of use, and relatively simple to standardize and execute. Equilibrium binding assays require overnight incubation at 4oC with AR isolated from castrated rat reproductive tissues ( e. g., epididymis, ventral prostate, seminal vesicle) with increasing concentrations of radio labeled ligand at different fixed concentrations of inhibitor or a fixed concentration of labeled androgen with increasing concentrations of unlabeled competitors. Following the incubation, hydroxyapatite or dextran coated charcoal is used to separate protein bound ligand from free ligand and specific binding is plotted in double reciprocal plots ( i. e., Lineweaver Burke) and as Scatchard plots as a function of competing inhibitor concentrations. Data analysis yields apparent equilibrium binding affinity constants for the inhibitor ( Ki), which reflects the affinity of the chemical for the AR. Ki values can be used to rank chemicals for their ability to bind AR and therefore for their potential to be endocrine active. IC50 values can be used to calculate Ki values and the relative binding affinity ( RBA) of the toxicant for AR, as compared to DHT or T, but this method is less accurate than experimental determination of the Ki. Within the last few years, a surprising number of chemicals in the environment of anthropogenic origin have been shown to act as AR ligands, including pesticides ( e. g., vinclozolin, procymidone), pesticide metabolites ( p, p' DDE and other DDT metabolites, methoxychlor metabolites), hydroxylated PCBs, and steroidal and non steroidal natural and synthetic estrogens ( Waller et al., 1996).

Advantages of the cell free binding assay include ease of use, low cost, the potential to standardize receptor preparations for distribution to many labs, and metabolism ( but not spontaneous degradation) of chemicals in the assay is minimized. The absence of metabolism is an important consideration as parent chemicals and/ or metabolites can be individually examined to determine which structure is responsible for AR binding, information that is critical if the data are to be used in a QSAR model. Disadvantages include the need for radio labeled ligands and that data are restricted only to ligand binding affinity with no information on agonist or antagonist activity, AR stabilization, or degradation or rates of association and dissociation from the AR.

b. AR Transcriptional Activation For AR mediated activity, stably transfected cell lines are under development, but not yet widely available. The AR transcriptional activation ( Cis Trans) assay, using monkey kidney CV 1 cells, is recommended. A MCF 7 cell stably transfected with wild type androgen receptor has recently become available; however, only a few androgen agonists and antagonists have been tested using this cell proliferation assay ( Szelei et al., 1997). Hence, like the CV 1, cell lines transiently cotransfected with hAR and a promoter construct with a Luc reporter are recommended at this time. It is noteworthy that as compared to MCF 7 cells, the CV 1 has some metabolic capability. Here again, the YAS is not acceptable as it is unable to detect the AR mediated activity of chlorinated pesticides.

Cells transiently transfected with hAR and reporter construct to detect transcriptional activation after receptor binding distinguish agonist/ antagonist. Such assays have been used extensively and can be EDSTAC Final Report Chapter Five August 1998 5  18 employed in a HTS mode for rapid screening. Transcriptional activation assays are used to determine whether chemicals which bind AR act as AR agonists or antagonists ( Zhou et al., 1994; Simental et al., 1991). CV 1 cells are transiently transfected with the hAR expression vector together with a reporter construct ( e. g., chloramphenicol acetyl transferase ( CAT), beta galactosidase, or firefly luciferase) containing an AR dependent promoter such as the mouse mammary tumor virus promoter. Transfected cells are cultured in the presence ( for antagonist activity) or absence ( for agonist activity) of a single concentration of androgen ( 0.1 nM DHT) together with increasing concentrations of inhibitor. Following a 48 hour culture period, cells are harvested and luciferase activity is measured in the resultant solubilized cell extract as an estimate of AR induced transcriptional activity.

Advantages of these types of assays are that they use human AR, they display some metabolic activity, and they establish whether a chemical that binds hAR acts as an agonist or antagonist. This information is critical in understanding the mechanism responsible for the induction of adverse endocrine mediated effects. Disadvantages of these assays are that they require the AR expression vector, reporter vectors, and transient cotransfections, which can be difficult. The assay requires close adherence to the standard operating procedure for reproducibility, and a 48 hour incubation during which time metabolism of the treatment chemicals may confound the data. In this regard, media from this assay, and other in vitro assays, should be analyzed before and after the incubation period to account for potential degradation and metabolism of the exogenous test chemicals and hormones.

3. Steroidogenesis Antiandrogens and antiestrogens act via a number of direct mechanisms in addition to those that directly involve the steroid hormone receptors. One prominent mechanism of antihormonal activity is inhibition of hormone synthesis by inhibiting the activity of P450 enzymes in the steroid ( and fungal) pathway. Such activity can be detected in vitro with a fairly simple in vitro procedure with minced testicular tissue obtained from adult male rats, because for many of the pesticides known to alter this pathway the parent material is active. Although aromatase, another P450 enzyme is present only at very low levels in the testis and male reproductive tract, it was proposed that inhibition of aromatase need not be included in vitro because it will be assessed in the in vivo pubertal female assay that follows. However, aromatase activity cannot be assessed in the recommended testis culture assay or in any of the in vivo assays using male rats.

The testis culture in vitro assay using minced ( 50 mg) pieces of single testis, which can be used to evaluate hormone synthesis with and without stimulation with cAMP, hCG, or substrates, is recommended. This assay assesses non receptor mediated effects on P450 steroidogenic enzymes. Incomplete metabolism in vitro is of concern, except for those classes of chemicals where the parent material is active ( e. g., certain classes of fungicides, drugs, and agricultural products). This assay has been used with fetal, neonatal, and adult testis, and is not limited to mammalian species, having been used to assess steroidogenesis in fish, reptile, avian, and amphibian systems as well.

It is also possible to use cultures of Leydig cells isolated from testicular tissue to perform steroidogenesis assays. Leydig cells are the cells, within the testis, responsible for steroid synthesis. The advantage of using these isolates is that they are enriched for the cells that synthesize testosterone. The disadvantage is that there are extra steps in the preparation of the EDSTAC Final Report Chapter Five August 1998 5  19 cells. Both approaches are expected to be comparable in their ability to detect steroidogenesis inhibitors. The utility of the minced testis culture is primarily based on data generated using Leydig cell cultures ( Klinefelter and Kelce, 1996).

Substances that interfere with steroidogenesis act primarily by inhibiting cytochrome P450 enzymes in the steroid pathway. For example, the mechanism of action of two major classes of herbicides, the imidazoles and the triazoles, involves inhibition of P450 enzymes in the sterol synthesis pathway for lanosterol, a vital component of fungal membranes ( Taton et al., 1988). Cytochrome P450 inhibitors tend to be non specific, and these fungicides can also inhibit other P450 enzymes such as those required for mammalian steroid hormone synthesis ( Murray and Reidy, 1990). Inhibition of mammalian steroid synthesis can potentially result in a broad spectrum of adverse effects in vivo, including abnormal serum hormone levels, pregnancy loss, delayed parturition, demasculinization of male pups, lack of normal male and female mating behavior, altered estrous cyclicity, and altered reproductive organ weights.

D. Scientific Basis for In Vivo Screening for Estrogen, Androgen, and Thyroid Activities The EDSTAC believes inclusion of in vivo methods in T1S can help reduce false negatives in the absence of knowledge of absorption, distribution, metabolism, and excretion. In vivo assays are often apical ( that is, while they incorporate endocrine specific endpoints, disruption of a number of hormone regulation/ delivery mechanisms can be evaluated in the same assay). Therefore, they are less specific, but more comprehensive, than in vitro assays. In vivo assays can be made more specific if accompanied by target organ/ cell dosimetry of biologically active metabolites. In vitro data are enhanced if the actual concentration of the chemicals in the media is determined, to account for metabolism, stability, and solubility, and to determine whether these concentrations compare to those that can be achieved in vivo. Cellular assays should determine viability, and the specificity and limitations of each assay should be defined. It is clear a combination of in vivo and in vitro assays is necessary in order to detect EAT alterations that act via the ER, AR, TR, inhibition of steroid hormone synthesis, and/ or alterations of the hypothalamic pituitary gonadal ( HPG) and HPT ( thyroid) axis.

More than 50 assays, and related endpoints, were considered by the STWG, including in vitro, in vivo, and ex vivo ( in vivo dosing followed by in vitro assessment of function) techniques. In vivo endpoints considered include reproductive organ weights and histology, serum hormone levels, in vivo gene activation, protein synthesis, behavior, growth, development, pregnancy maintenance, and anatomy/ morphology. For each endpoint, the sensitivity ( defined here as the response of the assay to low concentrations or dosage levels), specificity ( pathognomonic for a mechanism of action, since the lack of specificity leads to false positives), relative simplicity, difficulties encountered running the assay, confounding factors, and limitations, test duration, and costs were discussed. In addition, items such as degree of acceptance of the method, how many chemicals had been screened, and the relative " newness" of the assay ( state of the art) were considered.

Advantages of in vivo assays include: EDSTAC Final Report Chapter Five August 1998 5  20 a) account for absorption, distribution, metabolism, and excretion; b) well defined, acceptable methods used for decades; c) general acceptability in toxicity testing; d) some endpoints are toxicologically relevant and have been used in risk assessment; e) evaluate a broader range of mechanisms; f) provide a comprehensive evaluation of the whole endocrine system as a unit; and g) give comparative perspective to other endpoints of toxicity.

It is important to reiterate that the screening battery is being designed to minimize false negatives, based on an assessment of the ability of the battery to detect known EDCs that act via EAT. In this regard, the value of each individual assay cannot be considered in isolation from the other assays in the battery, as they have been combined in a manner such that limitations of one assay are complemented by strengths of another.

The EDSTAC believes the recommended screening battery, once validated, will detect all of the EDCs mediated by EAT including xeno( anti) estrogens ( that act via the ER or inhibition of aromatase by oral or parenteral administration), xeno( anti) androgens ( via AR or hormone synthesis), altered HPG axis, and antithyroid action ( via synthesis, metabolism, and transport, and the TR). However, results of even the most specific in vivo assays can be affected by endocrine mechanisms other than those directly related to ER, AR, and TR action. For example, uterine weight in the ovariectomized female rat is affected in an estrogen like manner by high doses of aromatizable and nonaromatizable androgens and growth factors like EGF. The age at puberty ( vaginal opening in the female or preputial separation in the male rat) can be affected by chemicals that act on the hypothalamus, pituitary, or thyroid or alter growth hormone secretion. If gonadally intact females are used, uterine weight can also be affected by toxicants that stimulate hypothalamic pituitary or gonadal endocrine secretions. Clearly, castration of the treated male or female markedly affects the specificity of the test. The lack of specificity of in vivo assays is a limitation if the goal is to only identify ER, AR, and TR alterations. In contrast, this lack of specificity could be considered an advantage if a broader, more apical screening strategy is desired.

1. Unique Thyroid Action Properties to be Considered in Design and Interpretation of T1S Thyroid dysfunction leads to abnormal development, altered growth patterns, and a variety of physiological perturbations in mammals ( Dussault and Ruel, 1987; Myant, 1971; Porterfield and Hendrich, 1993; Porterfield and Stein, 1994; Timiras and Nzekwe, 1989), as well as in birds ( Tsai and Tsai, 1997), reptiles ( Schrebier and Richardson, 1997), amphibians ( Brown et al., 1995; Tata, 1994), and fish ( Leatherland, 1994). Considering the consequences to wildlife populations and human health of the presence in the environment of synthetic compounds with thyroid disrupting activities, the EDSTAC has recommended a series of assays that will detect whether substances may interact with the thyroid.

The chemistry of thyroid hormone, the endocrine mechanisms governing its regulation, and the mechanisms by which thyroid hormone exerts its effects are surprisingly similar among vertebrates EDSTAC Final Report Chapter Five August 1998 5  21 ( Gorbman et al., 1983). The EDSTAC deliberations have, therefore, been guided by research focused on a variety of vertebrates to develop this series of screens. Despite the volume of literature reviewed, the rapid pace of research into thyroid hormone action makes it predictable that the present screens will become obsolete, both because more effective assays will likely be developed and because new information about thyroid hormone action may reveal mechanisms of thyroid disruption not identified by the recommended T1S battery. The following background information, about functioning within the thyroid axis and methods used to evaluate anti thyroid activities, is intended to provide a rationale for the recommended Tier 1 thyroid specific assays.

Endocrinology of the Vertebrate Thyroid:

Cells of the thyroid gland are arranged in follicles; the epithelial cells surround a fluid filled core containing proteinaceous material   the colloid ( Fawcett, 1986). Individual follicular cells respond to a pituitary hormone, thyrotropin ( TSH), by increasing the synthesis and release of thyroid hormones ( Wondisford et al., 1996). In mammals, TSH release from the pituitary is stimulated, in turn, by a neuroendocrine peptide, thyrotropin releasing hormone ( TRH) ( Greer et al., 1993; Morley, 1981; Taylor et al., 1990), and inhibited by the negative feedback effects of thyroid hormone itself ( Franklin et al., 1987; Mirell et al., 1987; Shupnik and Ridgway, 1987). In a redundant negative feedback loop, thyroid hormone also exerts an inhibitory effect on brain cells that manufacture TRH ( Koller et al., 1987; Zoeller et al., 1993). The functional relationships among levels of this endocrine axis are so tightly linked that perturbations within one level produce compensatory changes in the other levels. The details of the relationship within this axis have not been explored fully for all non mammalian vertebrates.

Thyroid Hormone Actions:

The majority of biological actions of thyroid hormones, including the regulation of brain development, are believed to be mediated by nuclear receptors for triiodothyronine ( T3) ( Lazar, 1993). Although the responsiveness to thyroid hormone requires the presence of nuclear TRs, the specific effects of thyroid hormone vary from tissue to tissue ( Schwartz, 1983). Pleiotropic effects of thyroid hormone may be in part attributable to different levels and combinations of TR isoform expression ( Lazar, 1993; Lazar, 1994). However, an important mechanism by which thyroid hormone effects can be regulated within cells, tissue, and across developmental stages is the interaction between receptors for thyroid hormone and those for retinoids ( Forman and Samuels, 1990; Kliewer et al., 1992; Mano et al., 1994; Yu et al., 1991; Zhang et al., 1992). The implication of these observations is that thyroid hormone action can be modified, even disrupted, by agents which interfere with retinoid metabolism.

Despite the recognition that thyroid hormone exerts its effects through nuclear receptors, there are very clearly defined endpoints of thyroid hormone action during development. There are a few genes expressed in mammals whose expression has been rigorously defined as directly regulated by thyroid hormone in the mammal. These include myelin basic protein ( MBP) ( Mitsubashi et al., 1988) neuroganin/ RC3 ( Iniguez et al., 1993), TRH ( Hollenberg et al., 1995), malic enzyme ( Song et al., 1986), thyrotropin ( Carr et al., 1993), and some neuron specific genes ( Thompson, 1996). In amphibians, a number of genes have been identified in frogs ( Xenopus EDSTAC Final Report Chapter Five August 1998 5  22 laevis), which are shown to mediate effects of thyroid hormone on metamorphosis ( Brown et al., 1996; Brown et al., 1995; Denver et al., 1997; Furlow et al., 1997; Kanamori and Brown, 1996), as well as more apical endpoints such as tail resorption.

Mechanisms of Antithyroid Activity:

A variety of environmental compounds are known to affect thyroid function or thyroid hormone action ( Gaitan and Cooksey, 1989; Green, 1996). Processes known to be affected and a brief description of the effects are included below:

a) Active transport of iodide into the thyroid gland. Inhibitors include complex anions ( e. g., ClO4, TcO4, thiocyanate). Reduces thyroid iodide uptake and can reduce thyroid hormone synthesis and circulating levels. Elevated TSH can overcome modest inhibition in the absence of other thyroid pathologies. b) Iodination of thyroglobulin ( by thyroid peroxidase ( TPO)). Inhibitors include thionamides ( e. g., propylthiouracil, methiazole, carbimazole), thiocyanate, aniline derivatives such as sulfonamides, substituted phenols ( resorcinol), flavonoids, and iodide. Reduces thyroid hormone synthesis and circulating levels, but can be overcome by elevated TSH. c) Coupling reaction. Iodinated tyrosine residues of thyroglobulin must be coupled by an ether linkage to form iodothyronines, which are released from the thyroid gland. Inhibition of this coupling reaction reduces thyroid hormone synthesis. This reaction may be controlled by TPO itself. Inhibitors include thionamides and other inhibitors of iodination, minocycline, and lithium salts. d) Hormone release. This is a cAMP dependent process stimulated by TSH. Inhibitors include iodide and lithium salts. e) Iodotyrosine deiodination. This process is important for recovery of iodide within the thyroid gland. Inhibition causes the reduction in thyroid iodide content and thus, inhibition of thyroid hormone synthesis. Inhibitors include nitrotyrosines. f) Iodothyronine deiodination. This reaction is important for conversion of thyroxine to the hormonally active tri iodothyronine, and for the conversion of T3 to the hormonally inactive T2. Inhibitors include thiouracil derivatives, oral cholecystographic agents, and amiodarone. g) Hormone excretion or inactivation. This process is affected by inducers of hepatic drugmetabolizing enzymes. Inhibitors include phenobarbital, phenytoin, carbamazepine, rifampicin, and organochlorines. h) Hormone action. Thyroid hormone action is largely mediated by binding to specific nuclear receptors. There is limited evidence that compounds such as phenytoin ( dilantin) and amiodarone can displace T3 from nuclear binding sites in vitro. However, there is little in vivo evidence that this interaction may compromise T3 action. In addition, there are predictions that specific PCBs may interfere with T3 binding to the nuclear receptor because of similarities in structure. However, these predictions have not yet been experimentally verified.

Recommended T1S Assays for Anti Thyroid Activity: EDSTAC Final Report Chapter Five August 1998 5  23 To the EDSTAC's knowledge, all known antithyroid compounds so far reported in vertebrates affect circulating levels of thyroxine ( T4). The physiological consequences of these effects are variable and may require considerable time to develop in a screening paradigm. In addition, they may represent endpoint measures that are not solely responsive to thyroid disruption. Therefore, the recommended T1S battery includes in vivo measures of circulating levels of thyroxine and TSH, and changes in the histopathology of the thyroid gland, as initial endpoints for antithyroid screening. These measures can be made in animals that are treated in the commission of other in vivo screens ( e. g., uterotrophic assay). Human serum based commercial kits are available to measure T4 and TSH by radio immunoassay, and these assays have been validated and standardized for some other species. It is important to recognize that a significant change in circulating thyroxine or TSH should be considered a positive finding. This determination is based upon the fact that some compounds, such as PCBs, have been reported to reduce circulating levels of T4, but leave TSH unaffected ( Goldey et al., 1995). In addition, weak antithyroid agents, especially those affecting some aspects of iodide metabolism in the thyroid ( Gaitan and Cooksey, 1989; Gaitan et al., 1989; Green, 1996), may be compensated for by elevated TSH. Thus, T4 may appear normal, but TSH would be elevated. Finally, the absence of an effect on circulating levels of T4 or TSH does not preclude the possibility that an agent is antithyroid. It is well known that goitrogens can affect thyroid function over long periods and not be manifested by significant changes in circulating levels of T4 or TSH measured by radio immunoassay ( Gaitan et al., 1989). These compounds would produce a measurable effect on the thyroid gland. For these reasons, the EDSTAC recommends thyroid histopathologic evaluations.

During their deliberations, the STWG extensively discussed the timing of exposure to a chemical substance or mixture. EDSTAC recommends evaluation of antithyroid effects in animals prepared for testing other actions ( either 14 day or 20 day exposure). Although no cases are known in which exposure to xenobiotics of greater than 14 days are required to significantly affect circulating levels of T4, TSH, or thyroid histopathology, EDSTAC believes longer periods may be required ( DeVito et al., 1998). The effects of duration of chemical exposure must be quickly evaluated in the validation phase.

These measures in mammals represent evaluation of thyroid function; there are no clear markers of thyroid hormone action that could be used within the context of a T1S assay. In contrast, tail resorption in amphibian metamorphosis represents an assay which utilizes specific thyroid hormone dependent effects as an endpoint for a T1S assay.

2. In Vivo Assays Using Other Vertebrates The T1S battery includes an amphibian and a fish assay, which fill important needs in the battery and complements the information from assays using mammals. These assays help the battery meet design criteria 2 and 4, which express the need for a sufficient range of taxonomic subjects and range of metabolic functions be evaluated in the battery. While the basic biochemical processes of receptor binding and cellular activation by hormones are known to be similar among many organisms, detailed comparative data do not exist to assess the extent of the homology across vertebrate classes. In particular, fish ER differs from mammalian ER more than the ER of other classes, and fish have some unique androgens. Hence, including fish as subjects makes sense as it EDSTAC Final Report Chapter Five August 1998 5  24 is the class most likely to show differences from mammals in EAT activity. In addition, there are known differences in the ability of organisms to metabolize xenobiotics, due partially to the route of exposure. Fish and amphibians receive more dermal exposure to chemical substances or mixtures than other vertebrate classes, and thus chemical substances or mixtures avoid immediate metabolism in the liver. These assays also meet the need for the battery to have a clear cut response to measure the effects of thyroid hormone, which frog metamorphosis does, and hence complements the data on serum concentration of thyroid hormones and thyroid gland histology derived from the pubertal female rodent assay.

Unlike the mammalian assays, the assays recommended for fish and frogs have not been used in regulatory testing, and hence they need more work before being implemented for that purpose. In fact, however, both procedures using these species and similar endpoints have been used to investigate endocrine activity of chemical substances or mixtures in the research literature. The work needed for standardization of protocols and validation of the assay with known endocrine disruptors should proceed as soon as possible as these assays play a crucial role in the T1S battery. In addition, EDSTAC encourages development of other assays in the event that either of these two fail to be adequately standardized and validated, so that a complete screening battery can be implemented.

E. In Vivo Assay Overviews Several measures of estrogenicity ( reviewed by Gray et al., 1997a; Reel et al., 1996; Parker, 1966, Chapter 30) have been used for over 70 years, including uterine size, vaginal cornification, female sexual receptivity, and age at puberty/ vaginal opening ( see Parker, 1966, for a thorough review). For example, Dodds et al., 1938, found that DES produced full estrus in ovariectomized rats, so far as vaginal, uterine, and mating reactions were concerned. These remain some of the most useful shortterm in vivo methods for screening for estrogenicity. Studies of xenoestrogens typically indicate that the sensitivity of these endpoints is as follows: uterine weight measured 5 hours after the last treatment, with fluid, is generally more sensitive than the age at vaginal opening or vaginal cornification; however, this is not always the case. Uterine histology and biochemical measures appear to be at least as sensitive to estrogens as uterine weight, but these endpoints are slightly more difficult to evaluate as they require specialized skills and equipment and are more expensive.

The sensitivity of the age at vaginal opening to methoxychlor appears to be about twofold greater than the onset of persistent vaginal cornification ( PVC) and at least equivalent to the sensitivity of the uterotrophic assay ( Gray et al., 1989; Gray and Ostby, 1998). However, in another study hydroxylated PCBs induced vaginal cornification at dosage levels that failed to induce an increase in uterine weight ( Gillesby and Zacharewski, 1996). PVC was not detected in a long term study of the estrogenicity of octylphenol that doubled uterine weight in long term ovariectomized rats after 10 weeks of oral administration and after three days of administration in juvenile rats ( Gray and Ostby, 1998). Hence, some of the original measures of estrogenicity, in use now for nearly three quarters of a century, are still regarded as the most useful indicators of estrogenic activity in vivo. EDSTAC Final Report Chapter Five August 1998 5  25 Recent studies to evaluate methoxychlor, 4 tert octylphenol, nonylphenol, bisphenol A, DES ( Reel et al., 1985), estrogens, and antiestrogens ( Conner et al., 1996) have demonstrated the utility of these biological assays, since: ( 1) pro estrogens/ metabolites may be detected following in vivo exposure; ( 2) agonistic/ antagonistic properties may be addressed; ( 3) bioaccumulation and/ or the development of tolerance to exposure may be evaluated; ( 4) multiple routes and lengths of exposure may be easily compared; and ( 5) acute exposure regimes may be used. However, care should be taken when interpreting results from these biological assays since: ( 1) some environmental chemicals do not test positive for all measures; ( 2) exposure route and time of assessment following exposure affect the results; and ( 3) the observed biological response may result from other mechanisms of action. For example, in the ovariectomized female, increased uterine weight can be induced by aromatizable and nonaromatizable androgens ( Salamon, 1938) and EGF ( Nelson et al., 1991).

In the intact ( ovaries present versus castrate or ovariectomized) juvenile female rat, the age at which treatment is initiated ( typically 19 21 days of age) and the duration of treatment are critical variables that affect uterine weight. Exposure duration longer than 3 or 4 days or the use of juvenile females 24 25 days of age at the start of the study are not recommended because of the potential confounding of the treatment effect with the onset of natural estrous cyclicity and its concurrent fluctuations in uterine weight and histology. As long as the uterine weight bioassay has been used, it still has not been completely standardized, a fact that leads to some variation in results from lab to lab. For example, there are differences with respect to how thoroughly the mesenteric fat along the uterine curvature is removed, and some labs weigh the uterus with its contents, while others remove the fluid before weighing. Uterine weight, serum hormone concentrations, and other evaluations in intact female rats are difficult to interpret unless great care is taken to assure that females are necropsied at the same stage of the estrous cycle. With regard to the measurement of serum hormones in the cycling female rat, the time of day is also critical, in addition to the day of the cycle. Effects on estrous cyclicity are not limited to ER mediated alterations; several other reproductive ( hypothalamic pituitary) and nonreproductive ( hypothyroidism) endocrine related alterations can alter estrous cyclicity in the female rodent. The detection of vaginal cornification in juvenile, and ovariectomized adult rodents is one of the original assays used to detect estrogenicity and, as indicated above, this assay appears to be relatively sensitive to weak estrogens. However, higher levels of xenoestrogens are required to disrupt estrous cyclicity and induce constant estrus PVC in intact female rats ( Gray et al., 1989).

1. Rodent 3 Day Uterotrophic Assay ( Subcutaneous)

Assay for estrogenicity An increase in uterine weight is generally considered to be one of the best indicators of estrogenicity when measured in the ovariectomized ( ovx) or immature female rat or mouse after 1 3 days of treatment. The recommended 3 day uterotrophic assay ( sc injection) uses the ovariectomized adult female rat ( the duration can be extended if so desired) with n = 10/ group. Subcutaneous treatment is recommended at this time because most of the historical data are collected in this manner and there is relatively few data concerning the effects of other routes of administration at this time. At necropsy one should carefully trim the uterus of fat and weigh with and without fluid and save uterus and vaginal tissues for histopathology. Most xenoestrogens have been examined in this assay. It also should be EDSTAC Final Report Chapter Five August 1998 5  26 executed in a manner to detect antiestrogens. In this regard, a control and xenobiotic treated group are coadministered with estradiol sc and necropsied.

2. Rodent 20 Day Pubertal Female Assay With Thyroid Assay for thyroid, HPG axis, aromatase, and estrogens that are only effective orally or after longer dosing than the uterotrophic assay.

The determination of the ages at " puberty" in the female rat is an endpoint that has already gained acceptance in the toxicology community. Vaginal opening ( VO) in the female is a required endpoint measured in the new EPA two generation reproductive toxicity test guideline. In this regard, this assay would be easy to implement because these endpoints have been standardized and validated and VO data are currently being collected under GLP conditions in most toxicology laboratories. In addition, VO data are reported in many recently published developmental and reproductive toxicity studies ( i. e., see studies from R. E. Peterson's, R. Chapin's and L. E. Gray's laboratories on dioxins, PCBs, antiandrogens, and xenoestrogens).

In the pubertal female assay, oral dosing is initiated in weanling rats at 21 days of age ( 10 per group, selected for uniform body weights at weaning to reduce variance). The animals are dosed daily, 7 days a week, and examined daily for vaginal opening ( one could also check for age at first estrus and onset of estrous cyclicity). Dosing continues until VO is attained in all females ( typically two weeks after weaning, unless delayed). Age at VO is also determined in the female rat. Rats are dosed by gavage with xenobiotic and examined daily for VO. The advantage over the uterotrophic assay is that one test detects both agonists and antagonists, it detects xenoestrogens like methoxychlor that are almost inactive via sc injection, it detects aromatase inhibitors, altered HPG function, and unusual chemicals like betasitosterol. In addition, at necropsy one should weigh the ovary ( increased in size with aromatase inhibitors, but reduced with betasitosterol), save the thyroid for histopathology, take serum for T4, and measure TSH.

Exposure of weanling female rats to environmental estrogens can result in alterations of pubertal development ( Ramirez and Sawyer, 1964). Exposure to a weakly estrogenic pesticide after weaning and through puberty induces pseudoprecocious puberty ( accelerated vaginal opening without an effect on the onset of estrous cyclicity) after only a few days of exposure ( Gray et al., 1989). Pubertal alterations also result in girls exposed to estrogen containing creams or drugs, which induce pseudoprecocious puberty and alterations of bone development ( Hannon et al., 1987).

Several examples of estrogenic chemicals affecting vaginal opening in rodents are known and include methoxychlor ( Gray et al., 1989), nonylphenol, and octylphenol ( Gray and Ostby, 1998). This endpoint appears to be almost as sensitive as the uterine weight bioassay, but the evaluation is easier to conduct and does not require that the animals be euthanized, so they can be used for additional evaluations. For example, treatment with methoxychlor at weaning ( 6 mg/ kg/ day or higher) caused pseudoprecocious puberty in female rats. Vaginal opening occurs from two to seven days earlier in treated animals than controls, in a dose related fashion, but methoxychlor did not alter estrous cyclicity at the low dosage levels, indicating a direct estrogenic effect of methoxychlor on vaginal epithelial cell function without an effect on hypothalamic pituitary maturation. Similar effects have been achieved EDSTAC Final Report Chapter Five August 1998 5  27 with chlordecone, another weakly estrogenic pesticide, and octylphenol. Chlordecone also induces neurotoxic effects ( hyperactivity to handling and tremors). In addition to estrogens, the age at vaginal opening and uterine growth can be affected by alteration of several other endocrine mechanisms, including alterations of the hypothalamic pituitary gonadal axis ( Shaban and Terranova, 1986; and Gonzalez et al., 1983). In rats, this event can also be induced by androgens ( Salamon, 1938) and EGF ( Nelson et al., 1991). In the last 20 years there have been over 200 publications which demonstrate the broad utility of this assay to identify altered estrogen synthesis, ER action, growth hormone, prolactin, FSH or LH secretion, or CNS lesions.

3. Rodent 5 7 Day Hershberger Assay Assay for Antiandrogens In the castrated male rat, the gonads have been removed and effects on androgen dependent accessory sex organs and tissues are likely to be direct and not a result of pituitary or gonadal secretion. The assay ( Hershberger, 1953) requires two stages as below:

· castrated male rat + T + Xenobiotic ( to detect antagonist) · castrated male + X ( to detect agonist)

In this in vivo test, sex accessory gland weights ( ventral prostate and seminal vesicle separately) are measured in castrated, testosterone treated adult male rats after 4 7 days of treatment by gavage with the test compound. The advantage of this assay is that it is fairly simple, short term, and relatively specific compared to other in vivo procedures. Although the androgens, testosterone, and dihydrotestosterone ( DHT), play a predominant role in the growth and maintenance of the size of these structures, several other hormones and growth factors can influence sex organ weights including the thyroid and growth hormones, prolactin, and epidermal growth factor ( EGF) ( Luke and Coffey, 1994). Exposure to estrogenic pesticides can also reduce sex accessory gland size; however, it is unclear to what degree these reductions result from direct versus indirect action of the chemical. Other useful endpoints that help reveal the mechanism of action include serum hormone levels of T, DHT, LH, AR distribution, TRPM2/ C3 gene activation, ODC, and 5 alpha reductase activity in the prostate. The prostate and seminal vesicles should be weighed separately because these organs differ with respect to the androgen that controls their growth and differentiation. The prostate is dependent upon enzymatic activation of T to DHT, whereas the seminal vesicle is less dependent upon this conversion. Hence, effects on 5 alpha reductase can be distinguished from AR mediated mechanisms by determining whether the prostate is preferentially affected. Growth of the levator ani muscle is T dependent, having little capacity to convert T to the more potent androgen DHT. Weight of this muscle is useful in identifying anabolic androgens and antiandrogens, and for this reason has been used extensively in the pharmaceutical industry. In order to detect androgenic rather than antiandrogenic action one would simply delete the hormone administration from the protocol.

Data from this assay ( often with slight modifications), using drugs and xenoantiandrogens, are widely available in the literature. For a non in utero assay, this assay robustly detects androgens and antiandrogens with a dynamic response that typically exceeds that of the intact adult male ( Raynaud, 1984). Most of the studies are able to detect significant effects with only five animals per group. In EDSTAC Final Report Chapter Five August 1998 5  28 fact, in one study which used 10 15 drugs, the Hershberger assay was more responsive than was the intact male for every chemical examined ( Wakeling et al., 1981). The power of the castrate male assay arises from the fact that castration creates a " fetal like" endocrine system with regard to the regulation of androgen secretion, as the HPG axis can no longer compensate for the effect of the chemical on the AR. For example, p, p' DDE reduces sex accessory gland weights in this assay, but not when administered to intact male rats ( Kelce et al., 1997).

4. Frog Metamorphosis Assay This assay employs intact larval ( tadpole) stages of the African clawed frog ( Xenopus laevis) exposed over a 14 day time period, 50 64 days of age, to observe the rate of tail resorption ( Fort and Stover, 1997). Tail resorption can be easily quantified with computer aided video image processing ( Fort and Stover, 1997). The molecular mechanisms involved in tail resorption are well characterized ( Brown et al., 1995; Hayes, 1997a) and this assay is, therefore, considered to be a simple and specific assay for thyroid action. It will detect thyroid ( increase in tail resorption rate) and antithyroid ( decrease in tail resorption rate) effects. Because evidence also suggests that thyroid action on tail resorption is regulated by corticoids, estrogens, and prolactin ( Hayes, 1997b), this assay will address distinctive modulating pathways and, in tandem with the 14 day mammalian pubertal assay, a comprehensive screen for thyroid hormone activity is achieved.

5. Fish Gonadal Recrudescence Assay Intact mature fish maintained under simulated " winter" conditions ( short day length, cool temperatures) exhibit regressed secondary sex characteristics and gonad maturation. In this assay, intact fish of both sexes ( fathead minnow, Pimephales promelas, or other appropriate species) are simultaneously subjected to an increasing photoperiod/ temperature regime and test substance to determine potential effects on maturation from the regressed position ( recrudescence). The primary endpoints examined in the assay include morphological development of secondary sexual characteristics, ovary and testis development ( weight increases), gonadosomatic index ( ratio of gonadal weight to body weight), final gamete maturation ( ovulation, spermiation), and induction of vitellogenin. This assay is sensitive to HPG axis effects in addition to androgen and estrogenrelated activity.