· polymers which will be placed into a " hold" status ( with some exceptions) pending a review of their monomers and oligomers; · chemicals for which insufficient data exists to proceed to either T2T or hazard assessment and will, therefore, need to be prioritized for T1S; · chemicals for which sufficient data exists, or voluntary bypass of T1S, to go to T2T; and · chemicals for which sufficient data exists to go to hazard assessment.

B. Tier 1 Screening to Detect Interactions With the Endocrine System The purpose of T1S is to obtain a minimum, yet sufficient, set of valid and reliable data to detect whether a chemical substance or mixture may interact with the endocrine system. Included in T1S is a battery of assays designed to detect effects that enhance, mimic, or inhibit estrogen, androgen, and thyroid hormone related processes. In contrast to the more refined and detailed tests of Tier 2, the T1S assays should:

· be inexpensive, quick, and easy to perform; · be validated and standardized as soon as possible, defining characteristics such as sensitivity and specificity against a clearly defined standard, once it is identified; · be more " sensitive" than they are " specific," meaning they should have as their primary objective the minimization of false negative or ( Type II) errors, while permitting an as of yet undetermined, but acceptable, level of false positive or ( Type I) errors; · capture multiple endpoints and reflect as many modes of endocrine action as possible; · be broadly predictive across species, gender, and age; and · yield data capable of being interpreted as either positive or negative for the purpose of determining whether and how to conduct T2T. EDSTAC Final Report Chapter Three August 1998 3  10 Information gathered during T1S should be used to make initial judgments about areas of concern and should direct and focus T2T. The interpretation of T1S results should be consistent with best scientific judgment, formed on the basis of considerations such as " weight of evidence," consistency of the data set, and methodological strengths and limitations. Based on the evaluation made during this phase, one of two decisions is possible:

1. Proceed to T2T   If the interpretation of results from the full battery of T1S assays is determined to be " positive" ( i. e., the screens produced evidence of interaction with the endocrine system, within the scope of endocrine functions addressed by the program), the chemical substance or mixture will enter T2T to characterize the nature of any endocrine disrupting effects.

2. Hold Screening and Testing   If the interpretation of results from the full battery of T1S assays is determined to be " negative" ( i. e., the screens have not produced evidence of interactions with the endocrine system, within the scope of endocrine functions addressed by the program), and these results are not contravened by the " weight of evidence" developed during the prioritization phase, no additional screening or testing is necessary unless:

a) existing statutes require periodic review ( e. g., FIFRA re registration); b) new statutory requirements mandate review; c) new screens for endocrine disruption are incorporated into the strategy and it is determined that these new screens may either generate significant new information or they invalidate prior screens and therefore warrant the re screening of chemical substances and mixtures that have already been subjected to T1S; and/ or d) new information on the endocrine disrupting potential of the chemical substance or mixture becomes available which warrants the re screening of the chemical substance or mixture.

C. Tier 2 Testing to Determine and Characterize Endocrine Disruption The purpose of T2T is to determine and characterize the nature, likelihood, and dose response relationship of estrogen, androgen, and thyroid related effects in humans and wildlife. Selection of Tier 2 tests should be based upon T1S results and other relevant information. An underlying principle of T2T is that it should provide information useful for human/ ecological hazard assessment. The T2T scheme should be flexible enough to allow for scientific judgment in the selection of the most appropriate tests and endpoints, and costs should be practical. T2T should be aimed at determining whether the chemical substance or mixture is an endocrine disruptor. In addition, these tests should be designed to establish the relationship between different exposure levels, timing and duration of exposure, and adverse effects, including developmental and reproductive effects on the individual and its progeny. EDSTAC Final Report Chapter Three August 1998 3  11 In comparison to the components of the T1S phase, T2T should ideally be both sensitive and specific. In other words, it should be designed to minimize both false positive ( Type I) and false negative ( Type II) errors. Additionally, this battery of tests should:

· include assessment of endpoints identified as relevant from T1S; · include parental/ offspring developmental endpoints ( e. g., two generation studies) in order to adequately evaluate all life stages; · include the life cycle of both viviparous ( live birth) and oviparous ( egg laying) organisms; · be conducted at a range of doses that allows full characterization of the adverse effects of the chemical substance or mixture being tested; · be conducted in accordance with Good Laboratory Practice ( GLP) regulations to the degree consistent with resources and the goal of timely decisions; and · be validated, if need be, as soon as possible against a clearly defined standard.

Interpretation of results from T2T should reflect current scientific judgment, including considerations such as " weight of evidence" and consistency of the data set.

A " negative" result in T2T should abrogate any additional endocrine screening and testing for that particular chemical substance or mixture within the scope of endocrine functions defined by the program unless:

a) existing statutes require periodic review ( e. g., FIFRA re registration); b) new statutory requirements mandate review; c) new screens or tests for endocrine disruption are incorporated into the EDSTAC strategy which will generate significant new information, or invalidate prior screens or tests upon which decisions have been made to stop screening and testing; d) new information on the endocrine disrupting potential of the chemical substance or mixture becomes available and it is determined that this new information warrants additional testing; and/ or e) there are changes in the use and expected exposure patterns upon which the initial selection of tests was made.

In the event of a " positive" outcome, the chemical substance or mixture will proceed to the hazard assessment phase of decision making, whereupon it may be decided that additional T2T is required before a final determination of hazard can be made.

IX. Additional Components and Clarifications to the Original EDSTAC Conceptual Framework During the course of its deliberations, the EDSTAC identified an additional concept, the incorporation of " high throughput pre screening," to be considered in the context of the original Conceptual Framework. In addition, the EDSTAC clarified conditions under which a chemical substance might be permitted to bypass T1S assays and, instead, go directly to T2T. These two EDSTAC Final Report Chapter Three August 1998 3  12 issues are closely connected to other issues discussed later in the report, including testing at low doses and the definitiveness of T2T.

A. High Throughput Pre Screening During its deliberations the EDSTAC concluded that biological effects data were incomplete or lacking for most chemical substances. In the absence of biological effects data, EPA would be left with the choice of either raising or lowering the priority of a chemical based on the lack of effects information.

To address this problem, the EDSTAC recommends that a subset of the in vitro assays recommended for inclusion in the T1S battery should be conducted with the assistance of automated technology, in advance of the priority setting step of the overall sorting and priority setting process. The EDSTAC came to refer to this technology, which uses robotics and other automated processes, and the role that it will play in the overall endocrine disruptor screening and testing program, as " high throughput pre screening" ( HTPS).

HTPS results, by themselves, will not be sufficient to make a determination about whether a chemical may interact with the endocrine system of an intact animal. Such determinations will require additional screening and testing.

The primary purpose of the HTPS is to provide preliminary biological activity information for a large number of chemicals in a relatively short period of time. This information will simultaneously be of value for: ( a) detecting hormonal activity ( as a component of T1S); and ( b) providing at least some biological effects related information for the estrogen, androgen, and thyroid hormonal systems to assist in the effort to set priorities for T1S. The in vitro assays that would be performed as part of the HTPS include transcriptional activation assays. Performance of these assays would still be required as part of T1S for all chemicals that do not go through HTPS.

The EDSTAC recommends that all chemicals currently produced in an amount equal to or greater than 10,000 pounds per year ( estimated to be about 15,000 chemicals) be subjected to HTPS. Also, it is expected that all pesticides ( i. e., both active ingredients and formulation inerts) will be subjected to HTPS. Any chemicals subjected to the assays conducted in the HTPS step would not be required to repeat the ER binding/ transcriptional activation assay and the AR binding/ transcriptional activation assay as part of T1S. On the other hand, for any chemicals not subjected to HTPS ( e. g., production volumes less than 10,000 pounds per year), the assays in T1S would result in information equivalent to that which would have been provided from HTPS. The HTPS concept is explained in more detail in Chapter Four, Section V, and referred to often in Chapter Five. EDSTAC Final Report Chapter Three August 1998 3  13 B. Alternative Means of Meeting Versus Bypassing Tier 1 Screening The EDSTAC expects the vast majority of chemicals included in the EDSTP to go through the program in the logical, hierarchical manner in which the program was designed. Notwithstanding this expectation, the EDSTAC recognizes there will be circumstances where it may be inefficient to follow all steps of the EDSTP. For example, the EDSTAC Conceptual Framework allows chemicals that have already been subjected to tests that are the " functional equivalents" of the T2T two generation reproductive toxicity tests, endocrine disruptor endpoints, taxa, and dosing considerations recommended by the EDSTAC, to bypass both T1S and T2T and proceed directly to hazard assessment.

The EDSTAC has identified two other circumstances where a chemical substance or mixture may not be required to be evaluated in the assays included in the recommended T1S battery. Both of these scenarios are discussed below and then in further detail in Chapters Four and Five.

1. Alternative Means to Meet T1S Information Requirements The EDSTAC recommends that it should be permissible to complete the information requirements of T1S through the submission of data that are " functionally equivalent" to the data that would be generated from the recommended T1S battery. Further, functionally equivalent information could be submitted for one or more of the recommended T1S assays or for the entire battery. The EDSTAC believes it is helpful to distinguish this scenario, which is in essence an alternative means of meeting the information requirements associated with T1S, from two other scenarios, which are considered bypassing T1S.

2. Bypassing T1S There are two scenarios in which the EDSTAC recommends that the owner of a chemical should be permitted to bypass T1S. Each of these two scenarios has different implications for the information requirements associated with completing T2T and hazard assessment following T2T.

a) Chemicals That Have Previously Been Subjected to Two Generation Reproductive Toxicity Tests The first scenario includes those chemicals that have previously been subjected to mammalian and wildlife developmental toxicity and/ or reproductive toxicity testing, but such testing may not include additional EAT endpoints for T2T, as specified in Chapter Five, Section VI. The EDSTAC expects that food use pesticides will fall into this category, given the requirements of FIFRA and FQPA, as well as a small number of other types of pesticides and industrial chemicals. The EDSTAC agrees that chemicals which meet this criterion for bypassing T1S would still be subjected to the assays that will be part of the HTPS. In addition, chemicals which meet this criterion will also be the most likely candidates for the alternative approaches for completing T2T, as discussed in Chapter Five, Section V, C, 2. EDSTAC Final Report Chapter Three August 1998 3  14 b) Chemicals for Which There is No Prior Toxicity Testing The second scenario includes those chemicals for which the owner of the chemical has decided to voluntarily go to T2T without having completed the full T1S battery or any prior two generation reproductive toxicity testing. These chemicals must be evaluated in the HTPS assays. In addition, chemicals that bypass T1S under this second scenario must be evaluated in all the tests of the T2T battery ( i. e., the mammalian and non mammalian multi generation tests with all the recommended endpoints), consistent with the principles governing T2T, which are set forth in Chapter Five, Section V, C.

X. Compilation of Chapter Three Recommendations 1. The EDSTAC developed a tiered Conceptual Framework that formed the basis for its screening and testing strategy and all subsequent recommendations. The tiered framework consists of the following three major activities:

· Priority setting includes the sorting and prioritization of chemical substances and mixtures based on existing information. The existing information would be used to sort chemicals into four categories. An evaluation and analysis of this information will lead to sorting chemicals into one of four categories:

· Polymers, which are placed into a " hold" status ( with some exceptions) pending a review of their monomers and oligomers. · Chemicals for which there is insufficient data regarding endocrine disruption and will therefore need to be prioritized for Tier 1 Screening. · Chemicals for which sufficient data exists to proceed to Tier 2 Testing. · Chemicals for which sufficient data exists to go to hazard assessment.

· Tier 1 Screening ( T1S) to detect chemical substances and mixtures capable of acting on endocrine systems.

· Tier 2 Testing ( T2T) to determine, characterize, and quantify the nature of the endocrine disrupting properties of the chemical substances and mixtures identified by prior information and/ or T1S.

2. The EDSTAC recommended the adoption of several principles to guide the use of the Conceptual Framework.

· A chemical may bypass one or more tiers when warranted by appropriate information ( e. g., sufficient prioritization data on endocrine disrupting properties to initiate T2T or hazard assessment). · If information is inadequate to determine if a chemical should move to the next tier, an active process should be developed for generating the needed information to make such a EDSTAC Final Report Chapter Three August 1998 3  15 decision. · Criteria and default assumptions for deciding whether chemicals move from one tier to the next should be developed in advance of initiating screening and testing.

3. Within the context of the Conceptual Framework, the EDSTAC recommends that the overall scope of the screening and testing strategy should:

· be relevant to both human health and ecological effects; · initially emphasize identifying and characterizing effects that enhance, mimic, or inhibit estrogenic, androgenic, and thyroid hormone related processes; · consider tests that detect multiple hormone interactions, address endpoints in multiple species, and predict long term or delayed effects; · be periodically revisited to permit inclusion of additional hormone mediated effects or new screens and tests as they become available; · be capable of evaluating the endocrine disrupting properties of chemical substances and common mixtures; and · allow determination of possible additive, synergistic, or antagonistic effects caused by interactions among the components of mixtures.

4. The EDSTAC recommends nine broad principles to guide the implementation of the endocrine disruptor screening and testing program. The screening and testing program should:

· require the minimal number of screens and tests necessary to make sound decisions, thereby reducing the time needed to make these decisions; · examine existing screens and tests for their potential to predict, detect, and/ or characterize endocrine disruptors, ensuring that any modification to existing screens and tests does not compromise their ability to predict other toxicity endpoints; · systematically examine existing screening and testing data not only for adverse endpoints in high dose groups, but also for physiological changes in low dose groups; · not detract from current and new efforts to assess the toxicity of compounds and mixtures through mechanisms other than endocrine disruption; · provide data that can be used for a broad range of management and regulatory programs in a form that supports international harmonization of their use; · include periodic review of new scientific information; · use a performance based approach to the selection of screens, tests, and species, including the use of more appropriate screens, tests, and species as they are developed and validated; · be dynamic in order to stay current with the rapidly evolving science related to the endocrine system; and · be conducted at the minimum cost necessary to make the decisions within the EDSTAC Conceptual Framework.

5. The EDSTAC also recommended six guiding principles specific to the screens and tests EDSTAC Final Report Chapter Three August 1998 3  16 themselves.

· To facilitate making decisions within the EDSTAC Conceptual Framework, all screens and tests should have well defined endpoints. · The use of animals should be reduced to the minimum level needed to obtain scientifically valid results and interpretations. · The results of screens and tests should support further research on effects of endocrine disruptors on populations, communities, and ecosystems. · In interpreting screening and testing results, a " weight of evidence" approach should be used, but should be consistent with a principle of prudence in protecting human health and the environment. In the case of T1S, this means that the strategy will err on the side of false positive identifications rather than false negatives. · Screening and testing results should be reported in a format that facilitates database development and analysis by a broad array of scientific, regulatory, and management organizations. · Decision criteria, such as those for determining statistical significance ( e. g., necessary confidence intervals) and biological plausibility, should be clearly defined.

6. The EDSTAC recommends that T1S provide the minimum, yet valid and reliable, data to detect interactions with the endocrine system. In contrast to Tier 2 tests, T1S assays should:

· be inexpensive, quick, and easy to perform; · be validated and standardized as soon as possible, defining characteristics such as sensitivity and specificity against a " gold standard," once it is identified; · be more " sensitive" than they are " specific," meaning that they should have as their primary objective the minimization of false negative or ( Type II) errors, while permitting an as of yet undetermined, but acceptable, level of false positive or ( Type I) errors; · capture multiple endpoints and reflect as many modes of endocrine action as possible; · be broadly predictive across species, gender, and age; and · yield data capable of being interpreted as either positive or negative for the purpose of determining whether and how to conduct T2T.

7. The EDSTAC recommends that T1S be used to make initial judgments about areas of concern in order to direct the focus of T2T. The interpretation of T1S results should be consistent with best scientific judgment, formed on the basis of considerations such as " weight of evidence," consistency of the data set, and methodological strengths and limitations.

8. The EDSTAC recommends that T2T be based upon T1S results and other relevant information. An underlying principle of T2T is that it should provide information useful for human and ecological hazard assessment. The T2T scheme should be flexible enough to allow for scientific judgment in the selection of the most appropriate tests and endpoints, and costs should be reasonable. Tests should be aimed at determining whether the chemical substance or mixture is an endocrine disruptor and whether the effects are a result of primary or secondary disturbances of endocrine function. In addition, these tests should be designed to establish the relationship between different exposure levels, timing and duration of exposure, and adverse EDSTAC Final Report Chapter Three August 1998 3  17 effects, including developmental and reproductive effects on the individual and its progeny.

9. In contrast to T1S, the EDSTAC recommends that T2T should be both sensitive and specific, and designed to minimize false positive ( Type I) and false negative ( Type II) errors. Additionally, this battery of tests should:

· include assessment of endpoints identified as relevant from Tier 1 screens; · include parental/ offspring developmental endpoints ( e. g., two generation studies) in order to adequately evaluate all life stages; · include the life cycle of both viviparous ( live birth) and oviparous ( egg laying) organisms; · be conducted at a range of doses that allow full characterization of the adverse effects of the chemical substance or mixture being tested; · be conducted in accordance with Good Laboratory Practice ( GLP) regulations to the degree consistent with resources and the goal of timely decisions; and · be validated, if need be, as soon as possible against a clearly defined standard.

10. The EDSTAC recommends that a subset of the T1S in vitro assays be conducted with the assistance of automated technology to provide biological effects information to assist in the overall sorting and priority setting process. Because of the role this technology will play in the overall EDSTP, the EDSTAC refers to it as " high throughput pre screening" ( HTPS). The EDSTAC recommends that all chemicals currently produced in an amount equal to or greater than 10,000 pounds per year ( estimated to be about 15,000 chemicals) be subjected to HTPS. Also, it is expected that all pesticides ( i. e., both active ingredients and formulation inerts) will be subjected to HTPS. Any chemicals subjected to the assays conducted in the HTPS step would not be required to repeat the ER binding/ transcriptional activation assay and the AR binding/ transcriptional activation assay as part of T1S. On the other hand, for any chemicals not subjected to HTPS ( e. g., production volumes less than 10,000 pounds per year), the assays in T1S would result in information equivalent to that which would have been provided from HTPS.

11. The EDSTAC recommends that the vast majority of chemicals go through priority setting, T1S, and T2T in a sequential manner. However, the EDSTAC also recognizes there may be individual cases in which T1S is bypassed. Three situations were identified where a chemical may bypass T1S, each with different implications for information requirements in T2T.

· Alternative means to meet T1S information requirements through the generation of data which are " functionally equivalent" to data derived from the recommended screening battery. · Bypassing T1S for chemicals ( e. g., food use pesticides) that have previously been subjected to two generation reproductive toxicity tests. Such chemicals should still be subjected to high throughput pre screening assays. · Bypassing T1S for chemicals for which there is no prior toxicology testing but the owner has voluntarily decided to proceed directly to testing. Such chemicals must be evaluated in the high throughput pre screening assays, and all of the tests in the T2T battery. EDSTAC Final Report Chapter Three August 1998 3  18 XI. Literature Cited European Commission, " European Workshop on the Impact of Endocrine Disrupters on Human Health and the Environment," Environment and Climate Research Programme, DG XII, European Commission, Report EUR 17549, 1997. Kavlock, R. J. et al., " Research needs for risk assessment of health and environmental effects of endocrine disruptors: A review of the U. S. EPA sponsored workshop," Environmental Health Perspectives, 104, 1996, pp. 715 740. Tattersfield, L. et al. ( Ed.). " SETAC Europe/ OECD/ EC Expert Workshop on Endocrine Modulators and Wildlife: Assessment and Testing," SETAC Europe, Brussels, 1997. U. S. EPA, " Special Report on Environmental Endocrine Disruption: An Effects Assessment and Analysis. Office of Research and Development," EPA/ 630/ R 96/ 012, 1997. EDSTAC Final Report Chapter Four August 1998 Chapter Four Priority Setting EDSTAC Final Report Chapter Four August 1998 Table of Contents I. Introduction........................................................................................................................... 1 A. Charge to the PSWG ......................................................................................................... 1 B. The Need for Priority Setting ............................................................................................. 1 II. Overview of the Sorting and Priority Setting Recommendations ...................................... 4 A. Initial Sorting Step............................................................................................................. 4 B. Phased Approach ............................................................................................................... 5 C. Polymers............................................................................................................................ 6 D. Chemicals With Sufficient Data to go to T2T or Voluntary Bypass of T1S......................... 8 E. Chemicals With Sufficient Data to Proceed to Hazard Assessment...................................... 9 F. Chemicals with Insufficient Data to go to T2T or Hazard Assessment................................. 9 G. Priority Setting Information Categories and Criteria ......................................................... 10 H. Role of the Statutory Criteria ........................................................................................... 11 I. High Throughput Pre Screening ( HTPS) Step ................................................................... 12 J. Inclusion of Mixtures and Naturally Occurring Non Steroidal Estrogens and Recommendation for a Nominations Process......................................................................... 14 K. Introduction of the Endocrine Disruptor Priority Setting Database ( EDPSD) ................... 14 L. Overview of the Recommended Approach to Priority Setting ........................................... 15 III. Exposure Related Information and Criteria ................................................................... 15 A. Biological Sampling Data................................................................................................. 16 B. Environmental, Occupational, Consumer Product, and Food Related Data ....................... 18 C. Environmental Releases.................................................................................................... 19 D. Production Volume Data ................................................................................................. 20 E. Fate and Transport Data and Models................................................................................ 21 IV. Effects Related Information and Criteria........................................................................ 23 A. Toxicological Laboratory Studies and Databases.............................................................. 25 B. Epidemiological and Field Studies and Databases ............................................................. 26 C. Predictive Biological Activity or Effects Models............................................................... 27 V. High Throughput Pre Screening ....................................................................................... 28 A. Introduction..................................................................................................................... 28 B. Purpose of HTPS ............................................................................................................. 29 C. Which Assays Will be Conducted in HTPS? ..................................................................... 29 D. Limitations of the Assays to be Conducted During HTPS................................................. 31 E. Technical and Logistical Issues......................................................................................... 31 F. Which Chemicals Should be Subjected to HTPS?.............................................................. 32 G. How Will HTPS Results be Used? ................................................................................... 33 H. Practical Considerations and Constraints to Be Considered in HTPS Implementation ....... 35 VI. Recommendations for Handling Polymers....................................................................... 36 A. Introduction..................................................................................................................... 36 B. Key Issues Associated With the Prioritization of Polymers ............................................... 39 EDSTAC Final Report Chapter Four August 1998 C. Options Considered by the PSWG.................................................................................... 42 D. Recommendation for Handling Polymers.......................................................................... 44 VII. Recommendations for Handling Mixtures ..................................................................... 45 A. Introduction..................................................................................................................... 45 B. Definition......................................................................................................................... 45 C. Categorization Scheme for Mixtures ................................................................................ 45 D. Determining the Composition of Mixtures to be Considered............................................. 47 E. Criteria for Prioritizing Mixtures ...................................................................................... 47 F. Recommendations ............................................................................................................ 48 VIII. Recommendation to Screen Naturally Occurring Non Steroidal Estrogens ............... 51 A. Background ..................................................................................................................... 51 B. Recommendation ............................................................................................................. 52 IX. Recommendation for a Nominations Process .................................................................. 53 A. Introduction..................................................................................................................... 53 B. Description of the Nominations Process ........................................................................... 54 C. Criteria for Evaluating Nominated Chemicals ................................................................... 54 D. Submission of Nominations.............................................................................................. 55 E. Mixtures in the Context of the Nominations Process......................................................... 56 F. Ability to Track Nominations............................................................................................ 56 X. The Endocrine Disruptor Priority Setting Database ........................................................ 56 A. Introduction..................................................................................................................... 56 B. Recommendation and Principles to Guide the Continued Development, Utilization, and Maintenance of the Prototype EDPSD.................................................................................. 57 C. Description of the Prototype EDPSD ............................................................................... 57 D. Preliminary Recommendation for Data Fields to be Included in the EDPSD ..................... 59 E. Special Handling of Effects Data in the Context of the EDPSD ........................................ 59 F. Continued Development of the EDPSD ............................................................................ 63 G. Use by Multi Stakeholder Group ..................................................................................... 64 H. Maintenance .................................................................................................................... 65 XI. Recommended Approach to Priority Setting................................................................... 65 A. Introduction..................................................................................................................... 65 B. Obstacles to an Ideal Priority Setting System.................................................................... 65 C. Principles for Setting Priorities ......................................................................................... 66 D. Recommended Strategy for Setting Priorities for Tier 1 Screening ................................... 67 E. Examples of Compartments for the Recommended Priority Setting Strategy..................... 68 F. Numbers of Chemicals Prioritized and Associated Weightings of Compartments............... 72 G. Next Steps ( Reaching Closure) on Phase I Priorities for Screening and Testing ................ 74 H. Recommended Approach to Setting Priorities for Tier 2 Testing During Phase I of the EDSTP................................................................................................................................. 74 XII. Compilation of Chapter Four Recommendations .......................................................... 78 A. Summary and Scope of Effort .......................................................................................... 78 EDSTAC Final Report Chapter Four August 1998 B. The Universe of Chemicals and Initial Sorting .................................................................. 78 C. Polymers.......................................................................................................................... 79 D. Priority Setting Information Categories and Criteria ......................................................... 80 E. High Throughput Pre Screening ....................................................................................... 80 F. Mixtures........................................................................................................................... 81 G. Naturally Occurring Non Steroidal Estrogens ( NONEs)................................................... 82 H. Nominations .................................................................................................................... 83 I. Endocrine Disruptor Priority Setting Database ( EDPSD)................................................... 84 J. Recommended Approach to Priority Setting...................................................................... 85 XIII. Literature Cited ............................................................................................................. 87 Figures Figure 4.1 EDSTAC Conceptual Framework ........................................................................ 7 Figure 4.2 Example of Data Fields Arranged Into a Form as They Might Appear on a Computer Screen ............................................................................................... 60 Tables Table 4.1 Existing Files in the Endocrine Disruptor Priority Setting Database .................... 61 Table 4.2 Examples of File Types That Could be Placed in the Endocrine Disruptor Priority Setting Database.................................................................................... 62 Appendices Appendix F: References and Sources for Chapter Four   Priority Setting Appendix G: Exposure & Effects Data Source Matrices Appendix H: Fate and Transport Tables Appendix I: Feasibility Demonstration Project for HTPS EDSTAC Final Report Chapter Four August 1998 4  1 I. Introduction A. Charge to the PSWG This chapter of the EDSTAC report addresses the need to set priorities for endocrine disruptor screening and testing. It was developed by the Priority Setting Work Group ( PSWG) and was reviewed, refined, and endorsed by the EDSTAC. The PSWG consisted of nineteen individuals representing a wide diversity of perspectives and backgrounds including various sectors of industry; a variety of state and federal government agencies; national environmental, worker, and public health oriented organizations; and local citizen and environmental justice groups. A complete list of work group members is included in Appendix C. References and sources for this chapter can be found in the Literature Cited section at the end of the chapter and in Appendix F.

The charge given to the PSWG was to:

· specify types of information that should be gathered and analyzed to sort and prioritize chemical substances and mixtures for screening and testing; · develop criteria for evaluating the quality, adequacy, and reliability of the information that will be used in sorting and prioritizing chemical substances and mixtures for screening and testing; · develop criteria for sorting chemical substances and mixtures into four possible next steps, including: ( 1) hold screening and testing; ( 2) prioritize for Tier 1 Screening ( T1S); ( 3) go to Tier 2 Testing ( T2T); or ( 4) go to hazard assessment; · develop criteria for setting priorities for T1S. These criteria will address the relative order of priority in which chemical substances that are sorted into this category will actually proceed to T1S; and · suggest how information used for priority setting should be combined with screening and testing results to generate a " weight of evidence" determination for proceeding from screening to testing or from testing to hazard assessment.

B. The Need for Priority Setting Priority setting for endocrine disruptor screening and testing is not a trivial exercise. Industrial chemicals, pesticides, commercial products, and environmental contaminants have been subjected to various screening and testing regimes for decades ( Swanson and Socha, 1997). However, the existing regulatory screening and testing schemes do not specifically address endocrine disrupting mechanisms. The chemicals in commerce and the environment exhibit a range of physical and/ or chemical and toxicological properties, as well as varied production and use patterns. Only some chemicals are likely to cause endocrine disruption, and only some of these chemicals will be produced or used in such a fashion that humans or other living organisms will be exposed to them. Because screening and testing can be such a resource intensive process for both the public and private sectors, priorities must be set carefully to ensure that the chemicals of greatest concern are given priority over chemicals of little or no concern. EDSTAC Final Report Chapter Four August 1998 4  2 The challenge is daunting. Building on the information contained in Chapter Two, the EDSTAC recommends the universe of chemicals to at least be considered for endocrine disruptor screening and testing should include:

· all of the approximately 75,500 chemicals currently listed on the TSCA Inventory ( James Darr, U. S. EPA, personal communication); · all of the approximately 900 active ingredients ( approximately 500 of which are food use pesticides which will be prioritized for screening and testing according to the schedule and requirements of the FQPA ( see Chapter Four, Section XI, H) and approximately 2,500 inert ingredients that are used to formulate over 20,000 pesticide products ( Penny Fenner Crisp, U. S. EPA, personal communication); · approximately 8,000 chemicals regulated by the Food and Drug Administration ( FDA) including 5,000 ingredients in cosmetics and 3,000 food additives ( Bern Schwetz, FDA, personal communication); · naturally occurring non steroidal estrogens ( NONEs) and other naturally occurring or environmentally degraded chemicals; and · nutritional supplements, for which a number cannot be estimated because these chemicals are not currently regulated by the FDA or any other agency.

Thus, the number of individual chemical substances that should be considered for endocrine disruptor screening and testing exceeds 87,000. Later in this chapter the EDSTAC presents recommendations for screening and testing " commonly found mixtures" as per the scope of the recommended program set forth in Chapter Three. The EDSTAC recognizes that the evaluation of some of the individual chemical substances, if they are determined to be a priority for screening and testing, will require a cooperative effort among the responsible agencies.

In responding to the challenge, the PSWG grappled with a number of practical considerations:

· What scientific criteria should be used in establishing priorities? · What information is available with respect to these criteria and how readily can the information be analyzed? · What are the major gaps in information needed for setting priorities and how can these gaps be filled? · How should the priority setting system be designed to maximize " transparency" ( i. e., public understanding of the rationale underlying the established priorities)? · Should priorities be governed by existing statutory authorities? · How might priorities be set, without regard to EPA's statutory authority, to encourage voluntary private sector testing and to ensure compounds of concern are addressed?

The EDSTAC's efforts to develop a coherent, scientifically sound framework for setting screening and testing priorities have required EDSTAC members to carefully review the way in which EPA gathers information about new and existing chemicals. The Committee examined the authority provided to EPA by Congress which guide the Agency's data gathering efforts, and reviewed the Agency's management of the data available to it. The Committee also reached EDSTAC Final Report Chapter Four August 1998 4  3 beyond EPA in its quest for pertinent information sources to guide priority setting. Data on chemical hazards in the environment are also gathered by the Occupational Safety and Health Administration, the U. S. Department of Agriculture, the Food and Drug Administration, the U. S. Department of the Interior, and other federal and state agencies, as well as parties in the private and academic sectors.

Despite the multitude of data gathering authorities and databases on chemicals, information on exposure to, and the health and environmental effects of, most chemicals is incomplete, and inadequate with respect to endocrine disrupting effects. For example, much more information is available on the effects of pesticides regulated under FIFRA than is available on the effects of industrial chemicals addressed under TSCA. The EDSTAC's priority setting scheme attempts to address these information disparities.

The priority scheme recommended in this chapter reflects an integrated, scientifically driven concern for chemical exposures and effects that transcends the barriers that exist under current federal law. The priority setting scheme described in this chapter is noteworthy in several respects, including:

· First, even though the immediate impetus for endocrine screening and testing lies in provisions contained in the FQPA and the amendments to the SDWA, as described further below, the EDSTAC has not limited its priority setting scheme to chemicals addressed only under the endocrine disruptor screening and testing provisions contained in those two statutes. · Second, as described in the Conceptual Framework contained in Chapter Three, the EDSTAC has not limited its attention solely to the estrogen mimics that are explicitly mentioned in the FQPA and the SDWA, but is recommending that the initial screening and testing program also include androgen and thyroid related hormones. The Committee also recommends periodic review of the initial program to evaluate whether the inclusion of additional hormonal systems is warranted in the future. · Third, even though the FQPA and the SDWA focus on human health, the EDSTAC decided early in its deliberations that the Endocrine Disruptor Screening and Testing Program ( EDSTP) should address environmental impacts as well. · Fourth, the EDSTAC recommends that the EDSTP should address chemical mixtures in addition to single chemicals. · Fifth, the priority setting scheme, by promoting the use of robotic screening technologies ( referred to as High Throughput Pre Screening, or HTPS), is designed both to generate new information about chemicals and to help validate chemical modeling techniques that are used to judge hazards in the absence of empirical data. · Sixth, the Committee deliberately included so called NONEs ( e. g., phytoestrogens, mycotoxins)   substances that naturally occur in the environment   in its priority setting scheme.

It is important to note that the following discussion of the EDSTAC's recommended priority setting scheme does not reflect any interpretation by the EDSTAC of EPA's authority to implement these recommendations. The EDSTAC's priority setting scheme is driven by an overarching concern with exposures to and effects from chemicals. The Committee EDSTAC Final Report Chapter Four August 1998 4  4 acknowledges that EPA's screening and testing actions will be both heavily driven and constrained by its statutory authority.

II. Overview of the Sorting and Priority Setting Recommendations A. Initial Sorting Step As described in Chapter Three and graphically depicted in Figure 4.1, the EDSTAC Conceptual Framework consists of three major components: ( 1) the sorting and priority setting component; ( 2) the T1S component; and ( 3) the T2T component. Within the sorting and priority setting component, the EDSTAC has made a distinction between the tasks of " sorting" and of " priority setting."

The term " sorting" is used to refer to the initial effort to sort the universe of chemicals that will be considered for endocrine disruptor screening and testing into four distinct categories. Coming out of the " initial sorting" box, the four possibilities include:

1. polymers which will be placed into a " hold" status ( with some exceptions) pending a review of their monomers, oligomers, and other components; 2. chemicals for which insufficient data exist to proceed to either T2T or hazard assessment and will, therefore, need to be prioritized for T1S; 3. chemicals for which sufficient data exist to go to T2T; and 4. chemicals for which sufficient data exist to go to hazard assessment.

The term " priority setting" refers primarily to the need to set priorities for the chemicals that fall into the second category after the initial sorting stage   namely, those chemicals for which insufficient data exist to proceed to either T2T or hazard assessment and will, therefore, need to be prioritized for T1S.

The remainder of this section provides an explanation of the phased approach to screening and testing, a brief overview of each of the four categories of chemicals that flow from the initial sorting step ( referred to above), and some of the other key features of the priority setting system recommended by the EDSTAC. The rest of the chapter builds upon this overview section. EDSTAC Final Report Chapter Four August 1998 4  5 B. Phased Approach The EDSTAC agreed that the EDSTP should be implemented in a phased manner. In general, this means the chemicals determined to be a high priority should be screened and, if necessary, tested prior to those determined to be a lower priority. Two of the reasons for this general agreement on the use of a phased approach to implementation are: ( 1) to ensure that the program does not get bogged down by taking on too much too fast, both in terms of laboratory capacity and the administrative challenges with implementing a program of this magnitude; and ( 2) to ensure that periodic programmatic level evaluations occur that includes the incorporation of new scientific findings, new screens and/ or tests, etc. However, the EDSTAC did not have a sufficient amount of information nor the time to develop more refined recommendations about precisely how many phases there should be, how long each phase should be, or the number of chemicals that should be screened and/ or tested in each phase. The EDSTAC understands that some of these issues will be addressed by EPA when it issues its detailed implementation plan after the conclusion of the EDSTAC.

Given the elements of the screening and testing program upon which EDSTAC was able to agree, it is clear there are a number of activities which will need to occur immediately following the conclusion of the EDSTAC process and prior to the actual screening and testing of compounds. These activities include the validation and standardization of the recommended T1S assays and Tier 2 tests, the completion of the HTPS assays ( assuming they are shown to be technically feasible and are validated), and the completion of the T1S priority setting process. Therefore, if " Phase I" of the program is defined as the start of the actual screening and testing of chemicals, its start date is dependent upon the completion of these preliminary activities. EPA and some industry representatives have indicated that they may wish to make use of a screening assay or test as soon as it is validated, rather than waiting for all screens and tests to be validated. Thus, the start of Phase I may be staggered depending upon the results and timing of the validation process.

During " Phase I" of the program ( as defined in the preceding paragraph) T1S will only include those chemicals determined to be a high priority, and T2T will only include those chemicals that bypass T1S. During the second phase of the program, those chemicals that were determined to be positive in T1S will move into T2T, and a new set of priority chemicals will then be subjected to T1S.

Finally, as noted above, one of the reasons for recommending a phased approach to implementation is to ensure that EPA conducts periodic programmatic level evaluations of the EDSTP. The EDSTAC has stated in several places in this report that the design of the EDSTP needs to be flexible to account for the newly emerging and rapidly evolving scientific investigation of endocrine disruptors. Although the EDSTAC's recommendations regarding flexibility are meant to imply that new scientific findings and new screens and/ or tests should be incorporated into the program as they emerge, the EDSTAC believes it is critically important to include an explicit evaluation step into the program. The use of a phased approach to implementation can help to ensure that such evaluations occur. Some of the issues that should be evaluated at the EDSTAC Final Report Chapter Four August 1998 4  6 conclusion of one phase and prior to the start of another relate to the criteria for chemicals coming back into the program when they are placed in the " hold box." These include an evaluation of whether new screens and tests have been developed for the EAT hormonal systems, including, for example, in utero/ developmental assays and whether new screens and tests have been developed for other hormonal systems.

C. Polymers In an effort to grapple with the very large number of chemicals that the PSWG had identified as candidates for endocrine disruptor screening and testing, the group spent considerable time addressing the question of which, if any, chemicals should be placed in the " hold box" as part of the initial sorting step. It was thought that a class, or classes, of chemicals with a very low probability of being endocrine disruptors for the hormonal systems addressed by the screening and testing program could be set aside so as to avoid " clogging up" the system.

The group initially identified polymers as one type of chemical that warranted consideration for exclusion because of their molecular size. It was initially thought that polymers would not pose a threat to the endocrine systems of humans and other biota. Subsequently, the group learned there are instances where polymers could be absorbed, particularly in neonates.

Consequently, the EDSTAC recommends that:

1. All monomer and oligomer components of polymers should be prioritized for and subjected to endocrine disruptor screening and testing.

2. All " new" polymers ( i. e., those produced after the Initial TSCA Inventory, which was published in 1979) with number average molecular weight ( NAMW) less than 1,000 daltons should also be prioritized for and subjected to endocrine disruptor screening and testing. Throughout this document, the term " number average molecular weight," or " NAMW" of polymers is utilized. This term indicates a numerical mean, with the actual MW of the polymers ranging about this mean. The EDSTAC recommends embracing the language in the 1995 Final TSCA Polymer Rule ( 60 FR 16333) which uses a NAMW cutoff of 1,000 daltons, provided that the polymer does not contain other than certain specified reactive functional groups and that the polymer contains less than 10% oligomers with MW less than 500 daltons and less than 25% oligomers with a MW of less than 1,000 daltons.

3. All previously manufactured polymers ( regardless of NAMW) and all " new" polymers with a NAMW greater than 1,000 daltons should be set aside pending the outcome of the screening and testing of their monomer and oligomer and other components.

4. If the component is determined to have endocrine disrupting properties, the component should proceed to hazard assessment. EDSTAC Final Report Chapter Four August 1998 4  7 1 This flow chart represents a more detailed description of the sorting and priority setting components, and how they relate to the Tier 1 Screening ( T1S), Tier 2 Testing ( T2T), and Hazard Assessment components. 2 See Chapter Four, Section I. B. 3 See Chapter Four, Section VI. A. 2. 4 See Chapter Four, Section II. K. Essentially, this number results from subtracting 25,000 polymers from the total universe of 86,000 chemicals. 5 See Chapter Four, Section VI. A. 1. 6 See Chapter Four, Section V. F. Polymers ( Est. 25,000) 3 Hold Polymers Pending Screening and Testing and Exposure Assessment of Their Components Sufficient Data to go to Hazard Assessment Sufficient Data or Voluntary Bypass of T1S to go to T2T Insufficient Data to go to T2T or Hazard Assess. ( Est. 62,000) 4 Initial Sorting Total Universe of Chemicals ( Est. 87,000) 2 High Throughput Pre Screening Set Priorities for T1S Using Both Exposure and Effects Information Tier 1 Screening ( T1S)

Tier 2 Testing ( T2T)

Hazard Assessment Figure 4.1 Detailed Depiction of EDSTAC Conceptual Framework1 Phase I Currently Produced in Quantities > 10,000 lbs/ yr ( Est. 15,000) 6 Yes No Is It a " New" Polymer w/ NAMW < 1000 Daltons? 5 No Yes Phase II Phase III Hold No Further Analysis Required at This Time EDSTAC Final Report Chapter Four August 1998 4  8 5. As with any chemical shown to have endocrine disrupting properties, an exposure assessment should be performed. It is at this stage, that all potential exposure routes for a component would be determined, including the potential for the component to be available from the polymer. Implications with respect to the polymer itself would be dependent upon the results of such an exposure assessment.

As indicated in Figure 4.1, if this approach is utilized it will place approximately 25,000 polymers of the approximately 87,000 chemicals being considered for endocrine disruptor screening and testing into a " hold box" pending a review of their monomers and oligomers ( James Darr, U. S. EPA, personal communication). The rationale for these recommendations, as well as the recommendations themselves, are elaborated upon in Section VI of this chapter.

D. Chemicals With Sufficient Data to go to T2T or Voluntary Bypass of T1S As noted in Chapter Three, there are two scenarios in which the EDSTAC recommends the owner of a chemical should be permitted to voluntarily bypass T1S. Each of these two scenarios has different implications for the information requirements associated with completing T2T.

1. Chemicals That Have Previously Been Subjected to Two Generation Reproductive Toxicity Tests The first scenario includes those chemicals that have previously been subjected to mammalian and wildlife developmental and/ or reproductive toxicity testing, but where such testing may not have included additional endpoints for T2T, as specified in Chapter Five, Section V, C. The EDSTAC expects that food use pesticides will fall into this category, given the requirements of FIFRA, as will a small number of other types of pesticides and industrial chemicals. The EDSTAC agrees that chemicals that meet this criterion for bypassing T1S would still be subjected to the assays that will be part of the HTPS, for the reasons outlined in Chapter Four, Section V, G, 2.

In addition, chemicals that meet this criterion will also be the most likely candidates for the alternative approaches for completing T2T, as discussed in Chapter Five, Section VII, C. As described in more detail in Chapter Four, Section XI, H, the recommended approach for setting priorities for T2T of food use pesticides is basically to follow the schedule for pesticide reregistration and tolerance reassessments for these chemicals, as per the schedule and requirements of the FQPA. Also in Section XI, H, the EDSTAC discusses the need for special treatment of those pesticides that are likely to complete their tolerance reassessments prior to the completion of the validation and standardization of recommended Tier 2 tests. EDSTAC Final Report Chapter Four August 1998 4  9 2. Chemicals for Which There is no Prior Toxicity Testing The second scenario includes those chemicals for which the owner of the chemical has decided to voluntarily complete T2T without having completed the full T1S battery or any prior twogeneration reproductive toxicity testing. The EDSTAC recommends that these chemicals must also be evaluated in the HTPS assays. In addition, chemicals that bypass T1S under this second scenario must be evaluated in all the tests of the T2T battery ( i. e., the mammalian and nonmammalian multi generation tests with all the recommended endpoints), consistent with the principles governing T2T, which are set forth in Chapter Five, Section V, C. Finally, as discussed in Section XI, H of this chapter, these chemicals should retain their T1S priority ranking for T2T even though they will voluntarily bypass the screening tier. In other words, if these chemicals are deemed to be a high priority for T1S and the owner of the chemical decides to voluntarily bypass T1S, they should also be a high priority for T2T.

E. Chemicals With Sufficient Data to Proceed to Hazard Assessment The EDSTAC recommends that chemicals for which there are sufficient data to conduct a hazard assessment should be permitted to bypass both T1S and T2T and proceed directly to the hazard assessment step of the process. This option should be available for chemicals that have sufficient data to make either a definitive positive or negative determination that the chemical either does or does not have endocrine disrupting properties for the estrogen, androgen, and thyroid hormonal systems addressed by the program.

This step in the process will require a case specific review and determination that the same type and quality of information exist for the chemical as would be necessary to move from T2T to hazard assessment. The owner of such a chemical ( i. e., the company or companies that produced the chemical) or EPA in the case of an " orphan" chemical ( i. e., one that has no owner) would need to show that the screens and tests conducted yielded data that are the " functional equivalent" of data that would have been produced from T1S and T2T. Such functional equivalency will certainly include sufficient dose response relationship clarification before proceeding to the hazard assessment phase.

The EDSTAC believes that only a small number of chemicals will meet this criterion; however, it did not attempt to identify these chemicals. Rather, the Committee has appropriately deferred this determination to EPA as part of the implementation of the EDSTP. As noted above, such a determination will need to be made on a case specific basis. When EPA formally proposes its approach to implementing the EDSTP, the Agency should publish more detailed decision making criteria, data and reporting requirements, and procedures that should be followed to provide the degree of clarity necessary to implement this recommendation.

F. Chemicals with Insufficient Data to go to T2T or Hazard Assessment EDSTAC Final Report Chapter Four August 1998 4  10 A very large number of chemicals will remain after the initial sorting step has separated out polymers ( with some exceptions), food use pesticides, and any chemicals that have functionally equivalent data to bypass T1S, and the small number of chemicals that will be ready for the hazard assessment step. The EDSTAC estimates the number of chemicals that will fall into this category to be approximately 62,000. The EDSTAC has developed a set of recommendations to guide the T1S priority setting process for these chemicals.

As discussed in more detail below, the EDSTAC recommends an approach to setting priorities for T1S without specifying the precise number of chemicals that should ultimately be subjected to T1S. Building on the discussion of the phased approach to implementing the EDSTP, it should, however, be noted that even though approximately 62,000 chemicals will remain after sorting out polymers and the relatively small number of chemicals that will meet the criteria for bypassing T1S and/ or T2T, the EDSTAC does not expect that 62,000 chemicals will be subjected to T1S. ( See Chapter Four, Section XI, F)

G. Priority Setting Information Categories and Criteria When the PSWG began its deliberations, the group sought to address the following questions:

· What information is relevant to the task of priority setting? · Is this information readily available? · If so, how easily can the information be accessed? · What is the quality, variability, and reliability of this information? · Can the information be used as the basis for criteria to determine priorities for endocrine disruptor screening and testing?

In grappling with these questions, the PSWG established three main categories for organizing information and criteria related to priority setting: exposure related, effects related, and statutory criteria. The exposure and effects categories and information are consistent with those in Swanson and Socha, 1997. Under each of these main headings, the group identified a number of subheadings:

1. Exposure Related Information and Criteria a) Biological sampling data i. Human ii. Other biota b) Environmental, occupational, consumer product, and food related data i. Air ii. Water ( including surface water, groundwater, and drinking water) iii. Soil/ Sediments iv. Consumer products v. Food c) Environmental releases EDSTAC Final Report Chapter Four August 1998 4  11 d) Production volume e) Fate and transport data and models 2. 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., SAR, QSAR) d) Results of high throughput pre screening 3. Statutory Criteria a) Pesticides, as per FQPA b) Chemicals found in sources of drinking water affecting significant populations, as per SDWA c) Chemicals that may have a cumulative effect with pesticides, as per FQPA For the exposure and effects criteria, the PSWG identified a significant number of data sources, evaluated the quality and strengths and limitations of these data sources, and determined how to best utilize these data sources to accomplish the task of priority setting. The results of this effort are set forth in Section III for the exposure related criteria, and in Section IV for the effectsrelated criteria. Appendix G includes a series of detailed matrices containing a list and preliminary evaluation of data sources organized under the exposure and effects subheadings.

H. Role of the Statutory Criteria The PSWG of the EDSTAC discussed the proper role of the statutory criteria listed above in relation to the other criteria. The EDSTAC understands that the screening and testing requirement for pesticides ( both active and " inert" ingredients) contained in the FQPA is mandatory. However, the EDSTAC also understands the screening and testing of chemicals found in sources of drinking water affecting significant populations under the SDWA and chemicals that may have a cumulative effect with pesticides under the FQPA to be discretionary.

While recognizing the importance of the statutory criteria in relation to EPA's implementation authorities, the Committee has developed its priority setting recommendations based on public health and environmental concerns rather than on existing regulatory requirements. Thus, the Committee recommends that the statutory criteria should not be used as a sole basis for establishing priorities for endocrine disruptor screening and testing. The Committee recognizes that this recommendation might result in a chemical substance or mixture being identified as a high priority for endocrine disruptor screening and testing for which EPA does not have authority to require such screening and testing under FQPA. Nevertheless, the Committee believes it is important to have priorities driven by scientific considerations and explicit value judgments, rather than by existing regulatory requirements. EDSTAC Final Report Chapter Four August 1998 4  12 The Committee is hopeful that when a chemical is identified as a high priority for T1S that falls outside the scope of the FQPA and the SDWA, the owner of the chemical would voluntarily conduct T1S and, if necessary, T2T. The Committee acknowledges, however, that reliance on authority other than FQPA may affect the timing of actually conducting T1S, notwithstanding the priority ranking of the chemical.

I. High Throughput Pre Screening ( HTPS) Step One problem the PSWG identified early on in its deliberations is the lack of endocrine disruptor effects related data on the vast majority of chemicals and their breakdown products. The PSWG considered recommending the use of published and available Quantitative Structure Activity Relationship ( QSAR) models to obtain predictions for the endocrine disrupting potentials of untested compounds. Although promising, available QSAR models are generally thought to be insufficiently validated for the diversity of chemicals that will be included in endocrine disruptor screening and testing ( Ankley et al., 1997). Therefore, it was the PSWG's determination that QSARs were currently incapable of providing accurate predictions for this highly diverse universe of chemicals. To rectify this problem, the work group recommended, and the plenary endorsed ( subject to a demonstration of feasibility), incorporating into the EDSTAC Conceptual Framework the use of " high throughput pre screening," or the use of automated processes ( robotic and specialized instrumentation) to aid in the screening of compounds ( discussed in more detail in Chapter Four, Section V). The feasibility demonstration effort for HTPS is described more fully both in Section V of this chapter and in Appendix I.

The primary purpose of HTPS would be to address the fact that there is very little, if any, biological effects information for humans, and even less for other species, on the vast majority of chemicals to be considered for endocrine disruption screening and testing. The assays that will be conducted during the HTPS step of the process are transcriptional activation assays for the three hormonal systems ( estrogen, androgen, and thyroid hormone related). Two of the HTPS assays ( ER and AR binding/ transcriptional activation) are part of the T1S battery. Any chemicals subjected to the assays conducted in the HTPS step would not be required to repeat the ER and AR binding/ transcriptional activation assays as part of T1S. On the other hand, any chemicals which are subjected to T1S but not to HTPS ( e. g., production volumes less than 10,000 pounds per year) would go through the in vitro assays on the bench as part of T1S, thereby resulting in information equivalent to that which would have been provided from HTPS.

However, the assays in the HTPS step will be far from comprehensive or definitive. The HTPS assays will certainly provide valuable information on the potential of a chemical to bind to the relevant receptor in cell culture and result in transcriptional activation, which is information that is missing for a large number of chemicals. However, the results of HTPS will not be sufficient EDSTAC Final Report Chapter Four August 1998 4  13 by themselves to support the conclusion that a chemical is or is not an endocrine mediated toxicant.

Data resulting from HTPS will be combined with exposure related information, and with any other effects related information that is available, for each chemical for the purpose of setting priorities for T1S. In other words, HTPS data will not be used in isolation of other relevant data, nor will it become the de facto determinant of priorities for T1S.

Although the use of robotic technology will greatly expand the " throughput" of chemicals over a given period of time for the selected assays, the EDSTAC does not recommend that all chemicals be subjected to HTPS. Rather, the EDSTAC recommends that the estimated 15,000 chemicals currently produced in an amount equal to or greater than 10,000 pounds per year and all pesticides be subjected to HTPS. The EDSTAC makes this recommendation to help EPA avoid a task that might never be completed if a higher number of chemicals were to be recommended for HTPS. Also, the EDSTAC believes that 15,000 chemicals is not an insignificant number of chemicals, especially given the history of TSCA.

The EDSTAC further recommends that chemicals permitted to bypass T1S and go directly to T2T, as well as those permitted to bypass both T1S and T2T and go directly to hazard assessment due to functional equivalency of data, also be subjected to HTPS. There are several generic reasons why the EDSTAC recommends conducting HTPS assays on these chemicals which include: ( 1) the data generated from the HTPS assays will be valuable in and of themselves, even though they are limited to receptor binding mechanisms and cannot be used by themselves to determine whether a chemical is or is not an endocrine disruptor; ( 2) as an ancillary benefit, the data can be used to improve and validate QSARs; and ( 3) beyond these generic benefits, in the case of food use pesticides that will complete tolerance reassessments prior to the availability of validated Tier 2 tests, HTPS data can be used along with other relevant information to help prioritize whether and, if so, when these chemicals should be subjected to any additional endocrine disruptor testing. The rationale for recommending that food use pesticides complete HTPS assays is further elaborated upon in Chapter Four, Section XI, H.

The EDSTAC recommends that existing QSAR models be rederived and supplemented with data from the HTPS assays, thereby expanding the predictive ability of these models. Existing QSARs are derived using data from cell free receptor binding and cellular proliferation assays. These assays are part of the T1S battery, as specified in Chapter Five, Section III. New QSARs using HTPS data and transcriptional activation potencies from whole cell assays will need to be developed. These new models will likely be expansions of existing QSARs if the same chemical compounds are included in both.

Thus, when it comes time to set priorities for the first phase of T1S, HTPS data ( as well as improved QSARs) should be used along with other relevant exposure and effects data. Chemicals not subjected to HTPS ( because they are produced in amounts less than 10,000 pounds per year), but which are selected for T1S during the first phase of the program, would still have to complete the transcriptional activation assays as part of the T1S battery. EDSTAC Final Report Chapter Four August 1998 4  14 It is envisioned that the process of QSAR model expansion and improvement will continue in a cyclical feedback manner, thus providing the opportunity to validate the QSAR models using external data sets for screens and tests of compounds not subjected to HTPS. Eventually, predictions of endocrine disruption potentials obtained from validated QSAR models could be used as surrogates for HTPS data in the case of compounds for which effects data are not available.

J. Inclusion of Mixtures and Naturally Occurring Non Steroidal Estrogens and Recommendation for a Nominations Process The EDSTAC recommends in subsequent sections of this chapter that EPA include a discrete number of mixtures ( Section VII) and naturally occurring non steroidal estrogens ( Section VIII) in the EDSTP. In addition, the EDSTAC recommends that a process, separate and distinct from the core priority setting process, be conducted to allow affected communities and members of the public to nominate chemicals for screening and, if necessary, testing ( Section IX).

K. Introduction of the Endocrine Disruptor Priority Setting Database ( EDPSD)

The PSWG struggled with how to use the information sources and criteria it identified to sort and prioritize chemicals for endocrine disruption screening and testing. The EDSTAC, in response to work group information and queries, directed the PSWG to consider developing a computer database to electronically store information related to criteria that could be used for sorting and prioritizing. The EDSTAC was careful to instruct the PSWG not to develop a list of what were then referred to as " high priority chemicals for Phase I screening," but rather to develop a tool to illustrate different scenarios that could show the implications of alternative choices for setting priorities.

The PSWG asked two of its members to develop a relational database containing information sources associated with various criteria to facilitate the sorting and prioritizing processes. The resulting prototype database is referred to as the Endocrine Disruptor Priority Setting Database ( EDPSD). A preliminary version of the EDPSD was presented to the EDSTAC in December 1997.

The EDSTAC was impressed by the speed with which the EDPSD could provide different scenarios, and gave unanimous support for continued development of the EDPSD. However, it became clear that sufficient time and resources were not available to adequately develop the EDPSD within the time frame of EDSTAC's deliberations. Accordingly, the PSWG was told that EPA would complete and validate the EDPSD as a post EDSTAC exercise. Section X of this chapter provides a more detailed description of the prototype EDPSD, including the data fields that were included by the December 1997 plenary, the data fields the EDSTAC recommends that EPA include, and a process for using the EDPSD. EDSTAC Final Report Chapter Four August 1998 4  15 L. Overview of the Recommended Approach to Priority Setting In Section XI of this chapter, the EDSTAC presents its recommendations for how to set priorities: ( a) for chemicals that will need to be considered for T1S, and ( b) for chemicals that meet the criterion for bypassing T1S and going directly to T2T. The recommended approach for setting priorities for the approximately 62,000 chemicals that will need to be considered for T1S builds upon the EDSTAC's recommendations to: screen mixtures and naturally occurring nonsteroidal estrogens; establish a separate and distinct nominations process; separate out food use pesticides and other chemicals that have sufficient data to bypass T1S; and utilize a database tool to help analyze information relevant to priority setting. The recommended approach is one that would have EPA, with continued advice and assistance from a multi stakeholder group, use the EDPSD to help set priorities that flow from a simple and transparent application of the exposureand effects related information categories and criteria. The EDSTAC recommends that EPA apply the information categories and criteria outlined in Sections III and IV in a manner that would explicitly state the percentage of the total number of chemicals to be subjected to T1S in any one phase of the program to be drawn from the data sources for each criterion, or from the explicit combinations of criteria. This approach, which is referred to as a " compartment based" approach to priority setting, is described in more detail in Section XI.

The recommended approach for setting priorities for chemicals that meet the criterion for bypassing T1S and going directly to T2T, in the case of food use pesticides, is to use the schedule EPA has established for tolerance reassessments and pesticide re registration under the FQPA. All other chemicals that meet this criterion would be addressed on a case specific basis.

III. Exposure Related Information and Criteria This section describes in more detail the types of exposure related information and criteria that the EDSTAC recommends be used as the foundation for the priority setting process for T1S. Exposure related information and criteria consist of four exposure information categories and one fate and transport information category.

The four exposure related information categories are: ( a) biological sampling data for humans and other biota; ( b) environmental, occupational, consumer product, and food related data; ( c) data on environmental releases; and ( d) data on production volume. These four exposure related information categories can be viewed as a hierarchy or spectrum in an exposure chain. At one end of the exposure spectrum is the detection of chemicals in animal or human tissues and/ or fluids via biomonitoring studies. Such detection indicates that systemic exposure has actually occurred. Detection of a chemical in an environmental medium, or knowledge that a chemical is in food or a consumer product, indicates it is probable that exposure can occur. Knowledge that a chemical is released to the environment indicates that, depending upon its physical and/ or chemical properties, exposure is possible. Production volume data show that a given chemical is produced and could be released to the environment and exposure may occur. At the other end of the spectrum, some chemicals are entirely consumed in making a subsequent product ( e. g., in a EDSTAC Final Report Chapter Four August 1998 4  16 closed system) and, thus, are never released to the environment. Regardless of which data are used, special attention should be paid to chemicals for which there is evidence of embryonic, post partum or post hatch, early life stage, or pre maturation exposures.

A major limitation of the more direct measures of exposure is that data are available for only a limited number of chemicals. Human exposure information is not currently collected for the purpose of priority setting and/ or risk assessment. Data that do exist have been collected for other purposes. U. S. population exposure data that exist from the Centers for Disease Control and Prevention are limited to some heavy metals, volatile organic compounds, persistent organochlorines, and some non persistent pesticides. The ongoing National Health and Nutrition Examination Survey ( NHANES) family of surveys ( see Appendix G) does provide an opportunity to sample human tissue for additional chemicals. However, funding for analysis of the NHANES samples has not been secured. In contrast, while production data exist for a large number of chemicals, the link between production data and exposure is tenuous.

The fate and transport information category includes chemical and/ or physical properties that may be used to predict or estimate the medium or media where a chemical is likely to be found and whether or not a chemical is likely to remain in the environment over time. This information can be used in several ways. Since new chemicals will not have any data in the four exposure related information categories, the fate and transport information, along with estimates on production volumes or environmental releases can be used to estimate concentrations in environmental media. Fate and transport information can also be combined with known production volumes or environmental release information to estimate concentrations in environmental media. The more direct the measure of exposure that is combined with fate and transport information, the more likely one would anticipate the estimates to be of actual conditions. Unlike the other exposurerelated information categories which contain measurable empirical data, fate and transport information consists of estimations and predicted and/ or calculated data.

The remainder of this section describes in more detail the nature of the information included in each exposure related information category, the strengths and limitations of the type of information in each category, and a recommended set of guiding principles for how to use the information contained in each category to complete the task of setting priorities for endocrine disruptor screening and testing.

A. Biological Sampling Data Biological sampling refers to the monitoring of tissues or media from living or dead organisms for chemicals to document actual human or animal exposure. The biological sampling information category includes data that falls into two subcategories: ( 1) human biomonitoring, and ( 2) monitoring of other biota. Human biomonitoring refers to monitoring of human tissues and media ( e. g., blood, breast milk, adipose tissue, and urine). Monitoring of other biota encompasses the sampling of a very wide range of species ( invertebrates, vertebrates such as fish, and other wildlife) and sample matrices ( e. g., carcass, liver, kidney, egg, feathers, etc.) for exposure to environmental contaminants. EDSTAC Final Report Chapter Four August 1998 4  17 Strengths Human · Data are evidence of actual human exposure · Many data sets are representative of large populations; other data sets are representative of disproportionately exposed populations · Can be used to provide data to address mixtures · Generally good quality data; however, this must be determined on a case specific basis · May be used to identify trends · For those substances monitored, can evaluate frequency and magnitude of exposure detections relative to each other to help prioritize · Addresses multiple routes of exposure Other Biota · Data document actual exposure · Analytical data sets are generally of high quality · Multiple routes of exposure are addressed · Broad coverage of phylogenetic groups ( e. g., fish, reptiles, birds, wild mammals, shellfish and other invertebrates, etc.), habitats, and environmental matrices · Information on various animal species will substantially enhance understanding of the phenomenon of human effects · Many monitoring programs are spatially and temporally replicated Limitations Human · Limited number of compounds monitored; limited data available may not capture any short lived compounds or peak exposure · Biologic half life, metabolism, and tissue distribution vary from substance to substance · Limited opportunities to collect appropriate specimens · May not be representative with respect to time, population, or exposure distribution · Population surveys ( e. g., NHANES) may not characterize particularly susceptible or disproportionately or highly exposed subpopulations ( e. g., workers) · Identified compounds may not be traceable to a particular producer · Need to separate biomarkers of exposure from those of susceptibility or effect · Analyses often focus on the " usual suspects" and additional substances need to be measured EDSTAC Final Report Chapter Four August 1998 4  18 Other Biota · Limited number of compounds monitored; limited data available may not capture any short lived compounds or peak exposure · Biologic half life, metabolism, and tissue distribution vary from substance to substance · Limited opportunities to collect appropriate specimens · Population surveys may not characterize particularly susceptible or disproportionately or highly exposed subpopulations · Identified compounds may not be traceable to a particular producer Need to separate biomarkers of exposure from those of susceptibility or effect · " Exposure" or " potential exposure" are generally monitored; " biological effects" are not Guiding Principles for Using These Data for Priority Setting · The greater the relevance of the data set to large populations, disproportionately exposed subpopulations, or particularly susceptible subpopulations, the more weight the data set should be given. · Data sets with good quality assurance/ quality control ( QA/ QC) data should be given greater weight than those data sets with lower QA/ QC data. · The lower the detection limits and the greater the efforts to test organisms that are likely to be exposed, the greater the weight " non detect" data should be given.

B. Environmental, Occupational, Consumer Product, and Food Related Data Environmental, occupational, consumer product, and food related data include: ( 1) monitoring data for chemical contaminants found in a variety of environmental media to which humans and animals are exposed, such as water ( surface, ground, and drinking), air, soil, sediment, and food; and ( 2) use information for chemicals, when it is available.

Strengths · Provides data on likely exposures to humans and other biota · Databases exist for air, water, soil, and food · May be used to identify trends · Data can be used to identify relevant media for exposures ( e. g., food, air, and/ or water)

Limitations · Limited number of compounds monitored · Quantitative exposure levels must be inferred in many cases · " Detect" limits may vary from one data set to another · Use data sources are not comprehensive, are frequently secondary sources, and may not be independently verified. The highest quality, most comprehensive data sources are usually EDSTAC Final Report Chapter Four August 1998 4  19 maintained by fee for service organizations. Consequently, no use information databases for existing chemicals have been included in the EDPSD.

Guiding Principles for Using These Data for Priority Setting · The greater the relevance of the data set to large populations, disproportionately exposed subpopulations, or particularly susceptible subpopulations, the more weight the data set should be given. · The more likely a chemical is to be internalized by an organism from its environment, the greater weight it should be given. · Data sets with good QA/ QC data should be given greater weight than those data sets with lower QA/ QC data. · The lower the detection limits and the greater the efforts to test organisms that are likely to be exposed, the greater the weight " non detect" data should be given.

C. Environmental Releases Environmental release information includes data on chemicals released to the environment to which humans and animals may be exposed, such as permitted industrial discharges to air or water and accidental release or spill data. An example of the industrial discharge data is the Toxic Release Inventory ( TRI) reporting required by EPA. An example of accidental release or spill data is the Hazardous Substance Emergency Surveillance System maintained by the Agency for Toxic Substances Disease Registry ( ATSDR).

Strengths · Provides data on potential and known exposures to humans and other biota · Databases exist for air and water · May be used to identify trends · Data can be used to identify relevant media for exposures ( e. g., food, air, and/ or water) · TRI is updated annually · Databases include location specific data which are relevant to disproportionately exposed populations Limitations · Data exist for a limited number of industrial chemicals ( 528 in the case of the TRI) · Quantitative exposure levels are difficult to estimate in many cases · No data are available in the TRI for releases under 10,000 pounds per year from single sources EDSTAC Final Report Chapter Four August 1998 4  20 Guiding Principles for Using These Data for Priority Setting · The greater the relevance of the data set to large populations, disproportionately exposed subpopulations, or particularly susceptible subpopulations, the more weight the data set should be given. · The more likely the environmental releases are to lead to organism exposure, the greater the weight the release data should be given ( e. g., TRI releases to air and water should be given more weight than TRI releases to disposal such as permitted landfills, etc.).

D. Production Volume Data Production volume data include production information, primarily volume, for chemical substances and are mainly relevant to existing chemical substances. Such information can only be estimated for new products and is not relevant to environmental contaminants. The discussion of strengths and limitations which follows distinguishes among existing industrial ( i. e., TSCAregulated chemicals, existing pesticides ( i. e., FIFRA regulated), and new chemicals.

Strengths Existing Industrial Chemicals ( TSCA Regulated) · Quick, easy way to obtain a rough estimate of exposure potential · Readily available ( to EPA) for chemicals other than polymers and inorganics produced or imported in amounts greater than 10,000 pounds per year · Reliable and comprehensive · Identifies site limited chemicals · Excludes non isolated intermediates · Includes data on imported chemicals Existing Pesticides ( FIFRA Regulated) · Production data available at national level ( but not state level) for all covered products only as composite, not manufacturer specific · Available to EPA and the public New Chemicals · Estimated production volume data available to EPA for all new chemicals · Comprehensive EDSTAC Final Report Chapter Four August 1998 4  21 Limitations Existing Industrial Chemicals ( TSCA Regulated) · TSCA inventory update identifies site limited intermediates, but does not contain information on uses of individual chemicals · TSCA inventory data may be protected as Confidential Business Information ( CBI), which means that they are available to the EPA but not to the public · Does not contain data on degradates, mixtures of chemical substances, inorganics, polymers, or chemicals produced/ imported in amounts less than 10,000 pounds per year Existing Pesticides ( FIFRA Regulated) · Often lacking information on number of potentially exposed workers, " fence line" concentrations, and environmental release pathways · Currently contains information on currently registered and used products only New Chemicals · TSCA data for new chemicals may be protected as CBI, which means that they are available to the EPA but not to the public · Production data are estimates · Many Pre Manufacture Notification ( PMN) chemicals are never commercialized; fewer are commercially successful Guiding Principles for Using These Data for Priority Setting · Production volume provides only a very rough indication of potential human and ecological exposure. Combining production data with other data ( e. g., effects data) minimizes, to a certain extent, some of the inherent weaknesses of using production data as a surrogate for exposure. Production information should not be used to prioritize between existing industrial chemicals and pesticides or between new chemicals and pesticides because production volume ranges are too divergent. For example, production volumes for high volume industrial chemicals are several orders of magnitude higher than those for either new chemicals or pesticides.

E. Fate and Transport Data and Models Environmental fate and transport information is available from various reference sources, including databases, textbooks, and monographs ( e. g., Swanson and Socha, 1997; Cowan et al., 1996). Although the data source matrix for environmental fate and transport data and models included in Appendix G highlights a number of specific sources of information, no single source is really superior to another in that each is a collection of data. Because there is a lot of environmental fate and transport data from which to choose, the challenge is to identify the critical fate and transport data useful for sorting and prioritization purposes.

The EDSTAC recommends that EPA focus on three subcategories of environmental fate and EDSTAC Final Report Chapter Four August 1998 4  22 transport information: persistence, mobility, and bioaccumulation. Each of these factors can affect the bioavailability of a chemical substance because each is directly correlated to potential exposure. The definitions used by the EDSTAC for these terms are as follows:

Persistence is the tendency of a chemical substance or its degradation products to persist ( survive) in the environment without transformation into another chemical form.

Mobility is the tendency of a chemical substance to move within environmental media ( e. g., air or water) or between media ( e. g., to migrate from soil to groundwater).

Bioaccumulation is the capacity of a chemical to accumulate ( be stored in the tissue) in an organism as a result of uptake from all environmental sources.

Strengths · Environmental fate and transport tests pertaining to the three categories are already in place and have a long history of use for many chemicals · EPA has identified thresholds for various environmental fate and transport tests that trigger regulatory concern; however, at this time, the quantification of these potential thresholds ( or " triggers") and their application to determine the potential for endocrine disruption may be lacking or subjective · Modeling can also be used to estimate environmental fate and transport characteristics of persistence, bioaccumulation, and mobility when test data on specific substances are lacking Limitations · No single source of information on fate and transport includes all chemical substances · There are gaps in the data sources, making direct comparisons between chemical substances difficult · Most fate and transport estimating procedures have not been validated over the range of possible chemical substances that will need to be considered for endocrine disruptor screening and testing · Test data for the three selected parameters may not be available for all chemical substances · At this time, there are no generally established or accepted environmental fate or transport criteria directly related to endocrine disruption · Fate and transport of chemical substances may vary widely depending on environmental conditions; arbitrary standard conditions are established for regulatory and comparative purposes Guiding Principles for Using These Data for Priority Setting · For each of the three environmental fate and transport characteristics   persistence, mobility, and bioaccumulation   the tables contained in Appendix H specify relevant physicochemical criteria along with their corresponding threshold ( or " trigger"). These " triggers" are those which EPA generally takes into consideration when evaluating a pesticide or chemical for EDSTAC Final Report Chapter Four August 1998 4  23 registration. However, it should be noted that EPA does not rely solely on " trigger" values, but considers other environmental effects ( e. g., wildlife toxicology) before granting product registrations. · The use of fate and transport data to help set priorities for T1S should take into account all three environmental compartments   air, water, and soil. · Fate and transport characteristics should be based on laboratory or field tests when good quality data are unavailable. If laboratory or field data are lacking for a chemical, EPA should calculate the predicted fate and transport data for use in priority setting by means of reliable methodology or by use of an algorithm. · The physicochemical measures recommended for each of the three environmental fate and transport characteristics identified above   persistence, mobility, and bioaccumulation   are listed below:

· Hydrolysis half life   persistence; · Biodegradation   persistence; · Photooxidation   persistence; · Volatility ( Henry's Law)   mobility; · Absorption Coefficient ( Koc)   mobility; and · Octanol: Water Partition Coefficient ( Kow/ LogP)   mobility and bioaccumulation.

· Fate and transport measures that provided redundant information were eliminated. Some measures, such as photolysis, must be determined experimentally. A surrogate measure, photooxidation in this case, can help to fill gaps in the data ( photooxidation is one estimate of the atmospheric half life of a parent compound due to reaction with photochemicallyproduced hydroxyl radicals).

IV. Effects Related Information and Criteria In addition to HTPS, which is described separately in Chapter Four, Section V, the effects related information categories the EDSTAC recommends as the foundation for the priority setting process include: ( a) toxicological laboratory studies and databases; ( b) epidemiological and field studies and databases; and ( c) predictive biological activity or effects models, commonly referred to as Structure Activity Relationship ( SAR) and/ or Quantitative Structure Activity Relationship ( QSAR) models.

Toxicological laboratory studies and databases include all published, publicly available, or otherwise useable information related to the laboratory study of toxic effects of chemical substances and mixtures on living organisms or cell systems, including humans, wildlife, and ecological systems.

Epidemiological and field studies and databases range from hypothesis generating descriptive studies, such as case reports and ecological field analyses, to prospective cohort studies and rigorously controlled hypothesis testing clinical trials or community interventions. The most EDSTAC Final Report Chapter Four August 1998 4  24 common studies are descriptive.

Empirical toxicological and epidemiological data are reported in a large number of peer reviewed scientific journals. Published studies are conducted with varying degrees of methodological rigor and data are reported in widely varying detail. Consequently, information obtained from the general literature must be reviewed in detail in order to determine its applicability and adherence to generally acceptable investigatory practices. Some positive reproductive effects data are included in several regularly updated databases which are described in more detail in Appendix G.

Predictive biological activity or effects models attempt to identify correlation between properties that can be derived from the chemical structure or properties of molecules and biological activities, including those that can be identified through in vitro or in vivo screens and tests. SAR and QSAR models are also used to predict physicochemical properties such as solubility, volatility, and lipophilicity ( LogP). QSARs are useful for estimating or predicting how a chemical may behave when empirical toxicological or epidemiological data are unavailable.

General Guiding Principles for Effects Related Criteria · The EDSTAC believes that using published toxicological laboratory, epidemiological, or field studies for priority setting without first narrowing the universe of chemicals subject to detailed review would be virtually impossible in the appropriate time frame and with available resources. Accordingly, the EDSTAC recommends that data from the general scientific literature, which is not organized into logical databases, be used to help set priorities after an initial selection is made based on effects related data organized into logical databases. This issue is discussed in more detail in Chapter Four, Section X, E. · QSAR models should not override HTPS information. Rather, HTPS data should be used to improve the QSAR database as described more fully in Chapter Four, Section V, G, 3. · Positive epidemiological studies should be considered of higher value for priority setting purposes even in the presence of negative toxicological studies. · EPA has provided considerable guidance on how to interpret the results of toxicity, epidemiology, and other relevant data. This guidance should be relied upon in interpreting the available database for prioritizing effects information. The most relevant guidance for endocrine disruptor information are the Guidelines for Developmental Toxicity Risk Assessment ( U. S. EPA, 1991), the Guidelines for Reproductive Toxicity Risk Assessment ( U. S. EPA, 1996), and the Guidelines for Neurotoxicity Risk Assessment 1998.

The remainder of this section describes in more detail the nature of the information included in each effects related information category, the strengths and limitations of the type of information in each category, and a recommended set of guiding principles for how to use the information EDSTAC Final Report Chapter Four August 1998 4  25 contained in each category to complete the task of setting priorities for endocrine disruptor screening and testing.

A. Toxicological Laboratory Studies and Databases Strengths · For a few chemicals, particularly those regulated under FIFRA, a wide variety of whole animal studies have been completed using modern protocols with some endocrine sensitive endpoints ( e. g., developmental toxicity, reproductive toxicity) and conducted under Good Laboratory Practices ( GLPs) · Allows for testing of single agents and/ or mixtures to establish cause and effect relationships · Studies are likely to provide useful dose response data for the endpoints and species studied · Good coverage for a few chemicals and/ or substances ( e. g., petroleum crudes, organochlorine pesticides) with respect to aquatic species, birds, and wildlife Limitations · Toxicological database for industrial chemicals is less complete than that for pesticides · Ability to extrapolate endocrine related knowledge in test species to other species, including humans, is limited by the lack of knowledge about interspecies comparative endocrinology · Effects at very low doses and the presence of an inverted " U shaped" dose response curve or the " inverted J shaped" curve indicative of hormesis have generally not been examined in toxicological studies · Studies may not be designed to detect the relevant endpoints · Relevance of in vitro data to organisms and populations is not well characterized · Very little data on TSCA related chemicals especially for effects on birds and fish · Little is known of endocrine disruptor effects in wild mammals, lower vertebrates, and invertebrates · Relatively few studies have looked at subtle and multi generation effects Guiding Principles for Using These Data for Priority Setting · Whenever possible, in vivo studies with relevant endpoints and with wide dose response data should be viewed as more relevant for priority setting than in vitro studies. This is especially true when considering in vitro studies featuring receptor mediated mechanisms, which typically do not correlate well with endocrine mediated in vivo effects. · Studies that have any or all of the following characteristics should be valued greater than those that do not: · inclusion of relevant endpoints sensitive to endocrine disruption · indication of a dose response for endocrine disruptor effects · receipt of peer review · GLP compliance EDSTAC Final Report Chapter Four August 1998 4  26 B. Epidemiological and Field Studies and Databases Strengths · These data may provide actual observation of impacts upon humans, organisms, or ecological communities, removing many of the uncertainties inherent in assessing risk based on laboratory studies · When these data include biomarkers of exposure or effects, they can serve to document a completed exposure pathway · Data from these studies can provide information on vulnerable populations or high exposure subgroups, such as the occupationally exposed Limitations · Human disease and organ system dysfunction is multifactorial in causality, making it difficult to identify the contribution of individual factors unless they are dominant causes · The mechanisms which lead to specific human diseases are often unknown, and the specific endocrine disruption mechanisms which cause specific diseases are poorly understood · Small excess risks for common health outcomes may be difficult to identify without appropriate consideration of study power. · Studies of highly exposed individuals may not be relevant to much lower population exposures or to more vulnerable subpopulations; extrapolation of high exposure effects to low exposure circumstances or between subpopulations introduces uncertainty and decreases the utility of the data · Studies often address only one route of exposure; this route, however, may not be the most relevant route for the general population · Human and ecological communities are seldom exposed to only one compound; it is difficult to identify and examine the effects of multiple exposures and their possible interactions Guiding Principles for Using These Data for Priority Setting · Despite the many limitations inherent in epidemiological and field study data, statistically positive studies should be a priority indicator for additional screening and testing. · When multiple studies exist and there is a consistently positive association between exposure and an effect, but individually the studies do not reach statistical significance, this finding should be given weight when determining the priority for screening and testing. · Weight given to statistically negative studies should be dependent upon the study design, quality of the data, and the power of the study to detect an effect. Negative human epidemiological studies and ecological field studies should be considered, but should not necessarily override positive toxicological studies when determining priority for screening and testing. · When multiple studies exist, weight should be given to those studies that have received peer review and which are of high design quality. A checklist of issues important to evaluating a EDSTAC Final Report Chapter Four August 1998 4  27 study should be developed to assist in the review. Such a checklist would include likelihood of misclassification of exposure or disease, likelihood of introduction of bias, and utilization of standardized tools or methods. · Descriptions of studies should include a characterization of their design. Study design is important in determining the inferences that can be drawn from the results. Commonly used descriptors include: descriptive ( case reports, case series, calculations of rates of prevalence, incidence, mortality); observational ( ecological, cross sectional, case control, cohort, proportionate morbidity/ mortality ratio); and experimental ( clinical trial, community trial).

C. Predictive Biological Activity or Effects Models Strengths · SARs/ QSARs can be used to rapidly and relatively inexpensively predict biological activities of large numbers of compounds, thereby avoiding the need to prioritize on the basis of " no data" · Current SAR/ QSAR models developed for application to endocrine disruption analysis predict binding affinity and, therefore, have the same advantages and disadvantages as the in vitro models upon which they are based · The use of SARs/ QSARs in sorting and prioritizing allows for transparency and comparative consistency and avoids the problem of comparing different experimental data types against each other ( e. g., two generation reproduction study versus in vitro binding)

Limitations · No models are perfect, and the current receptor binding models suffer both from the imperfections of receptor binding modeling and the ability of receptor binding to predict in vivo activity · Not all mechanisms of endocrine disruption are known or have enough data to model; it is, therefore, not possible to generate models for all possible ways in which the endocrine system can be disrupted Guiding Principles for Using These Data for Priority Setting · Guiding principles applicable to the biological effects data used as the basis for the SAR, as well as to the QSAR itself, should be applied to the results of the SAR/ QSAR. · The applicable chemical domain of the SAR/ QSAR should be as diverse as possible. · SARs/ QSARs should be developed using the most complete and accurate data sets available. · SARs/ QSARs should be validated and used only within the range of conditions for which they are validated. EDSTAC Final Report Chapter Four August 1998 4  28 V. High Throughput Pre Screening A. Introduction During the course of its investigations the EDSTAC realized that, with the exception of food use and consumer pesticides with regulatory mandates requiring developmental and two generation reproductive toxicity testing, substantial endocrine effects data were lacking for most chemical substances. Developmental and reproductive toxicity screening and testing data are available in the literature for an estimated 5,000 chemicals, a large fraction of which are pesticides and pharmaceuticals ( John D. Walker, U. S. EPA, personal communication). In addition, existing QSAR methods for endocrine mediated effects are presently insufficiently validated to be universally accepted as a source of effects data ( Ankley et al., 1997).

In the absence of biological effects data, the scientists and officials within EPA charged with carrying out the priority setting process will be left with the choice of either raising or lowering the priority of a chemical based on a lack of effects information. Raising the priority seems to make sense from a public health protection standpoint, but in reality it will accomplish nothing because the vast majority of chemicals being evaluated are likely to be in the " no data" category for endocrine mediated effects. In essence, if a lack of data became a rationale for making a chemical a high priority for screening and testing, it could render the biological effects portion of the prioritization process meaningless.

To address the problem of having little or no endocrine disruptor effects data on the majority of chemicals that will need to be screened and possibly tested, the EDSTAC recommends that EPA use " high throughput pre screening" ( HTPS). As the term is used throughout this document, HTPS refers to the use of automated processes ( robotic and specialized instrumentation) to aid in the screening of compounds. These automated processes involve a number of preparatory operations, some of which are also associated with traditional screening approaches, such as sample preparation ( weighing and dissolving in the appropriate medium), screening, and the reading of screening results. However, in the case of HTPS, the process of placing the samples into a microliter plate, the sampling process itself, and the reading of sampling results, are all automated. Since all processes are automated and can be programmed to run continuously, it is possible for large numbers of samples to be assayed in a relatively short period of time using this technology.

High throughput screening technology is used extensively in the pharmaceutical and agrochemical industries to identify chemicals that have commercial potential or that may have desirable or undesirable biological effects ( Christopher Waller, OSI Pharmaceuticals, Inc., personal communication). The EDSTAC proposes that high throughput screening technology be employed as a prioritization tool   hence the term " pre screening"   for the endocrine disruptor screening and testing program. HTPS results, although limited in the scope of information they generate, will be useful in identifying chemicals that have an affinity for the estrogen, androgen, or thyroid hormone receptor. This information could be used in conjunction with other exposureand effects related information to determine the priority by which chemicals should be advanced EDSTAC Final Report Chapter Four August 1998 4  29 to the screening and testing tiers of the program. However, HTPS results will not be sufficient to make a definitive determination about whether a chemical does or does not have endocrine disrupting properties. This is the function of T2T.

The EDSTAC's recommendations regarding HTPS are contingent upon the demonstration of feasibility of this process in the context of the EDSTP. Appendix I contains information on the demonstration of feasibility proposal. The remainder of this section explains the purposes of HTPS and how it will be used to improve the endocrine disruptor priority setting, screening, and testing processes. Chapter Five describes some of the HTPS assays in more detail and their relation to the other assays in the T1S battery.

B. Purpose of HTPS First and foremost, HTPS will provide a baseline of systematically gathered data for the endocrine hormones that are currently addressed in the program   estrogen, androgen, and thyroid. This is especially important for those chemicals for which such data on endocrine relevant effects are otherwise lacking, namely most chemicals on the TSCA Inventory. The use of HTPS data should make screening more productive, as it is likely that a higher proportion of chemicals sent to T1S during the early phases of the program will have some evidence of biological activity.

Second, given the exploratory nature of HTPS, it is important to gain some perspective on the effectiveness of this methodology compared to other methodologies, such as QSARs, that can be used to identify compounds for screening. There is some concern that pre screening chemical substances   especially some pesticides, for which substantial reproduction and developmental ( whole animal) testing data may already exist   is a redundant exercise.

The EDSTAC recognizes that the inappropriate use of HTPS data could result in a certain stigma or in product de selection. This potential is not unique to HTPS, but is a broader communication issue related to endocrine disruptor screens and tests in general. This issue is addressed in Chapter Six. EDSTAC members believe that if communication of the results of HTPS is handled effectively, inappropriate use of the data and potential adverse marketplace reactions to such inappropriate use will be minimized.

C. Which Assays Will be Conducted in HTPS?

As noted in Chapter Two, one of the key mechanisms by which chemicals affect the endocrine system is by interacting with receptors. There is substantial evidence to support this statement for estrogen and androgen receptors. Existing data suggest that receptor binding may not be a significant mechanism for thyroid related effects. As discussed in Chapter Five, both the transcriptional activation and receptor binding assays for estrogen alpha and androgen hormones are recommended for inclusion in the standardization and validation program for T1S. If transcriptional activation assays can be standardized, validated, and shown to be as reliable as receptor binding assays, the EDSTAC recommends that they be included in the screening battery EDSTAC Final Report Chapter Four August 1998 4  30 as the preferred assay to detect receptor interactions. The receptor binding assay is an acceptable alternative that has decades of use, but may be less informative than the transcriptional activation assays in terms of the nature of the interaction ( agonism or antagonism). The potential contributions of both types of assays in the context of priority setting are discussed below.

Receptor binding assays are cell free biochemical preparations in which one determines the amount of chemical that binds to the hormone receptor as a function of the concentration of the chemical in solution, thus determining the affinity of the chemical for the receptor. Because receptor binding assays only measure binding, whether a substance is an agonist ( turns on or turns off gene expression like the natural ligand) or an antagonist ( which has the ability to block the action of the natural hormone) cannot be determined.

Transcriptional activation assays are conducted with intact cells that have been genetically modified to contain a hormone receptor and a reporter gene. The reporter gene produces a protein that can be quantitatively measured to reflect the ability of a chemical to act like a hormone, or to block the action of a hormone. The chemical may bind to the receptor and the resulting receptor ligand complex binds to a specific place on the reporter gene called the hormone response element. Subsequent steps include transcription of DNA of the gene to form RNA and translation of the RNA to form the marker protein. There are several different kinds of marker proteins that have been used in these assays. The common property is that they produce detectable signals that gene expression has taken place. For example, one marker protein, luciferase, is derived from fireflies and causes the emission of light when acting on luciferin, which is introduced into the culture medium. Thus, the activity of a hormone mimic is detected by the amount of light produced by the cell. In practice, the amount of light produced can be compared with that produced when the natural hormone or a reference substance is added to the culture.

Transcriptional activation assays incorporate receptor binding, but may be more relevant to responses in whole animals because they use intact cells and measure biological processes that result from receptor binding. However, relevance must be balanced with the fact that, because of the added complexity inherent in these processes, it is possible for the marker protein to be expressed by actions of the chemical unrelated to receptor binding. The cells used may have some ability to metabolize tested chemicals. This metabolic competence can be enhanced by genetically incorporating the ability to make one or more of the enzymes typically involved in metabolism of exogenous chemicals. This may provide the assay with the ability to detect compounds which must be metabolically altered in order to bind to the receptor. These enzymes can also be added to the receptor binding assays.

Both the transcriptional activation and receptor binding assays can be run automatically at several concentrations to determine an EC 50 ( the concentration at which 50% response is obtained). The EC 50 can be used to compare potencies of chemicals within each assay, which is a useful index for setting priorities among chemicals for additional screening. EPA has selected the transcriptional activation assay utilizing the luciferase reporter gene for demonstration purposes and, if shown to be technically feasible and valid, intends to use it for the HTPS. In this assay system the test material is run in the assays listed below with and without metabolic activation for agonist and antagonist potential. Multiple doses ( probably five plus a EDSTAC Final Report Chapter Four August 1998 4  31 control) would be run so that an EC 50 for transcriptional activation can be determined as a measure of potency as discussed above.

1. Estrogen Alpha Receptor Transcriptional Activation Assay ( no metabolism) 2. Estrogen Alpha Receptor Transcriptional Activation Assay ( metabolism) 3. Estrogen Beta Receptor Transcriptional Activation Assay ( no metabolism) 4. Estrogen Beta Receptor Transcriptional Activation Assay ( metabolism) 5. Androgen Receptor Transcriptional Activation Assay ( no metabolism) 6. Androgen Receptor Transcriptional Activation Assay ( metabolism) 7. Thyroid Receptor Transcriptional Activation Assay ( no metabolism) 8. Thyroid Receptor Transcriptional Activation Assay ( metabolism)

D. Limitations of the Assays to be Conducted During HTPS There are two noteworthy limitations to the types of assays being considered for the HTPS step. First, these assays cover only one of the possible mechanisms of action for endocrine mediated toxic effects. At present, this includes biological activity resulting directly from the binding of a chemical to the hormone receptor. On the other hand, existing data on thyroid active substances ( other than the natural ligand) have not shown that thyroid receptor binding/ activation is a key component of the mode( s) or mechanism( s) of action by which that substance exerts its thyroidrelated effects. Nonetheless, the HTPS will include the thyroid receptor transcriptional activation assays. This will be done for two reasons: ( 1) to do so will constitute only a minor increase in cost and effort; and, perhaps more importantly, ( 2) to confirm or refute the current hypotheses. If the results of HTPS show that thyroid receptor binding/ activation is a key component of the mechanism( s) of action, then thyroid receptor assays would be included in the basic T1S battery.

Assays that assess the activity of enzymes involved in hormone synthesis are technically possible to conduct using high throughput technologies but are not being recommended for inclusion in HTPS by the Committee. Despite this limitation, there are good scientific reasons to believe that most androgen and estrogen mediated toxicants capable of eliciting adverse effects at low doses do so by binding to a receptor. Therefore, the overarching goal of protecting human and ecological health is likely to be served by evaluating this mechanism early in the EDSTP.

The second significant limitation of the assays being considered for use in the HTPS step of the process is that they are unlikely to produce the same spectrum of metabolites that an intact animal produces. That is, chemicals that need to be metabolized in order to be active may not be detected by HTPS. Again, this limitation will be addressed in the screening tier. Both of these limitations will also need to be considered in the interpretation and utilization of the results of HTPS for purposes of priority setting.

E. Technical and Logistical Issues Estimates of the speed of using high throughput technology are encouraging. Once the EDSTAC Final Report Chapter Four August 1998 4  32 preliminary collection and handling of the chemicals are completed, it is not out of the question for several thousand assays to be run in one month, depending on whether confirmatory assays are also run ( Christopher Waller, OSI Pharmaceuticals, Inc., personal communication). However, there are technical and logistical constraints, as well as policy issues, that will need to be addressed in determining the number of chemicals that can or should be subjected to HTPS.

With regard to the technical constraints, some compounds have physical and/ or chemical characteristics such as insolubility, high volatility, and high reactivity that are not amenable to any in vitro screening system. There are, however, scientific reasons to assume that highly insoluble and highly reactive chemicals are unlikely to be endocrine disruptors.

There are also some significant, but not insurmountable, logistical hurdles to be overcome. One so called hurdle includes validation of the assays for the significantly diverse kinds of chemicals that will be subjected to HTPS. While it is intended, and expected, that HTPS will provide some false positives while minimizing false negatives, there is currently no history of use for HTPS methodology to evaluate large numbers of diverse chemical substances for potential endocrinemediated effects. Until now, HTPS endocrine assays have been used mainly as a tool to identify new leads or to assess biological activity of an existing lead. The possibility exists that HTPS may not provide sufficient effects data to warrant continued use, or that it may result in an unacceptable number of false negatives. However, all screens, whether automated or not, must undergo the process of validation.

In addition to validation, the problem of obtaining the chemicals must be overcome before HTPS can be implemented. This process involves not only collection but quality assurance of the collected samples. Some chemicals in the environment ( e. g., NONEs) are simply not commercially available. Moreover, since there is no registrant or chemical manufacturer for such " orphan" chemical substances, the ownership and responsibility to shepherd them through the screening and testing processes will rest with EPA or other government agencies. Obviously, if chemical substances cannot be procured they must either be isolated or synthesized in order to be screened and, if necessary, tested. At this time, the EDSTAC is not aware of how many compounds could fall into this category.

EPA has launched a feasibility demonstration effort designed to ensure that the types of assays being considered for HTPS can be used on the wide range of chemicals that will need to be subjected to this step in the process. For more information on the HTPS feasibility demonstration project, see Appendix I.

F. Which Chemicals Should be Subjected to HTPS?