1. EDSP Validation Gary E.Timm Senior Technical Advisor Office of Science Coordination and Policy U.S. Environmental Protection Agency

2. Framework EPA proposed a Two Tier screening process: Screening (Tier 1) –Identifies substances for further testing Testing (Tier 2) –Identifies adverse effects and establishes dose-response relationship for hazard assessment

3. TIER 1 SCREENING Detect potential for endocrine disruption Screens should be –inexpensive, quick, simple –standardized and validated –more sensitive than specific –able to detect multiple endpoints –readily interpretable Use information to –make initial judgments –direct and focus tier 2 tests

4. Modes of Action Hormone synthesis and clearance Hormone storage and release Hormone transport Receptor binding Altered post-receptor activation

5. PROPOSED SCREENING BATTERY (Tier 1) In vitro Screens –ER Binding / Reporter Gene Assay* –AR Binding / Reporter Gene Assay* –Steroidogenesis Assay with minced testis In vivo Screens –Rodent 3-day Uterotrophic Assay (sc) –Rodent 20-day Pubertal Female Assay with Thyroid –Rodent 5-7 day Hershberger Assay –Frog Metamorphosis Assay –Fish Reproduction Screening Assay * These assays are in the HTPS

6. ALTERNATE SCREENING ASSAYS Rodent 20-day Pubertal Male Assay with Thyroid Placental aromatase Rodent in utero through Lactation Assay

7. Screening Battery Example 1 In vitro Screens –ER Binding or Reporter Gene Assay* –AR Binding or Reporter Gene Assay* –Placental aromatase + –Steroidogenesis Assay with minced testis # In vivo Screens –Rodent 3-day Uterotrophic Assay (sc) –Rodent 20-day Pubertal Male Assay with Thyroid + –Rodent 20-day Pubertal Female Assay w/ Thyroid # –Rodent 5-7 day Hershberger Assay # –Frog Metamorphosis Assay –Fish Gonadal Recrudescence Assay + Assays in yellow replace assays in red #

8. Screening Battery Example 2 In vitro Screens –ER Binding or Reporter Gene Assay* –AR Binding or Reporter Gene Assay* –Steroidogenesis Assay with minced testis # In vivo Screens –Rodent 3-day Uterotrophic Assay (sc) # –In-utero Assay + –Rodent 20-day Pubertal Female Assay w/ Thyroid # –Rodent 5-7 day Hershberger Assay # –Frog Metamorphosis Assay –Fish Gonadal Recrudescence Assay + Assays in yellow relace assays in red #

9. TIER 2 TESTING Confirm and characterize endocrine effects Tests should –determine if effects are a primary or secondary disturbance of endocrine function –establish exposure/concentrations/timing and effects relationships –be sensitive and specific –assess relevant endpoints –include life cycle of live-bearing and egg-laying species –include a dose range for full characterization of effects –be conducted in accordance with GLP –be validated

10. PROPOSED TIER 2 TESTING BATTERY Multigeneration reproduction and development studies –Rodents –Birds –Frogs –Fish –Shrimp

11. Validation Process Stakeholder Involvement Standardization and Validation Task Force –Technical group made up of major stakeholders –3 expert workgroups: in vitro, mammalian, ecotoxicity –To be replaced by FACA EDSPVAC –To be chartered by September 2000 –Will advise EPA on Standardization and validation issues

12. Validation of Test Methods ICCVAM What is it? Interagency Coordinating Committee for the Validation of Alternative Methods What does it do? – Establises criteria for validation and regulatory acceptance of alternative test methods –Set up process for regulatory acceptance of alternative methods

13. Validation Process 1. Research/Method Development 2. Demonstration of Relevance 3. Standardization of Protocol 4. Validation in multiple laboratories 5. Scientific Peer Review

14. Validation Process Statutory Requirements All screens and tests in the EDSP must be validated The FIFRA Scienctific Advisory Panel (SAP) must review the EDSP The SAP must peer review all test guidelines to be used under FIFRA

15. Validation Process ICCVAM Role ER and AR binding assays to be validated by ICCVAM –no laboratory work required –generic performance criteria to be established rather than specific methods All other assays to be validated in EPA process with extensive ICCVAM coordination

16. EPAValidation Process ICCVAM Role Method development and preparation of Background Review Document (BRD) Pre-validation--demonstration of relevance and development of standardized protocol Determination of readiness for validation in consultation with ICCVAM Validation Peer review by SAP/SAB and review by ICCVAM

17. VALIDATION APPROACH Human health related assays are highest EPA priority (2- 3 year time frame ) –in vitro assays –Uterotrophic, Hershberger –Female and male pubertal assays –Rodent in utero screen –Rodent multigeneration demonstration Ecotoxicological assays EPA second order priority (3 -5 year time frame ) –frog metamorphosis assay –fish reproductive screen –fish, frog, and invertebrate reproduction and development studies

18. VALIDATION APPROACH Select chemicals specific to each assay, validate assay rather than battery More chemicals in pre-validation to establish relevance of assay and standardize the protocol than in validation phase Battery validation will be a paper exercise

19. Ongoing EPA Work Completed initial demonstration of relevance –Female Pubertal Development Assay (4/2000) –Male Pubertal Development Assay (4/2000) Need to refine and standardize protocols Initial developmental protocol trials –Frog metamorphosis assay (8/99) –Fish Reproduction Screening Assay (8/99) Additional effort needed on frog assay before a determination of relvance/usefulness can be made

20. OECD ENDOCRINE DISRUPTOR TESTING AND ASSESSMENT WORKGROUP Framework for Screening and Testing Standardizing/validating three screens: –Uterotrophic –Hershberger –28-Day repeat dose study (OECD Method 407) Updating existing guidelines –Example: Mammalian multi-generation reproduction study (OECD Method 416) Developing new guidelines –Avian multi-generation reproduction study –Fish partial life-cycle reproduction research

21. Status of Assays

23. Tier I Validation Program Projections

26. Summary Priority Setting Tier I Validation Tier II Validation Phase I Screening