Ellen Mihaich, p H .D ., DABT Environmental and Regulatory Resources ISRtp Workshop December 13, 2010

1. EDSP Test Guidelines and Guideline Modifications Ellen Mihaich, pH.D., DABTEnvironmental and Regulatory ResourcesISRtp WorkshopDecember 13, 2010

2. Courtesy of Tim Ward-ABC Laboratories

3. 890 Series In Vitro Screens

4. 890 Series In Vivo Screens

5. Test Order Restriction Requirement to perform screens according to 890 series guideline “…you may not deviate from an approved testing protocol unless you first consult with the Agency and obtain Agency approval of any planned deviation.”

6. General and Specific Guideline Issues • Primarily hitting the high points of some of the screens today • For more information please see the Test Guideline comments sent by the EPF in the EPA docket: EPA-HQ-OPP-2009-0634-0135

7. General Guideline Issues *No public review of guidelines prior to publication • Very prescriptive and inflexible • Test validity criteria too stringent • Sensitivity and specificity issues • Typographical errors • No standard evaluation procedures • No defined Weight of Evidence Procedures for the battery

8. ER and AR Binding Assays Shortcomings • No metabolic capability • Impacted by pH, denaturation, precipitates and particulates • Cannot distinguish agonists/antagonists Validation and Guideline Issues • Inconsistency in rat uterine cytosol preparation • Inconsistency in cytosolic prostate gland preparation • Numerous typographical errors

9. ER Transcriptional Activation Shortcomings • No metabolic capability Validation and Guideline Issues • Only validated for ER agonists • Limited validation and false positive rate high • EPA’s recommendation for positive response (PC10) deviates from peer review recommendation • EPA’s acceptable lower level of dynamic range of 4-fold induction raises issue of distinguishing from background noise

10. Aromatase (Human Recombinant) Shortcomings • No metabolic capability • Impacted by denaturation of protein Validation and Guideline Issues • Limited validation • Numerous typographical errors

11. Steroidogenesis (Human Cell Line) • Shortcomings • Cytotoxicity confounds results • Presence of any detergent residue on glassware can confound assay results • Validation and Guideline Issues • High variability between laboratories during validation make the results difficult to interpret • The guideline is poorly organized with no stated purpose – more of a protocol than guidance • Very small pipetting volumes could lead to errors • Parallel evaluation of cytotoxicity needed. Guideline permits up to 20% cytotoxicity while ICCVAM recommended only 10%

12. Uterotrophic • Shortcomings • Route of administration and animal model compared to relevance • Validation and Guideline Issues • While there are references to the anti-estrogenic component of the study, there has been no validation of this and anti-estrogenicity should not be assessed • Dose route and animal model preferences not harmonized with OECD - environmental relevance and metabolism should be considered

13. Hershberger • Validation and Guideline Issues • Wide inter-laboratory variation in the mean age at which preputial separation occurs • Clarity needed for the interpretation of study with and without optional endpoints • EPA recommends use of multivariate analyses of all accessory sex organ weights in cases where only a single tissue gives a significant response; such post-hoc analyses are useful for hypothesis generation, but should not be used in hypothesis testing • Anesthetic agent and euthanizing method should be chosen carefully to avoid artifacts if performing optional steroid measurement • CV’s for control and high dose organ weights should be reported. Deviation of more than 3 could result in study rejection

14. Pubertals • Shortcomings • High sensitivity but low specificity • Apical endpoints provide only limited information on the mode of action for potential endocrine-active chemicals • Significant inherent biological variability in the endpoints (puberty onset, estrous cycle, organ weights) complicates interpretation • Validation and Guideline Issues • Validation studies did not demonstrate a negative response using a true negative control agent • Dose selection is critical to avoid non-specific outcomes

15. Amphibian Metamorphosis • Shortcomings • Apical endpoints with unknown specificity • Poorly soluble or unstable compounds difficult to test • Not a short screen • Validation and Guideline Issues • No known negative compounds • Dose setting guidance needed • Background levels of iodide in food and water may make comparison between labs difficult • Developmental staging

16. Fish Short-Term Reproduction • Shortcomings • Multiple modes of action detected – apical endpoints • Medium sensitivity, low specificity • Long and expensive “screen” • Validation and Guideline Issues • High variability observed in plasma sex steroids suggests they are not particularly robust endpoints • Statistical power of the fecundity endpoint is low • Limited quantities of blood plasma may require prioritization of most robust measurements, such as vitellogenin, rather than sex steroids • Dose setting is critical to avoid confusing systemic toxicity with genuine endocrine-mediated effects

17. Sensitivity and Specificity The Series 890.1350 TG (fish assay) states: "It is recognized that some endpoints may be responsive to nonendocrine stresses in addition to endocrine-mediated pathways, particularly fecundity. Although reductions in fecundity indicate adverse organismal and, potentially, population level effects (i.e., reproductive toxicity), these cannot be definitively distinguished from direct endocrine-mediated effects by this assay when changes in other core endpoints are not present. Nevertheless, reductions in fecundity are considered a positive effect in this assay because they may be endocrine-mediated ..."

18. Staging the Conduct of the Screens

19. Summary • The current US EPA EDSP falls short in meeting many of the attributes of an efficient and effective screening program. • Many of the assays are not mechanistic, and some have yet to be shown to meet the basic requirement of distinguishing an endocrine active substance from a negative control or differentiating potential endocrine-mediated responses from responses via other modes of action (e.g. hepatotoxicity) or systemic toxicity. • The prescriptive nature of the guidelines combined with typographical errors, overly conservative validity criteria, and inflexible test order requirements likely mandate protocol approval prior to conducting the tier 1 battery

20. Summary-cont. • A staged approach to the performance of the screens may improve the interpretability of the results, increasing the efficiency of the work and clarity of the results. • Efficiency and clarity are essential because interpretation of the entire battery is the determinant for proceeding to Tier 2. • While staging the EDSP ESB may improve interpretation as to whether a substance may interact with components of the estrogen, androgen, and thyroid hormone systems, such activities cannot overcome inherent limitations of the tier 1 screens.

21. EPA EDSP ESB Test Guidelines can be accessed at http://www.epa.gov/ocspp/pubs/frs/publications/Test_Guidelines/series890.htm