RTI International is a trade name of Research Triangle Institute The International Society of Regulatory Toxicology and Pharmacology (ISRTP) 2009 Endocrine Workshop Lister Hill Auditorium, NIH Campus, Bethesda Maryland Session 1: Strengths and Weaknesses of the EDSP Screen Assays (September 9, 2009) Challenges in Conducting and Interpreting Tier 1 EDSP Screening Assays: CRO Perspective Rochelle (Shelley) W. Tyl, Ph.D., DABT Distinguished Fellow, RTI International

2 Tier 1 Screening to Detect Interactions With the Endocrine System (EDSTAC, 1998) 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 (mechanism of action). In contrast to the more extensive and detailed tests of Tier 2, the T1S assays should: (continued)

3 Be inexpensive, quick, and easy to perform Be validated and standardized, defining sensitivity and specificity against a clearly defined standard Be more “sensitive” than they are “specific,” with their primary objective the minimization of false negative (or Type II) errors, while allowing an acceptable level of false positive (or Type I) errors Capture multiple endpoints and reflect as many modes/mechanisms of endocrine action as possible; Be broadly predictive across species, gender, and age Yield data capable of being interpreted as either positive or negative to determine whether and how to conduct T2T

4 Selected Overarching Principles for Development of the EDSP (EDSTAC, 1998) The screening and testing (S&T) strategy should: Require the minimal number of S&T necessary to make sound decisions, to reduce the time and cost needed to make these decisions Examine existing S&T data for adverse endpoints in high-dose groups and also for physiological changes in low-dose groups Not detract from assessments of toxicity of chemical substances/mixtures via mechanisms other than endocrine disruption Provide data that can be used for a broad range of management and regulatory programs to support international harmonization of the data’s use Use a performance-based approach to select better S&T as they are developed and validated Be conducted at a minimal cost necessary to make the decisions within the EDSTAC Conceptual Framework

5 Six Specific Principles (EDSTAC, 1998) 1.To make decisions within the EDSTAC Conceptual Framework, all S&T should have well-defined endpoints. 2.The use of animals should be reduced to the minimum needed to obtain scientifically valid results and interpretations. 3.The results of S&T should support further research on effects of endocrine disruptors on populations, communities, and ecosystems. 4.In interpreting S&T results, a “weight-of-evidence” approach should be used, consistent with a principle of prudence in protecting human health and the environment. 5.S&T results should be reported in a format that facilitates database development and analysis by a broad array of scientific, regulatory, and management organizations. 6.Decision criteria, such as those for statistical significance (e.g., necessary confidence intervals) and biological plausibility, should be clearly defined.

6 Tier 1 In Vitro Assays EDSTAC in 1998EDSP in 2009Alternatives Estrogen receptor (ER) binding (cell free) or binding and transcriptional activation (transfected cell lines) SameNone Androgen receptor (AR) binding (cell free) or binding and transcriptional activation (transfected cell lines) SameNone Steroidogenesis assay with minced testes Steroidogenesis assay with H295R cell line (human female adrenocortical carcinoma) Placental aromatase (converts androstenedione or testosterone to estradiol)

7 Tier 1 Screening In Vitro Assays (continued) The in vitro assays, whether done “at the bench” or through the high throughput prescreening, should: Evaluate binding to estrogen, androgen, and perhaps thyroid nuclear receptors (cell free) Evaluate binding to the receptor in the presence and absence of metabolic capability (e.g., one or more of the P450 isozymes, CYP1A1, CYP3A4, etc.) in cell lines Distinguish between agonists and antagonists in functional assays Yield dose responses for relative potency of chemical substances/mixtures with endocrine activity

8 Tier 1 Screening In Vitro Assays (EDSTAC, 1998) Advantages: Sensitivity to low concentrations increases detectability High specificity of response Low cost Small amount of chemical substance/mixture required In vitro assays can be automated, including use of robotics High throughput assays can be developed Results can be coupled with QSAR models and for database screening Can be used for complex mixtures (sludge, water contaminants) Reduces or replaces animal use (continued)

9 Tier 1 Screening In Vitro Assays Disadvantages: Cell-free ER/AR binding assays cannot differentiate between agonists and antagonists Steroidogenesis assay with H295R cell line (only recent access is expensive and very restrictive) In vitro assays do not include ADME of the whole animal, so they can (and will) result in false positives and (worse) false negatives They cannot be used as “decision nodes” In vivo assays always trump in vitro assays

10 Tier 1 In Vivo Assays EDSTAC in 1998EDSP in 2009Alternatives Rodent 3-day uterotrophic assay (subcutaneous), adult ovariectomized Modified rodent 3-day uterotrophic assay (intraperitoneal or subcutaneous injection; OECD TG 440) Intact weanling rodent 3-day uterotrophic assay (validated; OECD TG 440); intact HPG axis, animal welfare concerns Rodent 20-day pubertal female assay with thyroid None Rodent 20-day pubertal male assay with thyroid Rodent 5-7 day Hershberger assay (adult castrate; OECD TG 441) None Intact stimulated weanling rodent Hershberger assay (less sensitive); intact HPG axis, animal welfare concerns Rodent 14-day intact adult male assay with thyroid (EPA SAP: not acceptable alternative) Modified OECD TG 407 (with endocrine endpoints) (continued)

11 Tier 1 In Vivo Assays (continued) EDSTAC in 1998EDSP in 2009Alternatives Nonmammalian Frog metamorphosis assay (thyroid) None Fish gonadal recrudescence assay (phylogenetically farthest from mammals) Fish short-term reproduction None

12 Tier 1 Screening In Vivo Assays (EDSTAC, 1998) Advantages: Account for absorption, distribution, metabolism, and excretion (ADME) Well-defined, acceptable methods used for decades General acceptability in toxicity testing Many endpoints are toxicologically relevant and are used for risk assessment Evaluate a broader range of mechanisms Provide a comprehensive evaluation of the whole endocrine system as a unit Give comparative perspective to other endpoints of toxicity

13 Tier 1 Screening In Vivo Assays Disadvantages: Phytoestrogens in the feed and bedding can confound estrogenic results Varying levels of “metabolizable energy” in feed can confound estrogenic results in intact animals Incomplete removal of ovaries in the ovx uterotrophic assay can result in endogenous estrogen production (check at necropsy) Castrate/ovariectomized animal assays only detect ER/AR agonists or antagonists (dependent on receptor binding) and will not detect EDCs with other mechanisms (e.g., phthalates) or indirect EDCs Intact animal assays detect EDCs with broader range of mechanisms (direct and indirect), but the intact weanling Hershberger assay is less sensitive to weak anti-androgens There are no T1S assays with in utero or in ovo exposures.

14 Interpretation In the best of all possible worlds, all of the in vitro and in vivo screens are negative, so the test chemical does not go to T2T or All the in vitro and in vivo screens are positive, so the test chemical must go to T2T (at least it is very clear) In the real world, it is much more likely that some screens are positive, some negative, and some equivocal. Now What? “WEIGHT OF EVIDENCE ASSESSMENT”!

15 “Weight-of-Evidence” Considerations At the end of T1S, to determine whether the evidence warrants a particular conclusion (the substance does or does not have endocrine activity for EAT) and to determine progression (or not) to T2T and Consideration on whether results from T1S should affect the conclusions from T2T (should T1S assay results be “weighted into” the determination of whether a substance passes or fails the Tier 2 tests; is it or is it not an EDC?)

16 “Weight-of-Evidence” Considerations (continued) Information considered in determining “weight-of- evidence” for T1S includes: The balance of assays that gave positive/negative results Results of in vitro versus in vivo assays The nature of the biological effects induced The range of effects observed The slope and shape of the dose-response curves The level, magnitude, and/or severity of the effects induced The presence/absence of response in multiple taxa (least important?)

17 “Weight-of-Evidence” Approach Results of some assays in some taxa, at some level of severity, are intrinsically worth more than others and should, therefore, carry more weight in decisions following T1S and T2T. Is there consistency in results of assays with same mechanism/mode of action (intentional redundancy)? The focus of T1S is on sensitivity rather than specificity. Therefore, the focus is to minimize false negatives (the risk we miss an EDC) and allow a certain level of false positives (but problem for registrants, unnecessary animal usage, costly, etc.)

18 Final Comments In vitro assays are more useful for determining mechanism than for determining endocrine activity (I think that they should be done last). Most estrogen EDCs to date have been estrogen agonists or estrogenic (estrogen mimics). Most androgen EDCs to date have been androgen antagonists or anti- androgenic (one exception: an androgen agonist, trenbolone, to fatten cattle found in feed lot effluents). The number of animals per group are rather small (typically six), but the endpoints are almost all organ weights and are therefore objective, not subjective. The 15-day intact adult male assay relies heavily on hormone assays (organ weights and histopathology are included, but the exposure period to an intact adult is brief), so the timing of necropsy and blood sampling is exquisitely important (many/most of the hormones are cyclically released with different peak timings: hourly, diurnally, etc.).

19 Final Comments (continued) Analyze the feed and bedding (phytoestrogens can be major confounders)! Make sure the necropsy team is trained, experienced, and consistent. Routes of administration were not considered terribly important at the time of EDSTAC. Now we know that ADME of xenobiotics is absolutely dependent on route with both quantitative and qualitative differences. Rodents have enterohepatic circulation, so even if the xenobiotic is metabolized (detoxified) in the gut, it can lose the glucuronide or sulfate in the liver and pass via the bile duct back into the gut (prolonging exposure to the parent compound). Metabolism to a toxic metabolite can also occur and can be route dependent.

20 Final Comments (continued) EDSTAC never got around to in vitro thyroid receptor assays, but we know a lot more now about the roles and regulation of the thyroid gland (TSH, T3, T4, rT3, etc.). Adult surgical models versus intact weanling models: strengths and weaknesses. One outstanding deficit in T1S: no evaluation of consequences of prenatal/prehatch exposures (not in EDSTAC or EDSP); T2T does include these. The more the CROs know about your chemical and results of other tests, the more they can assist you in study design, conclusions, and interpretation.

21 Take-away Points T1S assays should be interpreted as part of the screening battery EPA should define an optimal T1S battery EPA should provide guidance on interpreting T1S battery results There is intentional redundancy in T1S in vivo assays Some endpoints have more inherent variability than others (e.g., body weights, organ weights, age at vaginal patency and preputial separation, hormone levels, etc.)

22 Take-away Points (continued) Should use appropriate statistical tests Use “worst case scenarios” to detect endocrine active substances/mixtures If we accept “false positives,” it will be expensive, unnecessary use of many more animals, and a waste of time T1S is not about assessing risk T1S is not about endocrine disruption T1S is designed to identify those chemicals/mixtures capable of interacting with the endocrine system to go on to T2T (or not) THANK YOU!