Wildlife Screens What Do They Tell Us? Dr. Pat Guiney Manager Global Safety, Regulatory & Environmental Assessment S.C. Johnson & Son, Inc. Racine, WI

Summary Introduction Screening Tier – Ecotoxicity Assays –Fish Short-Term Reproduction –Amphibian Metamorphosis Study Considerations Weight of Evidence Conclusion

Tier 1 Screening A battery of short-term assays that will identify the potential for a substance to interact with the estrogen, androgen, and thyroid hormonal systems Mechanistic in scope? –4 in vitro screens –4 in vivo mammalian screens –2 ecotoxicity screens

Fish Short-Term Reproduction Assay Purpose To assess reproductive performance as the primary indicator for potential endocrine disruption. Endpoints include morphology, histopathology, and biochemistry to ensure that potential toxicological and endocrine mechanisms of concern are detected for the test chemical.

Fish Short-Term Reproduction Assay Design –Reproductively mature fathead minnows (Pimephales promelas) –Flow-through exposure system –Minimum of three concentrations of a test chemical and appropriate control(s) –Four replicates per concentration/control, four females and two males per replicate –14 day pre-exposure to establish successful spawning –21 day exposure

Fish Short-Term Reproduction Assay Endpoints –Adult survival –Reproductive behavior –Fecundity –Fertility –Secondary sexual characteristics –Gonadal histopathology –GSI –Vitellogenin –Plasma sex steroids

Fish Short-Term Reproduction Assay Timeframe –6 months to rear test population, 5 weeks of pre-exposure and in-life and 4-6 weeks for biochemical analyses, histopathology, report Cost –$100,000 - $120,000 excluding the cost of sex steroid radioimmunoassay (cost unknown) –Histopathology $35,000 - $50,000 –Analytical method validation, development of solvent-free delivery system

Amphibian Metamorphosis Assay Purpose A screening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic-pituitary- thyroid (HPT) axis. Amphibian metamorphosis is a thyroid-dependent process which responds to substances active within the HPT axis.

Amphibian Metamorphosis Assay Design –Xenopus laevis tadpoles at NF stage 51 –Flow-through exposure system preferred –Minimum of three concentrations of a test chemical and appropriate control(s) for 21 days –Four replicates per concentration/control –Larval density at test initiation is 20 tadpoles per replicate

Amphibian Metamorphosis Assay Endpoints –Mortality –Snout-vent length (days 7 and 21) –Hind limb length (days 7 and 21) –Wet weight (days 7 and 21) –Developmental stage (days 7 and 21) –Histology - Thyroid gland (day 21)

Amphibian Metamorphosis Assay Timeframe Approximately days to rear acceptable testing population, 3 weeks of in-life and 4-6 weeks for histopathology Cost –$100,000 - $110,000 –Histopathology $45,000 - $55,000 –Analytical method validation, development of solvent-free delivery system

Amphibian Metamorphosis Decision Logic Advanced development Thyroid Activity Asynchronous developmentThyroid Activity Remarkable histological effects Thyroid Activity Thyroid Inactive No Yes

Summary of Modes of Action Identified By EDSP Screens Current EDSP AssaysMode of Action Covered by Assay Steroidogenesis EAnti-EAAnti-ATEHPGHPT Fish Short-Term Reproductionxxxxxxx Amphibian Metamorphosisx ER Binding or Transactivationxx AR Bindingxx Steroidogenesisxx Aromatasex Uterotrophicxx Hershbergerxx Pubertal Malexxxxx Pubertal Femalexxxxx

Consideration: Histopathology The 2006 Histopathology guideline document offers good instructions but does not discuss the relevance of any findings in the context of the study (  Use toxicological histopathologist with small fish experience –Spermatogonia, testis-ova, testicular degeneration, interstitial cell and perifollicular cell abnormalities, oocyte atresia, decreased yolk formation, change in gonadal staging, and a host of secondary endpoints are all required  No guidance in protocol as to what is required and what the findings will mean in the context of the study.

Consideration: Weight of Evidence The weight of the evidence both within a study and between studies must be considered before requiring further testing  When there is good mechanistic information, the “potential” to interact with the endocrine system is fairly straightforward to identify. This is not the case for screens that are apical in nature. In those cases the results should be considered only in the context of other studies that are mechanistically based.

Consideration: Reportability Adverse effect reporting (FIFRA and TSCA) is required however…  What is the definition of adverse in a screening study?  Mechanistic versus apical endpoints  The order of the performance of the screens could be very important

Guidance on Identifying Endocrine Disrupting Effects ECETOC Technical Report No. 106, June 2009

Conclusion Guidance by OECD for establishing test guidelines New and updated TGs should reflect scientific progress, address animal welfare aspects and improve cost-effectiveness of test methods

Conclusion The resources required for the ecotoxicity screens are not trivial. There must be a willingness to critically evaluate individual endpoints for relevance and robustness, as well as the inclusion of the study in a weight of evidence Be willing to remove endpoints or screens that are not informative or relevant for the purposes of potential endocrine activity screening

Acknowledgements Ellen Mihaich – ER 2 Lisa Ortego – Bayer CropScience Ron Biever – Springborn Smithers