Wildlife ScreensWhat Do They Tell Us?

Dr. Pat GuineyManager Global Safety, Regulatory & Environmental

AssessmentS.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.

http://www.epa.gov/endo/pubs/att-f_fish_assay_protocol.pdf

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

TimeframeApproximately 12-17 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 development Thyroid Activity

Remarkable histological effects Thyroid Activity

Thyroid Inactive

No

No

No

Yes

Yes

Yes

Summary of Modes of Action Identified By EDSP Screens

Current EDSP Assays Mode of Action Covered by Assay

Steroidogenesis

E Anti-E A Anti-A T E HPG HPT

Fish Short-Term Reproduction x x x x x x x

Amphibian Metamorphosis x

ER Binding or Transactivation x x

AR Binding x x

Steroidogenesis x x

Aromatase x

Uterotrophic x x

Hershberger x x

Pubertal Male x x x x x

Pubertal Female x x x x x

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(http://www.epa.gov/endo/pubs/att-h_histopathologyguidlines_fhm.pdf)

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 IdentifyingEndocrine 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 – ER2

• Lisa Ortego – Bayer CropScience• Ron Biever – Springborn Smithers