Wildlife Screens What Do They Tell Us?
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Transcript of Wildlife Screens What Do They Tell Us?
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