# Presentation: 295210

## Comments EDSP Tier 1 Fish Short-Term Reproduction Assay

- Reinhard Fischer, Ph.D.
- Bayer CropScience

## Background for Comments 

- Review of proposed EPA design
- Currently no information available how results will be used within the screening battery.
- Assay by itself to provide data that can be clearly interpreted as either positive or negative for endocrine disruption.

## Is the basic design of the assay scientifically sound and suitable as a Tier 1 screen?

- Study design is scientifically sound
- Does not fulfill _in its current design_ requirements of a _screen_ for _endocrine disruption_ in fish
*“... **some endpoints may be responsive to non-endocrine stresses in addition to endocrine-mediated pathways, particularly fecundity.” *(Technical Review Document for Endocrine Disruptor Screening Program (EDSP): Proposed Tier 1 Screening Battery, EPA, 2008)
*“... **any significant effect in one or more of the core endpoints of this assay (fecundity, histopathology, GSI, sex steroid measurements, vitellogenin, and secondary sex characteristics) should be considered a positive response.”*
- False positives possible by design

## Is the basic design of the assay rapid and cost effective? 

- Not Rapid
- Exposure duration 21 days + pre-exposure period 14 - 21 days
- plus time for a 4 – 7 day range-finding test
- Labor extensive
- Expensive
- incl. analytic, histopathology, plasma sex steroids ~ $100,000
- Focus on OECD endpoints (VTG, SSC) may offer option for reduction in test duration and costs

## Is the basic design of the assay mechanistic? 

- Vitellogenin, secondary sex characteristics mechanistic, BUT can be also impacted by systemic toxicity
- e.g. liver toxicity affecting VTG
- Fecundity, fertility, gonadal histopathology not necessarily mechanistic
- May require additional information for interpretation

## Are the results straight forward to interpret? 

- “Some endpoints may be responsive to non-endocrine stresses in addition to endocrine-mediated pathways, particularly fecundity”
-  	(Technical Review Document for Endocrine Disruptor Screening Program (EDSP): Proposed Tier 1 Screening Battery, EPA, 2008)
- Interpretation will require additional data and efforts

## Has the test method been sufficiently  standardized ?

- Considerable variability during validation
- Variability of histopathology and plasma sex steroids measurements open
- Performed only by single laboratory each
- Some technical aspects should be revisited and modified as needed.
- e.g. survival criteria, age of animals, minimum requirement for control spawning, pre-exposure in the same tanks as exposure?

## For its intended purpose is the method ...  

- Sensitive?
- Yes, validation exercise identified _all_ chemicals
- Specific?
- Not in current design.
- Predictive?
- Depends on evaluation method
*“... **any significant effect in one or more of the core endpoints of this assay (fecundity, histopathology, GSI, sex steroid measurements, vitellogenin, and secondary sex characteristics) should be considered a positive response.”*
- Reproducible?
- Yes, although more work needed to reduce variability

## What are the strengths and limitations?

- Strengths
- Sensitive
- Able to detect _reproductive toxicants_ in fish
- Limitations
- Depending on endpoints and evaluation method, can be unspecific
- False positives by design
- Significant variability in validation exercise
- Worldwide laboratory capacity limited

## Overall conclusions

- EPA assay in design and extent beyond requirements of a screen for detection of potential endocrine disruption in fish
- Biological test procedure adequately demonstrated
- Some of the measurement endpoints not validated
- Some of the protocol parameters need to be refined or clarified
- Limited laboratory capacity worldwide for such a complex study
- EPA and OECD test methodologies for fish screen to be harmonized
- OECD focus on vitellogenin and secondary sex characteristics

## Measurement Endpoints in OECD FSA and EPA FRSA

**OECD ****Fish Screening Assay**
- Vitellogenin (VTG)
- Secondary Sex Characteristics

**EPA Fish Reproduction Screening Assay**
- Vitellogenin (VTG)
- Secondary Sex Characteristics
- Gonadal-Somatic Index (GSI)
- Fecundity
- Fertility
- Histopathology
- Plasma Sex Steroids

## Thank you very much for your attention