## Review of ER/AR Binding & Estrogen Receptor Transcriptional Assay (ERTA) Methodology and Performance
Presented By: Steve Levine, Ph.D. Senior Science Fellow Ecotoxicology Monsanto Company On Behalf of the Endocrine Policy Forum May 2013

# Overview of Comments

• Comments on the AR/ER competitive binding assays

• Approaches and challenges to the ERTA HeLa

- assay (OECD 455)

• Advantages of “new” BG1 Assay (OECD 457)

• ERTA Assay performance

• Comments on cell line assays and impacts of cytotoxicity

• Closing remarks

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# General Comments for AR/ER

• Current assays are very large and require many test tubes with cytosolic preparations

– Advancements with recombinant receptors in HTP format

• Solubility issues at the high concentrations and assay should be limited to a maximum concentration of 100 uM not 1000 uM. Validation was only performed to max of 100 uM

• There is a desire from testing facilities to harmonize AR/ER

• Screens generally appear to be specific but some compounds can result in false positives

• These assays could eventually be replaced with reliable and validated transcriptional activation assays

• EPA developed SAR model to predict potential estrogenic activity

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# ERTA Model

Translation New Polypeptides Increased Estrogen Responsive Gene Product Estrogens and Xenoestrogens

Luciferase Activity mRNA

EREs Estrogen Responsive Gene

EREs Luciferase Gene hsp90 Estrogen Receptor

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# ERTA Methodology

• In 890.1300, cells treated with compound across a dose range only 3 hours after plating in the presence of stripped serum

• Activity and cytotoxicity assessed ~24 h after exposure

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# PC10 and Inducibility

6 At the highest level of induction during the validation, the PC10 values only correspond to 1.6

- fold induction

# OECD Panel Comments on the PC10

• The OECD validation review Panel had several reservations about the reliability of the PC10 value

• Review stated: “ If the PC10 value were to be used as an indicator of a positive response, it may lead to identification of a lot of false positives when testing a broad array of environmental compounds. ”

• ICCVAM Appendix B7 stated “ An efficacy of 25% of the positive control should be considered a positive response for that test chemical in that assay .”

• Consistent with the ICCVAM recommendation, the BG1 assay uses a PC20 as the endpoint

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# Assay Performance

• Overall poor concordance between ER binding assay and ERTA

• ERTA had 5 positive out of 19 compounds evaluated to 0 for the ER binding assay

• In the BG1assay concordance between the two assays was >95%

• High concordance between the ER and uterotrophic

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# Cytotoxicity

• ICVAAM recommended that only dose levels not associated with >10% cytotoxicity should be included in the analysis.

• The TGs permits ≤ 20% cytotoxicity which is to high particularly if the assay will be used to assess antagonism

• Cytotoxicity assessment should be quantitative rather than visual categorical rating

– BG1 allows for this

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# Closing Remarks

• Retrospective analysis needed for studies using the PC10 value rather than the PC20 in the WoE evaluations for compounds judged to be positive with a value >PC10 but <PC20.

• Further consideration that cytotoxicity should not be >10%

• Cytotoxicity particularly important if antagonism is evaluated

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