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

New Polypeptides Translation

Increased EstrogenResponsive 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

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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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