Citation:

Laws, S. AND V. Wilson. Integrated Summary Report: Validation of Two Binding Assays Using Human Recombinant Estrogen Receptor Alpha (hrERa). U.S. Environmental Protection Agency, Washington, DC, 2014.

Impact/Purpose:

This Integrated Summary Report (ISR) was prepared at the request of OCSPP (OSCP) and allowed OSCP to meet an OMB deadline for the Agency's Endocrine Disruptor Screening Program. Purpose: This ISR summarizes the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays, and will be used as the primary document for a letter peer review to support the final step in the validation process. It is anticipated that once these assays are fully validated they will provide an updated alternative for the Agency’s current test guideline for an ER binding assay (OPPTS 890.1250) and will likely also lead to the development of a performance-based test guideline for ER binding assays in collboration with the OECD.

Description:

This Integrated Summary Report (ISR) summarizes, in a single document, the results from an international multi-laboratory validation study conducted for two in vitro estrogen receptor (ER) binding assays. These assays both use human recombinant estrogen receptor, alpha subtype (hrERα), to identify chemicals that may impact estrogen signaling through binding to the ER. The purpose of the ISR is to support the peer review of the findings obtained during the validation process.The two assays evaluated during this validation process are: The Freyberger-Wilson Assay (FW) using a full length human ER, and The Chemical Evaluation and Research Institute (CERI) Assay using a ligand-binding domain of the human ER.The two assays are mechanistically and functionally similar in that each measures the ability of a test chemical to competitively inhibit binding of [3H]17β-estradiol to the human recombinant ER. The essential elements of the FW and the CERI assays were developed at the laboratories of Bayer Pharma AG, Wuppertal, Germany (Freyberger et al., 2010) and CERI, Tokyo, Japan (Akahori et al., 2008), respectively.The ER competitive binding assay has long been in use, and is a well characterized approach, but historically uses rodent or other animal tissues as a source of the ER. Validation of the FW and CERI assays using human recombinant estrogen receptors ( subtype) will provide an updated alternative for the Agency’s current test guideline (OPPTS 890.1250 (USEPA, 2009b) that does not require the use of animals as the source of ER and also permits a higher through-put capability.The U.S. EPA served as the lead for an international validation study using two optimized protocols for the CERI and FW hrER binding assays. The assays each contain the following two major components: 1) saturation binding and 2) competitive binding. Saturation binding is important because it confirms the specificity and activity of the receptor preparations. The competitive binding experiment is used to evaluate the ability of a test compound to bind to hrER and is the key component of the assay. Transferability and reproducibility of the optimized test methods were successfully demonstrated by having a total of six laboratories for the FW assay and five for the CERI assay participate in the validation study. Overall, a total of 29 compounds were tested in both assays which included three control chemicals, nine uncoded chemicals and 17 coded chemicals across 3 subtasks. ER binding assays are designed to identify chemicals that have the potential to impact the estrogen signaling pathway. The overall goal of the validation study was to demonstrate the ability of each of two assays to reliably and reproducibly classify the test chemical set into binders and non-binders. Laboratories had little trouble with the binders which produced a full binding curve using either the CERI or FW assay. As consistent with all ER binding assays, the weak binders proved to be more challenging for the laboratories because it is not always possible to test weak binders at concentrations high enough to produce a full binding curve. Those runs typically need closer scrutiny to determine if additional analysis is required before classification. Finally, the results from both the FW and CERI assays were consistent and in agreement with expected classifications even though there were differences in the form of the hrER (full length ER for FW vs. ligand binding domain for CERI) and subtle differences in the protocols. Further, reproducibility of both assays is comparable to the existing ER binding guideline OPPTS 890.1250 without the need for the use of animal as the receptor source.

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