National Toxicology Program

National Toxicology Program
http://ntp.niehs.nih.gov/go/40417

Validation of the BG1Luc Transactivation Test Method

U.S. Federal agencies have agreed with ICCVAM recommendations on the use of human cell-based test methods to screen for substances with the potential to interact with the estrogen receptor. ICCVAM evaluated the scientific validity of the BG1Luc estrogen receptor (ER) transactivation (TA) agonist and antagonist assays and recommended how they could be used to identify substances that induce or inhibit human ER activity in vitro. The U.S. Environmental Protection Agency (EPA) responded that they regard the BG1Luc ER TA test method as an alternative to the ER TA test method currently used in their Endocrine Disruptor Screening Program. Several other Federal agencies also indicated that they would communicate the ICCVAM recommendations to stakeholders and encourage appropriate use of the recommended methods.

Test Method Evaluation Report (February 2012) with ICCVAM recommendations

ICCVAM developed the recommendations on the BG1Luc ER TA assays after considering the comments of an international independent peer review panel that met in 2011, as well as comments from the public and the Scientific Advisory Committee on Alternative Toxicological Methods. ICCVAM recommended that the BG1Luc ER TA test methods may be used as screening tests to identify substances with in vitro ER agonist or antagonist activity.

ICCVAM concluded that the accuracy of the BG1 ER TA agonist assay is at least equivalent to the only ER TA test method currently in a U.S. regulatory test guideline, the EPA’s “OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903))”. The BG1Luc ER TA test method was found to offer several advantages over the existing ER TA method, including (1) validation for use over a wider concentration range of test substances, (2) potential to detect a wider range of ER-active substances, (3) ability to identify both substances that induce and inhibit the estrogen receptor, and (4) availability of the cell line used for the test from more than one source.

See below for letters to Federal agencies communicating ICCVAM recommendations and agency responses


Background on the BG1Luc ER TA Test Method Validation Study

In 2003, ICCVAM evaluated the validation status of in vitro ER and androgen receptor (AR) binding and TA test methods for potential use in the EPA's Endocrine Disruptor Screening Program. The evaluation indicated that there were no in vitro ER- or AR-based test methods that were adequately validated for this purpose. In response to an ICCVAM request for nominations of test methods, the BG1Luc ER TA was nominated to ICCVAM for a validation study.

The BG1Luc ER TA test method (also known as the LUMI-CELL® test method) was developed by Xenobiotic Detection Systems, Inc., with support from a Small Business Innovation Research grant from the National Institute of Environmental Health Sciences. The test method measures the extent to which a substance induces or inhibits TA activity via ER-mediated pathways in recombinant BG1Luc4E2 cells.

The BG1Luc4E2 cell line was derived from BG-1 immortalized adenocarcinoma cells that endogenously express both human ER forms, ERα and ERβ and that have been stably transfected with the plasmid pGudLuc7.ERE. This plasmid contains four copies of a synthetic oligonucleotide containing the estrogen response element upstream of the mouse mammary tumor viral promoter and the firefly luciferase gene. BG-1Luc4E2 cells emit measurable light in response to estrogen and estrogen-like substances.

NICEATM coordinated an international interlaboratory validation study of the BG1Luc ER TA test method. The goal of the validation study, which was completed in 2010, was to evaluate the usefulness of the BG1Luc ER TA test method as a screening test to identify substances with in vitro ER agonist and antagonist activity. The international validation study of the BG1Luc ER TA test method included participating laboratories located in Italy, the U.S., and Japan. It was the first validation study sponsored jointly by ICCVAM, the European Centre for the Validation of Alternative Methods (now known as the European Union Reference Laboratory for Alternatives to Animal Testing), and the Japanese Center for the Validation of Alternative Methods.

Peer Review of the BG1Luc ER TA Validation Study

In a March 2011 public meeting, an independent international peer review panel agreed with ICCVAM draft test method recommendations stating that an in vitro test method may be used as an initial screen to identify substances with the potential to enhance or inhibit activation of the estrogen receptor. The panel of 16 expert scientists from six countries reviewed data from the validation study, made recommendations on future studies that could further enhance the usefulness of this test method, and commented on draft performance standards that could be used to expedite the evaluation of similar test methods.

Read the peer review panel report (May 2011)

Corrections to the Peer Review Panel Report (August 2011)
This document lists changes made to the report by the peer review panel members that were inadvertently omitted in the printed version of the report. The electronic version of the report available above includes these corrections. If you have a printed copy of this report, please print this document and keep it with the printed report. We apologize for any inconvenience.

Read a summary of the March 2011 peer review panel meeting

View agenda for the peer review panel meeting

BG1Luc ER TA Submitted to Tox21

NICEATM also nominated the BG1Luc ER TA test methods for evaluation in Tox21. The assays have now been adapted to a high throughput format using 1536-well plates by the National Center for Advancing Translational Sciences and have been used to screen all compounds in the Tox21 10K chemical library. More about the Tox21 program


Transmittal of Recommendations to Federal Agencies


Other Materials Relevant to the BG1Luc ER TA Test Method Validation Study

Test Method Nomination: Submission of XDS’s LUMI-CELL® ER High-Throughput System for Screening Estrogen-Like Chemicals for Review by ICCVAM (February 2005)

Draft Evaluation: Nomination of the LUMI-CELL® ER High-Throughput System for Screening Estrogen-Like Chemicals for Validation Studies (August 2004)

Protocols for the International Validation Study:

Links to Home Pages of Laboratories Participating in the Conduct of the BG1Luc ER TA test method validation study

The links listed below connect to pages outside the ICCVAM website. These links are for the convenience of visitors to this page, who may find them useful. ICCVAM is not responsible for the availability or content of these external sites, nor do we endorse, warrant or guarantee any information or any products or services described or offered at these other sites.

Xenobiotic Detection Systems, Inc.

Hiyoshi Corporation

ECVAM Laboratories

The NTP is located at the National Institute of Environmental Health Sciences, part of the National Institutes of Health.