National Toxicology Program

National Toxicology Program

Endocrine Disruptor Test Method Evaluations

Integrated Testing Strategies to Identify Potential Endocrine Disruptors

An integrated testing strategy is a limited type of integrated approach to testing and assessment that relies on:

  • Input data generated from identified methods
  • A computational model or other evaluation protocol through which the data is run

More about integrated approaches to testing and assessment

NICEATM and U.S. Environmental Protection Agency (EPA) scientists developed and validated an integrated testing strategy that runs data from 18 high throughput screening assays through a computational model to identify chemicals with the potential to interact with the estrogen receptor (ER). Use of this integrated testing strategy has been accepted by the EPA as an alternative to three assays currently used in its Endocrine Disruptor Screening Program (EDSP) Tier 1 battery.

Reference: Browne et al. 2015. Screening chemicals for estrogen receptor bioactivity using a computational model. Environ Sci Technol 49:8804-8814.

NICEATM and EPA are developing a similar strategy to identify chemicals with the potential to interact with the androgen receptor.

Please note that the terms “integrated testing strategy” and “integrated approach to testing and assessment” represent evolving concepts. NICEATM has adapted these definitions from a preliminary draft guidance document being prepared by the Organisation for Economic Co-operation and Development.

Reference Data for Evaluation of In Vitro Test Methods and Computational Models

To support the testing strategy described above, NICEATM created a comprehensive database of high-quality in vivo testing data from over 1000 articles from the scientific literature describing over 1500 uterotrophic assay experiments to identify chemicals with ER interaction potential. This database is available to support other validation efforts of in vitro test methods and computational models of estrogenic activity. Current projects using the database include:

  • Evaluation of uterotrophic assay study design and variability
  • Comparison of data from high-throughput screening (HTS) assays in the EPA ToxCast program to high-quality in vivo data

These analyses will provide insights about the reproducibility and variability of uterotrophic data and allow for the evaluation of in vitro assay data utility, including HTS data, for predicting in vivo responses.

Reference: Kleinstreuer et al. 2015. A curated database of rodent uterotrophic bioactivity. Environmental Health Perspectives DOI:10.1289/ehp.1510183

NICEATM is currently contributing to the construction of a similar reference database to support validation of high throughput assays to identify androgen-active chemicals.

Validation Study of Test Method to Identify Androgen-active Chemicals

NICEATM is collaborating with the test method developer CertiChem, Inc., to validate an in vitro test method that uses a human breast cancer cell line (MDA-Kb2 cells) to measure androgen receptor (AR) agonist and antagonist activity. The study will test 67 reference chemicals to characterize the reliability and relevance of the method, and 30 consumer products to evaluate its utility beyond single chemicals. The study is planned to run through summer 2017.

NICEATM Activities Contributing to the Prediction of Endocrine Activity

NICEATM is using data generated by the multiagency Tox21 research initiative to develop models and other resources to predict potential endocrine activity, including:

  • Domain specific quantitative structure–activity relationship (QSAR) models; for example, models to predict specific activity and relative potency of phenolic compounds that act on the estrogen pathway (with collaborators at the University of North Carolina at Chapel Hill)
  • Mathematical models to distinguish true AR pathway activity from technology-specific assay interference using data from 11 Tox21 and ToxCast assays; the results from this model will be compared to data from low throughput in vitro AR assays and in vivo Hershberger test results
  • QSAR models to predict binding and antagonism activity against the AR pathway for binding and antagonism; these models will be used to help prioritize future chemicals testing in the EDSP.
  • List of reference chemicals to validate high throughput assays used to identify androgen active chemicals (with collaborators at EPA)
  • Physiologically based pharmacokinetic models to quantitatively correlate in vitro and in vivo dosimetry for ER reference chemicals using Tox21 ER data; this approach highlights the importance of pharmacokinetic considerations in assessing and ranking endocrine-active chemicals based on in vitro HTS assays, and is being expanded to extrapolate ToxCast data for a wider range of environmental chemicals
    Reference: Chang et al. 2014. Application of Reverse Dosimetry to Compare In vitro and In vivo Estrogen Receptor Activity. Applied In Vitro Toxicology, 1(1): 33–44.

Validation of the BG1Luc Estrogen Receptor Transactivation Test Method

Upon evaluation of the BG1Luc ER transactivation (TA) agonist and antagonist assays, ICCVAM recommended that they could be used to identify substances that induce or inhibit human ER activity in vitro (2012). Federal agencies concurred with the ICCVAM recommendation; EPA responded that they regard the BG1Luc ER TA test method as an alternative to the ER TA test method included in the EDSP Tier 1 battery.

Please note: Beginning in June 2016, the BG1Luc ER TA assay will be referred to as the VM7Luc ER TA assay. This reflects new information regarding the identity of the cell line used in the assay. Refer to the webpage summarizing the assay validation for more details.

The ICCVAM recommendations were based on data from an international interlaboratory validation study of the BG1Luc ER TA test method (also known as the LUMI-CELL® test method) coordinated by NICEATM. The study included three laboratories in the U.S., Italy, and Japan, sponsored respectively by NICEATM, 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.

The ICCVAM recommendations on the BG1Luc ER TA test method formed the basis for test guidelines issued in 2012 and updated in 2015 by the Organisation for Economic Co-operation and Development (OECD). Adoption of these guidelines means that the BG1 agonist and antagonist assays can be used in the 34 OECD member countries to identify substances that induce or inhibit human ER activity in vitro.

The BG1Luc ER TA agonist and antagonist assays have also been adapted to an HTS format and used in the Tox21 high-throughput screening program.

Validation of CertiChem Inc. MCF-7 Cell Proliferation Test Method

NICEATM coordinated an international interlaboratory validation study of a MCF-7 cell proliferation test method, developed by CertiChem, Inc., for the detection of estrogenic activity. The test method uses a human cell line to screen for substances that may induce cell proliferation via ER-mediated pathways. The cell line (MCF-7 WS8) is a subclone of the MCF-7 cell line, an immortalized human breast adenocarcinoma cell line that endogenously expresses both human ER forms, ERα and ERβ.

The study included participating laboratories located in the U.S., Japan, and Korea. It was sponsored jointly by ICCVAM, the Japanese Center for the Validation of Alternative Methods, and the Korean Center for the Validation of Alternative Methods.

The validation study was completed in 2011. Although accuracy of the ER agonist protocol was high at the lead laboratory and sufficient in the partner labs, transferability, as indicated by interlaboratory reproducibility, was insufficient for the method to proceed further. The test method protocols, especially the antagonist protocol, require additional development to enhance interlaboratory reproducibility before this method can be considered validated.

NICEATM Draft Validation Study Report (June 2012)

NICEATM Pre-Screen Evaluation of the In Vitro Endocrine Disruptor Assay (Robotic MCF-7 Cell Proliferation Assay of Estrogenic Activity) (October 2006)

Review of In Vitro Endocrine Disruptor Screening Assays

ICCVAM evaluated the validation status (2003) of four types of in vitro methods proposed as possible components of the EDSP. NICEATM and ICCVAM prepared background review documents that detailed the available data and information needed to evaluate the current validation status of each test method type. An independent expert panel review of the ICCVAM background review documents concluded that there were no adequately validated in vitro ER- or AR-based test methods. ICCVAM subsequently developed test method recommendations that included minimum procedural standards and a list of reference substances that should be used to standardize and validate in vitro ER and AR binding and TA test methods.

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