National Toxicology Program

National Toxicology Program
https://ntp.niehs.nih.gov/go/iccvam-endocrine

Endocrine Disruptor Test Method Evaluations


Integrated Testing Strategies to Identify Potential Endocrine Disruptors

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

  • Input data generated from specific sources
  • A data interpretation procedure such as a machine-learning model, flowchart, or decision tree, through which the input data is run
  • A simultaneous assessment of the input data to arrive at either a hazard prediction or a decision that more testing is needed

More about defined approaches to testing and assessment

NICEATM and U.S. Environmental Protection Agency (EPA) scientists developed an integrated testing strategy that combines data from 18 high throughput screening (HTS) assays to identify chemicals with the potential to interact with the estrogen receptor (ER) pathway. 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.

Similarly, NICEATM and EPA scientists developed an integrated testing strategy that combines data from 11 HTS assays with a mathematical model to identify chemicals with the potential to interact with the androgen receptor (AR) pathway. The EPA is currently considering whether this approach is potentially useful for replacement of existing tests currently required in the EDSP.

Reference: Kleinstreuer et al. 2017. Development and validation of a computational model for androgen receptor activity. Chem Res Toxicol 30(4):946-964.

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

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

  • Evaluation of uterotrophic assay study design and variability
  • In vitro to in vivo extrapolation to facilitate direct comparison of data from 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 on rodent Hershberger studies. This work will support validation of high throughput assays to identify androgen-active chemicals.

Validation Study of Test Method to Identify Chemicals with Androgenic Activity

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 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:

  • Physiologically based pharmacokinetic models to quantitatively correlate in vitro and in vivo dosimetry for ER reference chemicals using Tox21 ER data
  • Quantitative structure–activity relationship (QSAR) models to predict ER pathway binding and antagonism activity to help prioritize future testing of chemicals in the EDSP
  • QSAR models to predict AR pathway binding and antagonism activity to help prioritize future testing of chemicals in the EDSP
  • A reference chemical list to validate high throughput assays used to identify chemicals with androgenic or antiandrogenic activity (with collaborators at EPA)
  • Mathematical models to distinguish true AR pathway activity from technology-specific assay interference using data from 11 Tox21 and ToxCast assays
  • A reference in vivo database to validate high throughput assays used to identify chemicals with androgenic or antiandrogenic activity (with collaborators at EPA)
  • Domain specific QSAR models, such as models to predict specific activity and relative potency of phenolic compounds that act on the ER pathway (with collaborators at the University of North Carolina at Chapel Hill)

Validation of the LUMI-CELL Test Method

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

Please note: In June 2016, the generic name of the LUMI-CELL assay changed from the BG1Luc ER TA assay to 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 LUMI-CELL test method (also known at the time as the BG1Luc ER TA 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 LUMI-CELL test method formed the basis for Test Guideline 455 issued in 2012 and updated most recently in 2016 by the Organisation for Economic Co-operation and Development (OECD). Adoption of Test Guideline 455 means that these ER TA 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 LUMI-CELL agonist and antagonist assays have also been adapted to an HTS format and used in the Tox21 HTS 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 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, interlaboratory reproducibility was insufficient for the method to proceed further. The study indicated that the test method protocols, especially the antagonist protocol, required additional development before this method could 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 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 2002 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.