An integrated testing strategy is a type of defined approach to testing and assessment that relies on:
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.
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:
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.
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 is using data generated by the multiagency Tox21 research initiative to develop models and other resources to predict potential endocrine activity, including:
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.
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 Pre-Screen Evaluation of the In Vitro Endocrine Disruptor Assay (Robotic MCF-7 Cell Proliferation Assay of Estrogenic Activity) (October 2006)
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.