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In the endocrine system, hormones produced by glands throughout the body act as chemical messengers to control a variety of body functions. Examples of hormones include estrogens, androgens, and thyroid hormones.
Endocrine disruptors include a wide range of compounds that can interfere with normal hormone function by mimicking or blocking their action, which may cause adverse health effects. Evidence suggests that environmental exposure to endocrine disruptors may cause reproductive and developmental problems in animals; the effect of endocrine disruptors in humans is less clear.
The Food Quality Protection Act of 1996 directed the EPA to screen pesticides and other substances for their potential to affect the endocrine systems of humans. EPA subsequently initiated the Endocrine Disruptor Screening Program and began efforts to standardize and validate test methods to include in the program. EPA and other ICCVAM agencies are currently exploring how high-throughput screening (HTS) approaches can identify potential endocrine disruptors without using animals.
Receptor-based in vitro transactivation assays are effective tools for screening large numbers of samples for endocrine activity. DOI adapted two commonly used human breast luciferase transactivation cell bioassays, the estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and an androgen glucocorticoid screening assay using the MDA-Kb2 human breast cancer cell line, to 384-well formats for screening of endocrine-active substances. The adaptations include a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay, which enhances precision and accuracy of the results and allows identification of cytotoxic agents. Additionally, use of the fluorescamine assay confirmed improved accuracy of luciferase activity in wells along the edge of the plate (the so-called edge effect), thereby increasing usable wells to the entire plate, not just interior wells. Overall, this method increases the utility of these cell bioassays for screening of endocrine-active substances.
NICEATM and EPA scientists developed and validated a defined approach that combines data from 18 HTS assays in an integrated testing strategy to identify chemicals with the potential to interact with the estrogen receptor pathway. Use of this integrated testing strategy was accepted by the EPA in 2015 as an alternative to three assays used in the Endocrine Disruptor Screening Program Tier 1 battery, including the rodent uterotrophic assay.
Similarly, NICEATM and EPA scientists developed an integrated testing strategy that combines data from 11 HTS assays with a computational model to identify chemicals with the potential to interact with the androgen receptor pathway. The EPA is currently considering whether this approach, described in a 2017 publication, is potentially useful for replacement of existing Endocrine Disruptor Screening Program tests. EPA anticipates further progress during 2018-2019 to develop and adopt new approach methodologies for Endocrine Disruptor Screening Program testing.
To support the estrogen receptor testing strategy described above, NICEATM created a comprehensive database of high-quality in vivo testing data from rodent uterotrophic studies. 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, and 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.
NICEATM, EPA, and OECD are currently compiling a similar reference database on rodent Hershberger studies, which will be described in two journal articles to be submitted for publication in 2018. This database 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 MDA-Kb2 cells to measure androgen receptor agonist and antagonist activity. Testing of 67 reference chemicals will be completed in early 2018 to characterize the reliability and relevance of the method, and subsequent testing of 30 consumer products will evaluate the utility of the method for testing chemical formulations. The study is planned to run through the end of 2018.