Use of New Approach Methodologies
Learn more about the future of the Endocrine Disruptor Screening Program (EDSP):
Preface
The ability to screen chemicals rapidly for bioactivity in several endocrine pathways, and reducing the use of animals in testing, have been EDSP goals since 1998, when the program was first adopted.
As previously noted, when the first Tier 1 orders (for List 1 chemicals) were issued in 2009, EPA had not confirmed the reliability and relevance of the ToxCastTM results so that they could be cited as “other scientifically relevant information” to satisfy the Tier 1 order. However, since that time, EPA has reached a critical juncture, determining that the science has progressed such that reevaluation of EPA's earlier position is warranted.
Based on scientific advances, EPA intends to implement the use of high throughput screening assays and computational models to evaluate, and to a significant extent, screen chemicals. The in vitro high throughput and computational model alternatives provide an accurate quantitative measure of specific endocrine receptor binding bioactivity and mechanisms that can serve as alternatives to the current Tier 1 estrogen receptor (ER) binding, ER transactivation (ERTA), uterotrophic assays and androgen receptor (AR) binding in vitro assay.
Learn more about TOX21 and the future of evaluating chemical safety with this short video. For additional information concerning high throughput screening assays and computational models, please refer to the document: Availability of New Approach Methodologies (NAMs) in the Endocrine Disruptor Screening Program (EDSP).
High Throughput Assays
High throughput assays are automated methods that allow for a large number of chemicals to be rapidly evaluated for a specific type of bioactivity at the molecular or cellular level. This approach, which can help identify compounds that may modulate specific biological pathways, was initially developed by pharmaceutical companies for drug discovery. The results of these methods provide an initial understanding of a biochemical interaction or possible role of a chemical in a given biological process.
In vitro high throughput assays are usually conducted using a microtiter plate: a plate containing a grid with a large number of small divots called “wells.” The wells contain chemical and/or biological substrate (e.g., living cells or proteins). Depending on the nature of the experiment, changes can be detected (e.g., color, fluorescence, etc.) when the chemical is added to indicate whether there is bioactivity. High throughput microtiter plates typically come in multiples of 96 wells (96, 384, or 1536), so that through the use of robotics, data processing and control software, liquid handling devices, and sensitive detection methods, an extremely large number of chemicals can be evaluated very efficiently.
High throughput assays can be run for a range of test chemical concentrations and produce concentration-response information representing the relationship between chemical concentration and bioactivity. The concentration-response data from multiple assays can be mathematically integrated in a computational model of a biological pathway, providing values representative of a chemical's bioactivity in that pathway (e.g., estrogen receptor pathway).
To reduce non-specific results, the computational model can use results from multiple assays and technologies to predict whether a chemical is truly bioactive in the pathway being evaluated. The most prominent cause of non-specific results (activity in an assay that is likely not due to bioactivity of the chemical in the pathways) is cytotoxicity in cell-based assays. In other cases, chemicals influence the assays through a manner dependent on the physics and chemistry of the technology platform (i.e.,“assay interference”).