Gaining Confidence in Advanced Methodologies for Studying Challenging Chemicals
Wednesday, February 22, 2023
11:00 a.m - 12:30 p.m ET
Integrating High Throughput Transcriptomics into a Tiered Framework to Prioritize Chemicals for Toxicity Testing
Presented by Jesse Rogers, PhD
US EPA is developing a tiered assessment strategy for chemical toxicity testing by integrating multiple data streams. Pairing high content assays such as high-throughput transcriptomics (HTTr) with high-throughput screening (HTS) of specific molecular targets may improve confidence when assessing key hazards. Here, we used HTTr screening data to generate new signatures representing known molecular targets, and signature-level potency estimates were integrated with orthogonal HTS assays as a proof-of-concept framework for chemical prioritization.
Transcriptomic profiles generated via the TempO-Seq platform in both HepaRG and U-2 OS cell lines were used to develop signatures comprised of genes selectively responsive to reference chemicals for one of 13 distinct molecular targets. Of 1,218 chemicals screened in HTTr to date, 232 chemicals demonstrated selective potency in at least one reference signature versus non-selective potency estimates. In examining these chemicals using available orthogonal HTS assays from US EPA’s ToxCast program, 74 chemicals were confirmed as selective AHR, GR, or RAR/RXR nuclear receptor agonists.
Our work demonstrates that HTTr data can inform putative molecular targets and identify chemicals for further screening in a framework to support chemical risk assessment.
The views expressed in this abstract are those of the authors and do not necessarily reflect the views or policies of the US EPA.
Evaluation of Per- and Poly fluoroalkyl Substances (PFAS) in vitro toxicity testing for developmental neurotoxicity
Presented by Kelly Carstens, PhD
Per- and poly fluoroalkyl substances (PFAS) are a diverse set of commercial chemicals widely detected in humans and the environment. However, only a limited number of PFAS are associated with epidemiological or experimental data for hazard identification. To provide developmental neurotoxicity (DNT) hazard information, the work herein employed DNT new approach methods (NAMs) to generate in vitro screening data for a set of 160 PFAS. The DNT NAMs battery was comprised of the microelectrode array neuronal network formation assay (NFA) and high-content imaging (HCI) assays to evaluate proliferation, apoptosis, and neurite outgrowth. The majority of PFAS (118/160) were inactive or equivocal in the DNT NAMs, leaving 42 active PFAS that decreased measures of neural network connectivity and neurite length. Analytical quality control indicated 43/118 inactive PFAS samples and 10/42 active PFAS samples were degraded; as such, careful interpretation is required as some negatives may have been due to loss of the parent PFAS, and some actives may have resulted from a mixture of parent and/or degradants of PFAS. PFAS containing a perfluorinated carbon (C) chain length ≥8, a high C:fluorine ratio, or a carboxylic acid moiety were more likely to be bioactive in the DNT NAMs. Of the PFAS positives in DNT NAMs, 85% were also active in other EPA ToxCast assays, whereas 79% of PFAS inactives in the DNT NAMs were active in other assays. These data demonstrate that a subset of PFAS perturb neurodevelopmental processes in vitro and suggest focusing future studies of DNT on PFAS with certain structural feature descriptors. (This abstract does not reflect U.S. EPA Policy).
A Modern Framework to Establish Scientific Confidence in New Methods
Presented by Anna van der Zalm, MChem
Current processes to validate new non-animal methods are costly, time-consuming, and do not necessarily produce methods that are fit for regulatory purposes. This talk will build on previous efforts from the Organisation for Economic Co-operation and Development and the International Cooperation on Alternative Test Methods to propose a modern, flexible framework comprising five essential elements to establish scientific confidence in non-animal methods for regulatory use: fitness for purpose, human biological relevance, technical characterization, data integrity and transparency, and independent review.
Updates to the current process are based on the recognition that (a) the relevance of the results of the new method need not be determined through direct alignment with the results of the traditional animal test method, and instead may be determined through alignment with, or fidelity to, human biological understanding; (b) the new method should not be required to replace the traditional animal test method one to one, nor produce the same information generated by the traditional animal test method; (c) the currently accepted levels of reproducibility in traditional animal test methods can be used to inform performance benchmarks for new methods; (d) ring trials may not be necessary for the assessment of the reproducibility of a new method; and (e) preferably before a method is developed, its purpose should be clearly defined and discussed amongst the method developer, regulators, and the regulated industry to ensure the production of new non-animal methods that are fit for purpose.