How does toxicological testing deal with the ever-increasing number of new chemicals in an ethical, safe, cost-effective, and coordinated manner? Over the last two decades, there has been a paradigm shift in thinking, which has pushed forward the need to replace or reduce conventional animal in vivo toxicity studies with new alternative methodologies (NAMs). Much progress has been made in this regard. However, from a risk assessment and regulatory perspective, there remains many questions that need to be addressed empirically and strategies to discuss before NAMs-based approaches can be effectively implemented.
Presentations revolving around these questions and aligned topics were central to the 2024 SOT Annual Meeting Workshop Session “Application of Early Molecular Measurements to Develop Points of Departure for Risk Assessment.” The Workshop helped bring forth several complementary strategies for early evaluation of risks associated with chemicals that, if properly utilized, may help cut down the time and monetary costs associated with toxicity studies. Connie Mitchell, Health and Environmental Science Institute (HESI), and Leah Wehmas, US Environmental Protection Agency (US EPA), chaired the workshop, which was endorsed by the SOT Molecular and Systems Biology, Risk Assessment, and Regulatory and Safety Evaluation Specialty Sections.
Dr. Wehmas articulated that identification of a point of departure (POD) for chemicals was essential to chemical risk assessment and had historically been derived from chronic animal toxicity studies. Upon chemical exposure, the toxicity endpoints that were historically measured were apical endpoints. However, molecular changes induced by chemicals in a cell precede cellular-, organ-, and organism-level changes, often in a dose-dependent manner, and may be equally predictive of chronic apical changes.
Integration of NAMs and ’Omics for Determining PODs
The aforementioned idea was reinforced by the first speaker, David Rouquie, Bayer, who pointed out that the current testing paradigm for toxicity studies was a top-down approach with chronic apical studies being carried out first and in silico or in vitro studies being carried out last to identify probable mechanisms of toxicity. However, the next generation of risk assessment studies aim to reverse this approach and use in vitro studies to predict human safety by implementing quantitative in vitro to in vivo extrapolation methods. For measurement of early molecular changes to be predictive of apical endpoints, he mentioned the importance of establishing causality and that early molecular initiating events and primary key events in an adverse outcome pathway needed to be identified.
Dr. Rouquie referred to the OASIS (Omics for Assessing Signatures for Integrated Safety) Consortium, a working group within the HESI Emerging Systems Toxicology for the Assessment of Risk (eSTAR) Committee. OASIS aims to generate confidence in chemical safety assessment from early high-throughput cell phenotypic measurements, transcriptomics, and proteomics studies by integrating and utilizing existing in vivo data from rat hepatotoxicity studies. Among some of the challenges that exist in this approach, Dr. Rouquie particularly focused on the need to identify the extent to which the in vitro data reflected the mechanisms associated with apical toxicity and be mindful of false negatives. He emphasized that global collaboration between stakeholders in academia, industry, and regulatory bodies was required to build trust in the next generation of risk assessment.
Evaluating the Impact of Cell Line Choice on Determination of Human Health Protective Molecular PODs
Joshua Harrill, US EPA, was the second speaker and described how molecular PODs (mPODs) may be calculated from NAM-based in vitro assays based on changes in gene expression or cellular morphology. He explored how administered equivalent doses that were associated with mPODs derived from in vitro NAMs, compared to traditional PODs, and how the choice of cell line may influence these mPODs for a wide range of chemicals. The data that he shared demonstrated that 89% of tested chemicals had PODNAM that was lower than traditional in vivo POD, and therefore, PODNAM could serve as a substitute to in vivo POD in terms of human protective measures. However, mPODs were not always predictive of traditional POD values for some chemicals, such as organophosphates and carbamates, and other integrated analyses, such as chemotype analyses based on chemical structures, could be useful in this context. Even when chemotype analyses were implemented, the derived mPODs could differ based on the cell type or assay used. Dr. Harrill reflected on how these factors needed to be considered and integrated when determining mPODs for chemicals.
Incorporating Extracellular Vesicle Mediators of PFAS Toxicity into NAMs-Based PODs: A Comparison against Transcriptomic-Based Risk Values
Celeste Carberry, a doctoral student at the University of North Carolina at Chapel Hill, introduced the concept of measurement of microRNA (miRNA) in extracellular vesicles as a risk assessment tool. Data from her studies demonstrated that exposure to per- and polyfluoroalkyl substances (PFAS), especially perfluorooctanesulfonic acid (PFOS), leads to concentration-dependent changes in miRNA signatures in extracellular vesicles released from HepG2 liver cells. miRNA alterations could be linked to global transcriptomic deviations in cells and reflect changes in cellular states. Thus, miRNA measurement could serve as a useful biological endpoint for calculation of mPOD in NAM-based assays.
Derivation of Metabolic POD using High-Throughput In Vitro Metabolomics: Investigating the Importance of Sampling Time Points on Benchmark Concentration Values in the HepaRG Cell Line
Julia Malinowska, European Commission Joint Research Centre, introduced to the audience the use of metabolomics as an alternative ’omics-based approach for determining chemical PODs from high-throughput in vitro experiments. Metabolomics-associated changes are perhaps more closely related to apical endpoints but have been largely underutilized in deriving in vitro PODs for chemicals. Dr. Malinowska pointed out that several challenges exist for successful incorporation of this approach in toxicity testing, with some major bottlenecks being that of analytical sensitivity in detecting metabolites and lack of annotation and difficulty in identification of metabolites. She shared results that demonstrated good concordance between three approaches to derive benchmark concentration modeling (BMC) values for most of the chemicals used in her metabolomics experiments with the HepaRG liver cell line. She also demonstrated the effects of sampling time on BMC values for the chemicals which showed that temporal fluctuations in BMC values were chemical-specific as temporal trends were apparent for one chemical but not for others. While this does bring forth the complexity associated with deriving mPODs for chemicals from in vitro experiments, Dr. Malinowska mentioned that it was important to be cognizant of the role of multiple factors that influence mPODs to enable standardization of approaches. This will accelerate the successful utilization of in vitro metabolomics data for regulatory purposes.
Using ’Omics for Decision-Making and Regulatory Applications by Industry
Current ’omics-based approaches and their use for risk assessment and regulatory adoption was the focus of the presentation by Richard Currie, who is a senior fellow at Syngenta. He mentioned that integration of different ’omics-based methods for risk assessment purposes remains a challenge. He presented a case associated with acute toxicity testing of herbicides where, at a particular concentration, there was no appreciable change in in vivo transcriptomics data that would signify toxicity but surprisingly had changes in the metabolomics profile. Therefore, gene changes alone may not always be predictive for some toxicity studies, and calculation of PODs from short-term transcriptomics and metabolomics studies in combination with measurement of phenotypic changes are perhaps more predictive of long-term chemical-induced toxicity.
Evaluating In Vitro Transcriptomics Data for Regulatory Testing and Assessment: Are We There Yet?
Tara Barton-Maclaren, Health Canada, discussed the intricacies associated with adoption of transcriptomic PODs (tPODs) for regulatory risk assessment. She talked about several factors that need to be considered in deriving optimum in vitro tPODs for chemicals that can serve as surrogates for apical PODs. These include factors such as duration of chemical exposure, cell lines and types of in vitro models that are used, and bioinformatic approaches involved in deriving tPODs. The challenge lies in characterizing the uncertainty associated with these factors when deriving tPODs for use in a regulatory context. Dr. Barton-Maclaren mentioned some ongoing efforts to address these uncertainties as well as reduce false positive genes when deriving tPODs. She concluded by highlighting the need for standardized data collection and common data reporting frameworks to facilitate regulatory decisions based on the use of tPODs.
Panel Discussion
A panel discussion that followed the speakers’ presentations brought into focus many uncertainties that need to be addressed to ensure confidence in utilizing mPODs for chemical risk assessment. Questions to the panel revolved around, among others, the strategy to identify uncertainty factors for tPODs and extrapolation of tPODs to human-equivalent doses. The importance of selecting relevant cell lines and including more time points for in vitro experiments was considered. Overall, the Workshop was highly relevant from toxicology and regulatory perspectives and addressed, in detail, the existing challenges for incorporating mPODs in risk assessment decisions.
This blog was prepared by an SOT Reporter and represents the views of the author. SOT Reporters are SOT members who volunteer to write about sessions and events in which they participate during the SOT Annual Meeting and ToxExpo. SOT does not propose or endorse any position by posting this article. If you are interested in participating in the SOT Reporter program in the future, please email SOT Headquarters.
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