Functional Read-Out Enabling High Compound Throughput Toxicokinetic Assays

Summary: This Phase I project will implement and demonstrate the feasibility and utility of a new “universal” assay readout to provide high throughput in vitro toxicokinetics (HTTK) data without having to develop specific analytical methods to measure each compound. The amount of available compound from standard in vitro toxicokinetic modes of plasma binding, metabolic clearance, and bidirectional permeability will be quantified using the microplate-based TempO-Seq® gene expression platform, the S1500+v2 surrogate whole transcriptome assay which measures every known molecular pathway, and a HepaRG cell system to first identify a gene expression signature that is characteristic of each test compound and generate dose response data, then use this and benchmark concentration (BMC) analysis of signature genes and pathways as a calibration curve to quantify the amount of compound available from each in vitro toxicokinetic model. The in vitro TempO-Seq HTTK data will also identify potential safety issues and provide data useful for read-across comparisons, making the assay highly efficient by providing both the in vitro safety and in vitro toxicokinetic data necessary to make in vitro-to-in vivo extrapolations (IVIVE) and prioritize compounds for follow-up based on exposure safety risk. Reference compounds will be tested and we will benchmark the TempO-Seq HTTK data against published data obtained using analytical methods, and also demonstrate that TempO-Seq HTTK results are equivalent to IVIVE results determined using analytical methods. Unlike a functional assay using a single endpoint specific for the compound being measured, the TempO-Seq S1500v2 assay has been shown by numerous labs to provide a signature of many robustly differentially expressed genes for all compounds tested to-date, unique for each compound. Thus, this content represents a “universal” assay for any test compound or mixture (e.g. as produced in industrial chemical processes), producing compound specific signatures for use in the TempO-Seq HTTK assay. By enabling the high throughput in vitro assessment of toxicokinetics of thousands of compounds, the TempO-Seq HTTK assay will enable programs such as ToxCast to achieve the objective of providing IVIVE assessment of the tens of thousands of compounds humans and the environment are exposed to for which there are no safety data. It will also provide the pharma, agricultural, cosmetics, and petroleum/chemical industries comparative in vitro toxicity and toxicokinetic data supporting IVIVE analysis to assist synthetic chemists in design and development decisions for advancing chemical series and leads or protection of employees from exposure, and has the potential to spare animals by preselecting the compounds put into animal models.Narrative: This Phase I project will implement and demonstrate the feasibility and utility of an assay based on the high throughput TempO-Seq® platform and S1500v2 surrogate whole transcriptome assay (the TempO-Seq HTTK assay) to provide in vitro toxicokinetics data from standard models without having to develop an analytical method to measure each compound individually. By enabling the high throughput in vitro assessment of toxicokinetics of thousands of compounds the TempO-Seq HTTK assay will enable programs such as ToxCast to carry out in vitro-to-in vivo extrapolations (IVIVE) for the tens of thousands of compounds humans and the environment are exposed to for which there are no safety data. Because the TempO-Seq HTTK assay will be used to establish a dose response calibration curve for each compound, the assay will also provide an in vitro indication of safety as well as the data required for read-across comparisons to other compounds, providing broad utility to ToxCast and also the pharma, agricultural, cosmetics and industrial chemicals industries.