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.
