Progression of new approach methods relies on broadening experimental data quantifying in vitro disposition for use in high-throughput (HT) screening assays of thousands of chemicals. Nominal concentrations are often used for potency estimation; however, physicochemical parameters contributing to a chemical’s bioavailability in vitro (e.g., vapor pressure, water solubility, Henry’s Law Constant (HLC)) may artifactually skew potency estimates, heightening uncertainty with HT assay bioactivity endpoints. This study investigated ten chemicals with HLC values spanning eight orders of magnitude to indicate chemical suitability in a HT testing platform. Each chemical was incubated in a modified ToxCast assay using live or lysed T-47D breast cancer cells in a 96-well plate format with time course samples collected from 0-80h. The samples were analyzed by either gas or liquid chromatographic separation combined with targeted mass spectrometry to determine test chemical concentration in various compartments (media, lysate). Preliminary data showed the test chemical concentrations heavily depended on its HLC, with HLC values exceeding 5.7e-3 atm-m³/mol displaying a rapid decrease in concentration (70% nominal concentration loss after 1h) in both live and lysed cellular assay preparations.

Mass balance in silico models are often used to adjust point of departure concentrations based on a chemical’s physicochemical properties and assay conditions (e.g., well number, media composition). The Armitage model compared to experimental concentrations suggests that a properly parameterized in silico mass balance model may be used to establish suitability cutoffs for chemical testing in HT assays based on their physicochemical parameters. This abstract does not represent US EPA policy.