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Using in vitro data to estimate point of departure (POD) values is an essential component of new approach methodologies (NAMs)-based chemical risk assessments. In this case study, we evaluated a NAM for hepatotoxicity based on rat primary hepatocytes, high-content imaging (HCI), and toxicokinetic modeling. First, we treated rat primary hepatocytes with 10 concentrations (0.2–100 µM) of 51 chemicals that produced hepatotoxicity in repeat-dose subchronic and chronic exposures. Second, we used HCI to measure endoplasmic reticulum stress, mitochondrial function, lysosomal mass, steatosis, apoptosis, DNA texture, nuclear size, and cell number at 24, 48, and 72 h and calculated concentrations at 50% maximal activity (AC50). Third, we estimated administered equivalent doses (AEDs) from AC50 values using toxicokinetic modeling. AEDs using physiologically based toxicokinetic models were 4.1-fold (SD 6.3) and 8.1-fold (SD 15.5) lower than subchronic and chronic lowest observed adverse effect levels (LOAELs), respectively. In contrast, AEDs from ToxCast and Tox21 assays were 89.8-fold (SD 149.5) and 168-fold (SD 323.7) lower than subchronic and chronic LOAELs. Individual HCI endpoints also estimated AEDs for specific hepatic lesions that were lower than in vivo PODs. Lastly, AEDs were similar for different in vitro exposure durations, but steady-state toxicokinetic models produced 7.6-fold lower estimates than dynamic physiologically based ones. Our findings suggest that NAMs from diverse cell types provide conservative estimates of PODs. In contrast, NAMs based on the same species and cell type as the adverse outcome may produce estimates closer to the traditional in vivo PODs. [ABSTRACT FROM AUTHOR]

'Cell Painting' is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ∼100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies. • The percentage of ToxCast chemicals active in Cell Painting was 49.3%. • Pharmaceuticals and pesticides were more active than food additives and inerts. • 4.4% of chemicals were bioactive at predicted human exposure concentrations. • Pyrene produced a profile similar to known glucocorticoid receptor agonists. • Diniconazole bioactivity differed from other conazoles and structurally related chemicals. [ABSTRACT FROM AUTHOR]