Citation:

Shah, I., T. Antonijevic, B. Chambers, J. Harrill, AND R. Thomas. Estimating Hepatotoxic Doses Using High-content Imaging in Primary Hepatocytes. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 183(2):285-301, (2021). https://doi.org/10.1093/toxsci/kfab091

Impact/Purpose:

This retrospective case-study is aimed at evaluating a quantitative new approach methodology (NAM) for risk-based prioritisation of environmental chemicals by integrating in vitro and in silico approaches. The approach is based on calculating the in vitro potency (AC50) of 51 chemicals using HTS data, extrapolating this potency to administered equivalent doses (ADEs) using toxicokinetic modeling and comparing the ADEs (which are assumed to be putative point of departure (POD) estimates) with lowest observed adverse effect (LOAELs) from repeat-dose toxicity testing studies. A key question explored in this work is whether the choice of species, cell type and assays have an impact on POD estimates. To address this question, PODs are estimated using AC50 values from rat primary hepatocytes via high-content imaging and from a range of in vitro assays (multiple species and cell types) from ToxCast. The POD estimates are compared with rat hepatic LOAELs from subchronic and chronic repeat-dose guideline testing studies. In addition, we systematically explore the impact of different toxicokinetic assumptions (physiologically based toxicokinetics vs steady-state concentration, different dose-metrics, etc.) on in vitro to in vivo extrapolation (IVIVE). Our findings suggest that using rat hepatocytes and PBTK modeling produce POD estimates that under-estimate vivo LOAELs for rat subchronic and chronic hepatotoxicity by 4.1-fold (SD 6.3) and 8.1-fold (SD 15.5), respectively. On the other hand, diverse assays from ToxCast under-estimate rat subchronic and chronic hepatotoxicity LOAELs 89.8-fold (SD 149.5) and 168-fold (SD 323.7), respectively. While ToxCast assays provide more health-protective estimates of PODs, using cells from the same species (rat) and target organ (liver) result in a greater consistency with in vivo toxicity values.

Description:

Using in vitro data to estimate point of departure (POD) values is an essential component of new approach methodologies (NAM)-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 to 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 end-points 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.

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