Systematic Evaluation of In Vitro Intestinal Permeability Measurements Across Diverse Chemicals: Insights for Advancing New Approach Methods for Chemical Risk Assessment

A mechanistic understanding of chemical permeation across biological membranes, e.g., the intestinal epithelium, is essential for evaluating the exposure and toxicokinetics of environmentally relevant chemicals in the era of new approach methods (NAMs). Intestinal transmembrane permeability is commonly assessed using in vitro assays, including the cellular Caco-2 transwell assay and the noncellular parallel artificial membrane permeability assay (PAMPA). Here, we systematically investigate some longstanding assumptions about Caco-2 and PAMPA permeabilities by analyzing the most comprehensive data sets to date on measured Caco-2 permeability, PAMPA permeability, and partition coefficients at physiological pH. We confirm the widely held assumption that hydrophobicity correlates with PAMPA permeability and that PAMPA permeability correlates with Caco-2 permeability. However, these correlations are only moderate and hence insufficient for reliable prediction or read-across in chemical risk assessments. Furthermore, the lack of standardized PAMPA protocols or inconsistent adherence to standardized Caco-2 protocols makes literature reported permeability measurements highly sensitive to test setup and conditions. Using PAMPA permeability data without considering methodological appropriateness and data quality may result in misleading conclusions. We reveal that hydrophobicity and permeability are not inherently interdependent, and therefore, they should be treated as equally critical and fundamental parameters in chemical risk assessments.