Challenges integrating skin sensitization data for assessment of difficult to test substances

Difficult to test substances, including poorly soluble, mildly irritating, or UVCBs (unknown or variable composition complex reaction products or biological materials), producing weak or borderline in vivo results, face additional challenges in in vitro assays that often necessitate data integration in a weight of evidence (WOE) approach to inform skin sensitization potential. Here we present several case studies on difficult to test substances and highlight the utility of the toxicological priority index (ToxPi) as a data visualization tool to compare skin sensitization biological activity. The case study test substances represent two poorly soluble substances, tetrakis (2-ethylbutyl) orthosilicate and decyl palmitate, and two UVCB substances, alkylated anisole and hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2 undecanone. Data from key events within the skin sensitization adverse outcome pathway were gathered from publicly available sources or specifically generated. Incorporating the data for these case study test substances as well as data on chemicals of a known sensitization class (sensitizer, irritating non-sensitizer, and non-sensitizer) into ToxPi produced biological activity profiles which were grouped using unsupervised hierarchical clustering. Three of the case study test substances concluded to lack skin sensitization potential by traditional WOE produced biological activity profiles most consistent with non-sensi­tizing substances, whereas the prediction was less definitive for a substance considered positive by traditional WOE. Visualizing the data using bioactivity profiles can provide further support for WOE conclusions in certain circumstances but is unlikely to replace WOE as a stand-alone prediction due to limitations of the method including the impact of missing data points.