Mapping ToxCast/Tox21 Assay Endpoints to Key Characteristics of Carcinogens to Support Cancer Hazard Evaluation

Abstract The Environmental Protection Agency’s Toxicity Forecaster (ToxCast) and the Toxicity Testing in the 21st Century federal consortium (Tox21) programs provide vast high-throughput screening data on thousands of chemicals, capturing a range of molecular and cellular responses relevant to toxicity. However, their application to cancer hazard identification requires a structured framework. The ten key characteristics of carcinogens (KCCs) offer a systematic approach for organizing this mechanistic evidence. We present an updated, comprehensive mapping of 1,570 ToxCast/Tox21 assay endpoints to the KCCs, developed through an expert-driven consensus process. A novel tiering approach was introduced to characterize each assay’s level of mechanistic relevance: Tier A indicates a direct effect linked to a KCC, while Tier B signifies an indirect or downstream effect. Additionally, we incorporated response directionality to correctly identify events contributing to, rather than merely associated with, a KCC. Of the 1,570 assay endpoints available in ToxCast/Tox21, 838 assay endpoints (53%) were mapped to the KCCs with some assay endpoints mapping to multiple KCCs. The majority of mapped assay endpoints were associated with KCC8 (modulates receptor-mediated effects) and KCC10 (alters cell proliferation, cell death, or nutrient supply). Some assay endpoints were mapped on different KCCs depending on the directionality of the response. No assay endpoints were mapped to KCC1 (is electrophilic or can be metabolically activated to electrophiles) or KCC9 (causes immortalization). This publicly available tiered mapping provides a crucial resource for interpreting high-throughput screening data in the context of carcinogenesis. It enables researchers and risk assessors to more systematically utilize such data for cancer hazard evaluation and chemical prioritization.