Cytotoxicity Burst? Differentiating Specific from Nonspecific Effects in Tox21 in Vitro Reporter Gene Assays

BACKGROUND: High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the “cytotoxicity burst,” which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death. OBJECTIVES: We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity. METHODS: Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure–activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to [Formula: see text] chemicals that were tested in six of the high-throughput Tox21 reporter gene assays. RESULTS: Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%–87% of the active hits were presumably caused by the cytotoxicity burst ([Formula: see text]) and only 2%–14% were specific with [Formula: see text] against experimental cytotoxicity but 75%–97% were specific against baseline toxicity. This difference was caused by a large fraction of chemicals showing excess cytotoxicity. CONCLUSIONS: The specificity analysis for measured in vitro effects identified whether a cytotoxicity burst had likely occurred. The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative in vitro–in vivo extrapolation and risk assessment of chemicals. https://doi.org/10.1289/EHP6664