High-Throughput Testing for Unknown Mutagens and Cytotoxica via Duplex Planar Ames–Cytotoxicity Bioassay Including Metabolic S9 Activation

Current nontarget effect-directed analysis of complex samples for mutagens is hampered by matrix effects, associated cytotoxicity, diffusion effects, insufficient sensitivity, and a lack of selectivity. Non-target analysis may overlook highly potent, unknown mutagens at trace levels. To overcome these limitations, a duplex planar Ames mutagenicity-cytotoxicity bioassay was developed to sensitively and selectively detect individual mutagens and cytotoxic compounds with or without metabolic activation. Key innovations included high-throughput testing of samples, either directly or as raw extracts, separated in parallel by planar chromatography, substance zone fixation to prevent diffusion during long incubation times, integration of the human versus rat liver S9 enzyme systems for metabolic de/activation, and use of a tetrazolium salt substrate that provides a dual end-point read-out. Compared to the state of the art, time to result was reduced 5-fold, manual work was reduced 330-fold, and costs for nearly plastic-free consumables were reduced 651-fold. The selective, sensitive, and quantitative unmasking of previously unknown mutagens and cytotoxica was shown for highly complex samples, such as teas, cosmetics, skin care creams, and perfumes. An exemplary daily exposure to 11.5 g of skin care products exceeded the half-maximal effective mutagenicity dose by at least 4 orders of magnitude. Using the open-source and sustainable 2LabsToGo-Eco, the new duplex planar bioassay method can be applied worldwide to serve as a valuable tool for hazard minimization and to support regulatory safety and risk assessments, industrial quality control, and drug development.