High-Throughput Assessment of the Abiotic Stability of Test Chemicals in In Vitro Bioassays

Abiotic stability of chemicals is not routinely tested prior to performing in vitro bioassays, although abiotic degradation can reduce the concentration of test chemicals leading to the formation of active or inactive transformation products, which may lead to misinterpretation of bioassay results. A high-throughput workflow was developed to measure the abiotic stability of 22 test chemicals in protein-rich aqueous media under typical bioassay conditions at 37 °C for 48 h. These test chemicals were degradable in the environment according to a literature review. The chemicals were extracted from the exposure media at different time points using a novel 96-pin solid-phase microextraction. The conditions were varied to differentiate between various reaction mechanisms. For most hydrolyzable chemicals, pH-dependent degradation in phosphate-buffered saline indicated that acid-catalyzed hydrolysis was less important than reactions with hydroxide ions. Reactions with proteins were mainly responsible for the depletion of the test chemicals in the media, which was simulated by bovine serum albumin (BSA) and glutathione (GSH). 1,2-Benzisothiazol-3­(2H)-one, 2-methyl-4-isothiazolinone, and l-sulforaphane reacted almost instantaneously with GSH but not with BSA, indicating that GSH is a good proxy for reactivity with electrophilic amino acids but may overestimate the actual reaction with three-dimensional proteins. Chemicals such as hydroquinones or polyunsaturated chemicals are prone to autoxidation, but this reaction is difficult to differentiate from hydrolysis and could not be simulated by the oxidant N-bromosuccinimide. Photodegradation played a minor role because cells are exposed in incubators in the dark and simulations with high light intensities did not yield realistic degradation. Stability predictions from various in silico prediction models for environmental conditions can give initial indications of the stability but were not always consistent with the experimental stability in bioassays. As the presented workflow can be performed in high throughput under realistic bioassay conditions, it can be used to provide an experimental database for developing bioassay-specific stability prediction models.