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High-throughput in vitro bioassays are becoming increasingly important in the risk characterization of anthropogenic chemicals. Large databases gather nominal effect concentrations (C) for diverse modes of action. However, the biologically effective concentration can substantially deviate due to differences in chemical partitioning. In this study, we modeled freely dissolved (C), cellular (C), and membrane concentrations (C) in the Tox21 GeneBLAzer bioassays for a set of neutral and ionogenic organic chemicals covering a large physicochemical space. Cells and medium constituents were experimentally characterized for their lipid and protein content, and partition constants were either collected from the literature or predicted by mechanistic models. The chemicals exhibited multifaceted partitioning to proteins and lipids with distribution ratios spanning over 8 orders of magnitude. Modeled C deviated over 5 orders of magnitude from C and can be compared to in vivo effect data, environmental concentrations, and the unbound fraction in plasma, which is needed for the in vitro to in vivo extrapolation. C was relatively constant for chemicals with membrane lipid-water distribution ratios of 1000 or higher and proportional to C. Representing a sum parameter for exposure that integrates the entire dose from intracellular partitioning, C is particularly suitable for the effect characterization of chemicals with multiple target sites and the calculation of their relative effect potencies. Effective membrane concentrations indicated that the specific effects of very hydrophobic chemicals in multiple bioassays are occurring at concentrations close to baseline toxicity. The equilibrium partitioning model including all relevant system parameters and a generic bioassay setup is attached as an excel workbook to this paper and can readily be applied to diverse in vitro bioassays.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity, activation of the estrogen, aryl hydrocarbon and peroxisome proliferator-activated receptors and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street run-off were detected. The unexpectedly high estrogenic effects in many samples indicated impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (<8 %) of the measured effects. The experimental effects of designed mixtures of detected chemicals that were expected to dominate the mixture effects of detected chemicals were consistent with predictions for concentration addition by a factor of two for 94 % of the mixtures.Overall, the burden of chemicals and effects were much higher than previously detected in surface water during dry weather with the effects often exceeding effect-based trigger values. In-vitro bioassay test batteryEndocrine effects • ER Metabolism • AhR • PPARy Adaptive stress response: • Oxidative stress Chemical analysis Sampling triggered by rain events Mixture models

Most studies using high-throughput in vitro cell-based bioassays tested chemicals up to a certain fixed concentration. It would be more appropriate to test up to concentrations predicted to elicit baseline toxicity because this is the minimal toxicity of every chemical. Baseline toxicity is also called narcosis and refers to nonspecific intercalation of chemicals in biological membranes, leading to loss of membrane structure and impaired functioning of membrane-related processes such as mitochondrial respiration. In cells, baseline toxicity manifests as cytotoxicity, which was quantified by a robust live-cell imaging method. Inhibitory concentrations for baseline toxicity varied by orders of magnitude between chemicals and were described by a simple quantitative structure activity relationship (QSAR) with the liposome-water partition constant as a sole descriptor. The QSAR equations were remarkably similar for eight reporter gene cell lines of different cellular origin, six of which were used in Tox21. Mass-balance models indicated constant critical membrane concentrations for all cells and all chemicals with a mean of 69 mmol·kglip –1(95% CI: 49–89), which is in the same range as for bacteria and aquatic organisms and consistent with the theory of critical membrane burden of narcosis. The challenge of developing baseline QSARs for cell lines is that many confirmed baseline toxicants are rather volatile. We deduced from cytotoxicity experiments with semi-volatile chemicals that only chemicals with medium-air partition constants >10,000 L/L can be tested in standard robotic setups without appreciable loss of effect. Chemicals just below that cutoff showed crossover effects in neighboring wells, whereas the effects of chemicals with lower medium-air partition constants were plainly lost. Applying the “volatility cut-off” to >8000 chemicals tested in Tox21 indicated that approximately 20% of Tox21 chemicals could have partially been lost during the experiments. We recommend applying the baseline QSARs together with volatility cut-offs for experimental planning of reporter gene assays, that is, to dose only chemicals with medium-air partition constants >10,000 at concentrations up to the baseline toxicity level.

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 QSARpredicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to ∼ 8,000 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 (SR < 1) and only 2%-14% were specific with SR ≥ 10 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.