BACKGROUND: Risk assessment of chemical mixtures or complex substances remains a major methodological challenge due to lack of available hazard or exposure data. Therefore, risk assessors usually infer hazard or risk from data on the subset of constituents with available toxicity values. OBJECTIVES: We evaluated the validity of the widely used traditional mixtures risk assessment paradigms, Independent Action (IA) and Concentration Addition (CA), with new approach methodologies (NAMs) data from human cell-based in vitro assays. METHODS: A diverse set of 42 chemicals was tested both individually and as mixtures for functional and cytotoxic effects in vitro. A panel of induced pluripotent stem cell (iPSCs)-derived models (hepatocytes, cardiomyocytes, endothelial, and neurons) and one primary cell type (HUVEC) were used. Bayesian concentration-response modeling of individual chemicals or their mixtures was performed for a total of 47 phenotypes to derive point-ofdeparture (POD) values. Probabilistic IA or CA was conducted to estimate the mixture effects based on the bioactivity profiles from the individual chemicals and compared with mixture bioactivity. RESULTS: All mixtures showed significant bioactivity, even though some were constructed using individual chemical concentrations considered "low" or "safe." Even though CA is much more accurate as a predictor of mixture effects in comparison with IA, with CA-based POD typically within an order of magnitude of the actual mixture, in some cases, the bioactivity of the mixtures appeared to be much greater than that of their components under either additivity assumption. DISCUSSION: These results suggest that CA is a preferred first approximation for predicting mixture toxicity when data for all constituents are available. However, because the accuracy of additivity assumptions varies greatly across phenotypes, we posit that mixtures and complex substances need to be directly tested for their hazard potential. NAMs provide a practical solution that rapidly yields highly informative data for mixtures risk assessment.
Plant growth can be inhibited by herbicides and is strongly limited by the availability of nutrients, which can influence human health through the food chain. Until now, however, cross talk between the enantioselectivity of herbicides and nutrient stresses has been poorly understood. We analyzed trace element and macroelement contents in shoots of Arabidopsis thaliana treated by the chiral herbicide imazethapyr (IM) and observed that multiple-nutrient stress (trace elements Mn, Cu, and Fe and macroelements P, K, Ca, and Mg) was enantioselective. The (R)-IM treatments resulted in Mn 23.37%, Cu 63.53%, P 30.61%, K 63.70%, Ca 34.32%, and Mg 36.14% decreases compared with the control. Interestingly, it was also found that herbicidally active (R)-IM induced notable aggregation of nutrient elements in leaves and roots compared with the control and (S)-IM. Through gene expression analyses, it was found that herbicidally active (R)-IM induced the up- or down-regulation of genes involved in the transport of nutrient elements. We propose that (R)-IM affected the uptake and translocation of nutrient elements in A. thaliana, which destroyed the balance of nutrient elements in the plant. This finding reminds us to reconsider the effect of nutrient stresses in risk assessment of herbicides.
that encourage the use of alternative test methods for hazard and risk assessment applications, such as read-across, prioritization, and screening (ECHA, 2016; US EPA, 2018;Taylor et al., 2014). Novel analytical and in vitro data, now commonly referred to as new approach methodologies (NAMs), are being used in support of regulatory decisions (Kavlock et al., 2018;Paul Friedman et al., 2020); however, concerns about the limitations of NAMs in decision-making also have been voiced (Gocht et al., 2015;Berggren et al., 2015). The US Environmental Protection Agency (EPA) is developing a strategic plan to reduce the use of vertebrate animals in testing chemical substances and promote the development of alternative test methods; the goal is to eliminate animal testing from regulatory requirements for pesticides and industrial chemicals by 2035 (US EPA, 2019).The efforts to expand the portfolio of NAMs and test their utility in decision-making are most prominent in the European
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