Ten years of research on synergisms and antagonisms in chemical mixtures: A systematic review and quantitative reappraisal of mixture studies

Several reviews of synergisms and antagonisms in chemical mixtures have concluded that synergisms are relatively rare. However, these reviews focused on mixtures composed of specific groups of chemicals, such as pesticides or metals and on toxicity endpoints mostly relevant to ecotoxicology. Doubts remain whether these findings can be generalised. A systematic review not restricted to specific chemical mixtures and including mammalian and human toxicity endpoints is missing.We conducted a systematic review and quantitative reappraisal of 10 years' of experimental mixture studies to investigate the frequency and reliability of evaluations of mixture effects as synergistic or antagonistic. Unlike previous reviews, we did not limit our efforts to certain groups of chemicals or specific toxicity outcomes and covered mixture studies relevant to ecotoxicology and human/mammalian toxicology published between 2007 and 2017.We undertook searches for peer-reviewed articles in PubMed, Web of Science, Scopus, GreenFile, ScienceDirect and Toxline and included studies of controlled exposures of environmental chemical pollutants, defined as unintentional exposures leading to unintended effects. Studies with viruses, prions or therapeutic agents were excluded, as were records with missing details on chemicals' identities, toxicities, doses, or concentrations.To examine the internal validity of studies we developed a risk-of-bias tool tailored to mixture toxicology. For a subset of 388 entries that claimed synergisms or antagonisms, we conducted a quantitative reappraisal of authors' evaluations by deriving ratios of predicted and observed effective mixture doses (concentrations).Our searches produced an inventory of 1220 mixture experiments which we subjected to subgroup analyses. Approximately two thirds of studies did not incorporate more than 2 components. Most experiments relied on low-cost assays with readily quantifiable endpoints. Important toxicity outcomes of relevance for human risk assessment (e.g. carcinogenicity, genotoxicity, reproductive toxicity, immunotoxicity, neurotoxicity) were rarely addressed. The proportion of studies that declared additivity, synergism or antagonisms was approximately equal (one quarter each); the remaining quarter arrived at different evaluations. About half of the 1220 entries were rated as "definitely" or "probably" low risk of bias. Strikingly, relatively few claims of synergistic or antagonistic effects stood up to scrutiny in terms of deviations from expected additivity that exceed the boundaries of acceptable between-study variability. In most cases, the observed mixture doses were not more than two-fold higher or lower than the predicted additive doses. Twenty percent of the entries (N = 78) reported synergisms in excess of that degree of deviation. Our efforts of pinpointing specific factors that predispose to synergistic interactions confirmed previous concerns about the synergistic potential of combinations of triazine, azole and pyrethroid pesticides at environmentally relevant doses. New evidence of synergisms with endocrine disrupting chemicals and metal compounds such as chromium (VI) and nickel in combination with cadmium has emerged.These specific cases of synergisms apart, our results confirm the utility of default application of the dose (concentration) addition concept for predictive assessments of simultaneous exposures to multiple chemicals. However, this strategy must be complemented by an awareness of the synergistic potential of specific classes of chemicals. Our conclusions only apply to the chemical space captured in published mixture studies which is biased towards relatively well-researched chemicals.The final protocol was published on the open-access repository Zenodo and attributed the following digital object identifier, doi: https://doi.org//10.5281/zenodo.1319759 (https://zenodo.org/record/1319759#.XXIzdy7dsqM).