Mixture toxicity revisited: A translational review of experimental evidence from animal models to human health risk assessment

This review examines research investigating how simultaneous exposure to multiple chemicals affects biological systems, highlighting deficiencies in conventional single-substance risk evaluation frameworks. Living organisms in their natural habitats face continuous exposure to diverse chemical combinations, which frequently result in interactive effects, including synergism and antagonism, that diverge from the straightforward additive outcomes anticipated by traditional toxicological approaches. Research using animal and aquatic experimental models has shown that these exposures depend on numerous variables, such as the specific chemicals involved, their dose relationships, duration of contact, measured biological outcomes, and underlying mechanistic processes. This review examines fundamental methodological frameworks, particularly concentration addition (CA) and independent action (IA) models, used for predicting mixture toxicity, with most mixture effects falling within a two-fold range of additivity predictions though important deviations occur. Additional complicating factors include the timing of exposure and the specific biological traits of test species. The present review also addresses difficulties in applying findings from animal research to human populations, given differences in toxicokinetic processes and genetic makeup across species. To navigate these complexities, this review supports the adoption of mechanism-based frameworks incorporating high-throughput omics technologies, computational approaches, and standardized protocols for evaluating environmentally realistic mixtures. The review advocates for implementing tiered, cumulative risk-assessment methodologies that accurately represent real-world exposure conditions and emphasize protection of susceptible populations. This transformation is vital for advancing predictive toxicology and strengthening protections for public and environmental health. Ultimately, the review argues for moving beyond the obsolete single-substance paradigm toward comprehensive, evidence-driven approaches equipped to handle the multifaceted nature of chemical exposures. This review critically evaluates experimental animal studies in chemical mixture toxicology, emphasizing the complexities and prospects of applying animal findings to human health risk assessment. It identifies key gaps linking controlled experiments with epidemiological data and proposes research directions to advance risk evaluation and management of real-world chemical co-exposures.