This paper addresses major developments in the safety evaluation of chemical mixtures during the past 15 years, reviews today's state of the art of mixture toxicology, and discusses challenges ahead. Well-thought-out tailor-made mechanistic and empirical designs for studying the toxicity of mixtures have gradually substituted trial-and-error approaches, improving the insight into the testability of joint action and interaction of constituents of mixtures. The acquired knowledge has successfully been used to evaluate the safety of combined exposures and complex mixtures such as, for example, the atmosphere at hazardous waste sites, drinking water disinfection by-products, natural flavouring complexes, and the combined intake of food additives. To consolidate the scientific foundation of mixture toxicology, studies are in progress to revisit the biological concepts and mathematics underlying formulas for low-dose extrapolation and risk assessment of chemical mixtures. Conspicuous developments include the production of new computer programs applicable to mixture research (CombiTool, BioMol, Reaction Network Modelling), the application of functional genomics and proteomics to mixture studies, the use of nano-optochemical sensors for in vivo imaging of physiological processes in cells, and the application of optical sensor micro- and nano-arrays for complex sample analysis. Clearly, the input of theoretical biologists, biomathematicians and bioengineers in mixture toxicology is essential for the development of this challenging branch of toxicology into a scientific subdiscipline of full value.
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