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When two toxicants overtly producing similar effects are given together, the effect of the combination may either be equal to the algebraic sum of their individual effects or may be more than or less than the algebraic sum of their individual effects. Therefore, to ascertain the nature of interaction precisely and accurately, whether it is additive, supra-additive (synergistic), or infra-additive (antagonistic), appropriate experimental designs are required to be followed. Factorial experiments requiring large numbers of animals are not only uneconomical, but also fail to reveal the exact nature of interaction with regard to addition, synergism, or antagonism. Scientists have therefore devised several simple experimental designs that are based on the dose-response curves of individual interacting substances and the dose-response curve of fixed-proportion combinations of these toxic substances. Among the reported experimental designs, the most efficient methods include the isobolographic method and the interaction index method. The present review aims to discuss the principle and methodology behind the experimental designs used to study interaction of binary toxic mixtures, to make it comprehensible for researchers engaged in the field of toxicology. A procedure by which nature of interaction can be ascertained with the help of the interaction index using Student t-test has also been explained. The review has been supported with appropriate illustrations.

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Our results explicitly indicate the synergistic nature of the interaction between NOS and COX inhibitors in formalin-induced nociceptive behavior in mice, and provide an alternative approach for controlling pain.