Evaluation of experimental combined toxicity by use of dose–frequency curves: comparison with theoretical additivity as well as independence

Abstract

Dose–frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve–fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the χ²-square goodness-of-fit test. The theoretical dose–frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose–response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.Key words: combined toxic effects, statistical evaluation, dose–frequency curves, additivity, independence.