Statistically significant deviations from additivity: What do they mean in assessing toxicity of mixtures?

• Available models interpreted small random deviations in responses as interactions.
• Deviation patterns proved to be non-reproducible over independent experiments.
• Finding a deviation can be the starting point of further mechanistic research.

There is increasing attention from scientists and policy makers to the joint effects of multiple metals on organisms when present in a mixture. Using root elongation of lettuce (Lactuca sativa L.) as a toxicity endpoint, the combined effects of binary mixtures of Cu, Cd, and Ni were studied. The statistical MixTox model was used to search deviations from the reference models i.e. concentration addition (CA) and independent action (IA). The deviations were subsequently interpreted as ‘interactions’. A comprehensive experiment was designed to test the reproducibility of the ‘interactions’. The results showed that the toxicity of binary metal mixtures was equally well predicted by both reference models. We found statistically significant ‘interactions’ in four of the five total datasets. However, the patterns of ‘interactions’ were found to be inconsistent or even contradictory across the different independent experiments. It is recommended that a statistically significant ‘interaction’, must be treated with care and is not necessarily biologically relevant. Searching a statistically significant interaction can be the starting point for further measurements and modeling to advance the understanding of underlying mechanisms and non-additive interactions occurring inside the organisms.