Time-dependence in mixture toxicity with soft-electrophiles: 2. Effects of relative reactivity level on time-dependent toxicity and combined effects for selected Michael acceptors
Toxicity assessments for organic chemical mixtures are often described as being approximately additive. Recent mixture studies with soft electrophiles have suggested that agents with less-than fully time-dependent toxicity (TDT) may actually induce toxicity by more than one mode of toxic action within the same series of concentrations. To evaluate this concept further, four Michael acceptor electrophiles, each with a different rate of in chemico reactivity and different level of TDT, were tested with each other and in sham combinations (a single chemical tested as if it were a binary mixture) using the Microtox system. For each binary combination, each agent was tested alone and in a mixture, with toxicity assessed as inhibition of bioluminescence at 15-, 30- and 45-min of exposure. Each single agent and mixture test included seven duplicated concentrations and a duplicated control treatment. To evaluate relative reactivity, each agent was also tested with the model nucleophile glutathione (GSH). Agents with greater in chemico reactivity (mean RC(50) mM) showed greater toxicity (mean 45-min EC(50) - mM) but these were inversely related to the TDT levels of the agents. Combined effects for the sham combinations, as quantified by additivity quotient values for the EC(50) of the mixture, tended to be close to 1.00 (i.e., the dose-addition EC(50)-AQ). For true binary combinations (i.e., two chemicals tested together), the EC(50)-AQ tended to be increasingly above 1.00 when TDT levels of the agents in the mixture were more disparate. The results of this study with Michael acceptors suggested that: (i) when reactivity was fast, there was most likely a single prominent mode of toxic action, i.e., electro(nucleo)philic reactivity, leading to time-dependent toxicity at the full or high levels, (ii) when the reaction rate for a chemical was slower, two modes of action, electro(nucleo)philic reactivity and narcosis, were apparent such that the time-dependent toxicity level was lower as well, (iii) mixtures of the former agents show a combined effect that was strictly dose-additive, whereas (iv) mixtures which included one (or more) agent with a lower reaction rate had a combined effect that was approximately additive rather than strictly dose-additive.