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

Abstract

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 RC₅₀ mM) showed greater toxicity (mean 45-min EC₅₀ – 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 EC₅₀ of the mixture, tended to be close to 1.00 (i.e., the dose-addition EC₅₀-AQ). For true binary combinations (i.e., two chemicals tested together), the EC₅₀-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.

Keywords:

• Reactive toxicity
• time-dependent toxicity
• dose-addition
• Michael acceptors
• molecular sites of toxic action
• Microtox

Acknowledgments

This publication was made possible, in part, by grant 2 R15 ES08019-03 from the National Institute of Environmental Health Sciences (NIEHS), NIH, USA. Its contents are solely the responsibility of the authors and do not represent the official views of the NIEHS, NIH.

This paper was presented in the SETAC Asia/Pacific 2006 conference held at Peking University, China on September 18–20, 2006.

Notes

^a Reactivity as measured with GSH as the model nucleophile; values are the means of 2 replicate tests each.

^b Mean value obtained from five separate tests of each agent singly.

^c Based on a mean TDT_{15−45 min} value of 1 = 10–39%, 2 = 40–69%, 3 = 70–99%, 4 ≥ 100%.

^a A sham combination is a mixture prepared from two separate stock solutions of the same chemical, that is then tested as if it were a binary combination. Expected EC₅₀ and slope AQ values for a sham combination are 1.00.

^a Mean time-dependent toxicity values were calculated as described in the text; TDT level definitions are given in the text and shown in Table 1.

^b Chemicals tested were: ethyl propiolate (EP), methyl vinyl ketone (MVK), ethyl acrylate (EA), methyl crotonate (MC).

^a TDT levels of the individual agents (A:B) in the mixture as noted in the text and in Table 1.

^b Chemicals tested were: ethyl propiolate (EP), methyl vinyl ketone (MVK), ethyl acrylate (EA) and methyl crotonate (MC).

^a See the text for TDT calculation equations and Table 1 for TDT level definitions.

^b Calculated by summing EC50-AQ values for a given pairing (i.e., 4:4, 4:3, etc.) across 15-, 30-, and 45-min exposure times, then dividing by three, and taking the difference from 1.00 (i.e., the AQ value for dose-addition).

^c Sham combination.