Time Dependence in Mixture Toxicity with Soft Electrophiles: 1. Combined Effects of Selected SN2- and SNAr-Reactive Agents with a Nonpolar Narcotic

Abstract

Frequently the toxicity of an organic chemical mixture is close to dose-additive, even when the agents are thought to induce toxicity at different molecular sites of action. These findings appear to conflict with the hypothesis that a strictly dose-additive combined effect will be observed for agents sharing a single molecular site of toxic action within the organism. In this study, several SN2-reactive (alpha-halogen) or S(N)Ar-reactive (halogenated dinitrobenzene) soft electrophiles were tested with a model nonpolar narcotic (NPN) to determine the toxicity of the combinations. A sham combination of the model NPN (3-methyl-2-butanone) was also tested as a positive control. The study design incorporated time-dependent toxicity (TDT) determinations at 15, 30, and 45 minutes using a Microtox (Vibrio fischeri) protocol that included testing seven duplicated concentrations for each single agent and mixture per combination. Additionally, in chemico reactivity was determined for each compound using thiol in glutathione as a model nucleophile. The model NPN alone lacked reactivity and TDT. The SN2-reactive agents individually showed varying levels of both reactivity and TDT alone, while the SNAr-reactive chemicals alone were reactive and had toxicity that was fully time-dependent between 15 and 45 minutes of exposure. Data analyses indicated that the sham combination was dose additive, as expected, whereas three of four SN2:NPN combinations showed effects close to that predicted for dose addition but with some differences. The fourth SN2:NPN combination, which included an alpha-halogen with full TDT, showed a less-than-dose-additive combined effect as did both of the SNAr:NPN pairings. By incorporating TDT values, shapes of the dose-response curves, chemical reactivity data with thiol, reactive mechanisms for the soft electrophiles, and quantitative structure activity relationship information on whether the toxicity of the individual soft electrophiles did or did not exceeded that predicted for baseline narcosis, the results suggested that the alpha-halogens elicited two toxic effects at the concentrations tested (reactivity and narcotizing effects), whereas toxicity induced by the halogenated dinitrobenzenes was essentially limited to reactive effects. Collectively, these results provide experimental evidence consistent with previous explanations as to why binary mixtures of industrial organic chemicals often show combined effects that are close to dose additive, even when the chemicals are thought to induce toxicity at different molecular sites of action.

Mixture Toxicity of SN2-Reactive Soft Electrophiles: 3. Evaluation of Ethyl α-Halogenated Acetates with α-Halogenated Acetonitriles

“…Earlier works have demonstrated the utility of incorporating time-dependent toxicity evaluations (e.g., Gagan et al 2007) and an asymmetry parameter in concentration-response curve-fitting (Dawson et al 2012) when evaluating mixture toxicity. Two recent studies examining toxicity of xAN-containing (Dawson et al 2010) and ExAC-containing binary mixtures (Dawson et al 2011) included: 1) both sham (i.e., A:A) and true combinations (i.e., A:B) for each chemical group and 2) combinations of each of those chemicals with a model nonpolar narcotic, 3-methyl-2-butanone (3M2B).…”

Evaluation of consistency for multiple experiments of a single combination in the time-dependence mixture toxicity assay

“…To quantify changes in toxicity over exposure time, TDT values were calculated at the 25, 50 and 75% effect-levels (Gagan et al 2007) and for the 15 to 30-min, 30 to 45-min and 15 to 45-min time-intervals using the appropriate time-factors for each. Time-factors (0.5 for 15 to 30-min, 0.333 for 30 to 45-min and 0.667 for 15 to 45-min) were calculated via the following equation: $$(t_2-t_1)/t_2$$ with t 2 being the later time of the exposure interval and t 1 the earlier time of that interval.…”

“…These studies have examined toxicity of selected mixtures of industrial organic chemicals (e.g., Gagan et al 2007, Dawson et al 2008), developed and evaluated the 5PL-1P function for concentration-response curve-fitting (Dawson et al 2012, 2016) and examined the viability of using time-dependent toxicity (TDT) of the individual chemicals for predicting TDT of the mixtures (Dawson et al 2014a). The design of these studies has included multiple tests of individual chemicals (Dawson et al 2010, 2011, 2014b, 2016) thereby allowing for assessment of test-to-test consistency of those data, but none of these studies had evaluated such consistency over several tests of the same mixture.…”

“…Chemicals lacking TDT tend to be unreactive (Gagan et al 2007), thereby exerting reversible toxicity primarily via effects on the cell membrane (e.g., non-polar narcosis). Additionally, it was determined that neither chemical should have fully time-dependent toxicity (i.e., TDT ≥100%) as those agents appear to exert only irreversible toxic effects.…”

“…Additionally, it was determined that neither chemical should have fully time-dependent toxicity (i.e., TDT ≥100%) as those agents appear to exert only irreversible toxic effects. Previous testing had suggested that there would be higher test-to-test variability in TDT for chemicals with mid-range TDT (i.e., TDT between about 20 and 80%), as such chemicals appear to have two modes of toxic action –reversible toxicity, especially at lower concentrations during earlier exposure times, and some form of irreversible action, most notably at higher concentrations and later in exposure (Gagan et al 2007). For these reasons 3C2B and MC were selected as the single-chemicals to be used in this study as they have mid-range TDT.…”