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

Test-to-test consistency was evaluated for a single binary combination of organic chemicals using an assay that examined toxicity over multiple exposure times. Six experiments were conducted. The toxicities of 3-chloro-2-butanone (3C2B), methyl crotonate (MC) and the mixture of both (MX) were evaluated in each experiment at 15, 30 and 45 min of exposure using the Microtox® system. Concentration-response (x/y) curves were generated via the five-parameter logistic minus one-parameter (5PL - 1P) curve-fitting function and were used to develop predicted x/y curves for the dose-addition (DA) and independence (I) models of combined effect. A variety of toxicity (e.g. effective concentration, EC50) and time-dependent toxicity (TDT) endpoints, 5PL - 1P parameters and various combined-effects metrics (e.g. MX/DA) were calculated. Test-to-test consistency was evaluated via the coefficient of variation (CV) or, for TDT, the standard deviation of mean values. In the study, CVs obtained for single-chemical and mixture toxicity endpoints (EC25, EC50 and EC75) at each exposure time were <20, as were those for the predicted DA and I curves. For the MX/DA metric, mixture toxicity was consistent with that predicted for DA at each exposure time in each experiment with CVs <6, despite some substantial variation in TDT for MC-alone at the EC25 and for the 30-45 min time-interval. There was a lower variation in TDT for 3C2B and MX. Mean and CV values for 5PL - 1P-derived slope and asymmetry parameters were also assessed to provide bases for comparisons in future reports.