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

Abstract

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.

Keywords:

• Microtox^®
• methodology
• dose-addition
• bioluminescence
• time-dependent toxicity

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was made possible in part by grants 2 [grant number R15 ES08019] – 03/04 from the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH). The content is solely the responsibility of the investigators and does not represent the official views of NIEHS, NIH.