The aim of this study was to use different toxicity indices to investigate the effect of N, N-Dimethylacetamide (DMA), Polyethylene glycol 400 (PEG 400), Methyl-Pyrrolidone/aromatic hydrocarbon (MPH), Cremophor((R)) EL, and Dimethylsulfoxide (DMSO) on Calu-3 cells. Membrane perturbation and cytotoxicity were investigated using lactate dehydrogenase (LDH), 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolinium bromide (MTT), transepithelial electrical resistance (TEER) assessment, sodium fluorescein (SF) permeation, and phalloidin actin staining. The MDH activity of cells treated with DMSO (<or= 4.0 v/v), Cremophor EL (<or= 10% v/v), and PEG 400 (<or= 10% v/v) was not significantly altered (p > 0.5). Similarly, DMSO (<or= 8.0% v/v), Cremophor((R)) EL (<or= 16.0% v/v) and PEG 400 (<or= 32.0% v/v) had no significant effect on LDH (p > 0.05). Conversely, MPH and DMA at very low concentrations (<or= 0.25% v/v) induced cell toxicity. However, up to 2.0% v/v MPH and (DMA) had no effect on TEER and SF permeation. No obvious change in actin staining was observed for DMSO (15%), Cremophor((R)) EL (15%), and MPA (1%). However, the actin architecture for cells treated with DMA (1%) and PEG 400 (15%) appeared significantly different from control. Based on this study Cremophor (<or= 10%), PEG 400 (<or= 5%), and DMSO (<or= 5%), and low concentrations of DMA and MPA (<or= 0.25%) were suitable for in vitro studies with Calu-3 cells.