Cytotoxicity and genotoxicity in vitro and irritation potency in vivo of two red phosphorus-based pyrotechnic smokes

Two red phosphorus (RP)-based smokes (P60 and RPB), differing from each other mainly in RP content and in type of additive, were evaluated for in vitro cytotoxicity (cell viability by the trypan-blue exclusion method) and genotoxicity (comet assay) by exposing BEAS 2B human bronchial epithelial cells to the smokes in a laboratory-scale chamber for 5 min. The irritation potency of RPB smoke was studied in mice. A hexachloroethane-based smoke (HC/Zn/TNT) was used as a reference in the studies. A 5-min exposure of BEAS 2B cells to P60 smoke (1.1, 2.2 and 4.4 g/m3, measured as H3PO4) did not induce any cytotoxic effects, while RPB smoke (1.3, 2.6 and 5.1 g/m3, measured as H3PO4) caused a mild decrease in cell viability at higher concentrations, without a clear dose-dependent effect. Neither of the RP smokes showed a genotoxic response in the comet assay with BEAS 2B cells, while HC/Zn/TNT was clearly genotoxic (0.9–3.5 g/m3 as ZnCl2). In the mouse bioassay, head-only exposure to RPB smoke (20–450 mg/m3 for 30 min as a single exposure, or 65–90 and 25–110 mg/m3 – measured as H3PO4 – for 30 min/day during 5 days) caused a concentration-dependent sensory irritation, which was evident as a decrease in respiratory rate and an increase in time-of-pause after inspiration, in a similar manner as with HC/Zn/TNT smoke. The concentration that caused a 50% decrease in respiratory frequency (RDmax50) was calculated to be 1140 mg/m3 for the RPB smoke and 145 mg/m3 for the HC/Zn/TNT smoke. No pulmonary irritation was observed.