In Vivo and In Vitro Evaluation of the Acute Toxicity, the Genotoxicity, and the Irritation Potency of Two Hexachloroethane-based Pyrotechnic Smokes

Abstract

The two hexachloroethane (HC)-based smoke formulations studied consisted of HC/Zn/2,4,6-trinitrotoluene (TNT) and HC/Zn. In the in vitro tests, human bronchial epithelial cells were exposed to the smokes at various concentrations. The responses studied were acute toxicity (viability of cells, trypan blue exclusion method) and genotoxicity (DNA single-strand breaks, COMET assay). The tests were conducted in a laboratory-scale chamber (V = 150 L) and in a container (V = 55 m³). Both smoke formulations appeared to be acutely toxic and genotoxic. For the 0.5- and 1-g burning experiments the responses were more pronounced with HC/Zn/TNT than with HC/Zn smoke. To study the irritation potency of the smokes, the mouse bioassay according to ASTM E 981-84 was applied. The respiratory parameters measured were tidal volume (VT), airflow during expiration at 0.5 VT (VD), time of pause after expiration (TP), time of breaking after inspiration (TB), and the respiratory frequency (BPM; breaths per minute). In the single-exposure experiments, HC/Zn/TNT smoke induced concentration-dependent sensory irritation in mice and the occupational exposure limit (TLV) was estimated to be 4 mg/m³. In the repeated-exposure experiments, HC/Zn/TNT smoke induced sensory irritation at the beginning of the exposure. Pulmonary irritation tended to dominate when the exposures were repeated. With HC/Zn smoke we were unable to generate sufficient high exposure concentrations. In the repeated-exposure experiments, indications of sensory and pulmonary irritation were seen at concentrations used. No evidence of apoptotic cell death was found in caspase-3-like protease activity assay.

Our deepest gratitude is expressed to the Scientific Advisory Board of National Defence for financing this study. We wish to thank Kirsti Nieminen, Satu Suhonen, Satu Lätti, and Kari Reinola for their skilful technical assistance in different phases of the research, Anja Suonurmi-Virtanen and Ph.Lic Jaakko Toivanen with their teams for the chemical analysis of the smokes, Dr. Jonne Naarala for the apoptosis studies, Dr. Matti Harkoma for the technical merits in the planning and construction of the aerosol chamber, Tapio Heininen for his advice on the statistical analysis of the results, and Jukka Nenonen and the fire brigade soldiers of the NBC school for their help in the heavy and dirty work in relation to the container tests. Finally, Professor Erkki Kantolahti and the reviewers are gratefully acknowledged for their valuable advice and criticism of the manuscript.