Air/liquid interface (ALI) technique for toxicity testing of gaseous compounds on human lung cells

With the new European Regulation (REACH) in place there is an increasing need for alternative toxicity testing techniques other than in vivo. Currently animal experiments are the main method for testing acute inhalation toxicity. This study is aiming for an alternative to animal experiments by providing an in vitro model system for acute lung toxicity testing of inhalable, gaseous compounds. Therefore an extended pre-validation study was performed including laboratories from academia, governmental institutions and research organizations: Fraunhofer ITEM, BAuA, UFZ, and BfR. Human lung epithelial type II cells (A549) were cultured on microporous membranes at the air/liquid interface and were exposed to various test gases (i.e. nitric oxide, sulphur dioxide, acetaldehyde, ammonia gas, tetrafluoroethane, argon and synthetic air). Cytotoxicity was measured by counting the viable cell numbers (CASY® technology). Gas-mediated genotoxicity was elucidated by the COMET assay. Non toxic, inherent gases (synthetic air, tetrafluoroethane and argon) displayed no cyto- or genotoxicity. Toxic gases such as nitric oxide, sulphur dioxide, acetaldehyde, and ammonia gas showed dose dependent effects on the viability of the exposed cells. EC50 values obtained from the experiments were compared to LD50 literature based values from mice and rat acute inhalation experiments. Furthermore all data have been cross validated amongst the participating laboratories. Tail-moment values retrieved by the COMET assay showed a dose dependent correlation for nitric oxide, sulphur dioxide, and ammonia gas indicating dose dependent DNA damage. This extended pre-validation study across laboratories presents a promising, novel, and non-invasive method for acute inhalation toxicity screening