Acute inhalation toxicity in vitro and ex vivo test battery prior to regulatory OECD 403 studies

New crop protection agents are usually tested for acute inhalation toxicity in vivo according to OECD Test Guideline 403. In order to reduce the amount of animals and test compound needed, this project aimed at the implementation of ex vivo and in vitro screening tests that can be used to estimate the acute inhalation toxicity prior to regulatory OECD 403 studies. Therefore, two reference compounds with known toxicity, chlorothalonil (CAS 1897-45-6) and mancozeb (CAS 8018-01-7), were tested applying two complementary methods: the rat isolated perfused lung (IPL) model and in vitro using human lung epithelial cells under air-liquid-conditions with the P.R.I.T.®-ALI Technology. For efficient aerosol generation and precise aerosol exposures, the PreciseInhale® technology was used. In order to determine pulmonary viability, the respiratory parameters tidal volume (VT), resistance (RL), dynamic lung compliance (Cdyn) and the relative increase in lung weight were determined in IPL experiments. In the in vitro assay analysis of cellular viability was carried out using the tetrazolium salt conversion assay (WST-1) following to a 24 hour post exposure incubation period. The exposures of IPLs to the two reference items led to moderate alterations of the respiratory parameters, but resulted in dose-dependent increase in lung weight and thus pulmonary oedema formation. In vitro exposure to the two test substances resulted in dose-dependent reductions of viability and establishment of dose response curves. In both assays mancozeb showed a significantly lower toxic potential than chlorothalonil and thus confirms the findings of the in vivo studies. In the next step both assays will be used to predict the acute toxic potential of further substances with known and unknown toxic potential to broaden the data base and verify the reliability of this test battery