A combination of in-vitro and ex-vivo test models as an upstream alternative to regulatory OECD 403 animal studies

Fraunhofer ITEM developed ex-vivo and in-vitro screening tests for estimation of acute inhalation toxicities prior to regulatory OECD 403 in-vivo studies. The two complementary methods applied included a rat isolated perfused lung (IPL) model and a cell-based invitro approach using human lung epithelial cells under air-liquid-conditions based on the P.R.I.T.®-ALI technology. Sodium lauryl sulfate (SDS, positive control in-vitro), chlorothalonil and mancozeb represent compounds with well-known toxicity that were used to establish dose-response relationships in-vitro and to assess acute changes in respiratory parameters ex-vivo. The Precise Inhale® technology was used for efficient aerosol generation and precise aerosol exposures. 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-vitro analysis was carried out by application of a tetrazolium salt assay (WST-1) for estimation of cell toxicity following to a 24 hour post exposure incubation period. The exposures of IPLs to chlorothalonil or mancozeb led to moderate alterations of respiratory parameters, but resulted in dose-dependent increases in lung weight and thus pulmonary oedema formation. In-vitro exposures to all three test substances resulted in dose-dependent reductions of viability and establishment of EC50 values. Consistently in both assays, mancozeb showed a significantly lower toxic potential than chlorothalonil and results were, thus, confirming the findings of in-vivo studies. The combination of both assays for the prediction of acute toxic potential will be used with further substances exhibiting known or unknown toxic potentials to broaden the data base and verify the reliability of this test battery.