Etablierung eines Modellsystems mit Aerosolen zur Charakterisierung von Expositionssystemen für die Untersuchung inhalierbarer Substanzen in vitro

The study of substances, concerning their potential inhalative toxicology, traditionally follows from in vivo animal experimentation studies. For financial, temporal and ethical reasons even in this field of toxicology an increasing search of alternative test methods, which can generate reliable data, takes place. Conventional in vitro methods provide the testing of substances, which are dissolved in medium. This test method, termed as submerged exposure, neither matches the physiological conditions in the lung, nor describes the inhalative exposure appropriately. The aim of this bachelor thesis was to establish and verify an experimental approach for the study of inhalable test substances with selected model substances on the basis of a novel, device-based in vitro test method. Developed at the Fraunhofer ITEM, the P.R.I.T.®-ExpoCube enables the exposure of a biological test system to airborne substances, simulating in vivo-like conditions. The human originated A549 cell line served as biological test system, whereas it has characteristics of alveolar lung epithelial cells. The target cells were cultured under so-called air/liquid interface (ALI-) conditions on microporous cell culture inserts, in which the cells were supplied basolaterally with culture medium and exposed apically with a test aerosol. Previously the model substance aerosols were physically characterised by using a cascade impactor. Based on the data of the submerged testing, SDS (positive substance) and NaCl (negative substance) were selected from the six model substance candidates SDS, CuSO4, LiCl, 5-Fluorouracil, NaCl and lactose. These two model substances were used in the device-based method. After aerosolisation and deposition on the target cells under ALI-conditions, dose-response curves were established. Resulting from these, effective dose (ED) values were determined for the aerosolised model substances and compared with the ED values of the submerged testing. The particle sizes of the generated model aerosols ranged between 389 - 1580 nm. A toxicity ranking was derrived from the results of the conventional test method with dissolved model substances. This corresponded to the results of the ranking, obtained with the same model substances after aerosolisation and deposition under ALI-conditions and proved as a consequence the plausibility of this method. The results of the present study attest the general feasibility and the high potential of this in vitro test method for applications in the field of inhalation toxicology. An expansion of the database, including further substances and target cells of the respiratory tract (tracheal, bronchial), could be the next step that will be needed for further evaluation of the method aiming at the risk assessment of substances based on in vitro methods