In vitro genotoxicity test using metabolic competent 3D human bronchial epithelial model as alternative to in vivo genotoxicity studies with inhalation exposure.

EU policy, including REACH, demands reliable in vitro alternatives to replace costly and time-consuming animal tests required for the safety evaluation of compounds. In safety evaluation, the genotoxic potential of compounds is one of the parameters that needs to be evaluated to predict a compounds’ carcinogenic potential. The current in vitro cell systems to test the genotoxicity of compounds have several drawbacks including: 1) The current in vitro systems typically produce large numbers of false positive results. In the present testing strategy this unavoidable leads to in vivo testing to confirm or disapprove these findings. 2) Most of the applied cell systems lack xenobiotic metabolic capacity and hence require the addition of an artificial P450 system (i.e. rat liver S9-mix). 3) Most systems make use of non-human cells or cell-lines derived from human carcinomas. The relevance of such cell systems for the human situation is questionable. 4) Although inhalation is a major route of exposure there are no in vitro systems available to test the genotoxic potential of gasses, volatiles and particulate matter (or nanoparticles). The genotoxic potential of these air-borne substances can currently only be estimated by the analysis of micronuclei (MN) induced in the bone marrow or DNA damage in the liver of exposed animals. One can argue whether these organs are relevant to study the genotoxicity of inhaled compounds. 5) The current testing is primarily based on hazard identification, realistic chronic exposure to low doses is usually omitted. The LUMC has developed robust air-exposed 3D tissue cultures generated from human bronchial epithelial cells. This in vitro 3D tissue system has several important characteristics including: 1) The cell cultures gradually differentiate into 3D tissue with mucociliary characteristics (markers of mucin production and active ciliary beating) that closely resemble the pseudostratified human lung epithelium in vivo, the target tissue for inhaled compounds. 2) The differentiation requires culture at the interface between liquid (the culture medium) and air. This provides the unique opportunity to expose these in vitro cultures to air-borne substances. 3) The differentiated cultures can be maintained for several weeks making realistic chronic exposures a possible option. 4) During differentiation the cultures gradually acquire the activity of the typical human pulmonary cytochrome P450 enzymes. Consequently, the system should be able to detect compounds that require metabolic activation. 5) Immortalized human epithelial cells have been generated and pilot studies show that they retain most of the characteristics of the primary cells, which makes a sustainable system a realistic possibility. 6) Although a large proportion of cells in the mature tissue cultures is differentiated there are still sufficient proliferating cells to allow sensitive measurement of genotoxicity by the integration of the MN-assay within these tissue cultures. Recent pilot studies have actually demonstrated the feasibility of this integration. TNO has established expertise in the validation of various in vitro genotoxicity assays, including the in vitro MN-assay (OECD accepted). Furthermore, TNO has an inhalation facility that is unique in Europe and that allows well-controlled exposure of in vitro cultures to air-borne substances. The aim of this project is to examine the applicability and to pre-validate the 3D human lung model as an in vitro system for screening the genotoxic potential of compounds including air-borne substances. The plan of investigation can be divided in three phases: In Phase I the integration of the MN-assay within the 3D tissue cultures will be optimized. In parallel, the performance of immortalized cells to generate functional in vitro epithelium will be investigated and the final protocols will be established. In Phase II the in vitro assay will be challenged with a set of 6 model compounds consisting of 2 non-genotoxic compounds, 2 direct acting genotoxic compounds and 2 compounds that require metabolic activation. After the initial screenings at the LUMC, the assay is transferred to TNO to examine the robustness and transferability of the assay. In Phase III the cultures will be exposed to air-borne substances. Obviously, this reflects much better the natural exposure of airway-epithelium via inhalation. At least three genotoxic compounds will be tested. Finally the feasibility of chronic exposures will be explored. Ultimately this should lead to a relevant in vitro 3D human lung tissue assay that can replace the existing in vivo genotoxicity studies with inhalation exposure.