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Validation of the CULTEX® RFS procedure and optimization of a prediction model

Subject: Validation of the CULTEX® RFS procedure and optimization of a prediction model for the assessment of the acute inhalative toxicity of dusts


Project Partner
Institute for Pharmacology and Toxicology of the German Armed Forces, Munich
seh consulting + services, Paderborn
Walther-Straub-Institute for Pharmacology and Toxicology, Munich
Cultex® Laboratories GmbH, Hannover


Exposure to airborne particles is gaining increasing importance due to the ubiquitous use of loose solids in the fields of industry and pharmaceuticals. Depending on the type of substance, particle size and concentration, serious health disorders – even death – could result from an inhalative burden.

Within the EU, the European Regulation REACH deals with the Registration, Evaluation, Authorisation and (where necessary) Restriction of Chemicals. A test strategy is predefined for toxicological evaluation that demands the performance of exposure tests for inhalable substances (gases, aerosols and particles).

Data on the inhalative toxicity are, however, often not available and are then based mostly on animal experiments. The estimates for the numbers of animals used for these tests differ considerably (up to 10 million animals), and approx. 1-2% of these (i.e. 100,000-200,000 animals) are used for experiments on acute inhalation toxicity. Alternative methods to animal experiments are urgently needed both for REACH and other regulatory measures, but are currently not available for the field of pulmonary toxicology.

Even though human cell-based test systems are already used in a large number of studies on the toxicological evaluation of substances, their validity with regard to the evaluation of airborne particles is limited due to the non-physiological exposure modalities (submersed cultures). The CULTEX® RFS - Radial Flow System closes this gap. It is based on the direct exposure of human lung epithelial cells at the air-liquid interface to the substances to be tested. This method allows the direct particle exposure of cellular surfaces.

In a research project funded by the BMBF (BMBF 0315710), this approach was validated in various laboratories with regard to its technical establishment and performance. The test methods were defined in standard operating procedures (SOPs) (ECVAM Module 1) and could be successfully transferred in two naïve respectively unacquainted laboratories (ECVAM Module 3). The reproducibility of the method in the laboratories (ECVAM Module 2) and between the laboratories (ECVAM Module 4) could be confirmed here. ECVAM Module 5 (establishment of a prediction model) and 6 (definition of the test field of application) could not, however, be completed in the preceding research project.

The current collaborative research project continues immediately on from this point. By optimizing the CULTEX® RFS procedure, further validation and refinement of the prediction model for the assessment of the acute toxicity of inhalationally effective substances should be achieved.

In the first phase, the cell exposure systems were established at all sites and the formal requirements for the project created. The cellular test system (A549 cells) was established in all laboratories and the culture conditions harmonized. The first experimental block contains tests with clean air, which, as negative control, may not impair the viability of the cells, so that in the later exposure experiments the effect of the test particles can be faultlessly determined. The efficiency of the test procedure was confirmed by analyzing a set of known substances (Lactose (negative substance), copper(II) oxide nano, copper(II) sulfate and Pural 200). After the successful completion of this project phase we are now entering the “blind testing phase” by using a variety of coded substances (up to 25 compounds) for establishing at the end of the project a relevant prediction model for the assessment of the acute inhalative toxicity of dusts.