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.