Epithelix Sàrl, 1228 Plan-les-Ouates, Geneva, Switzerland
Keywords: human; epithelia; epithelia; lung; toxicology; cell cultures: 3d cultures; replacement; toxicity testing: aerosols; validation
Duration: 1 year Project Completion: 2009
Background and Aim
Even though the alternative methods and models for addressing respiratory problems exist (Farmen et al., 2005; Matsui et al., 2005; Ostedgaard et al., 2005; Tarran et al., 2006; Geiser-Kamber, 2004; Kalberer et al., 2006), none of them have gone through the validation process. In this situation, it would be very difficult for industrial partners to use these alternative methods: Due to the legal constraints, the chemical industry, for example, cannot register products which are not tested with validated methods. As a result, animals will still be used in the future. With the implementation of REACH, more than 30’000 chemicals will have to be tested within the next 11 years. Thus, the standardization and validation of alternative methods is becoming a matter of urgency.
The compagny Epithelix, has developed and marketed a novel in vitro cell model of the human airway epithelium (MucilAir) which has some unique features:
Method and Results
According the ECVAM principles on test validity (Curren et al., 1995; Hartung et al., 2004), in order to validate an alternative method, a pre-validation step must be performed. This step includes several criteria:
1: Test definition:
- A definition of the scientific purpose of the test:
Test of the acute and chronic toxicity of the chemical compounds by inhalation.
- A description of the mechanistic basis of the test in view of the broader current scientific knowledge of the test endpoint: MTT, Resazurin test and TEER
- A definition of the protocol compliant with the Good Laboratory Practice.
2: Within-laboratory variability:
this addresses the variability over time and for different operators, but using the same laboratory set-up. Concretely, we propose to work out the following end-points:
a) Basal cytotoxicity tests: MTT and Resazurin test.
There are several traditional endpoints for evaluating the cytotoxic effects of chemical compounds, such as the MTT, Resazurin test. The protocols for these two tests are well established.
b) Integrity of the epithelium: Trans-epithelial Electric Resistance (TEER): Cellular electrophysiological properties, such as trans-epithelial resistance, are excellent indicators of the integrity of the epithelium. Furthermore, the TEER measurement is a non-destructive method. Therefore, it can be used as an endpoint alone for long-term toxicity tests or combined with other endpoints (MTT, Resazurin).
The preliminary results of cytotoxicity tests:
In order to perform routine toxicity tests, measurable end-points have to be defined. We chose the MTT measurement as an endpoint evaluating the cyto-toxicity effects of the chemical compounds. Preliminary tests have been performed on MucilAir to establish a standard protocol, as well as to assess reproducibility. A protocol has been established. The results are reproducible when tested on 24 inserts, with a very small standard deviation (see Table in Figure 1).
B: Relevance - Cytotoxic Effect of Acrolein on MucilAir:
Next, we tested the cyto-toxic effects of Acrolein on MucilAir. The results obtained (Fig. 2) correlated very well with the published data (Haddad-Romet, 1993, Official Data of OCDE).
Conclusions and Relevance for 3R
Actually, there is no alternative method for testing the chronic toxicity chemicals. All tests have to be done on animals such as rats. We would like to offer a validated alternative method/model which will have profound implications in relation to 3R’s animal protection principles.
First, unlike most of the airway epithelial cell models which can survive for only several days or weeks, our in vitro cell model has a shelf-life of one year. This unique feature makes it possible now to test the chronic effects of drugs or chemicals.
Secondly, the mission of Epithelix is not research per se; rather, Epithelix is actively applying and promoting the 3R principles by offering a ready-for-use in vitro cell model as a full package. Therefore, the impact on the animal experimentations is not limited to one research laboratory as it was often the case; instead, its impact is worldwide. If its full potential could be reached, then the life of a lot of animals might be saved.
(see also 3R-INFO-BULLETIN Nr. 41)
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Figure 1: To test the reproducibility of the MTT test on MucilAir, 24 inserts of fully differentiated airway epithelium were submitted to MTT test, using a protocol that we optimized for MucilAir. As shown above, the MTT test using MucilAir is highly reproducible and reliable.
Cytotoxicity effects of Acrolein, a well-known toxic compound for the airway epithelium, were tested on MucilAir (incubation period of 24h). The “dose-effect” response is between 0.5 and 3.0 mM. The EC50 obtained (1.5 mM) was in good correlation with the published results (N=24).