List of selected 3R Methods (ordered according to topics)

Organ culture assay as a replacement for the Draize test

April 2011

Author: Felix Spöler, Institute of Semiconductor Electronics, RWTH Aachen University, Sommerfeldstrasse 24, 52074 Aachen, Germany
E-Mail: spoeler@iht.rwth-aachen.de

Interaction of chemicals and pharmaceuticals with the eye can be investigated using an organ culture in vitro assay based on rabbit corneas obtained from abattoir waste. Full metabolic function is maintained over a culturing period of up to 21 day enabling observation of recovery after chemical or mechanical trauma and undesired side effects during pharmaceutical treatment without the need for animal experiments.

Keywords: rabbit; cornea; eye; organ culture; reduction; replacement; toxicity testing: chronic exposure

Method
The new organotypic model system is published under the name Ex Vivo Eye Irritation Test (EVEIT). A short term approach of this test uses full eye globes to evaluate the acute toxicity of chemicals. This is achieved with high effectiveness by combination of the short-term EVEIT with time resolved analysis of the damaging process using optical coherence tomography. Thereby chemical trauma can be assessed via penetration velocity and depth of damage (Spöler et al., 2007). The short term approach is also applicable to quantify decontamination procedures. This was demonstrated by example using different recommended rinsing solutions after hydrofluoric acid burn (Spöler et al., 2008). The long-term approach of the EVEIT enables direct observation of recovery in vitro. Also examination of repeated exposure is accessible. The test system was used to investigate the effect of Benzalkonium chloride – a widely used preservative in pharmaceuticals – on the healing process of the corneal epithelium after mechanical trauma (Frentz et al., 2008). Another similar study examined the side effects of buffer substances used in eye drops (Schrage et al., 2010). Furthermore, it is feasible to use the EVEIT test system to model corneal diseases for pharmaceutical studies. This was investigated for the dry eye syndrome, one of the most common diagnoses in ophthalmology (Spöler et al., 2010).

3R effect
Ocular toxicity is an independent hazard class in the globally harmonized system (GHS) for the classification and labeling of chemicals. Therefore, numerous in vivo studies, most of them with rabbits, are necessary to date to manage that risks from chemicals and to provide safety information on substances, which are put into circulation. Additional in vivo tests are conducted during the development and authorization of ophthalmological products. Current in vitro tests like the Bovine Corneal Opacity and Permeability (BCOP) test and the Isolated Chicken Eye (ICE) assay are capable to identify corrosives and severe irritants only. As the GHS classification of eye irritants is based on the reversibility of observed effects the Ex Vivo Eye Irritation Test is a promising assay to analyze the full spectrum of eye irritation potency without the need for animal tests. Additional, many pharmacological questions can already be addressed by the test system today.

References/ Links
1. Spöler F, Först M, Kurz H, Frentz M, Schrage NF. Dynamic analysis of chemical eye burns using high-resolution optical coherence tomography. J Biomed Opt. 2007; 12: 041203. PubMed, Article [PDF]
2. Spöler F, Frentz M, Först M, Kurz H, Schrage NF. Analysis of hydrofluoric acid penetration and decontamination of the eye by means of time-resolved optical coherence tomography. Burns. 2008; 34: 549-55. PubMed, Article [PDF]
3. Frentz M, Goss M, Schrage N.F. Reim M: Repeated exposures of benzalkonium chloride to the Ex Vivo Eye Irritation Test (EVEIT). Short term hazard evaluation on the isolated cornea observing healing and damage: ATLA 2008; 36: 1-12. PubMed, Article [PDF]
4. Schrage NF, Frentz M, Reim M: Changing the composition of buffered eye-drops prevents undesired side effects. Br J Ophthalmol 2010; 94: 1519-1522. PubMed, Article [PDF]
5. Spöler F, Frentz M, Schrage NF. Towards a New in vitro Model of Dry Eye. The ex vivo Eye Irritation Test. Research Projects in Dry Eye, Developments in Ophthalmology vol. 45, Ed. Brewitt H, Karger 2010: 93-107. PubMed, Article [PDF]

Figures
The combination of the short-term EVEIT approach and cross-sectional imaging by optical coherence tomography allows for direct analysis of the depth of damage. This is demonstrated here for different concentrations of aqueous sodium hydroxide solutions which were applied locally onto a single cornea. The left image shows the fluorescein stained bulbus directly after application of the sodium hydroxide solutions. Images (1) - (5) demonstrate cross sectional images of the marked areas obtained by optical coherence tomography. Increasing depth of damage with increasing concentration of sodium hydroxide is clearly observed.

Figure 1