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This cell-based in vitro test method assesses the effects of test compounds on the integrity/permeability of epithelial cells and therefore may be useful as a measure of eye irritancy potential.

The method was validated in an EURL ECVAM retrospective validation study and is considered ready for use within a top-down approach in tiered eye irritation testing strategies.

In a wider testing strategy, this means it can be used to identify potential ocular corrosives and severe irritants from all other classes of water-soluble chemicals.

The results and full reports will be soon available on TSAR, the Tracking System for Alternative methods towards Regulatory acceptance.

Eye irritation


In eye irritancy, an initial physiological event is the loss of impermeability of the cornea, conjunctiva and/or conjunctival epithelium. Specifically, tight junctions between cells form a seal that effectively prevent the passage of most molecules.

Loss of impermeability (due to exposure to a chemical that can break these tight junctions) in epithelial cells in the eye mean that irritants can access the underlying stroma (scaffold-like support for cells) and might result in corneal opacity.

Thus, a method that can assess the effect of a chemical on the strength of this barrier and the tight junctions may provide relevant information on the eye irritancy potential of a chemical or substance.[/collapse]

The Fluorescein Leakage (FL) test method


Various cell types can form tight junctions when cultured in vitro. In the case of the fluorescein leakage test method, Madin Derby Canine Kidney (MDCK) cells can do this in a manner that is very similar to tight junctions found in epithelial cells in the eye and specifically the cornea. This creates a largely impermeable barrier that most chemicals cannot cross.

This is also applies to fluorescein, which is a dye that is normally blocked by MDCK cells. In the method, the MDCK cells are grown to confluence (i.e. there are tight junctions between cells) on porous filters and then exposed to a test chemical. Leakage is then assessed by measuring how much fluorescein passes through the cell layer.

As such the method tries to model the disruption of the integrity of the corneal epithelial barrier by chemicals which might have eye irritancy potential.[/collapse]

Animal testing replacement


The test method can be used to assess chemicals and substances that might disrupt tight junctions between epithelial cells that make up the cornea and similar constructs within the eye.

According to the ESAC statement and related outcomes of the retrospective validation study conducted by EURL ECVAM (see below) it can be used to identify potential ocular corrosives and severe irritants within a top-down Approach and as part of a wider testing strategy.

As such, the method addresses one part of many wider pathways that can cause eye irritation. It can thus be considered as only a partial replacement. Indeed, limitations of the method also mean it can only be applied to certain classes of chemicals.

At present, no alternative (in vitro) method is considered a full replacement of the in vivo Draize eye test.[/collapse]

Validation study


The retrospective validation study of four cytotoxicity and cell function-based assays, i.e. the neutral red release test, the red blood cell test, the fluorescein leakage assay and the cytosensor microphysiometer test, took place between May 2006 and October 2008.

The study was based on the retrospective collection of existing data compiled according to the EURL ECVAM modular approach to validation and weight-of-evidence principles.[/collapse]

Validation study outcomes


After peer review, two assays, the cytosensor microphysiometer test and the fluorescein leakage assay, were scientifically validated for being used as an initial step within a top-down approach to identify ocular corrosives and severe irritants from all other classes for the chemical applicability domain of water soluble chemicals.

Two other methods, the neutral red release and red blood cell haemolysis tests were considered not ready for use in regulatory assessments (based on available evidence at the time of review).[/collapse]

EURL ECVAM recommendations


EURL ECVAM recommended that the fluorescein leakage test method is considered scientifically validated and ready for consideration in regulatory assessments as an initial step in a wider top-down testing strategy.

Based on this assessment, it is unlikely this approach will replace the animal-based Draize test and is likely only of use if combined with further in vitro tests.[/collapse]