Bovine eyes recovered from a slaughterhouse are inspected and undamaged corneas are dissected and mounted in specially constructed holders. After 1 hour incubation in media, the basal opacity of each cornea is recorded using an opacitometer.
Two methodologies have been developed to adapt the protocol to the physico-chemical nature of the test compound. The first method (A) is used to test non-surfactant liquids and surfactants. Liquids are tested neat and surfactants, which may be liquid or solid, are diluted at 10% weight/volume (w/v). Both are applied for 10 minutes. Before reading the final opacity, the corneas are rinsed and incubated for 2 hours in fresh media to equilibrate.
The second method (B) is used with solids, tested at 20% (w/v) solution or suspension in 0.9% NaCl or deionised H2O. After 4 hours incubation, the corneas are rinsed and the final opacity measured. Then the permeability of each cornea is determined with a fluorescein solution after an incubation of 90 minutes. In Method A a fluorescein concentration of 4 mg/mL is used and in Method B - 5 mg/mL. An aliquot of the media from below the cornea is read in a spectrophotometer to determine the permeability of the cornea to the fluorescein solution. The opacity and permeability values are combined to obtain an In Vitro Irritation Score.
The following formula is used to determine the In Vitro Irritatin Score:
In Vitro Irritation Score = Corrected Opacity Value + (15*Corrected OD490 Value)
In Vitro Irritation Score: In Vitro Irritation Scale:
0−3 non eye irritant
3.1−25 milde eye irritant
25.1−55 moderate eye irritant
≥55.1 severe/corrosive eye irritant