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Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI without phenol red. Duration of post- treatment incubation in vitro : After the illuminance measurement was performed, the medium was removed from both chambers of the holder.

The posterior chamber was refilled with fresh complete RPMI. Number of animals or in vitro replicates: 3 corneas for the test item 3 corneas as negative controls treated with physiological saline 0.

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Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm 2b site- ul dating of sclera.

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The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders Duratec GmbH with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded.

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The anterior chamber was then positioned on top of the cornea and tightened with screws. The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

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Eyes that were noted to have defects were discarded and not used on the study. Exposure 4 hours ± 5 minutes.

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Once the medium was free of test item, the cornea was finally rinsed with complete RPMI without phenol red. Irritation parameter:.