Assessing ocular irritation potential using a modified ex vivo rabbit eye test

We have evaluated the ocular irritancy potential of an unknown environmental contaminant, para-toluene sulfonic acid (pTSA), compared with that of known irritants, 5% sodium dodecyl sulfate (SDS) and 10% acetic acid (AA), using a simplified, ex vivo rabbit eye test modified to measure cytotoxicity as a mechanistic correlate to the Draize rabbit eye test. Rabbit eyes were obtained fresh within 24 hours from an abattoir and then exposed to 50 mu L of test material. Eyes were then incubated intact for 3 hours or 1 day, and the corneas were removed, stained with calcein acetoxymethylester (AM)/ethidium homodimer (LIVE/DEAD assay, Invitrogen Corp., Carlsbad, CA, USA), and evaluated by laser scanning confocal microscopy. The number of dead cells was then quantified and the difference was statistically compared. For corneas exposed to 1 ppm and 1% pTSA, there was no significant difference in the number of dead cells compared with water-exposed, control corneas at either 3 hours or 1 day after exposure. However, corneas exposed to 10% and 50% pTSA showed significantly increased (p<.0001) numbers of dead cells, averaging (mean standard deviation) 486 +/- 133 and 1,052 +/- 101 cells/field of view (460 x 460 mu m). The level of cytotoxicity was comparable with that observed for 10% AA, which averaged 409 +/- 142 cells/field of view. The data suggest that pTSA is an innocuous irritant at exposure levels environmentally encountered, but that higher concentrations (10% and above) might be considered a slight to mild irritant. We conclude that this modified ex vivo rabbit eye test using the LIVE/DEAD assay may be a useful model for developing ocular irritation assays.