Optimized rinsing procedures for enhancing removal of residues of highly viscous and colored substances in the Bovine Corneal Opacity and Permeability (BCOP) assay

Two modifications of the rinsing procedure within the BCOP assay were proposed. Their ability to enhance the removal efficiency of highly viscous and colored samples was compared with an unmodified BCOP procedure (TG OECD 437). The first modification consisted of three-step washing of the applied chemicals from the cornea using Eagle's Minimum Essential Medium (EMEM), olive oil and EMEM, while the classical OECD TG 437 procedure prescribes only EMEM. Within the second modification, mechanical removal of the tested sample from the cornea surface prior to the two step washing procedure was performed. The in vitro irritation score (IVIS) exceeded the value of 55 for 9 out of 20 samples when a non-modified rinsing procedure was used. The first modification with the olive oil resulted in a decrease in IVIS for numerous samples, while an IVIS score drop below the threshold value of 55 was only observed for two of them. Mechanical removal of sample residua resulted in a further decline in the measured IVIS. Only the three samples treated by means of this procedure revealed an IVIS above 55. The decreases in IVIS observed during both modifications were mainly related to the reduced opacity, whereas the permeability mostly remained unaffected.