Effect of hydration on opacity in the bovine corneal opacity and permeability (BCOP) assay

John L. Ubels, Aileen M. Erickson, Uko Zylstra, Christopher Kreulen, Phillip L. Casterton

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


The bovine corneal opacity and permeability (BCOP) assay has been proposed an an in vitro method for predicting the irritancy or toxicity of chemical substances. A loss of corneal transparency, which reduces visual acuity, can be the result of an increase in corneal hydration or direct damage to corneal tissues. The BCOP assay as currently practiced does not differentiate between these mechanisms. The purpose of this study was to determine the effect of increased hydration on transparency of the bovine cornea and to measure the hydration levels of cornea treated with compounds and substances known, from previous BCOP studies, to cause corneal opacity. Corneas were treated according to the BCOP protocol and corneal opacity was determined by measurement of light absorbance at 570 nm (A570). Corneal hydration was determined by measurement of wet and dry weights. Hydration oh the intact cornea was 3.86 mg H2O/mg dry weight and A570 = 0.048. After removal of the epithelium and incubation in MEM, corneal hydration increases to 6.27 mg H2O/mg and A570 = 0.11. Hydration of the maximally hydrated corneal, following removal of the epithelium and endothelium and incubation in deionized water, is 16.33 mg H2O/mg and A570 = 0.67. The hydration of corneas treated with compounds that are ranked as moderate to severe irritants based on the BCOP assay is not correlated with hydration levels of these corneas, since the A570 of these corneas was usually higher than predicted from hydration levels. For example, after exposure to isopropanol hydration = 4.7, A570 = 0.59; acetone: hydration = 5.69, A570 = 1.38; 30% trichloroacetic acid: hydration = 4.28, A570 = 1.43; 1% NaOH: hydration = 8.22, A570 = 1.7. In contrast, after exposure to 30% sodium lauryl sulfate (SLS), which only removes epithelium, hydration = 5.38, A570 = 0.095. This suggests that opacity in excess of that caused by increased hydration is caused by damage to corneal tissue, including epithelium and stroma. This was confirmed by light and transmission electron microscopy. Determining the cause of an increase in corneal opacity in the BCOP assay is important since tissue damage may be irreversible, while the cornea can recover from moderate increases in hydration. On the other hand, since increases in hydration alone cause relatively low A570 readings, a potential irritant that causes a visually significant increase in cornea hydration might be ranked as a mild irritant. It is therefore proposed that measurement of corneal hydration be added to the BCOP protocol.

Original languageEnglish (US)
Pages (from-to)197-220
Number of pages24
JournalJournal of Toxicology - Cutaneous and Ocular Toxicology
Issue number4
StatePublished - Jan 1 1998
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Toxicology
  • Health, Toxicology and Mutagenesis


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