This assay directly assesses the effects of test chemicals on the opacity and permeability of bovine cornea.
It can be used as a measure for potential eye irritancy and as part of a wider testing strategy to classify chemicals and substances as ocular corrosives or severe irritants.
The method was assessed as part of a retrospective validation study that was conducted by ICCVAM-NICEATM in collaboration with EURL ECVAM.
Read more about BCOP method on ICCVAM-NICEATM website here.
Eye irritation
[collapsed]Eye irritation in relation to this test method refers to the transparency of the cornea as this is important for the proper functioning of the eye.
Corneal opacity usually indicates protein denaturation or precipitation in the epithelial or stroma layers due to exposure to an irritant.
Corneal permeability, corneal barrier function, thickness and hydration can also be assessed as part of the test method. As a result, the assay may conceptually be used to assess a range of parameters that relate to eye irritation.[/collapse]
The Bovine Corneal Opacity and Permeability (BCOP) assay
[collapsed]BCOP is an in vitro assay that directly assesses the effects of potential irritant substances on the opacity and permeability of freshly obtained bovine corneas (a by-product of abattoirs).
Following exposure to a test substance/chemical, corneal opacity is directly measured with an opacitometer.
Corneal permeability is determined using sodium fluorescein (a dye that usually cannot pass through epithelial cells of the cornea) and measured spectrophotometrically through changes in optical density.
An endpoint irritation score is calculated from these data, which can then be used to compare irritancy potential of different test substances/chemicals.[/collapse]
Animal testing replacement
[collapsed]The BCOP assay was developed as an alternative to the Draize rabbit eye irritation test. With certain limitations, it can be used to screen for potential ocular corrosives and severe irritants.
It can also be used as part of wider sequential testing strategies and as part of weight-of-evidence approaches.
In a non-regulatory context it can also be used to elucidate biological mechanisms in appropriate experimental designs. According to regulatory guidelines, a positive result is accepted as indicative of a substance being a severe irritant or ocular corrosive. In this case no further (animal-based) testing is required. In the case of a negative result, substances should be assessed with other in vitro methods for eye irritancy or finally in rabbits.
The method therefore has the potential to reduce the amount of animal-based testing required for eye irritation assessment, although it is not a full replacement (it only assesses effects of chemicals in cornea).
While the method uses corneas from freshly slaughter young adult cows, and therefore does involve an animal as part of the method, these corneas are a by-product of abattoirs and would under normal circumstances be disposed of.[/collapse]
Validation study
[collapsed]The retrospective validation study involved a re-analysis of pre-existing data relating to four organotypic assays: the bovine corneal opacity and permeability assay (BCOP), the isolated chicken eye test (ICE), the isolated rabbit eye test and the hen's egg test on the choir-allantoic membrane. The study was conducted by ICCVAM-NICEATM between 2003 and 2006. The study was subsequently peer reviewed in the US.
ESAC subsequently peer reviewed the same data for purposes of recommending the methods in the EU. The study specifically assessed whether the assays could detect severe eye irritants and ocular corrosives. Subsequently the same methods were assessed for whether they could identify mild or non-irritants.[/collapse]
Validation study outcomes
[collapsed]A total of eight studies and 158 substances were considered in the retrospective evaluation of the BCOP assay that was conducted by ICCVAM.
Both in vivo and in vitro data were included in the analysis and overall the essential components of a modular validation were considered to be satisfied. Specifically, high quality protocols/SOPs are available and endpoints of the assay are very clear.
Substances that have been previously evaluated and been subjected to ring trials have considerable data sets available and as a result the assay has a comprehensive applicability domain. However, there are some risks of false positives and negatives which should be considered.
Within- and between-laboratory reproducibility are high in most cases and the method is transferable between laboratories. After substances with high false positive/negative rates were excluded, predictive capacity ranged from 87-92%.
Following a shadow peer review by ESAC, it was agreed that ICCVAM recommendations should be endorsed.[/collapse]
EURL ECVAM recommendations
[collapsed]Following a shadow peer review by ESAC, it was agreed that ICCVAM recommendations should be endorsed by EURL ECVAM.
Subsequently an OECD test guideline was formulated and adopted 7 September 2009.[/collapse]