Validated Non-animal Alternatives
(a) Although not formally endorsed as valid, positive outcomes can be used for classifying and labeling substances as severe eye irritants (R41) in the European Union.
In 2007, ICCVAM made the following recommendations on four in vitro screening tests for identifying ocular corrosives and severe irritants: "BCOP and ICE recommended as screening tests for identifying corrosives and severe irritants, with certain limitations; HET-CAM and IRE not recommended for regulatory hazard classification purposes until further developed and evaluated."
The ESAC statement on the ICCVAM retrospective study of the four in vitro ocular screening assays endorsed the validity of the BCOP and ICE methods for use in a tiered strategy as part of the weight-of-evidence approach. The ESAC indicated further work is needed for the IRE and HET-CAM methods (ESAC Statement, April 27, 2007).
The specific limitations on the uses of the BCOP and ICE test methods are described in documents on the ECVAM and ICCVAM websites. OECD Proficiency Standards have also been published for the BCOP and ICE assays. A draft OECD guidance document on the use of ocular histopathology to improve test method predictivity for use in conjunction with BCOP and ICE OECD TGs to identify ocular corrosives and severe irritants is under consideration.
The BCOP assay uses cow eyes obtained from slaughterhouses. The cornea is isolated from the rest of the eye and maintained in a holder. A test substance is applied to this isolated cornea for a specified time, then removed, and the effect of the substance on the permeability of the cornea to fluorescein (a colored dye) and the increase in corneal opacity (transmission of light through the cornea) are determined. Both of these assay endpoints have been used in the in vivo Draize rabbit eye test and generally correlate well with the degree of corneal injury from a test material.
The ICE assay uses chicken eyes obtained from slaughterhouses. The eyes are placed in an apparatus where they can be kept moist and treated with the test substance. Three responses of the cornea are evaluated: corneal swelling, corneal opacity, and fluorescein retention. The irritation potential of a substance is then calculated from the mean values of these measurements.
In 2009, the ECVAM/ESAC endorsed two cell-based assays for eye irritation with specific limitations. The Fluorescein Leakage method can be considered for use in a top-down approach to identify ocular corrosives and severe irritants, and the Cytosensor Microphysiometer method can be used in testing for two of the three EU and GHS classification categories for ocular irritation. See the ESAC Statement for further clarification. ICCVAM also endorsed the cytosensor for very limited testing applications.
ICCVAM also evaluated an integrated testing strategy for assessing the eye irritation potential of antimicrobial cleaning products, which is composed of the following test methods: BCOP, EpiOcular, and Cytosensor Microphysiometer. ICCVAM decided that additional data are needed before this test battery can be endorsed as valid.
Several in vitro cell-based test methods are currently in interlaboratory validation studies to assess their performance in predicting eye irritation potential. Assays similar to the BCOP, but using pig eyes (PCOP), are also in development.
Additional information on in vitro methods being developed for eye irritation/corrosion toxicity testing can be found on AltTox at: Toxicity Endpoints & Tests: Eye Irritation/Corrosion.
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