Colworth, United Kingdom as an in vitro alternative to the in vivo Draize rabbit eye test method for the assessment of eye irritation

The IRE test was developed by Burton and his colleagues at Unilever Research Laboratory, Colworth, United Kingdom as an in vitro alternative to the in vivo Draize rabbit eye test method for the assessment of eye irritation (Burton et al. 1981). In the IRE test method, liquid test substances are spread using a syringe and solids are pulverized and applied as a powder over the corneas of enucleated rabbit eyes. The principal advantages of this test method are that the animals are euthanized prior to ocular irritancy testing, eyes from animals used for other toxicological purposes or from the food chain can be used, and testing is performed on the cornea, the part of the eye that is generally given the highest weight for scoring ocular irritancy in the Draize test. The effects of the test substance on the cornea of the isolated eye are measured quantitatively as an increase in thickness (swelling), subjectively as scores for corneal opacity, the area of corneal involvement, and fluorescein penetration, and descriptively as morphological changes to the corneal epithelium. However, the number of ocular parameters and the number of time points measured varies from study to study. Two additional refinements of the IRE test method may be incorporated into the protocol or used ad hoc to supplement existing data. One is the use of histopathology to confirm or identify the extent of irritancy at the cellular level, especially when the degree of irritancy falls between moderate and severe. Another is the use of confocal microscopy to determine the extent and depth of ocular injury (Maurer et al. 2002). Many studies using the IRE test method evaluate single or multiple ocular endpoints at various times and then assign irritancy classifications to the substances tested (CEC 1991; Köeter and Prinsen 1995; Cooper et al. 2001; Jones et al. 2001), while others use mean data from one or more ocular endpoints assessed at various times after application of the test substance, typically 0.5 to 4 hours (Balls et al. 1995; Gettings et al. 1996). One protocol for the IRE test method was designed to specifically identify severe eye irritants (Guerriero et al. 2004). In this study, cut-off values for each ocular parameter tested were predetermined. If these cut-off values were achieved or exceeded in any single parameter over a period of 0.5 to 4 hours, including a significant change in the corneal epithelium, the test substance was classified as a severe eye irritant with potential to cause serious or irreversible damage to the human eye. Protocols developed and used at SafePharm and Unilever in the United Kingdom were provided (Jones P and Whittingham A, personal communications) and information on additional IRE protocols was obtained from reports in the literature (Gettings et al. 1966; Burton et al. 1981; Price and Andrews 1985; INVITTOX 1994; Balls et al. 1995; Chamberlain et al. 1997; Cooper et al. 2001; Jones et al. 2001; Guerriero et al. 2004). These protocols are compared in Appendix A.