Alternatives to animal experiments for eye irritation

6. Evaluation of alternatives to ocular irritancy tests

Based on the results obtained in the EU/Home Office study, it was estimated that alternative methods replacing the Draize eye irritation test could only be established after 2005 (Purchase, 1997). In addition, the results of both the EU/Home Office study and the COLIPA study were characterized as disappointing in the EU Commission’s reports on alternatives as replacements of animal experiments for testing of cosmetics (EU Commission, 1995 and 1996b). On this basis, the ban against animal experiments on cosmetics implied in the EU cosmetics directive was postponed to January, 2000.

Recently, more optimistic view-points on the state-of-the-art of eye irritancy testing in vitro have been agreed upon on at workshops arranged by COLIPA (Bruner et al., 1998) and the EU Commissions center for validation of alternative methods (ECVAM)(Balls et al., 1999). The importance of understanding the mechanisms of eye irritation, particularly when attempting to improve in vitro prediction of in vivo eye irritancy, was emphasised at both workshops, and suggested research areas include:

	better characterisation of damage to the eye tissues, including the development of early markers for eye injury,
	increasing understanding of the effects of chemicals on the tear film and consequences of tear film disruption,
	characterisation of the acute and medium/chronic inflammatory response,
	validation of the area and depth of corneal injury as markers for eye injury,
	development of methods for assessing wound healing, pain, and the kinetics of the eye response, and
	development of methods for assessing persistence or reversibility of eye effects.

At the COLIPA workshop, there was a general agreement that the value of the Draize test for accurately assessing eye hazards in humans and for the development of alternative tests is limited. Therefore, the need for use of data on the human eye irritation response was stressed. It was recommended to identify a reference set of 10-20 model chemicals, selected on the basis of human eye irritation data when possible, for the development of mechanism based in vitro tests. It was recommended to assess the performance of the currently available in vitro tests with better in vivo data, and to develop and refine mechanism based in vitro methods for ocular irritancy testing.

The currently available in vitro methods, which are most likely to be relevant to eye irritation responses were identified as:

	cytotoxicity assays (including multilayered co-culture models),
	assays for measuring changes in epithelial function (e.g. the fluorescein leakage test),
	organotypic tests, as the BCOP test and tests with isolated chicken or rabbit eyes,
	CAM tests to model primary conjunctival effects, and
	long term models of the cornea (for example, cultured corneas).

An approach to the validation of in vitro tests for eye irritancy based on the use of reference standards was suggested at the ECVAM workshop, and ECVAM plans to validate further a test with isolated chicken eyes, the BCOP test, the combined used of the HET-CAM test and the neutral red uptake test, the red blood cell haemolysis test, and the EpiOcular^TM tissue model. In addition, it was recommended to compare the results from the EU/Home Office study and the COLIPA study with eye irritation classifications, and to evaluate the predictive abilities of testing strategies (combinations of in vitro tests.)

Prospects for progress in the replacement of eye irritancy studies

Today a very large body of data on the performance on various alternatives to eye irritancy tests has been generated. Various tissue models with topical application of the test substances have been demonstrated to be very good at predicting a broad spectrum of Draize test MMAS values, and the models appear to be able to accomodate all types of formulations and ingredients. Preliminary results also suggest that tissue models may be suited to studies of recovery from ocular irritancy. Tests with cellular monolayers being exposed to test substances dissolved in the culture medium appear to be limited to test of water soluble substances, and good concordances to eye irritation data have in general been obtained with surfactants. Results from various methods with isolated eyes or corneas have in several studies been shown to correlate well with in vivo data or to give good concordances to eye irritancy classes. The HET-CAM test appears to be able to identify some severe irritants, but in general it has been demonstrated to be less reproducible than the in vivo tests. In addition, very poor in vitro/in vivo concordances have in several studies been obtained with the EYTEX test.

Most of the alternative methods have, however, been documented to be far more reproducible than the in vivo eye irritancy tests. For this reason, much more weight should be given to an evaluation of the ability of the alternative methods to predict the in vivo response than to evaluations of their reproducibility. Test of a subset of chemicals may be a sufficient basis for an evaluation of the intra- and interlaboratory variability of the in vitro methods. Rigoristic demands to validation of in vitro methods may both lead to neglectance of valid alternatives and to acceptance of invalid tests..

The process of evaluating the information obtained appears to be much more complicated than expected, and a new, independent reevaluation of the existing data in line with the 1997 IRAG evaluation might very valuable. The establishment of prediction models for the alternative methods prior to their validation is crucial in order to obtain useable predictions of the in vivo response. For the majority of the alternative tests, it would be possible to develop more sophisticated prediction models based on advanced regression modelling on the relationships between historical in vitro and in vivo data.

The ban againts animal experiments on cosmetics implied in the EU cosmetics directive has been considered to be an impetus to the development and validation of alternative tests. However, the ban may be a major hinderance to the acceptance of alternatives, since a ban on products that have been subject to specific types of animal experiments in the European Union may be causing severe difficulties to the trade of cosmetics between the EU member states and other countries. The specific ban on animal tests on cosmetics and their ingredients appears also to be superfluous, since alternatives are to be used when available due to the EU directive on animal experimentation. An efficient move against improved possibilities to obtain a general acceptance of alternatives to the animal experiments required for safety assessements concerning cosmetics may be to delete the ban on animal experiments in the fortcoming revisions of the EU cosmetics directive.