Acute Eye Irritation Test. UNE-EN 10993-10: 2013; EPA (Environmental Protection Agency, U.S.A); OPPTS 870.2400:1998 (Office of Prevention, Pesticides and Toxic Substances); OECD 405:2012, or Directive 92/69/EEC, Julio 31, 1992, Annex B.5.

Test Accredited by ENAC (National Accreditation Entity).

Tedst with the Certificate of Good Laboratory Practices (GLPs).

The evaluation of the irritant and/or corrosive effect in the eye of animals is an important initial test to evaluate the toxic characteristics of a substance, since the information obtained with this test serves to indicate the possible risks that may occur after the exposure of the animals eyes and the associated mucous membranes. In the test, the ocular irritation effect is detected, corresponding to the reversible changes caused when the substance is applied to the anterior surface of the eye, or ocular corrosion, corresponding to the irreversible tissue damage after the application of the substance to the anterior surface of the eye.

This test should not be carried out when there is previous human or animal information, or from in vitro results, which indicate that the substance may damage the eyes, has caused eye irritation, has caused corrosion or intense irritation, or when there is evidence of structural analysis relationship of activity (SAR) that have demonstrated the harmful eye effects, neither with substances with a pH ≤ 2 or ≥ 11.5, or if tests of in vitro irritation or ocular ex vivo can be performed previously.

Healthy adult albino rabbits are used to perform the test, without signs of eye irritation, eye defects or previous corneal damage. The substance under test is initially applied as a single dose to one of the eyes of a single animal, using the unexposed eye as a control, and the effects are evaluated and scored at specified intervals.

The test substance is applied in the conjunctival sac, keeping the eyelids closed after application to limit the loss of substance. The eyes should not be washed for 24 hours, but they can be washed after 24 hours if deemed necessary. Liquid, solid, pasty or particulate substances can be applied, but in case of being solid or granulated they must be crushed to obtain a fine powder. Pressurized aerosols can be applied directly. For pumping sprays, the liquid in the container must be obtained and must be instilled.

The duration of the study should be sufficient to allow a complete evaluation of the reversibility or irreversibility of the lesions, but it must be at least 72 hours, if there are no signs of irritation and it does not need to be more than 21 days, so the observations they are performed at 1, 24, 48 and 72 hours after exposure, with prolonged observation at 7 and 21 days if there is corneal involvement or other signs of ocular irritation, in order to determine the progression of the lesions and their reversibility or irreversibility. If there is an intense (reversible) or corrosive (irreversible) irritant effect with the animal initially used, no further tests should be performed. If these effects are not observed, the test should be carried out on at least three animals.

A considerable difference between the standard EPA-OPPTS 870.2400: 1998 and the Directive 92/69/EEC, with respect to the OECD standard 405: 2012, is that the latter indicates the use of a systemic analgesic (buprenorphine) 60 minutes before the realization of the test, and of a local topical anesthetic applied in drops 5 minutes before exposing the eye to the test substance, as well as in the control eye. It also recommends the administration of another dose of two systemic analgesics (buprenorphine + meloxicam) at 8 hours. If eye lesions are observed, a systemic analgesic (buprenorphine) should be administered every 12 hours and another (meloxicam) every 24 hours until the eye lesions are resolved or there are no clinical signs of pain or distress.

The degree of irritation or corrosion is evaluated and quantified at the aforementioned intervals, evaluating the involvement of the cornea (opacity and affected area), iris (congestion, swelling, circumcorneal hyperemia, injection, light reactivity, hemorrhage, destruction) and the conjunctiva (reddening, vascular hyperemia, loss of discernible blood vessels, ecchymosis and exudation) assigning a numerical score according to the degree of each lesion observed, according to the tables included in the corresponding standards.