In vivo Eye Irritation / Serius Eye Damage Test (OECD 405: 2021 guideline). 

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

The OECD 405 guideline establishes a procedure to evaluate both irritation and severe eye damage, corresponding to irreversible tissue damage after the application of the substance to the anterior surface of the eye.

According to OECD 405 guideline, in vivo testing should not be considered until available data pertaining to possible eye irritation or serious eye damage of the test product have been obtained (existing data from in vivo, in vitro or ex vivo eye or skin tests, physical-chemical characteristics and any relevant information). In vivo serious eye damage/eye irritation test may be performed when required by authorities and the product has not been classified as eye irritant (GHS Category 1) or causing serious eye damage (GHS Category 2) by in vitro test methods, where it cannot be concluded with sufficient confidence whether the test substance is Category 1 or 2, and where the test substance cannot be tested with currently available in vitro test methods.

Healthy adult albino rabbits with no signs of eye irritation, eye defects or previous corneal damage are used to perform the test. The test substance is initially applied as a single dose to one eye of a single animal, using the unexposed eye as a control, and the effects are evaluated and scored at specified intervals. If no serious eye damage or irreversible effect is observed in this animal, the response is confirmed using two additional animals.

After pretreatment with a systemic analgesic and instillation of the appropriate topical anesthesia, the test substance is applied to the conjunctival sac, keeping the eyelids closed after application to limit substance loss. The eyes should not be flushed for 24 hours but may be flushed after 24 hours if deemed necessary. Liquid, solid, pasty or particulate substances can be applied, but if they are solid or granulated, they must be crushed to obtain a fine powder. For pressurized aerosols it can be applied directly. For pump sprays, the liquid must be obtained from the container and the liquid obtained must be instilled.

The duration of the study should be sufficient to allow full assessment of the magnitude and reversibility or irreversibility of the injuries but should be terminated at any time the animals show signs of severe pain or distress. If there are no signs of irritation, the minimum test duration is 72 hours. To determine the reversibility of effects, animals should be observed for up to 21 days after exposure. The evaluation of the effects is carried out at 1, 24, 48 and 72 hours after exposure, and at 7, 14 and 21 days if there is corneal involvement or other signs of eye irritation, in order to determine the progression of the lesions and their reversibility. or irreversibility.

The degree of irritation, corrosion or severe eye damage is evaluated and quantified at the aforementioned intervals, evaluating the involvement of the cornea (opacity and affected area), the iris (congestion, swelling, circumcorneal hyperemia, injection, reactivity to light, hemorrhage, destruction) and the conjunctiva (redness, vascular hyperemia, loss of discernible blood vessels, ecchymosis and exudation) assigning a numerical score according to the degree of each lesion observed, according to what is indicated in the OECD 405 Guideline.