PRINCIPLE OF THE IN VIVO TEST:
The substance to be tested is applied in a single dose to one of the eyes of the experimental animal; the untreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions of conjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects are also described to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate the reversibility or irreversibility of the effects. Animals showing continuing signs of severe distress and/or pain at any stage of the test should be humanely killed, and the substance assessed accordingly.
TEST PROCEDURE:
Application of the test substance: The test substance should be placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second in order to prevent loss of the material. The other eye, which remains untreated, serves as a control.
Irrigation: The eyes of the test animals should not be washed for at least 24 hours following instillation of the test substance, except for solids, and in case of immediate corrosive or irritating effects. At 24 hours a washout may be used if considered appropriate. Use of a satellite group of animals to investigate the influence of washing is not recommended unless it is scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washing should be carefully documented, e.g., time of washing; composition and temperature of wash solution; duration, volume, and velocity of application.
For testing liquids, a dose of 0.1 mL is used. Pump sprays should not be used for instilling the substance directly into the eye. The liquid spray should be expelled and collected in a container prior to instilling 0.1 mL into the eye.
Testing of solids: When testing solids, pastes, and particulate substances, the amount used should have a volume of0.1 mL or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume of solid material should be measured after gently compacting it, e.g., by tapping the measuring container. If the solid test substance has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment, the eye may be rinsed with saline or distilled water.
Testing of aerosols: It is recommended that all pump sprays and aerosols be collected prior to installation into the eye. The one exception is for substances in pressurised aerosol containers, which cannot be collected due to vaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance may vary depending on the pressure of the spray and its contents. Care should be taken not to damage the eye from the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential for “mechanical” damage to the eye from the force of the spray. An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance is sprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper. The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile substances, the dose may be estimated by weighing a receiving container before and after removal of the test material.
Observation period: The duration of the observation period should be sufficient to evaluate fully the magnitude and reversibility of the effects observed. However, the experiment should be terminated at any time that the animal shows continuing signs of severe pain or distress. To determine reversibility of effects, the animals should be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21 days, the experiment should be terminated at that time.
Clinical observations and grading of eye reactions.The eyes should be examined at 1, 24, 48, and 72 hours after test substance application. Animals should be kept on test no longer than necessary once definitive information has been obtained. Animals showing continuing severe pain or distress should be humanely killed without delay, and the substance assessed accordingly. Animals with the following eye lesions post-instillation should be humanely killed: corneal perforation or significant corneal ulceration including staphyloma; blood in the anterior chamber of the eye; grade 4 corneal opacity which persists for 48 hours; absence of a light reflex (iridial response grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjuctivae or nictitating membrane; or sloughing. This is because such lesions generally are not reversible. Animals that do not develop ocular lesions may be terminated not earlier than 3 days post instillation. Animals with mild to moderate lesions should be observed until the lesions clear, or for 21 days, at which time the study is terminated. Observations should be performed at 7, 14, and 21 days in order to determine the status of the lesions, and their reversibility or irreversibility. The grades of ocular reaction (conjunctivae, cornea and iris) should be recorded at each examination. Any other lesions in the eye (e.g. pannus, staining) or adverse systemic effects should also be reported. Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope, or other suitable device. After recording the observations at 24 hours, the eyes may be further examined with the aid of fluorescein. The grading of ocular responses is necessarily subjective. To promote harmonisation of grading of ocular response and to assist testing laboratories and those involved in making and interpreting the observations, the personnel performing the observations need to be adequately trained in the scoring system used.
DATA AND REPORTING:
The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions, and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard for the irritant properties of a material, as other effects of the test material are also evaluated. Instead, individual scores should be viewed as reference values and are only meaningful when supported by a full description and evaluation of all observations.