Toys Regulations — 2
2 (1) Use six albino rabbits for each test substance or stuffing material. Ensure that animal facilities for such procedures are designed and maintained so as to exclude sawdust, wood chips or other extraneous materials that might produce eye irritation. Examine both eyes of each rabbit before testing. Use only those rabbits without eye defects or irritation. Hold the rabbit firmly but gently until it is quiet. Place the test substance or stuffing material in one eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup into which the test substance or stuffing material is dropped. Gently hold the lids together for one second and then release the rabbit. The other eye, remaining untreated, serves as a control. For testing liquids, use 0.1 ml (2 drops) of the test substance or stuffing material. For testing solids or pastes, use 100 mg (1.5 grains) of the substance or stuffing material. For substances or stuffing materials in flake, granule, powder or other particulate form, use the amount that has a volume of 0.1 ml (2 drops) (after compacting as much as possible without crushing or altering the individual particles, such as by tapping the measuring container) whenever this volume weighs less than 100 mg (1.5 grains). In such a case, record the weight of the 0.1 ml (2 drops) test dose. Do not wash the eyes after instillation of the test substance or stuffing material except as noted in subsection (2). (2) Examine the eyes and record the grade of ocular reaction at 24, 48 and 72 hours. Use a binocular loupe, hand-held slit lamp or other expert means to facilitate observing the reactions. After the observations are recorded at 24 hours, the eyes may be further examined after fluorescein is applied. To do this, drop 0.05 ml (one drop) of fluorescein sodium ophthalmic solution U.S.P. or equivalent directly on the cornea. After the excess fluorescein is flushed out with sodium chloride solution U.S.P. or equivalent, injured areas of the cornea appear yellow. This is best visualized in a darkened room under ultraviolet illumination. The eyes may be washed with sodium chloride solution U.S.P. or equivalent after the 24-hour recording. (3) A rabbit is considered to exhibit a positive reaction if, at any of the observations, the test substance or stuffing material has produced one or more of the following reactions: (a) an ulceration of the cornea, other than a fine stippling; (b) an opacity of the cornea, other than a slight dulling of the normal lustre; (c) an inflammation of the iris, other than a slight deepening of the folds (or rugae) or a slight circumcorneal injection of the blood vessels; (d) an obvious swelling in the conjunctivae (excluding the cornea and iris) with partial eversion of the lids or a diffuse crimson-red with individual vessels that are not easily discernible. (4) The test is positive if four or more of the rabbits in the test group exhibit a positive reaction. If only one rabbit exhibits a positive reaction, the test is considered negative. If two or three rabbits exhibit a positive reaction, repeat the test using a different group of six rabbits. The second test is positive if three or more of the rabbits exhibit a positive reaction. If only one or two rabbits in the second test group exhibit a positive reaction, repeat the test with a different group of six rabbits. If a third test is needed, the substance or stuffing material is irritant if any rabbit exhibits a positive reaction.