Acute Irritation-Corrosion test. OECD 404: 2015.
Test Accredited by ENAC (Spanish National Accreditation Entity).
Test with the Certificate of Good Laboratory Practice (GLPs).
To avoid the unnecessary use of experimental animals and to protect animal welfare, before carrying out this test it is recommended to review all the possible relevant information regarding the potential irritant or corrosive of the chemical compounds to be evaluated. In this regard, the test should not be performed with strongly acidic or alkaline substances (pH ≤ 2 or ≥ 11.5); with products that have been shown to have a 50% lethal dose (LD50) of less than 200 mg/kg via dermal route or that have not shown irritation in a limit test with a dose of 2,000 mg/kg of body weight; with products that can be corrosive, predictable by their structure-activity relationships; or that have demonstrated corrosive properties by validated in vitro tests.
In this test the product under test is applied in an area of about 6 cm2 of shaved skin, avoiding the areas where there is sometimes accumulation of fur, of healthy adult albino rabbits, of any sex, without any skin lesion previous, using as control the skin of the same animal not exposed to the product. Animals must be acclimated to the conditions of the test in terms of temperature and humidity defined with periods of luminosity and darkness or controlled feeding and drinking. Single doses of 0.5 mL of liquids or 0.5 g of solid or pasty substances applied should ensure that it has intimate contact with the skin for which the liquid products are applied undiluted and the solids should be sprayed and moistened with water, physiological saline solution or another suitable vehicle that ensures a good contact with the skin, avoiding vehicles that could be irritating. As alternative vehicles, gum arabic, ethanol and water, carboxymethylcellulose, polyethylene glycol, vegetable oil or mineral oil can be used. During the exposure the application patch should be protected to avoid ingestion or inhalation of the product by the animal. The exposure period is 4 hours, which once elapsed must lead to the elimination of the product with water or another suitable solvent.
Skin irritation is defined as reversible skin damage after an application time of 4 hours. Skin corrosion is defined as the irreversible skin damage, characterized by the necrosis lesion that affects the epidermis and the dermis after the application of the product under test for 4 hours, with the appearance of ulcers, hemorrhages, or bloody scabs, that after the total observation period (14 days), they show areas of discoloration, complete alopecia and scarring.
If there is no previous evidence that the product is not corrosive, it should be started by carrying out initial tests to evaluate it. These initial tests can be ignored if there is evidence that the product is not corrosive. In these tests, a single animal is used to which three sequential patches are applied and placed after 3 minutes, 1 hour and 4 hours, respectively, applying the second and third patches, when no effect has been observed in the previous exhibition. If corrosion is observed in any of them, the test must be completed. When a corrosive effect is not expected, a patch can be applied to a single animal for 4 hours.
In case no irritant or corrosive effect is observed in the initial tests, confirmation tests may be performed. In these tests, two additional animals are used, which will be used sequentially if an irritant effect was observed in the initial test, or simultaneously if no irritant effect was observed in the initial test. In exceptional cases, in which the initial test is not performed because there is sufficient evidence that the product will not be corrosive, two animals can be exposed simultaneously with a single patch that is removed at 4 hours, so that if when exposing two animals, no effect was observed, the use of the third animal can be obviated, otherwise a third animal is used.
The observation period to determine the reversibility of the irritation, if observed, is 14 days after the removal of the patches, which may be shortened in case the irritation is resolved earlier, or it did not exist. When the animals show signs of intense pain or distress, the test must be completed, sacrificing the animals by a humanized method. The animals should be evaluated for the appearance of erythema and edema immediately, at 30-60 minutes, 24, 48 and 72 hours, after removal of the patch, qualifying between 0 and 4 the lesions according to their absence (0), or intensity (1 to 4).