Acute Dermal Toxicity test. OECD 402: 2017; ; EPA OPPTS 870.1200 OPPTS: 1998.
Test with the Certificate of Good Laboratory Practices (GLPs).
Acute dermal toxicity is the adverse effect that occurs within a short period of time after the application of one or multiple doses in the skin over a period of 24 hours. This test provides information to classify and label the products, and is an initial step for other studies of more durable exposure, as well as to know the absorption by this route. The assessment of acute dermal toxicity should include the relationship, if any, between the exposure of the animals to the substance and the incidence and severity of all observed abnormalities, both behavioral and clinical abnormalities, and their reversibility, macroscopic changes, body weight, the effect on mortality, or any other type of toxic effect. In the test, the number of animals used should be reduced, as far as possible, previously obtaining all available information on similar products for which toxicological information exists. When this information is not available, a "Limit Test" can be carried out. When rodents are used, a limit dose of 2,000 mg/kg is applied to a group of 5 animals of each sex, and if no lethality is observed in them, is sufficient to establish certain precautions with the product if the lethal dose is greater than 5,000 mg/kg. However, if the animals die with a dose of 2,000 mg/kg, a more complete study should be done to know the LD50.
Although in both standards there is the option to use rats, rabbits or guinea pigs, in our laboratory this test is performed using healthy adult albino rabbits of at least 12 weeks and weighing 2 to 3 kg, with healthy skin, with a maximum variation in their weights between them of ± 20%, which have not been used for other tests. Both standards (OECD and EPA-OPPTS) are very similar, almost identical in terms of the recommendation to obtain all the previous toxicological information of the products submitted to trials or their related ones; in the numbers of animals included in the tests; in terms of sex of the animals; ages and weights of the animals; the acclimatization preconditions; the conditions of temperature and humidity; food and drink; preparation and administration of the test substances; and results observations.
In the test, the substance is applied to several groups of at least 5 animals of the same sex per group, to apply a dose to each of the groups. It may be enough with three groups of doses properly separated to know the toxic effects and the mortality rates and elaborate a dose response curve, as well as estimate the LD50. Once the tests with the animals of one sex have been completed and evaluated, one of the groups with animals of the opposite sex is repeated to verify the identity of the results with another sex.
The test substance must take intimate contact with the skin for a period of 24 hours, on a surface ≥ 10% of the skin surface of the animal, which must be protected to prevent its loss or ingestion by the animals. If it is a solid substance, it must be sprayed and moistened with water or another vehicle. The liquid used as vehicle must be free of toxicity, must not alter the chemical or toxicological properties of the test substance, nor should it influence the reaction of the substance. Water, corn oil, gum arabic, ethanol and water, carboxymethyl cellulose, glycerol, polyethylene glycol (PEG), PEG with vegetable oil, mineral oil should be chosen, provided they are not irritating.
The recommended observation period is a minimum of 14 days, which can be varied according to the toxic reactions and the duration of the recovery period. The animals are observed several times on the first day and at least once a day in the following days to evaluate the state of the skin and its fur, the eyes and mucous membranes, the respiratory and circulatory effects, the effects on the autonomic nervous system such as salivation, effects on the central nervous system including the appearance of tremors and convulsions, changes in activity, changes in postures, their reactivity to handling them, the effects of sensory stimuli, the alteration of their strength, abnormal behaviors, as well as the weight comparing the weight before applying the substance, to the week after the application and when dying. If the animals die, or when they are sacrificed after completing the test, their necropsy should be performed to observe the existence, if any, of macroscopic lesions, which, if considered necessary, can be completed with microscopic observations. To correctly evaluate the results, a relationship is established between the observed effects and the doses administered and the LD50 is calculated.