Acute Oral Toxicity: Up and down procedure-UPD- (OECD 425: 2008).
Test with the Certificate of Good Laboratory Practices (GLPs).
This procedure of evaluation of acute oral toxicity allows the estimation of the mean oral lethal dose (LD50) with a confidence interval and classify the test substance according to the Globally Harmonized System (GHS), used for the classification of chemicals that cause acute toxicity. This procedure is designed for substances that cause the death of animals in 1 or 2 days, but it is not practical when the substances are expected to cause a considerably delayed death, after 5 days or more.
In this test preferably healthy young adult female rats are used. The test substance is administered in a single dose through a gastric tube or a cannula. In those situations where the available evidence indicates that the test substance is likely to have low toxicity, below the regulatory limit dose, a limit test may be performed. However, if there is little or no information available on the substance, the main test should be performed
The limit test is used when the available information indicates that the test material is probably not toxic. It is a sequential test in which a maximum of 5 animals are used. A test dose of 2,000 mg/kg or exceptionally 5,000 mg/kg may be used (only when justified by specific regulatory requirements). If the first dosed animal dies, or if in total 3 of the animals treated with the test substance die, the main test should be performed to determine the LD50.
The main test should be used when there is no information on the toxicity of the product to be tested, there is insufficient information or it is expected to be toxic. This test consists in the application of a dose progression in which the animals are dosed sequentially at intervals of at least 48 hours. When there is no information available to make a preliminary estimate of the LD50, it should be used with a dose close to 175 mg/kg, and use a progression with a slope of 2. The first animal receives the dose below the estimate of LD50, or close to 175 mg/kg if information on the possible LD50 is not available. If the animal survives, the next animal is inoculated with an increasing dose or if it dies with a decreasing one (the slope of the progression must be chosen between 1 and 8). According to the result obtained in the following animals, the test continues with increasing or decreasing doses. The trial ends when: 3 consecutive animals survive at the higher concentration; 5 reversals occur in 6 consecutive animals; at least 4 animals follow the first reversion and the probability ratios exceed the critical value. If none of the criteria for stopping the test occurs, the test will be terminated when 15 animals have been dosed.
The animals should be observed for a total of 14 days and should be informed of all toxic reactions observed and their duration. At the end of the test, all animals should be weighed and undergo a general necropsy, and if there are organs with evidence of pathology, microscopic examination of the organs should be considered.