Chronic toxicity tests (OECD 452: 2018)
Test with the Certificate of Good Laboratory Practices (GLPs).
The chronic toxicity study according to OECD 452 provides information on possible health hazards that are likely to arise from repeated exposure over a considerable part of the useful life of the species used. The study aims to provide information on the toxic effects of the substance under test, indicating the target organs and the possibility of accumulation, and can be used to establish safety criteria for human exposure. The objectives of this study are: to identify the chronic toxicity of a product; identify the target organs; characterize the dose-response relationship; identify a level of adverse effect not observed (NOAEL) or starting point for the establishment of a Reference Dose (BMD); predict the effects of chronic toxicity on human exposure levels; and obtain information to test hypotheses about the mode of action.
This study of chronic toxicity is carried out mainly in rodents (preferably rats), although other species of rodents or non-rodents can be used, justifying their choice. At least three dose levels and one control should be used, except in cases where a limit test is performed. For rodents, normally at least 20 animals of each sex should be used per group in each dose level, while for non-rodents a minimum of 4 of each sex per group is recommended. The test product is administered daily to the animals, usually orally, either with feeding, drinking water, or by gavage.
The test product is administered daily in graded doses to several groups of experimental animals, usually over a period of 12 months, although longer (e.g., 18 or 24 months) or shorter durations may also be chosen (e.g., 6 or 9 months) according to the regulatory requirements. The duration should be long enough to allow the effects of cumulative toxicity to manifest, avoiding confusion because of the effects of the changes due to the age of the animal. The dose levels used are usually based on the results of shorter-term toxicity studies and should take into account existing toxicological and toxicokinetic data available for the test substance or related materials. The highest dose level should identify the main target organs and the toxic effects, but the suffering, serious toxicity, morbidity or death of the animals should be avoided. The maximum dose should not exceed 1,000 mg/kg of body weight/day. After determining the highest dose level, a descending sequence of dose levels should be selected, generally reductions between 2 to 4 times are appropriate for these tests, although it will depend on the characteristics of the test product.
If from the information of preliminary studies, it can be anticipated that a test at a dose level, equivalent to at least 1,000 mg/kg body weight/day, using the procedures described for this study, is unlikely to produce adverse effects and if toxicity is not expected based on the data of structurally related substances, it can be considered that it is not necessary to conduct a complete study with three dose levels. In this case a limit test can be performed, with a maximum dose of 1,000 mg/kg of body weight/day, except when human exposure indicates the need to use a higher dose level.
The animals are observed daily and changes in body weight, water consumption and food should be recorded. In addition, hematological analysis, clinical biochemistry and urine analysis should be performed at 3, 6 and 12 months, in at least 10 females and 10 males of each group in the case of rodents. After the study period, the animals should be sacrificed and subjected to a complete necropsy, analyzing the weight of the organs and performing a histopathological examination of all the organs and body tissues.