Acute Oral Toxicity test – Fixed Dose Procedure. OECD 420: 2001 guidance.
Test with the Certificate of Good Laboratory Practices (GLPs).
The fixed dose method described by the OECD 420 guideline differs from the other methods in that it avoids using death of animals as an endpoint and relied instead on the observation of clear signs of toxicity at one of a series of fixed dose levels by gavage. To evaluate acute oral toxicity by the fixed dose method, groups of animals of a single sex are dosed in a stepwise procedure using fixed doses. First a sighting study is performed to allow selection of the appropriate starting dose for the main study, and then, the main study is performed. Depending on the outcome: death, evident toxicity or no toxicity, the test is ended, or animals are dosed with another concentration, to classify the test substance according to the Globally Harmonised System (GHS) for the classification of chemicals which cause acute toxicity. The preferred rodent species according to OECD Guideline is the rat, although other rodent species may be used. In our laboratory this test is performed using mice or rats.
In the sighting study the test substance is administered to single animals in a sequential manner. The sighting study is completed when a decision on the starting dose for the main study can be made, or if a death is seen at the lowest fixed dose. The initial dose level is selected as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. The starting dose for the sighting study is selected from the fixed dose levels of 5, 50, 300 and 2000 mg/kg as a dose expected to produce evident toxicity based on evidence from in vivo and in vitro data from the chemical or structurally related chemicals. In the absence of such information, the starting dose will be 300 mg/kg. Exceptionally, and only when justified by specific regulatory needs, the use of an additional upper fixed dose level of 5000 mg/kg may be considered.
In the main study, groups of 5 animals are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered). The initial dose level is selected on the basis of the sighting study. Depending on the presence or absence of signs of toxicity or mortality of one dose, further groups of animals may be dosed at higher or lower fixed doses. The test procedure continues until the appropriate hazard classification class is assigned, until the dose causing evident toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.
Furthermore, the guideline includes the option to perform a limit test when the test material is likely to be nontoxic. For this test, a sighting study with one animal and a starting dose of 2000mg/kg (or exceptionally 5000mg/kg) followed by a main study dosing further four animals.
The test animals should be observed for at least 14 days. Body weight determination and observation of clinical signs indicative of toxicity must be analysed. All test animals should be subjected to gross necropsy to assess gross pathological changes. Furthermore, microscopic examination of organs showing evidence of gross pathology may be performed.
With regard to the formulation of the dosing preparation, the use of an aqueous solution / suspension / emulsion is recommended whenever possible, followed in order of preference by a solution / suspension / emulsion in oil (for example, corn oil) and then possibly solution on other vehicles. In the case of vehicles other than water, a control must be carried out and therefore the test will carry an additional cost.