# Commission Directive 2004/73/EC of 29 April 2004 adapting to technical progress for the twenty-ninth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances (Text with EEA relevance), Division 1.5.3: Main study

Extent: E+W+S+N.I.

1.5.3.1 Numbers of animals and dose levels
The action to be taken following testing at the starting dose level is indicated by the flow charts in Annex 2. One of three actions will be required; either stop testing and assign the appropriate hazard classification class, test at a higher fixed dose or test at a lower fixed dose. However, to protect animals, a dose level that caused death in the sighting study will not be revisited in the main study (see Annex 2). Experience has shown that the most likely outcome at the starting dose level will be that the substance can be classified and no further testing will be necessary.
A total of five animals of one sex will normally be used for each dose level investigated. The five animals will be made up of one animal from the sighting study dosed at the selected dose level together with an additional four animals (except, unusually, if a dose level used on the main study was not included in the sighting study).
The time interval between dosing at each level is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. A period of 3 or 4 days between dosing at each dose level is recommended, if needed, to allow for the observation of delayed toxicity. The time interval may be adjusted as appropriate, e.g., in case of inconclusive response.
When the use of an upper fixed dose of 5000 mg/kg is considered, the procedure outlined in Annex 3 should be followed (see also section 1.6.2).
1.5.3.2 Limit test
The limit test is primarily used in situations where the experimenter has information indicating that the test material is likely to be nontoxic, i.e., having toxicity only above regulatory limit doses. Information about the toxicity of the test material can be gained from knowledge about similar tested compounds or similar tested mixtures or products, taking into consideration the identity and percentage of components known to be of toxicological significance. In those situations where there is little or no information about its toxicity, or in which the test material is expected to be toxic, the main test should be performed.
Using the normal procedure, a sighting study starting dose of 2000 mg/kg (or exceptionally 5000 mg/kg) followed by dosing of a further four animals at this level serves as a limit test for this guideline.

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Source: https://www.legislation.gov.uk/eudr/2004/73/annex/2B/division/B.1/division/1/division/1.5/division/1.5.3 (the official version)
Page: https://openlegislation.uk/eu-directive-2004-73/annex-2B/division-B.1/division-1/division-1.5/division-1.5.3
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.