# 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 1.3.2.3: Number of animals, dose levels and vehicle selection.

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

A minimum of four animals is used per dose group, with a minimum of three concentrations of the test substance, plus a negative control group treated only with the vehicle for the test substance, and, as appropriate, a positive control. In those cases in which individual animal data are to be collected, a minimum of five animals per dose group are used. Except for absence of treatment with the test substance, animals in the control groups should be handled and treated in a manner identical to that of animals in the treatment groups.
Dose and vehicle selection should be based on the recommendations given in reference (1). Doses are selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc. Existing acute toxicity and dermal irritation data should be considered, where available, in selecting the three consecutive concentrations so that the highest concentration maximises exposure whilst avoiding systemic toxicity and excessive local skin irritation (2)(11).
The vehicle should be selected on the basis of maximising the test concentrations and solubility whilst producing a solution/suspension suitable for application of the test substance. In order of preference, recommended vehicles are acetone/olive oil (4:1 v/v), dimethylformamide, methyl ethyl ketone, propylene glycol and dimethyl sulphoxide (2)(10), but others may be used if sufficient scientific rationale is provided. In certain situations it may be necessary to use a clinically relevant solvent or the commercial formulation in which the test substance is marketed as an additional control. Particular care should be taken to ensure that hydrophilic materials are incorporated into a vehicle system, which wets the skin and does not immediately run off. Thus, wholly aqueous vehicles are to be avoided.

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Source: https://www.legislation.gov.uk/eudr/2004/73/annex/2H/division/B.42/division/1/division/1.3/division/1.3.2/division/1.3.2.3 (the official version)
Page: https://openlegislation.uk/eu-directive-2004-73/annex-2H/division-B.42/division-1/division-1.3/division-1.3.2/division-1.3.2.3
Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives.