The impact of LLNA group size on the identification and potency classification of skin sensitizers: a review of published data.

The Organisation for Economic Co-operation and Development (OECD) Test Guideline 429 for the local lymph node assay (LLNA) indicates a minimum of 4 mice per dose group, or of 5 mice if statistics are required. Recent discussions at the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) have led to suggestions that there should be a change to LLNA protocol requirements to mandate a minimum of 5 mice per group. Although it is not certain that any such proposal will be made, the debate is an important one and prompts reconsideration of animal requirements in the LLNA. In this paper we have conducted an analysis of published data from our own laboratories to determine whether the use of 4 or of 5 mice has had any practical impact on the outcome of the assay. Of the data sets for 17 chemicals in the 4-animal assay (14 positive, 1 uncertain, and 2 negative), 16 results were identical in the 5-animal assay. A marginally positive result in the 4-animal assay was negative in the 5-animal assay. Where potency determinations were made, the outcomes were essentially identical in the 2 forms of the LLNA. Consequently, it is concluded that there is no scientific justification for removing the option to use a 4-animal version of the LLNA.