Local Lymph Node Assay LLNA

Strong allergens induce measurable changes in the draining lymph node weights when contact sensitizers are applied to the mouse ear. Rapid proliferation and pyroninophilia increase the node weight (Kimber and Weisenberger, 1989). To increase the sensitivity and reproducibility of the LLNA, a modified assay was developed using [3H]thymidine incorporation to measure proliferation.

In the LLNA, test material is administered to the ear over the course of several days. After a rest period of 5 days, mice receive a challenge dose on the ear and treated thymidine via an intravenous injection. A threefold increase in thymidine incorporation is considered a positive response.

This assay has been validated in several national and international inter-laboratory validation studies (Kimber et al., 1995; Chamberlain and Basketter, 1996; Scholes et al., 1992). With respect to moderate or severe sensitizers, data from the LLNA correlate well with the standard guinea pig assays. Often the LLNA assay fails to predict weak or moderate sensitizers that are positive in the GPMT. However, the GPMT is an artificial system bypassing the skin barrier and may overestimate the risk of sensitization. Occasionally, strong irritants will give a strong reaction in the LLNA. Thus, substances with exclusively irritating properties could falsely be classified as sensitizers by the method. Alternatively, chemicals with both sensitizing and irritating properties could be overestimated (Montelius etal., 1994).

Differences between irritant reactions and contact sensitivity can be ascertained by phenotyping cells from the lymph node (Sikorski et al., 1996). Sensitizers increase the percentage of CD4+ and CD8+ cells expressing CD6210 and CD44hi (Gerberick et al., 1992). Irritant reactions do not increase the number of cells in the preparation or the specific lymphocyte subsets.

The nature of the vehicle or the test material may influence the performance of the assay. For most test compounds, the LLNA uses a 4:1 acetone/olive oil vehicle (Basketter and Kimber, 1996). Other vehicles such as water or aqueous vehicles are ineffectual and often give false-negative results (Ikarashi et al., 1992). When metal salts are used in the assay, special vehicles must be used. Sensitization is only observed when the compounds are applied in dimethyl sulfoxide or aqueous ethanol solution.

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