Local lymph node assay

The murine local lymph node assay (LLNA) is an in vivo test for skin sensitisation. The murine local lymph node assay (LLNA) is an in vivo test for skin sensitisation. LLNA has largely superseded the guinea pig maximisation test and the Buehler test. It is considered more scientific and less cruel (lower number of animals; less suffering) and has found broad scientific and regulatory acceptance. The principle underlying the LLNA is that skin sensitizers induce growth of lymphocytes in the lymph nodes draining the site of application. Lymphocyte proliferation can be measured by radiolabeling (quantifying tritiaded thymidine), bioluminescence (quantifying ATP content in lymphocytes) or immunoassay (ELISA utilizing an antibody specific for BrdU). The test material is applied to the ears of mice. Optionally, a tracer substance such as 3H-Methyl-thymidine or BrdU is injected intraperitoneally for lymphocyte incorporation. The animals are euthanized and their lymph node cells are removed and analyzed. The ratio of tracer incorporation in lymph nodes from dosed animals is compared to control animals, giving a stimulation index (SI). When the stimulation index exceeds 3 (SI > 3), a relevant sensitizing potential is assumed. In contrast to the classical guinea pig tests, the LLNA provides a quantitative measurement of sensitizing potency of a tested chemical.