Test No. 442B: Skin Sensitization

The Local Lymph Node Assay: BrdU-ELISA (LLNA:BrdU-ELISA) is a non-radioactive modification to the LLNA method for identifying potential skin sensitizing test chemicals and measuring the proliferation of lymphocytes they induce in the auricular lymph nodes. The method described in mouse is based on the use of measuring 5-bromo-2-deoxyuridine (BrdU) content, an analogue of thymidine, as an indicator of this proliferation. A minimum of four animals are used per dose group, with a minimum of three concentrations of the test chemical, plus a concurrent negative control group and a positive control group. The experimental schedule lasts for 6 days. Thereafter, the animals are killed and a single cell suspension of lymph node cells (LNC) is prepared. The procedure for preparing the LNC is crucial, in particular for the small lymph nodes in control animals. Then the BrdU content in DNA of lymphocytes is measured by ELISA using a commercial kit, or by Flow Cytometry (FCM). This study includes: measurements (weighing, BrdU) and clinical daily observations. The results are expressed as the Stimulation Index (SI) obtained by calculation from the mean BrdU labelling index. The SI should be ≥1.6 for the ELISA method or ≥2.7 for the FCM method for identifying the test material as a potential skin sensitizer. The LLNA: BrdU-ELISA method also allow to discriminate between skin sensitisers sub-categories of 1A (strong sensitisers) and 1B (moderate sensitisers).