Test No. 442B: Skin Sensitization
Local Lymph Node Assay: BrdU-ELISA or –FCM
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 substances 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 is used per dose group, with a minimum of three concentrations
of the test substance, plus a concurrent negative control group and a positive control
group. The experimental schedule is during 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 NC animals.
Then the BrdU content in DNA of lymphocytes is measured by ELISA using a commercial
kit of 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.
Published on June 27, 2018Also available in: French
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