The Mouse Local Lymph Node Assay is now accepted by the EPA, OECD, and FDA as the preferred "stand-alone alternative" to the Guinea Pig Sensitization Test. MB Research offers a "Standard LLNA" based upon OECD TG429 and EPA OPPTS 870.2600 guidelines. In addition, for problem test substances and irritants, MB Research also offers an "Enhanced LLNA" using Flow Cytometry to allow the evaluation of optional immunophenotypic markers which discriminate "true sensitizers" from "false-positive irritants".
During the "induction phase" of sensitization, following exposure to a sensitizing test substance, lymphocyte proliferation occurs in the local lymph node. The LLNA measures increased proliferation of lymphocytes in the auricular lymph nodes (which drain the site of exposure; ears). Proliferation is assessed by determining the incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into the DNA of lymph node cells using flow cytometric method. This "Enhanced LLNA" reduces background noise and false positives, by quantitatively assessing irritation, (via ear swelling) and by incorporating optional immunophenotype analysis to resolve and discriminate false-positive irritants (%B220+, CD3+, CD69+, I-Ak+ cells).
Outline of the Assay:
- Determine optimal vehicle for test article (solubility testing)
- Conduct Dermal Irritation Pre-Screen to determine test concentrations
- Dose ears once daily for 3 consecutive days
- On Day 6, (2 day rest period) injection of mice with BrdU
- Sacrifice and isolation of the auricular lymph nodes
- Individual Animal Analysis ("Pooled Nodes" protocol also available)
- Processing/staining of lymph node cells (LNC)
- Flow cytometric measurement of proliferation of LNC
- Determination of the Stimulation Index (S.I.) for each group
- Calculate, if possible, the EC3 (Minimal Sensitizing Concentration; where SI=3), the OECD and EPA criteria for positive sensitizers