Local Lymph Node Assay (LLNA) - BrdU - Flow Cytometry Method

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

LLNA Study Timeline:

Pre study: Authorization, ordering and equilibration of mice
Week 1: Dosing and observation of animals.
Week 2: Lymph node cell analyses.
Week 2:End Verbal results available.
Week 3: Summary Table of Results Faxed or Emailed
Week 4-5: Complete report including all details and study results available between 4 and 5 weeks.



(includes n=4 mice/group @ 3 concentrations of Test Article plus Vehicle Control group; Historical Positive Control) Pooled Nodes from each group. Modified OECD TG429 does not include a Pre-Screen test for irritation/systemic toxicity.

* This is intended only as the most basic screening assay; dermal (ear) irritation will be assessed during the test; there will NOT be an irritation pre-screen unless requested and ammended to the Protocol. (Not recommended for new chemicals or mixtures without previous dermal irritation and sensitization data)


LLNA STANDARD EPA/OECD PROTOCOL +Basic Irritation Pre-Screen**

(OECD TG-429 and OPPTS 870.2600 compliant. Includes n=5 mice/group, Concurrent 25% HCA Positive Control; Vehicle Control + 3 concentrations of Test Article, n=5/group) Basic Dermal Irritation/Toxicity Pre-Screen
Discounts available for 2-3 Test Articles (As Above, Sent Together)

LLNA REGULATORY EPA/OECD Protocol +Full Irritation/Toxicity Pre-Screen**

(OECD TG 429 and OPPTS 870.2600 compliant. Includes Quantitative Range Finding irritation screen + 3 concentrations per TA in main study with Positive and Vehicle Controls n=5/group)
Discounts available for 2-3 Test Articles (As Above, Sent Together)
**OPPTS 870.2600 and OECD TG429 guidelines specify that "an irritation pre-study should be performed under identical conditions as the main test in order to determine the maximum (irritation threshold) test concentration."
Basic Range Finder Pre-Screen is 6 mice at 3 concentrations. See below for a more comprehensive quantitative range-finder test for suspected or known irritants.
Quantitative Range-Finder: If no mouse ear irritation data are available or provided, a quantitative range finder using 12 mice with n=2 at 6 concentrations (100%, 50%, 25%, 10%, 5%, 2.5%) should be performed first, these will allow proper choice of test concentrations for the main LLNA study. If the Test Article is irritating, a second range-finder at lower doses (1%, 0.5%, 0.25%, 0.1%, 0.05%, etc.) may be necessary to select appropriate test article concentration for use in the definitive study.

Options and "Enhanced" Endpoints:

A) Basic Dermal Irritation Pre-Screen: n=2 mice at 100%, 50%, 25% (for Non-Irritants)

B) Quantitative Dermal Irritation Pre-Screen:(Ear Swelling) Range-Finder with n=2 mice @ 6 concentrations (e.g. 100%, 50%, 25%, 10%, 5%, 2.5%)

C) Immunophenotyping: %B220, CD3, CD4, CD8, I-Ak (MHC), CD69, CD25, CD44+ CD62lo+ lymph node cells
Standard Study Groups:
Vehicle Control
5 mice
4 mice
Positive Control (moderate sensitizer)
5 mice
4 mice
Test Article (Low)
5 mice
4 mice
Test Article (Mid)
5 mice
4 mice
Test Article (High)
5 mice
4 mice

Total # of mice used in the standard assay:
25 mice
20 mice

Number of mice used in Irritation Pre-Screen 6-12 mice

Test Article (Low 2; as needed)
5 mice
4 mice
Test Article (Low 3; as needed)
5 mice
4 mice
Note: Although EPA/OECD Test Guidelines specify a minimum or 3 test concentrations, in the case of substances with dermal irritation properties, or in the absence of previous mouse ear dermal irritation data, the LLNA may require testing of 4-5 test concentrations to conduct a proper evaluation.

To discuss any aspect of this assay, or if you would like information on other in vivo and in vitro assays that are conducted at MB Research Laboratories, please contact our Client Services or our in vitro staff at 215-536-4110 or .