Nonradioactive Versions of the LLNA
The murine local lymph node assay (LLNA) is used to assess the potential of a test substance to induce allergic contact dermatitis in humans. ICCVAM has recommended that two modified versions of the LLNA that do not use radioactive isotopes can be used to identify substances as potential skin sensitizers or nonsensitizers, with certain limitations. The availability of LLNA methods that do not use radioactivity is expected to allow more institutions to take advantage of the animal welfare benefits of the LLNA and provides environmental benefits as well.
Federal agencies accepted or endorsed the ICCVAM recommendations on the modified versions of the LLNA in 2011.
LLNA: BrdU-ELISA: Test Method Evaluation Report (June 2010) containing ICCVAM recommendations
LLNA: DA: Test Method Evaluation Report (June 2010) containing ICCVAM recommendations
International Acceptance of the LLNA: DA and LLNA: BrdU-ELISA
NICEATM and ICCVAM, in conjunction with the Japanese Center for the Validation of Alternative Methods, drafted test guidelines for use of the LLNA: DA and LLNA: BrdU-ELISA and submitted them to the Test Guidelines Programme of the Organisation for Economic Co-operation and Development. The new test guidelines, designated Test Guidelines 442A (LLNA: DA) and 442B (LLNA: BrdU-ELISA) were adopted by the OECD in July 2010.
OECD Test Guideline 442A — Skin Sensitization: Murine Local Lymph Node Assay: DA
OECD Test Guideline 442B — Skin Sensitization: Murine Local Lymph Node Assay: BrdU-ELISA
Background and ICCVAM Evaluation
In January 2007, the U.S. Consumer Product Safety Commission (CSPC) requested that NICEATM and ICCVAM assess the validation status of new versions and applications of the LLNA. The CPSC nomination included a request that ICCVAM evaluate three modified versions of the LLNA not requiring the use of radioactive markers:
- The LLNA: BrdU-ELISA, which measures the nucleotide analog bromodeoxyuridine (BrdU) using an enzyme-linked immunosorbent assay (ELISA) to assess lymph node cell proliferation
- The LLNA: DA, which measures adenosine triphosphate content as an indicator of lymph node cell proliferation
- The LLNA: BrdU-FC, which measures BrdU using flow cytometry to assess lymph node cell proliferation
The ICCVAM interagency Immunotoxicity Workgroup (IWG), working with NICEATM, prepared draft background review documents (BRDs) and draft recommendations for the three modified versions of the LLNA. The draft BRDs and draft ICCVAM recommendations on these methods were reviewed in a public meeting of an international independent scientific peer review panel in March 2008.
The Panel agreed with the ICCVAM assessment that more data were needed to evaluate these new versions of the LLNA. Additional data were submitted to NICEATM; NICEATM and the IWG revised the BRDs, and updated test method recommendations were developed by ICCVAM. A second Panel meeting to consider the revised documents was held in April 2009, and the Panel's report was made available in June 2009. ICCVAM considered the Panel’s report along with all public and SACATM comments as they prepared final test method recommendations, which were forwarded to Federal agencies for their consideration in June 2010.
ICCVAM recommended that the LLNA: DA and the LLNA: BrdU-ELISA be used to identify substances as potential skin sensitizers or nonsensitizers, with certain limitations. ICCVAM deferred a formal recommendation on the validity of the LLNA: BrdU-FC until an independent audit of all data supporting the analysis has been conducted and until transferability has been demonstrated in an interlaboratory validation study.
Transmittal of Recommendations to Federal Agencies and Agency Responses
The ICCVAM recommendations on the use of nonradioactive versions of the LLNA were communicated to Federal agencies in letters from Dr. Linda Birnbaum, Director, NIEHS. These letters and the responses from ICCVAM member Federal agencies are posted below.
- National Institutes of Environmental Health Sciences (NIEHS)
- Agency for Toxic Substances and Disease Registry (ATSDR)
- Consumer Product Safety Commission (CPSC)
- Environmental Protection Agency (EPA)
- Food and Drug Administration (FDA)
- National Cancer Institute (NCI)
- National Institutes of Health (NIH)
- National Institute for Occupational Safety and Health (NIOSH)
- National Library of Medicine (NLM)
- Occupational Safety and Health Administration (OSHA)
- U.S. Department of Agriculture
- U.S. Department of Defense
- U.S. Department of Energy
- U.S. Department of the Interior