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ICCVAM Evaluations of the Murine Local Lymph Node Assay (LLNA)

ICCVAM Evaluation of the LLNA

ICCVAM evaluated the LLNA in 1998 and recommended it as an alternative to guinea pig tests for assessing the potential for chemicals to cause allergic contact dermatitis. Compared to guinea pig methods originally used to identify skin sensitizers, the LLNA:

  • Reduces the number of animals required for testing
  • Requires less time to perform
  • Provides dose-response information
  • Eliminates the pain and distress associated with an allergic response in animals
Materials from the 1998 LLNA Evaluation

Peer Review Report
The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds
The Results of an Independent Peer Review Evaluation Coordinated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
February 1999 — NIH Publication No. 99-4494

Summary Minutes of the September 1998 Peer Panel Meeting

Letter from Dr. Kenneth Olden, Director, NIEHS, to Ms. Carol Browner, Administrator, U.S. EPA, transmitting results of the independent peer review evaluation of the LLNA (sent February 16, 1999)

Response from Dr. Steven Galson, EPA, on behalf of Ms. Browner (received October 19, 1999)

NICEATM LLNA Database

Data from analyses of the LLNA conducted on behalf of ICCVAM by NICEATM are available to interested stakeholders as a reference for developing and evaluating alternative approaches to testing and assessment that replace, reduce, or refine the use of animals for identification of potential skin sensitizers.

NICEATM LLNA database

Database users please note:

  • The Excel spreadsheet contains three pages: (1) data, (2) references, and (3) abbreviations.
  • The database includes data published through 2010.

These data have been extracted from published and unpublished data sources with permission. Users of this database should consult the original data source for questions regarding data quality and/or authenticity.

ICCVAM Evaluation of New Versions and Applications of the LLNA

In response to a 2007 nomination from the U.S. Consumer Product Safety Commission (CPSC), ICCVAM evaluated several other versions of the LLNA that reduce animal use and eliminate the need to use radioactive materials. ICCVAM also recommended the use of the LLNA for testing a wider range of chemicals.

References Relevant to the 2007 LLNA Nomination to ICCVAM

Use of the LLNA for Potency Categorization

ICCVAM evaluated the use of the LLNA for potency categorization for chemicals that cause ACD in humans. ICCVAM concluded that the LLNA can be used to categorize substances as strong skin sensitizers (Globally Harmonized System of Classification and Labelling of Chemicals Subcategory 1A). However, substances that are not identified as strong sensitizers using the LLNA require additional information to categorize them as other than strong sensitizers (Globally Harmonized System of Classification and Labelling of Chemicals Subcategory 1B).

ICCVAM Test Method Evaluation Report: Usefulness and Limitations of the Murine Local Lymph Node Assay for Potency Categorization of Chemicals Causing Allergic Contact Dermatitis in Humans
NIH Publication 11-7709

Transmittal to Federal Agencies of ICCVAM Recommendations for Use of the LLNA for Potency Categorization

Nonradioactive Versions of the Murine Local Lymph Node Assay

ICCVAM made recommendations to federal agencies on the use of two nonradioactive versions of the LLNA. The availability of non-radioactive LLNA methods allows more institutions to take advantage of the animal welfare benefits of the LLNA.

ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay: BrdU-ELISA
A Nonradioactive Alternative Test Method to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products
NIH Publication No. 10-7552

ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay: DA
A Nonradioactive Alternative Test Method to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products
NIH Publication No. 10-7551

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

Transmittal to Federal Agencies of ICCVAM Recommendations for Use of Nonradioactive Versions of the LLNA

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.

Applicability Domain for the Murine Local Lymph Node Assay

The 1998 ICCVAM evaluation of the LLNA recommended the LLNA as a valid alternative to guinea pig test methods for assessing ACD hazard potential for most testing situations. The 2007 CPSC nomination noted above requested that NICEATM and ICCVAM evaluate the applicability of the LLNA for testing formulations, metals, substances in aqueous solutions, and other products with the expectation that a wider applicability domain for the LLNA would enable wider use. A subsequent ICCVAM evaluation recommended use of the LLNA to test any chemical or product for ACD hazard potential unless the chemical or product has properties that may interfere with the ability of the LLNA to detect sensitizing substances.

ICCVAM Test Method Evaluation Report on Using the Murine Local Lymph Node Assay for Testing Pesticide Formulations, Metals, Substances in Aqueous Solutions, and Other Products
NIH Publication No. 10-7512

National and International Acceptance of the Expanded Applicability Domain of the LLNA

In April 2011, the U.S. Environmental Protection Agency announced that it was expanding the existing dermal sensitization test guideline to include testing of end-use pesticide products using the LLNA. The EPA policy notes the animal welfare advantages of the LLNA as well as the ability of the LLNA to provide a more quantitative assessment of dermal sensitization than traditional guinea pig methods.

EPA policy document (April 2011)

NICEATM and the ICCVAM Immunotoxicity Workgroup also developed a revision to the test guideline issued by the Organisation for Economic Co-operation and Development (OECD), OECD Test Guideline 429, that describes the conduct of the LLNA. The revised Test Guideline 429, which was adopted by OECD in July 2010, incorporates the updated applicability domain for the LLNA. It also includes the updated protocol for the LLNA and provides for the use of the reduced LLNA method when appropriate.

Updated OECD Test Guideline 429 — Skin Sensitization: Murine Local Lymph Node Assay

Transmittal to Federal Agencies of ICCVAM Recommendations on the Expanded Applicability Domain of the LLNA

The ICCVAM recommendations on the applicability domain 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.

Performance Standards for the Murine Local Lymph Node Assay

Concurrently with evaluations of nonradioactive versions of the LLNA, ICCVAM prepared performance standards for the LLNA that can be used to evaluate the accuracy and reliability of new versions of the LLNA. These performance standards represent a set of internationally harmonized standards for the conduct of the LLNA (essential test method components, a minimum list of reference substances, and accuracy and reliability criteria). The performance standards also include an updated LLNA test method protocol that reduces animal use by 20% compared to the original ICCVAM‑recommended protocol.

Recommended Performance Standards: Murine Local Lymph Node Assay (January 2009)

Revision to OECD Test Guideline 429

NICEATM and the ICCVAM interagency Immunotoxicity Working Group subsequently developed a revision to OECD Test Guideline 429, "Skin Sensitization: Murine Local Lymph Node Assay." The revised Test Guideline 429, adopted by OECD in 2010, incorporates the harmonized performance standards and includes the updated protocol for the LLNA from the ICCVAM performance standards document. The updated protocol allows for a 20% reduction in animal use when compared to the original.

Updated OECD Test Guideline 429

Transmittal to Federal Agencies of ICCVAM Recommended Performance Standards for the LLNA

Reduced Murine Local Lymph Node Assay

One of the LLNA modifications evaluated by NICEATM and ICCVAM in response to the CPSC nomination was the reduced LLNA (rLLNA). When used to test a substance for ACD potential, the rLLNA uses fewer animals than the LLNA to provide a “yes-no” result. ICCVAM has recommended the use of the rLLNA rather than the standard (multidose) LLNA when a negative result is expected or when dose–response information is not required. Since the rLLNA uses only a single high-dose group along with concurrent negative and positive control groups, adoption of the rLLNA reduces the number of animals needed for each test by 40% compared to the standard LLNA.

ICCVAM Test Method Evaluation Report: The Reduced Murine Local Lymph Node Assay: An Alternative Test Method Using Fewer Animals to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products
NIH Publication No. 09-6439

National and International Acceptance of the Reduced LLNA

The U.S. Environmental Protection Agency adopted the rLLNA, noting in their announcement that the rLLNA allows for a 40% reduction in animal use compared to the multidose LLNA. April 2011 EPA policy document

NICEATM and ICCVAM subsequently developed a revision to Test Guideline 429, the LLNA test guideline issued by the Organisation for Economic Co-operation and Development (OECD). The revised Test Guideline 429 incorporates the rLLNA procedure. It was adopted by the OECD in July 2010. Revised OECD Test Guideline 429

Transmittal to Federal Agencies of ICCVAM Recommendations on the Reduced LLNA