In 1999, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended the mouse LLNA as a valid test method to assess the skin sensitization potential of most types of substances. Regulatory agencies around the world now commonly accept the LLNA.
It has several advantages over the traditional guinea pig assays (the Buehler assay and the Guinea Pig Maximization Test). First of all, the LLNA is run in mice which are lower on the phylogenetic ladder than guinea pigs and it uses fewer animals, which fulfill two of the animal welfare requirements (refinement and reduction). Also adjuvant is not required which reduces the pain and distress for the animals, colored products do not mask the results, and the results are quantitative and are obtained within a week.
In 2007 concern was raised about the use of the LLNA with formulated products (mixtures) which had not been considered in the original evaluation. ICCVAM convened an independent scientific peer review panel to review the use of the LLNA with mixtures and aqueous solution. In June of 2009 the panel made the recommendation that the LLNA was suitable for use with these types of materials.
The use of radioactivity in some cases can be an issue. Therefore, the peer review panel also evaluated 3 non-radioactive modified versions of the LLNA. Two of the assays, the LLNA:DA and the LLNA:BrdU-ELISA were found to be acceptable with certain limitations. The committee, however, concluded that the third assay, the LLNA:BrdU-FC, needed further evaluation since the results that were presented were only from one lab. An interlaboratory validation study is needed.
The committee also evaluated a reduced local lymph node assay (rLLNA). When appropriate, this method can reduce the number of animals used for each test by 40% compared to the traditional LLNA. The rLLNA was found acceptable for use in hazard identification of skin-sensitizing materials if dose-response information is not needed. It can also be used as an initial screen to determine the sensitizing potential of a material before conducting the traditional LLNA. The only difference between the rLLNA and the traditional LLNA is that for the rLLNA only 1 dose of the test material is used. The concentration should be the highest achievable concentration that does not cause overt systemic toxicity and/or excessive local irritation. When used as a screen, if the results are negative, no further testing is required. However, if the results are positive, the traditional LLNA should be conducted.
Calvert Laboratories has over 15 years of experience with the LLNA. Our standard protocol follows the ICCVAM recommendation and all regulatory guidelines. Contact Dr. Joan M. Chapdelaine at joan.chapdelaine@calvertlabs.com to discuss your next sensitization study.
