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J Pharmacol Toxicol Methods. 2008 Jul-Aug;58(1):11-26. doi: 10.1016/j.vascn.2008.05.001. Epub 2008 May 21.

Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

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  • 1Kyoto University School of Public Health, Japan.



The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA.


The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity.


The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations.


In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

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