Development of a Modified Local Lymph Node Assay using ATP Measurement as an Endpoint

Kunihiko Yamashita, Kenji Idehara, Norio Fukuda, Gaku Yamagishi and Naoki Kawada

Daicel Chemical Industries, Ltd., 1239, Shinzaike, Aboshiku, Himeji, Hyogo 671-1283, Japan

AATEX 11(2):136-144, 2005

Several proposed modified local lymph node assays (LLNAs) use a non-RI method, but such methods tend to yield lower stimulation indices (SIs) than those obtained with the standard LLNA when used to assess moderate sensitizers. To address this issue, we propose a modified LLNA in which mice are ex-posed to the test material four times, instead of three times, and adenosine triphosphate (ATP) content is used as a measure of proliferation of lymph node cells (LNCs). The chemicals tested were 2.4- dinitro-chlorobenzene (DNCB), eugenol, and a-hexyl cinnamic aldehyde (HCA), which are classified as strong-to-moderate sensitizers in the LLNA. Methyl salicylate (MS) was used as an example of a non-sensitizer. Using an SI of 3 as a cut-off, as recommended in the standard LLNA, DNCB, eugenol and HCA were classified as positive, while MS was classified as negative using the ATP-based non-RI method. Furthermore, there were no marked differences in SI levels for 10% eugenol and 10% HCA (a moderate sensitizer) between the non-RI method and the standard LLNA. Thus, it is reasonable to con-clude that the non-RI method proposed in the present study could be a valid alternative to the standard LLNA.

Key words: local lymph node assay, non-RI method, stimulation index, adenosine triphosphate (ATP), eugenol