Characterization and evaluation of a modified local lymph node assay using ATP content as a non-radio isotopic endpoint.
BACKGROUND
The murine local lymph node assay (LLNA) is an accepted and widely used method for assessing the skin-sensitizing potential of chemicals. Here, we describe a non-radio isotopic modified LLNA in which adenosine triphosphate (ATP) content is used as an endpoint instead of radioisotope (RI); the method is termed LLNA modified by Daicel based on ATP content (LLNA-DA).


METHODS
Groups of female CBA/JNCrlj mice were treated topically on the dorsum of both ears with test chemicals or a vehicle control on days 1, 2, and 3; an additional fourth application was conducted on day 7. Pretreatment with 1% sodium lauryl sulfate solution was performed 1 h before each application. On day 8, the amount of ATP in the draining auricular lymph nodes was measured as an alternative endpoint by the luciferin-luciferase assay in terms of bioluminescence (relative light units, RLU). A stimulation index (SI) relative to the concurrent vehicle control was derived based on the RLU value, and an SI of 3 was set as the cut-off value.


RESULTS
Using the LLNA-DA method, 31 chemicals were tested and the results were compared with those of other test methods. The accuracy of LLNA-DA vs LLNA, guinea pig tests, and human tests was 93% (28/30), 80% (20/25), and 79% (15/19), respectively. The estimated concentration (EC) 3 value was calculated and compared with that of the original LLNA. It was found that the EC3 values obtained by LLNA-DA were almost equal to those obtained by the original LLNA.


CONCLUSIONS
The SI value based on ATP content is similar to that of the original LLNA as a result of the modifications in the chemical treatment procedure, which contribute to improving the SI value. It is concluded that LLNA-DA is a promising non-RI alternative method for evaluating the skin-sensitizing potential of chemicals.
Idehara
Kenji
K
Analysis Service Center, Daicel Chemical Industries, Ltd., 1239, Shinzaike, Aboshi-ku, Himeji, Hyogo 671-1283, Japan. kn_idehara@daicel.co.jp
Yamagishi
Gaku
G
Yamashita
Kunihiko
K
Ito
Michio
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D017449:Dermatitis, Allergic Contact / Q000175:diagnosis / Q000209:etiology*; D004195:Disease Models, Animal; D023381:Endpoint Determination / Q000379:methods; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D007509:Irritants / Q000633:toxicity*; D023441:Local Lymph Node Assay; D051379:Mice; D008808:Mice, Inbred CBA; D015203:Reproducibility of Results; D013045:Species Specificity; D018675:Toxicity Tests
D007509:Irritants; D000255:Adenosine Triphosphate