Inter-laboratory validation of the modified murine local lymph node assay based on 5-bromo-2′-deoxyuridine incorporation

• Published in: Journal of applied toxicology 2011-01, Vol.31 (1), p.63-74
• Main Authors: Kojima, Hajime, Takeyoshi, Masahiro, Sozu, Takashi, Awogi, Takumi, Arima, Kazunori, Idehara, Kenji, Ikarashi, Yoshiaki, Kanazawa, Yukiko, Maki, Eiji, Omori, Takashi, Yuasa, Atsuko, Yoshimura, Isao
• Format: Article
• Language: English
• Subjects: 5-bromo-2′-deoxyuridine (BrdU); Animals; Biological and medical sciences; Bromodeoxyuridine - metabolism; Dermatitis, Allergic Contact - diagnosis; Dermatitis, Allergic Contact - etiology; Enzyme-Linked Immunosorbent Assay; Female; inter-laboratory validation study; Laboratories; Local Lymph Node Assay; local lymph node assay (LLNA); Medical sciences; Mice; Mice, Inbred CBA; Organic Chemicals - analysis; Organic Chemicals - toxicity; Quality Control; Random Allocation; Reproducibility of Results; Sensitivity and Specificity; skin sensitization; stimulation Index (SI); Toxicology
• ISSN: 0260-437X; 1099-1263; 1099-1263
• DOI: 10.1002/jat.1567

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 a drug, cosmetic material, pesticide or industrial chemical. Instead of radioisotope using in this method, Takeyoshi M. et al. (2001) has developed a modified LLNA based on the 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). The LLNA:BrdU‐ELISA is practically identical to the LLNA methodology excluding the use of BrdU, for which a single intraperitoneal injection of BrdU is made on day 4, and colorimetric detection of cell turnover. We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. In this study, 3 chemicals were examined in all laboratories and the remaining 7 were examined in 3 laboratories. The data were expressed as the BrdU incorporation using an ELISA method for each group, and the stimulation index (SI) for each chemical‐treated group was determined as the increase in the BrdU incorporation relative to the concurrent vehicle control group. An SI of 2 was set as the cut‐off value for exhibiting skin sensitization activity. The results obtained in the experiments conducted for all 10 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA:BrdU‐ELISA against those of GPMT/BT were 7/7 (100%), 3/3 (100%), and 10/10 (100%), respectively. Copyright © 2010 John Wiley & Sons, Ltd. Takeyoshi M. et al. (2001) has developed a modified murine local lymph node assay (LLNA) based on the 5‐bromo‐2'‐deoxyuridine (BrdU) incorporation (LLNA:BrdU‐ELISA). We conducted the validation study to evaluate the reliability and relevance of LLNA:BrdU‐ELISA. The experiment involved 7 laboratories, wherein 10 chemicals were examined under blinded conditions. The validation results indicate that the LLNA:BrdU‐ELISA is a promising new version LLNA.