实验室内部研究，以确定LLNA:BrdU-ELISA法预测化学品皮肤致敏潜力的可重复性。

Intra-laboratory study to determine the reproducibility of LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.

作者信息

Chen Wei, Xing Caihong, Hou Fenxia

机构信息

Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China.

出版信息

J Pharmacol Toxicol Methods. 2016 Nov-Dec;82:26-30. doi: 10.1016/j.vascn.2016.07.003. Epub 2016 Jul 22.

DOI:10.1016/j.vascn.2016.07.003

PMID:27456929

Abstract

INTRODUCTION

The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for identification the skin sensitization potential of new chemicals. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. An intra-laboratory reproducibility study for the LLNA:BrdU-ELISA was conducted to demonstrate its adequate performance in our laboratory.

METHODS

Ten independent LLNA:BrdU-ELISAs with the preferred positive controls (PCs), i.e., 25% hexyl cinnamic aldehyde (HCA) and 25% eugenol, were conducted within a period of less than one year. In addition, different concentrations of 2,4-dinitrochlorobenzene (DNCB, an extreme sensitizer) (0.01, 0.1 and 0.3%), HCA (10, 25 and 50%) and eugenol (10, 25 and 50%), were tested to determine the EC1.6 values. Special Pathogen Free female CBA/J mice of 8-10weeks old were randomly allocated to the groups, each group having 4 mice. 25μl of AOO (vehicle, acetone: olive oil=4:1, v/v) or HCA, eugenol, DNCB at the needed concentration was applied to the dorsum of each ear of the mice, daily for 3 consecutive days. A single intraperitoneal injection of 0.5ml of BrdU solution (10mg/ml) was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, and BrdU ELISA analysis was conducted.

RESULTS

The result for each group is expressed as the mean Stimulation Index (SI). The mean of the 10 mean SIs for 25% HCA (2.58±0.95) and 25% eugenol (3.51±1.25) was not significantly different to that from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (i.e., the data on the formal validation study for the LLNA:BrdU-ELISA by the ICCVAM) (3.03±2.00 for 25% HCA, 6.13±6.06 for 25% eugenol) (P>0.05), with even smaller Coefficient of Variations (CV) (36.8% for 25% HCA, 35.6% for 25% eugenol) than that from the ICCVAM (66.0% for 25% HCA, 98.8% for 25% eugenol). In addition, the EC1.6 values for HCA, eugenol and DNCB (15.2, 12.5 and 0.25%, respectively) were consistent with that from the ICCVAM (12.92, 8.85 and 0.34%, respectively).

DISCUSSION

The results indicate that the reliability for our laboratory to conduct the LLNA:BrdU-ELISA is successfully determined.

摘要

引言

局部淋巴结试验（LLNA）已被指定为鉴定新化学物质皮肤致敏潜力的首选体内试验。LLNA:BrdU-ELISA是对LLNA的一种经过验证的非放射性改良方法。开展了一项针对LLNA:BrdU-ELISA的实验室内部重现性研究，以证明其在我们实验室中的良好性能。

方法

在不到一年的时间内进行了10次独立的LLNA:BrdU-ELISA试验，采用了优选的阳性对照（PCs），即25%己基肉桂醛（HCA）和25%丁香酚。此外，测试了不同浓度的2,4-二硝基氯苯（DNCB，一种极强的致敏剂）（0.01%、0.1%和0.3%）、HCA（10%、25%和50%）以及丁香酚（10%、25%和50%），以确定EC1.6值。将8-10周龄的无特定病原体雌性CBA/J小鼠随机分配到各实验组，每组4只小鼠。每天将25μl的AOO（赋形剂，丙酮:橄榄油=4:1，v/v）或所需浓度的HCA、丁香酚、DNCB涂抹于小鼠每只耳朵的背部，连续3天。在第5天腹腔注射一次0.5ml的BrdU溶液（10mg/ml）。在第6天，切除每只小鼠的一对耳后淋巴结，并进行BrdU ELISA分析。

结果

每组结果以平均刺激指数（SI）表示。25% HCA（2.58±0.95）和25%丁香酚（3.51±1.25）的10个平均SI的平均值与机构间替代方法验证协调委员会（ICCVAM）的数据（即ICCVAM对LLNA:BrdU-ELISA正式验证研究的数据）（25% HCA为3.03±2.00，25%丁香酚为6.13±6.06）无显著差异（P>0.05），变异系数（CV）甚至比ICCVAM的更小（25% HCA为36.8%，25%丁香酚为35.6%）（ICCVAM的25% HCA为66.0%，25%丁香酚为98.8%）。此外，HCA、丁香酚和DNCB的EC1.6值（分别为15.2%、12.5%和0.25%）与ICCVAM的一致（分别为12.92%、8.85%和）。