Hou Fenxia, Xing Caihong, Li Bin, Cheng Juan, Chen Wei, Zhang Man
Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China.
Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China.
J Pharmacol Toxicol Methods. 2015 Mar-Apr;72:53-8. doi: 10.1016/j.vascn.2015.01.001. Epub 2015 Jan 16.
Allergic contact dermatitis (ACD) is a skin disease characterized by eczema and itching. A considerable proportion of chemicals induce ACD in humans. More than 10,000 substances should be tested for skin sensitization potential under the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACH) regulation. The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for sensitization testing by REACH. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. For both the LLNA and the LLNA:BrdU-ELISA, CBA/JN mouse is the preferred mouse strain recommended in the regulatory guidelines. However, the availability of CBA/JN mouse in China is only limited to a few animal suppliers, which makes the mouse difficult to obtain. BALB/c mouse, which is widely commercially available, is considered for alternative use but it can only be used in the assay after it has been evaluated by formal validation study. Thus, a validation study was conducted in our laboratory to determine if BALB/c mouse could also be used in the LLNA:BrdU-ELISA.
Forty-three test substances including 32 LLNA sensitizers and 11 LLNA non-sensitizers, their vehicles and each concentration used were the same as that used in the formal validation study for the LLNA:BrdU-ELISA using CBA/JN mouse. Female BALB/c mice of 8-10 weeks old were randomly allocated to groups (four mice per group). The test substance (25 μl) or the vehicle alone was applied to the dorsum of both ears daily for 3 consecutive days. A single intraperitoneal injection of 0.5 ml of BrdU (10mg/ml) solution was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, weighed and stored at -20°C until BrdU-ELISA was conducted.
This validation study for the LLNA:BrdU-ELISA using BALB/c mouse correctly identified 30 of 31 sensitizers and 8 of 11 non-sensitizers. The accuracy, sensitivity, specificity, false positive rate, false negative rate, positive predictivity values and negative predictivity values in this study, which could indicate the performance of the LLNA:BrdU-ELISA using BALB/c mouse, were not different statistically from that of the validation study for the LLNA:BrdU-ELISA using CBA/JN mouse.
This validation study indicates that BALB/c mouse could be used alternatively in the LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.
过敏性接触性皮炎（ACD）是一种以湿疹和瘙痒为特征的皮肤病。相当一部分化学物质可诱发人类发生ACD。根据《化学品注册、评估、授权和限制》（REACH）法规，超过10000种物质需进行皮肤致敏潜力测试。局部淋巴结试验（LLNA）已被REACH指定为体内致敏测试的首选方法。LLNA:BrdU-ELISA是对LLNA的一种经过验证的非放射性改良方法。对于LLNA和LLNA:BrdU-ELISA，CBA/JN小鼠是监管指南中推荐的首选小鼠品系。然而，CBA/JN小鼠在中国仅在少数动物供应商处有供应，这使得该小鼠难以获得。广泛商业化供应的BALB/c小鼠被考虑作为替代使用，但只有在经过正式验证研究评估后才能用于该试验。因此，我们实验室进行了一项验证研究，以确定BALB/c小鼠是否也可用于LLNA:BrdU-ELISA。
43种受试物质，包括32种LLNA致敏剂和11种LLNA非致敏剂，其赋形剂及各浓度与使用CBA/JN小鼠进行LLNA:BrdU-ELISA正式验证研究时所用相同。将8 - 10周龄雌性BALB/c小鼠随机分组（每组4只）。连续3天每天将受试物质（25μl）或单独的赋形剂涂于双耳背部。在第5天腹腔注射一次0.5 ml BrdU（10mg/ml）溶液。在第6天，切除每只小鼠的一对耳淋巴结，称重并储存在-20°C直至进行BrdU-ELISA检测。
这项使用BALB/c小鼠进行LLNA:BrdU-ELISA的验证研究正确识别出31种致敏剂中的30种和11种非致敏剂中的8种。本研究中的准确度、灵敏度、特异性、假阳性率、假阴性率、阳性预测值和阴性预测值，这些可表明使用BALB/c小鼠的LLNA:BrdU-ELISA的性能，与使用CBA/JN小鼠进行LLNA:BrdU-ELISA验证研究的结果在统计学上无差异。
这项验证研究表明，BALB/c小鼠可替代用于LLNA:BrdU-ELISA以预测化学物质的皮肤致敏潜力。
