LLNA: Brdu-FCM替代方法用于化妆品皮肤致敏性评价  

Experimental study on alternative method of local lymph node assay using bromodeoxyuridine with flow cytometry(LLNA:BrdU-FCM)for skin sensitization evaluation of cosmetics

作　　者：吕晓君 张菊 吴森 徐小玲 史梦婷 徐巾晶 潘望平 沈佳特 何开勇 LYU Xiao-jun;ZHANG Ju;WU Sen;XU Xiao-ling;SHI Meng-ting;XU Jin-jing;PAN Wang-ping;SHEN Jia-te;HE Kai-yong(Hubei Institute for Drug Control,Hubei Engineering Research Center for Drug Quality Control,Wuhan 430075,China)

机构地区：[1]湖北省药品监督检验研究院,湖北省药品质量检测与控制工程技术研究中心,湖北武汉430075

出　　处：《中国药理学通报》2025年第4期793-799,共7页Chinese Pharmacological Bulletin

基　　金：中国食品药品检定研究院化妆品安全技术规范制修订项目(No.2021-BZ-043)。

摘　　要：目的建立一种化妆品皮肤致敏性评价的替代方法——基于掺入溴脱氧尿嘧啶核苷(BrdU)和流式细胞术(fow cytometry,FCM)的小鼠局部淋巴结试验方法(LLNA:Brdu-FCM),并对该方法进行评价。方法(1)选择25%己基肉桂醛(HCA)作为阳性对照,丙酮∶橄榄油(4∶1,V/V,AOO)混合物作为溶剂对照进行试验:d 1、2、3涂抹小鼠双耳,d 5腹腔注射Brdu,d 6取双耳片和颌下淋巴结,流式细胞仪测定Brdu阳性细胞数,计算刺激指数(SI)是否≥3,以建立LLNA:Brdu-FCM试验方法。(2)选择OECD皮肤致敏性试验指导原则(No.TG429)中15种已知致敏信息的参考化学品,通过盲法进行LLNA:Brdu-FCM试验,每种受试物用AOO、N,N-二甲基甲酰胺(DMF)或二甲基亚砜(DMSO)溶解后配制成3种不同浓度,同法进行检测,计算SI和EC2.7,判定受试物是否具有致敏性,将判定结果与OECD TG429比较,验证该方法的可靠性和准确性。结果25%HCA的SI值为3.9,判断其致敏阳性,LLNA:Brdu-FCM试验方法可成功建立;9种受试物(2,4-二硝基氯苯、4-苯二胺、二氯化钴、2-氢巯基苯并噻唑、己基肉桂醛、丁香酚、苯甲酸苯酯、肉桂醇、咪唑烷基脲)为致敏阳性,6种受试物(甲基丙烯酸甲酯、氯苯、异丙醇、乳酸、水杨酸甲酯、水杨酸)为致敏阴性。该方法灵敏度为90%,特异性为100%,阳性预测率为100%,阴性预测率为83%,假阳性率为0,假阴性率为17%,准确度为93%,BrdU-FCM试验方法能够可靠地区分皮肤致敏剂和非致敏剂。结论LLNA:Brdu-FCM能较好地评价化学物的致敏性,可在化妆品安全性评价中作为补充化妆品原料的皮肤致敏性安全检测的替代方法。Aim To establish and evaluate an alternative method for detecting skin sensitization of cosmetics based on local lymph node assay using bromodeoxyuridine(BrdU)with flow cytometry(FCM).Methods(1)25%hexyl cinnamic aldehyde(HCA)was chosen as a positive control with an acetone:olive oil(4∶1,V/V,AOO)mixture as a vehicle control for the experiment.The dorsal sides of both ears of mice were treated with test solutions on day 1,day 2,and day 3.Brdu solution was injected inter-peritoneally on day 5.On day 6,the bilateral ears and mandibular lymph nodes were excised,and the number of Brdu positive cells was measured by flow cytometry.The stimulation index(SI)was calculated to identify whether it was≥3,in order to establish the method of LLNA:Brdu-FCM.(2)BrdU-FCM test was conducted using a blind method with the fifteen reference substances listed in OECD TG429 whose skin sensitization potentials were known.The test substances were dissolved in AOO,N,N-dimethylformamide(DMF)or dimethyl sulfoxide(DMSO)at three different concentrations.Tests were performed the same as above.SI and EC2.7 were calculated to evaluate whether the test substance was categorized as a skin sensitizer.The reliability and accuracy of the method were validated by comparing the classification of test substances with that in OECD TG429.Results The SI for 25%HCA was 3.9,showing positive in the skin sensitization test.It demonstrated that the LLNA:Brdu-FCM test method was properly implemented.Nine test substances(2,4-dinitrochlorobenzene,4-phenylenediamine,cobalt chloride,2-mercaptobenzothiazole,hexylcinnamaldehyde,eugenol,phenyl benzoate,cinnamic alcohol,imidazolidinyl urea)were positive,and six test substances(methyl methacrylate,chlorobenzene,isopropanol,lactic acid,methyl salicylate,salicylic acid)were negative.The method was evaluated with sensitivity of 90%,specificity of 100%,positive prediction rate of 100%,negative prediction rate of 83%,false positive rate of 0%,false negative rate of 17%and accuracy of 93%.The LLNA:BrdU-FCM assay could correc

关 键 词：致敏性 替代 LLNA:Brdu-FCM 刺激指数 化妆品 

分 类 号：R-332[医药卫生] R322.99R446.63R446.69R758.2