化妆品眼刺激性评价中角膜损伤生物标志物研究

Study on biomarkers of corneal injury in the evaluation of eye irritation of cosmetics

针对离体兔眼(IRE)试验只适用于筛选严重眼刺激性(UN GHS第1类)的缺陷,建立IRE试验结合基于细胞活性生物标记物的荧光染色方法用于评价化妆品眼刺激性。通过IRE试验条件的探索,包括测试浓度和后暴露时间,对市售化妆品进行眼刺激性检测,同时对角膜进行冰冻切片并染色,鬼笔环肽对丝状肌动蛋白进行染色,DAPI染色胞核后,测量损伤深度(depth of injury,DOI),并比较体外DOI、体内DOI及Draize试验结果,建立预测模型。结果显示通过DOI检测,可对化妆品无刺激性至中刺激性进行检测,表明在离体兔眼中,基于细胞活力的DOI测量可作为角膜损伤的生物标志物,适用于化妆品眼刺激性的评价。

To address the shortcomings of the isolated rabbit eyes(IRE)assay which is only suitable for screening for severe eye irritation(UN GHS Category 1),an IRE test combined with a fluorescent staining method based on the biomarkers of cell viability was developed for evaluating the eye irritation potential of cosmetic products.Cosmetic products all contain surfactants and other ingredients,and the surfactants may cause damage in a different way compared to other chemicals,in which the exposure procedures for eye irritation evaluation may be different.The IRE test conditions are explored,including the test concentration and post-exposure time.Eye irritation of the commercially available cosmetics were tested by the optimized IRE assay,processed for cryosectioning and staining using phalloidin to detect F-actin and DAPI staining to detect nuclei,and the depth of injury(DOI)were measured.To evaluate the applicability of eye irritation classification of cosmetics based on the measurement of DOI by live cell fluorescent staining,the correlation between in vitro and in vivo DOI measurements was evaluated,and pearson correlation analysis shows a good correlation between the above two methods(r=0.8511,p<0.001).Finally,we compared the in vitro DOI,in vivo DOI and Draize test results to establish a prediction model.The results show that the classification of eye irritation of cosmetic products from non-irritating to moderate level can be distinguished by DOI measuring,suggesting that in isolated rabbit eyes,DOI measurements based on cell viability can be used as a biomarker of corneal damage,and suitable for the evaluation of cosmetic eye irritation.