体内外方法检测化妆品用防腐剂的致敏性

In vitro and in vivo detection of allergenicity of preservatives for cosmetics

目的:采用豚鼠局部封闭涂皮试验和体外人细胞系活化试验检测两种常用化妆品防腐剂的皮肤致敏性,探讨两种方法组合用于检测皮肤致敏性的可行性。方法:豚鼠局部封闭涂皮试验参照化妆品安全技术规范(2015年版)的内容进行,试验设羟苯乙酯组、羟苯丁酯组,同时设甘油为阴性对照组、2,4-二硝基氯苯(DNCB)为阳性对照组,观察试验动物皮肤反应,通过积分评价皮肤致敏性。体外人细胞系活化试验参照经济合作与发展组织(OECD)毒理学测试中的442(E)方法,试验中设羟苯乙酯组、羟苯丁酯组,阴性对照组受试物为甘油、阳性对照组受试物为DNCB,采用CCK-8法测试细胞毒性,计算细胞存活率为75%时的暴露浓度(CV75)值,流式细胞术检测各试验组物质的CD86和CD54的相对荧光强度(RFI)值。结果:豚鼠局部封闭涂皮试验检测结果显示甘油为非致敏物,DNCB为强致敏物,羟苯乙酯为非致敏物,羟苯丁酯为中度致敏物。体外人细胞系活化试验检测结果显示羟苯乙酯的CV75值为126μg/mL,RFICD86为110%~140%,RFICD54为140%~260%;羟苯丁酯的CV75值为18μg/mL,RFICD86为75%~200%,RFICD54为90%~210%;甘油的CV75值大于5000μg/mL,RFICD86为75%~100%,RFICD54为60%~140%;阳性对照物质DNCB的CV75值为2.5μg/mL,RFICD86为185%~640%,RFICD54为100%~460%。由检测结果可见羟苯乙酯、羟苯丁酯在本试验中分别被判为弱致敏物和强致敏物,DNCB为极强致敏物,甘油为非致敏物。结论:在该对比试验中,体外人细胞系活化试验较局部封闭涂皮试验检测灵敏度更高,但也存在假阳性的可能,可以考虑用体外细胞方法作为筛选方法,结合动物体内试验方法或更多的体外组合试验进一步确定。

OBJECTIVE:To determine efficiency in using two assays,the human cell line activation test(h-CLAT)and the occluded patch test of Buchler(BT),in detecting skin sensitization of two preservatives commonly used in cosmetics.METHODS:BT was carried out according to“cosmetics safety technical specification(The 2015 Edition)”.Skin sensitizations to ethylparaben,butylparaben andglycerin were used as negative controls and to 2,4-dinitrochlorobenzene(DNCB)as positive control.In test animals,skin sensitization was evaluated by score.The h-CLAT was set up,according to the OECD No 442(E)toxicology test method,to evaluate the ethylparaben,butylparaben,glycerin and the DNCB groups.The CCK-8(cell counting kit-8)method was used to test evaluate cytotoxicity and to calculate the CV75 value.The RFI values of CD86 and CD54 were detected by flow cytometry.RESULTS:The results from the BT test show that glycerin was a nonsensitizer while DNCB was strong sensitizer,ethylparaben was a nonsensitizer and butylparaben was a moderate sensitizer.The results from h-CLAT show that the CV75 value of ethylparaben was 126μg/mL,RFICD86 was 110%-140%,RFI CD54 was 140%-260%;the CV75 value of butylparaben was 18μg/mL,RFICD86 was 75%-200%,RFICD54 was 90%-210%,the CV75 value of glycerol as more than 5000μg/mL,RFICD86 was 75%-100%,RFICD54 was 60%-140%,the CV75 value of DNCB was 2.5μg/mL,RFICD86 was 185%-640%and RFIcd54 was 100%-460%.The test results indicate that ethylparaben and butylparaben were weak and strong sensitizers,respectively,DNCB was a very strong sensitizer and glycerol was a non-sensitizer.CONCLUSION:In this comparative test,the sensitivity of h-CLAT was higher than BT.In addition,this cell culture method can be used as a screening method for some allergenicity evaluations.