体外微核试验方法的建立及其在医疗器械检测中的应用

The Micronucleus Test Method was Established in Vitro and Applied in Medical Devices Testing

目的:对体外哺乳动物细胞微核试验方法进行探索建立,并对聚氯乙烯(PVC)材质医疗器械的遗传毒性进行检测。方法:依据GB/T16886.3-2019和YY/T0870.6-2019等相关方法对样品进行体外哺乳动物细胞微核试验方法的探索建立,按照GB/T16886.12中规定的浸提比例(0.2g/mL)浸提样品,采用建立的微核试验方法检测样品有代谢性活化体系(3h)和无代谢性活化体系(3h、24h)的微核发生率(%)。结果:显微镜观察100%、50%、25%和12.5%样品浸提液均不超过20%的细胞呈圆缩、疏松贴壁、无胞浆内颗粒或显示形态学方面的改变,偶见细胞溶解,仅观察到轻微的细胞生长抑制现象,判定细胞毒性为1级,MTT结果显示100%、50%、25%和12.5%样品细胞毒性不超过2级。代谢性活化体系(3h)样品微核率为2.30%,与阳性对照微核率(15.70%)具有显著性差异(P<0.01);无代谢性活化体系(3h)样品微核率(0.90%),与阳性对照微核率(8.10%)具有显著性差异(P<0.01);无代谢性活化体系(24h)样品微核率(1.55%),与阳性对照微核率(11.80%)具有显著性差异(P<0.01);CBPI结果表明样品与阴性对照组细胞毒性相当,即细胞毒性为1级,与体外细胞毒性结果相符合。结论:通过上述试验方法建立的代谢性活化体系和无代谢性活化体系体外哺乳细胞微核试验结果稳定、可靠,对样品引起的遗传毒性的风险评价提供可行的检测试验方法。

Objective:To explore and establish the method of mammalian cell micronucleus test in vitro,and detect the genotoxicity of PVC medical devices.Methods:According to GB/T16886.3-2019 and YY/T0870.6-2019 and other relevant methods,the method of mammalian cell micronucleus test was explored and established in vitro.The samples were extracted according to the extraction ratio(0.2g/mL)specified in GB/T16886.12.The established micronucleus test method was used to detect the micronucleus incidence(%)of samples with metabolic activation system(3h)and without metabolic activation system(3h,24h).Results:Microscopic observation showed the sample extracts of 100%,50%,25% and 12.5% no more than 20% showed round,loose adherence,no cytoplasmic particles or morphological changes.Cell dissolution was occasionally seen,and only slight cell growth inhibition was observed.It was determined that the cytotoxicity was grade one.MTT results showed the cytotoxicity was no more than grade two.The micronucleus rate of metabolic activation system(3h)was 2.30%,which was significantly different comparded with positive control(15.70%)(P<0.01);The micronucleus rate of the sample without metabolic activation(3h)was 0.90%,which was significantly different comparded with the positive control(8.10%)(P<0.01);The micronucleus rate of the sample without metabolic activation(24h)was 1.55%,which was significantly different comparded with the positive control(11.80%)(P<0.01);CBPI results showed the cytotoxicity of the sample was equivalent to the negative control group,and the cytotoxicity was grade one,which was consistent with the cytotoxicity results in vitro.Conclusion:The results of mammalian cell micronucleus test with metabolic activation system and wthout metabolic activation system established in vitro are stable and reliable,and which provide a feasible detection methods for the risk assessment of the samples genotoxicity.