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This document is under the jurisdiction of the National Technical Committee on Biological Evaluation on Medical Device of Standardization Administration of China (SAC/TC 248).
Introduction
The normal keratinocytes obtained from healthy volunteers may be cultured at the gas-liquid interface on the film or filter paper for several days to form a three-dimensional skin model, including the basal layer, stratum spinosum, granular layer and functional stratum corneum, that is, the reconstructed human epidermis model. This model was originally developed to detect outer skin irritation of pure chemical objects. In recent years, such models have also been used to detect stimulating substance in medical devices.
In vitro skin irritation test for medical devices
1 Scope
This document specifies a method for test for medical devices using a reconstructed human epidermis (RhE) model.
This document is applicable to in vitro skin irritation test using RhE model to evaluate the potential skin irritation of medical devices.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 16886.10 Biological evaluation of medical devices — Part 10: Tests for irritation and skin sensitization
GB/T 16886.12 Biological evaluation of medical devices — Part 12: Sample preparation and reference materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in GB/T 16886.10 and GB/T 16886.12 apply.
4 Test principle
The polar and non-polar extracts of medical devices/materials or the devices/materials themselves may directly contact the upper surface of the RhE model; wash and remove the test samples on the epidermis after incubation for a certain time, and then detect the cell activity of the RhE model by methyl thiazolyl tetrazolium (MTT) test; compare with the negative control to obtain the tissue activity, and predict the irritation of the test samples according to the tissue activity.
5 Material
5.1 RhE model
The commercial RhE model is used for the test. See Annex A for instructions of RhE model.
Epidermal cells shall be taken from healthy volunteers whose antigens are negative for human immunodeficiency virus (HIV) 1 and 2 antibodies, HCV-Ab, hepatitis B. Users of this document should establish corresponding safety and health regulations to ensure biosafety.
The extracts of medical devices and/or materials shall be prepared according to GB/T 16886.12, and attention shall be paid to aseptic operation.
0.9% sodium chloride injection should be used as polar extraction vehicle to prepare polar extraction solution, and sesame oil should be used as non-polar extraction vehicle to prepare non-polar extraction solution. If other extraction vehicle is used, it shall be proved that the extraction vehicle will not affect the test system.
The extraction time and temperature should be demonstrated based on GB/T 16886.12.
When testing medical devices and/or materials that are not suitable for extraction, their suitability should be demonstrated.
6.1.2 Preparation of control solution
6.1.2.1 Negative control
Sterile DPBS (1 ×).
6.1.2.2 Vehicle control
The vehicle control shall be placed in the extraction container, and the same extraction procedure as the medical device and/or material shall be carried out.
6.1.2.3 Positive control
1% SDS solution. Take 0.5 mL of 20% SDS aqueous solution and 9.5 mL of corresponding extraction vehicle, and mix well with vortex oscillator.
Note 1: The positive control prepared freshly on the day of test is used, and it is recommended to use 20% commercial SDS solution to prepare positive control. SDS positive control may be prepared by sterile DPBS when the test samples are not suitable for extraction.
Note 2: If the test sample reacts with MTT reagent, or the tissues or cells are stained; and there is still a certain amount of residue after washing, the test results may be disturbed.
In this case, an appropriate control may be set to eliminate interference.
Foreword i Introduction ii 1 Scope 2 Normative references 3 Terms and definitions 4 Test principle 5 Material 6 Test procedure 7 Data calculation steps 8 Acceptance criteria for test 9 Result judgment 10 Test report Annex A (Informative) Description of the RhE model Bibliography
ICS 11.040.01 CCS C 30 YY 中华人民共和国医药行业标准 YY/T 1808—2021
医疗器械体外皮肤刺激试验 In vitro skin irritation test for medical devices
式中: VD——组织活度,%; ODx——PC、VC或试验样品组每块组织的OD值; ODNC——NC组OD值。 然后计算PC组3块组织的组织活度的平均值作为PC组的组织活度。 d) 同法计算试验样品和VC组每块组织的组织活度,然后计算各组3块组织平均组织活度作为各组的组织活度。 e) 根据各组每块组织的组织活度计算各组3块组织的组织活度的标准差(SD)。 注:若为排除试验样品干扰设置了相应对照,则在计算结果时扣除非特异性OD值。 8 试验接受准则 8.1 NC和VC接受准则 a) NC或VC的3块组织OD570平均值大于或等于0.7且小于3.0,则NC或VC的组织活度通常符合接受准则。 注:0.7和3.0是经济合作与发展组织化学品试验导则439(OECD TG439)中几种RhE模型NC和VC的最小和最大OD值,每种模型组织的范围可能有所差异。 b) 除了符合a)外,每种模型NC和VC的OD值范围应符合制造商各自的接受准则。 c) 孵育后NC或VC3块组织的组织活度的SD小于或等于20%。 d) VC组的组织活度应为NC的80%~120%。 8.2 PC接受准则 孵育后PC组的组织活度为NC的40%以下且3块组织的组织活度的SD小于或等于20%,则PC数据符合接受准则。 8.3 试验样品数据接受准则 试验样品孵育后3块组织的组织活度的SD小于或等于20%,则认为数据有效。若不符合要求则应重复试验。 9 结果判定 根据试验样品组的组织活度预测试验样品潜在刺激性。组织活度小于或等于阴性对照的50%即表示出现刺激反应。试验组至少一个浸提液组的组织活度小于或等于50%,则认为此医疗器械/材料有皮肤刺激性,见表1。 表1 皮肤刺激结果的判定 试验结果 判定 至少一个浸提液组的组织活度小于或等于阴性对照的50% 刺激性(I) 两个浸提液组的组织活度均大于阴性对照的50% 非刺激性(NI) 10 试验报告 试验报告应至少包括以下信息: ——试验材料或器械的描述; ——RhE模型的描述及其质控证书复印件; ——试验方法,包括样品制备过程的详细描述; ——试验结果; ——试验结论。 附录A (资料性) RhE模型的说明 OECD TG 439中收录了已经经过验证的6种RhE模型:EpiSkinTM (SM)、EpiDermTM SIT(EPI-200)、SkinEthicTM RHE、LabCyte EPI-MODEL24 SIT、epiCS和Skin+。另外,文件中还提供了模型验证方案与接受准则。其他模型若能通过对6种RhE模型比对试验相同的刺激及非刺激材料进行的实验室间(至少3个实验室)3轮(3个生产批号的RhE模型)盲法验证试验,证明其预测能力、实验室内及实验室间变异性与已收录某种模型有等同的表现,则可证明其与已收录模型的等效性。 ISO/TC 194相关工作组采用以上6种模型中的EpiDermTM (EPI-200)和SkinEthicTM RHE两种模型对医疗器械体外皮肤刺激试验进行了国际实验室间比对,验证了这两种模型的有效性。 其他模型如果通过了以上两种形式的比对试验即追随验证试验(catch-up validation),则认为可用于医疗器械体外皮肤刺激试验。 参考文献 [1] De Jong W. H., Hoffmann S., Lee M., Kandárová H., Pellevoisin C., Haishima Y. Toxicol. Round robin study to evaluate the reconstructed human epidermis (RhE) model as an in vitro skin irritation test for detection of irritant activity in medical device extracts. In Vitro. 2018, 50pp.439-449. [2] Kandarova H., Willoughby J.A., De Jong W.H., Letasiova s., Milasova T., Bachelor M.A. Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDermTM. Toxicol. In Vitro.2018,50 pp.407-417. [3] Olsen D.S., Lee M., Turley A.P. Toxicol. Assessment of test method variables for in vitroskin irritation testing of medical device extracts. In Vitro.2018 ,50 pp.426-432. [4] Pellevoisin C., Videau C., Briotet D., Grégoire C., Tornier C., Alonso A. SkinEthicTM RHE for in vitro evaluation of skin irritation of medical device extracts. Toxicol. In Vitro. 2018, 50 pp. 419-425. [5] Organization for Economic Cooperation and Development (OECD) .Guidelines for testing of chemicals. No.439.In Vitro Skin Irritation:Reconstructed Human Epidermis Test Method. OECD Publications, 2017. [6] Alépée N., Tornier C., Robert C., Amsellem C., Roux M.-H., Doucet O. A catch-up validation study on reconstructed human epidermis (SkinEthicTM RHE) for full replacement of the Draize skin irritation test. Toxicol. In Vitro.2010,24 pp.257-266. [7] Kandáová H., Hayden P., Klausner M., Kubilus J., Kearney P., Sheasgreen J. 2009):In Vitro Skin Irritation Test:Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol.AT-LA 37 ,671-669 ,2009. [8] Coleman K.P., Grailer T. P., McNamara L.R., Rollins B.L., Christiano N.J., Kandáová H. Toxicol. Preparation of irritant polymer samples for an in vitro round robin study. In Vitro.2018,50 pp.401-406. [9] Pellevoisin C., Tornier C., Bremond C., Rollins B., Briotet D., Turley A. Skin irritation of medical devices:In vitro assay with EPISKIN reconstructed human epidermis (RHE). Toxicol. Lett. 2016,258 (S63) p. vili. [10] Kandárová H., Bendova H., Letasiova S. , Coleman K.P., De Jong W. H., Jírova D. Toxicol. Evaluation of the medical devices benchmark materials in the controlled human patch testing and in the RhE in vitro skin irritation protocol. In Vitro.2018 ,50 pp.433-438. [11] Faller C., Bracher M., Dami N., Roguet R. Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics. Toxicol. In Vitro.2002 ,16 pp.557-572. [12] Mosmann T. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Methods. 1983, 65 pp.55-62. [13] SOP of in vitro skin irritation of medical devices extracts with SkinEthicTM kinEthic vitro skin irritation of medical devices extracts wepiskin. com/~/ media/Files/SOP SkinEthic RHE Irritation-Medical-devices ,2018. [14] Organization for Economic Cooperation and Development (OECD) Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment , No.34 ,OECD Publishing, Paris, 2005.