In vitro cytotoxicity tests. ISO 10993-5: 2009/11: 2025. Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests.
Test accredited by ENAC (Spanish National Accreditation Entity).
Test with the Certificate of Good Laboratory Practices (GLPs).
This standard describes the procedure to detect in vitro cytotoxicity of medical devices. The test can be performed with an extract of the device, with the entire device in direct contact with the cells, or with the entire device by an indirect application by agar diffusion or by filter diffusion.
The devices must be sterile, or they must be sterilized in the laboratory according to the manufacturer´s guidelines, as they must be contacted with sterile cell cultures. The test method requested and the manufacturer´s recommendations for the extraction solution should be provided in case the test should be performed with an extract of the device, and conditions that may degrade it to be avoided. If there is any preference for the use of a specific cell line, it should be indicated. The most commonly used cells available are Vero cells, BHK-21, 3T3 cells, ...etc. Unless the customer indicates another choice, the test will be performed with 3T3 cells.
The test must be carried out in triplicate and must include the corresponding positive and negative controls. The monolayers used will be in a state of partial confluence and after exposure to the device in any of its forms, will be followed for between 24 and 72 hours, depending on test method and cells requested.
Cytotoxicity can be determined by either qualitative or quantitative means. Quantitative evaluation of cytotoxicity is preferable. Qualitative means are appropriate for screening purposes. For tests with the medical device in direct contact or with the extract of a medical device, unless otherwise requested, both determinations are carried out, while for tests by indirect contact, by agar diffusion or filter diffusion, only the qualitative evaluation can be performed. The qualitative evaluation is performed by a microscopic exam of the cultures is performed giving a graduation of the effects (0 / 1 / 2 / 3 / 4), indicating grades 2 to 4 different intensity of product cytotoxicity (Annex B of thestandard). In quantitative evaluation, the rate of cell death should be determined by vital staining. The standard indicates three alternatives for liquid medical devices or extracts: the reduction of uptake of neutral red dye (NRU: Neutral Red Uptake) (Annex A of the standard); the reduction of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazoliumbromid] leading to the formation of formazan (Annex C of the standard) or the reduction of XTT [2,3-bis] (2-methoxy-4-nitro-5-sulfophenyl) -5 - [(phenylamino) carbonyl] -2H-tetrazolium hydroxide] leading to the formation of formazan (Annex D of the standard). When the calculated cell death rate is greater than 30%, it indicates cytotoxicity. At IVAMI, the NRU method is normally used for the evaluation of extracts and liquid medical devices, and the vital staining with trypan blue for the evaluation of cytotoxicity with solid medical devices by direct contact.