Citotoxicity test in dentistry. ISO 7405: 2018 - Evaluation of biocompatibility of medical devices used in dentistry. 

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The ISO 7405 standard specifies the test methods used in the evaluation of the biological effects of medical devices used in dentistry. It is to be used in conjunction with the ISO 10993 series of standards, but this document contains special tests, recognizing the special needs of dentistry. This document specifies the test methods used in the evaluation of the biological effects of medical devices used in dentistry.

The selection of test methods should be based on the intended use of the sanitary tissue, the tissue(s) with which the product may come into contact, and the duration of contact. The tests are classified into three groups, group 1 comprises in vitro cytotoxicity tests, which include: 1) the agar diffusion test, 2) the filter diffusion test, 3) the direct contact test or with an extract according to ISO 10993-5, and 4) the dentin barrier cytotoxicity test. While the first three tests can be used with all medical devices in dentistry, the dentin barrier test can only be used with external communication devices.

The agar diffusion test (section 6.2 of the standard) is designed to demonstrate nonspecific cytotoxicity of test materials after diffusion through agar. This test is not appropriate for leachables that do not diffuse through agar. On a cell culture of epithelial cells or fibroblasts, a layer of agar with culture medium and a solution of the neutral red vital dye is deposited. An appropriate number of test samples to perform the assay with each medical device, the negative control and the positive control, are applied to each plate and incubated for 24 hours at 37°C. Each test material must be analyzed in quadruplicate. The standard also includes a reference material as an optional control. If it is desired that the test be carried out with a reference material with similar characteristics to the test product, this must be provided by the client. The presence of areas of discoloration of the cell monolayer (not stained with neutral red) and cell lysis, around and below the samples, are evaluated by calculating the index of discoloration and lysis, respectively. The cellular response, calculated as the mean of the discoloration index and the lysis index, will determine whether the medical device is non-cytotoxic, moderately cytotoxic, moderately cytotoxic or strongly cytotoxic.

The filter diffusion test (section 6.3 of the standard) aims to demonstrate the non-specific cytotoxicity of test materials after diffusion through a cellulose acetate filter. A cell suspension of fibroblasts or epithelial cells is inoculated onto a cellulose acetate filter placed in a culture dish and incubated for 24 hours at 37°C. In culture plates a freshly prepared culture medium agar mixture is added and the filter is placed on top of the solidified mixture, cell culture side down. Three to 5 test samples are deposited on each filter and incubated at 37°C, evaluating evidence of cytotoxicity after 2 and 24 hours. Each test material is analyzed in at least quadruplicate. In addition, a positive control and a negative control are included on each plate, and other customer-supplied test method controls are tested if desired to be included. The standard also includes a reference material as a control. If the test is to be carried out with a reference material with similar characteristics to the test product, this must be provided by the client. After incubation, the filter is detached from the agarose and the area of ​​reduced activity of the enzyme succinate dehydrogenase (Method A) or non-specific hydrolase (Method B) is evaluated cytochemically by staining the filter. The assessment of cell damage will be based on the area of ​​discoloration on the surface of the filter, determining whether the medical device is non-cytotoxic, moderately cytotoxic, moderately cytotoxic or intensely cytotoxic.

The direct contact test or with an extract of the medical device in accordance with the ISO 10993-5 standard consists of putting a product or an extract of the product in contact with a cell monolayer (if the client does not indicate another preference, it will be of Vero cells). After 24 to 48 hours, cytotoxicity is evaluated by qualitative method (observation of microscopic effects, classifying reactivity as none, mild, mild, moderate or severe) and by quantitative method, determination of cell viability by counting viable cells for direct contact products or by one of the three methods indicated by the standard (NRU, MTT or XTT) to analyze extracted cells and determine the rate of cell death which, when greater than 30%, indicates cytotoxicity. At IVAMI, the neutral red method is commonly used. Request the specific information of the ISO 10993-5 standard if you want the test to be carried out under this method.

The dentin barrier cytotoxicity test (Annex B of the standard) is designed as an in vitro pulp chamber simulation of the filtration and diffusion of materials from a dental cavity preparation to the dental pulp. It is designed to demonstrate a change in component concentrations of test materials or extracts or sets when placed on one side of the perfusion chamber, on one side of a human or bovine dentin barrier, and allows them to diffuse to the opposite side. A mesh with cells is placed on the opposite side of the dentin. Cells whose physiology is similar to the tissues of the dental pulp should be used, for example, clonal cells by transfection of the T antigen of the SV40 virus, derived, for example, from the bovine dental papilla. After the assay, the cell viability of the mesh cells is determined using the MTT dye. Five to 10 replicates of the test material, positive control and negative control are made. The evaluation of cytotoxicity is based on a statistical comparison of the results obtained. Depending on whether or not the test material shows significant differences with the positive or negative control, the medical device will be classified as non-cytotoxic, moderately cytotoxic or intensely cytotoxic. This assay is not currently performed in our laboratory.