BS EN ISO 7405:2018 pdf download – Dentistry – Evaluation of biocompatibility of medical devices used in dentistry
6.1.4 Take the following factors into account, considering the final use of the chemically setting materials:
a) mixing: mix sufficient material to ensure that the preparation of each test sample is completed from one batch. Prepare a fresh mix for each test sample. The mixing shall be performed in accordance with the respective product standards, if applicable;
b) oxygen exposure: for materials that produce an oxygen inhibition layer during chemical curing, both ends of the mould shall be covered with oxygen barrier materials (e.g. a polyester film) during curing. If the material is recommended by the manufacturer for surface finishing after curing, the sample surfaces shall be ground and polished using the recommended clinical procedures. If there are no such instructions and if required for testing, the samples shall be ground on both ends, with a P2 000 paper in accordance with ISO 6344-1, after first being set against the oxygen barrier material.
6.1.5 Positive control material For in vitro tests and certain in vivo tests (e.g. pulp and dentine usage test), it is advisable to include a standard positive control material, which is handled and processed like the test materials (i.e. being plastic after mixing and then setting) and which is based on freely available chemicals or materials. Such a positive control material for in vitro testing of plastic filling materials is described in Annex B, Table B.1. The use of this specific positive control material is optional and other materials with a validated history and other well characterized positive control materials with reproducible data on toxicity can be used instead.
6.2 Agar diffusion test 6.2.1 Objective This test is designed to demonstrate the nonspecific cytotoxicity of test materials after diffusion through agar or agarose. This test method is not appropriate for leachables that do not diffuse through agar or agarose.
6.2.2 Cell line Use an established fibroblast or epithelial cell line, which is readily available [e.g. from the American Type Culture Collection (ATCC), see https://www.atcc.org] 1) . Specify in the report the identification number of the cell line, if applicable, the description and designation of the cell line used and a justification for its selection.
6.2.3 Culture medium, reagents and equipment Use the culture medium specified for the selected cell line. Sterilize by filtration. For the preparation of the agar, prepare a double-concentration of the culture medium. Sterilize by filtration. Prepare either 3 % agar or 3 % agarose. Sterilize by autoclaving. Prepare the vital stain by diluting a stock solution of 1 % aqueous neutral red solution (record source) 1:100 with 0,01 mol/l phosphate-buffered saline solutions [e.g. Dulbecco’s phosphate-buffered saline solution 2) ] immediately before use. Store neutral red solutions protected from the light. Use 6-well tissue culture plates (35 mm in diameter) or Petri dishes of 50 mm to 100 mm in nominal diameter suitable for tissue culture.
6.2.4 Sample preparation Prepare the samples in accordance with 6.1. The test shall be performed on either an extract of the material and/or the material itself, according to the guidance in ISO 10993-5.
a) For solid materials, prepare circular test samples of approximately 5 mm diameter, with a flat surface to ensure adequate contact with the agar overlay.
b) For setting materials, insert the freshly mixed material into rings of internal diameter 5 mm and height 2 mm. The material of the ring shall be stated in the test report. When testing materials in the freshly mixed state, place the rings on the agar prior to inserting the material. When testing after various setting periods, fill the rings so that the material is flush with the rim and allow it to set at (37 ± 2) °C and a relative humidity of (90 ± 10) % until ready for testing. c) For fluid test samples or extracts, imbibe 0,01 ml of the fluid on a borosilicate microglass filter disc of 5 mm diameter, placed on the agar.