Dear Participants I am now able to send a draft protocol for "TC194/WG5 Validation Study for SRMs on Cytotoxicity Test" to the participating laboratories. Please read following items and protocol, then make comments only if necessary by the end of this month (May 31, 1997). 1. Management The study is being planned by Management Members, Drs. M-F Harmond (WG5: Convener), A. Nakamura (sponsorship), and myself (N. Tanaka) based on the agreement of WG5 meeting in York. 2. Project Leader For conducting the TC194/WG5 validation study, there will be a Project Leader who will be responsible for the project. If you have any comments and questions, please feel free to ask by Fax or E-mail. Dr. Noriho Tanaka 729-5 Ochiai, Hadano Kanagawa 257, Japan Fax: +81-463-82-0773 E-mail: QZD02746@niftyserve.or.jp 3. The Time Schedule of the Study Protocol Reviewing: May 15 - May 31, 1997 Shipping of SRMs: June 1 - June 10, 1997 Starting of Experiment: June 10, 1997 Deadline of Report: October 31, 1997 4. Certification of the Data This study may or may not perform under GLP, however, the test results should be carefully checked by GLP spirit. Upon completion of the final report, all raw data and reports shall be achieved by the laboratory conducted. 5. Summarizing the Data All data will be sent to the Project Leader, then they will be circulated and summarized by the management members. 6. Future Work According to the data of present study, the "Optional Study" could be performed in a second step in order to validate the protocols of each laboratory with regards to the negative and positive controls, validated by extract test and direct contact test. 7. Sponsor The study is a partly supported by the Research Project (Project leader: Dr. A. Nakamura, NIHS) granted by the MHW (Ministry of Wealth and Welfare, Japan). Therefore, all participants will be received a set of SRMs with no charge, Finally, I hope that this validation study will be successful and fruitful for the international harmonization of biological testing on the medical devices. Yours sincerely, Noriho Tanaka, PhD Project Leader TC194/WG5 Validation Study DRAFT ---------------------------------------------------------------------- (PROTOCOL) The TC194/WG5 Validation Study for SRMs on Cytotoxicity Testing 1. Purpose According to the agreement of TC194/WG5 meeting in York (April 1997), we decided to perform an inter-laboratory validation study, "round robin test", on the Japanese Standard Reference Materials (SRMs) using the protocol arranged in WG5. The aim of this study is to assess the cytotoxicity of each SRM by using a protocol in compliance with ISO 10993-5. 2. SRMs In this study, three kinds of unsterilized SRMs are distributed. They should be autoclaved after cutting (121C, 20 min). The size of SRMs are 15 x 10 cm (thickness: approx. 0.3 mm). It can be tested almost 4-5 times with each SRM sheet. 2.1 Negative SRM: High density polyethylene sheet (lot No. 96001C) 2.2 Positive SRM-A (moderate): Polyetherurethane film containing 0.1% zinc diethyldithiocarbamate (ZDEC) (lot No. 96011A) 2.3 Positive SRM-B (weak): Polyetherurethane film containing 0.25% zinc dibutyldithiocarbamate (ZDBC) (lot No. 96011B) * Negative SRM is usable only for extraction method because of the floating nature. 3. Test System 3.1 Extraction method 3.2 Direct contact method 4. Culture Medium Eagle's MEM containing Earle's salts, L-glutamine and non-essential amino acids, adjusted with 2.2 g/L of NaHCO3 (Cat. No. 41500-034, GIBCO- BRL) are recommended. Antibiotics may be added if needed. MEM medium is supplemented with 5% FCS. 5. Cell Types L-929 cell line is requested. It shall be checked for mycoplasma contamination. 6. Outline of Experimental Design and Methodology 6.1. Extraction Method 6.1.1. Extract condition SRM shall be cut into a small pieces (2 mm x 15 mm) before extraction. The extraction (6 cm2/ml) of SRM is performed in MEM supplemented with 5% FCS at 37C for 24h. The original extract is defined as 100% extract. A reagent control (blank) is prepared in parallel. 6.1.2. Concentrations The following dilutions of original extract and original blank shall be studied: 1, 1/2, 1/4, 1/8, 1/16, 1/32 (or more lower doses by a factor 2). 6.1.3. Cell seeding and exposure L-929 cells are seeded in 24 well plates (5.2 x 10^4 cells/0.5 ml/well). Triplicate wells are prepared for each concentration of the extract or ofthe blank. After 24 h incubation in a humidified atmosphere containing 5% CO2 incubator at 37 +- 2ßC, culture medium is withdrawn and replaced with 0.5ml of the extract or blank. Exposure period shall be both 24h and 72h. 6.1.4. Measurement of cytotoxicity After 24h, or 72h incubation, with extract and blank, cell suspension is prepared by trypsin treatment, then viable cells are counted in each well using a hematocytometer or other acurate method. Staining with trypan blue, or a vital dye, is recommended for accurate counting. 6.1.5. Data analysis The mean cell numbers in the control wells and the treated wells in each group is calculated. The relative survival (% of control) versus the concentration of SRM extracts is plotted. The IC50 is calculated from the plot. 6.1.6. Report The report should include raw data and IC50 values obtained with each SRM independently. 6.2 Direct Contact Method 6.2.1. Cell seeding and treatment Triplicate cultures are prepared for each SRM and negative control. L-929 are seeded in 35-mm dish (2.6 x 10^5 cells/2 mL/dish). Following 24 h culture in a humidified atmosphere containing 5% CO2 incubator at 37C, the culture medium is replaced with 2 mL fresh medium. Test sample (approx. 1 cm2) shall be placed onto the center of each dish. If test sample is floating, medium can be reduced. All cultures are incubated for both 24h and 72 h at 37C. 6.2.2. Measurement of cytotoxicity Following 24h or 72h incubation, the test sample shall be carefully removed. The cell morphology around and under each test sample is examined under the microscope. Viable cells are counted in each dish using a hemocytometer or other acurate method. Staining with trypan blue or a vital dye, could be useful. 6.2.3. Data analysis The mean cell number of the control dishes and the treated dishes in each group is calculated. The relative survival (% of control) is evaluated. 6.2.4. Report The report should include raw data obtained with each SRM independently. ----------------------------------------------------------------------------- .