Document kmExKLjrxOQjgxMRb8MkoX9Zb

Lab Ng L 1538 SBRTC-2015-1052-6 TABLE OF CONTENTS SUMMARY 4 INTRODUCTION 5 MATERIALS 5-6 METHODS 6-7 RESULTS 8-9 CONCLUSION 9 RECORD STORAGE 9 PHOTOGRAPH OF THE TEST ARTICLE .10 Page 3 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, No 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: 0-- - ' - -- " - -3 Web site: www.sbrtc.com E-mail: b163.com Lab 1\~4 L 1538 SBRTC-20 15-1052-6 SUMMARY A Salmonella typhimurium reverse mutation standard plate incorporation study was conducted to determine whether the saline and DMSO extracts of Non-absorbable Surgical Suture (model:PTFE) would cause mutagenic changes in the average number of revertants for histidine-dependent Salmonella typhimurium strains TA97,TA98,TA100 ,TA102 and TA1535 in the presence and absence of S9 metabolic activation. This study was conducted to satisfy, in part, the genotoxicity requirement of the International Organization for Standardization ISO 10993-3:2014: Biological Evaluation of Medical Devices, Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity. ISO 10993-12:2012: Biological evaluation of medical devices, Part 12: Sample preparation and reference materials. The test article was extracted in 0.9% sodium chloride injection (SC) and DMSO respectively and then diluted into four concentrations by two-fold serial dilutions. Individual tubes containing 2 ml of molten top agar supplemented with histidine-biotin solution were inoculated with 0.1 ml of culture of tester strain and 0.1 ml of the test article extract. A 0.5 ml aliquot of 10% S9-mix (rat liver metabolizing system) was added when necessary. The mixture was poured on to Vogel-Bonner E agar plates in triplicate. Parallel testing was also conducted with negative controls and six positive controls. The mean number of revertants of the triplicate test plates was compared to those of the negative control plates for each of the five tester strains employed. There was no diminution of the background lawn in the four concentrations of the test extracts for tester strains TA97, TA98, TA100, TA102 and TA1535 The strains yield spontaneous revertant colony plate counts within the frequency ranges expected from the laboratory 's historical control data. In no case was there a twofold or greater increase in the mean number of revertants in the presence of the test article extracts. Under the conditions of this assay, the test article extracts were considered to be nonmutagenic to Salmonella typhimurium tester strains TA97, TA98, TA R), TA102 and TA1535. The negative and positive controls performed as anticipated. Study and Supervisory Personnel: JI Lina LU Hua Study Director: r SUN Jiao, Ph.D. Alet-v 2 3. d/s , Date Completed Page 4 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, N2 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: 0( 3 Web site: www.sbrtc.com E-mail: 163.com Lab Na L 1538 SBRTC-2015-1052-6 INTRODUCTION The purpose of the bacterial reverse mutation study was to evaluate whether the SC and DMSO test article extracts would cause mutagenic changes in the average number of revertants for Salmonella typhimurium tester strains TA97, TA98, TA100, TA102 and TA1535.The test was performed in the presence and absence of S9 metabolic activation. Bacterial reverse mutation tests have been widely used as a rapid screening procedure for the determination of mutagenic and potential carcinogenic hazards. The test article was received on Jun.16, 2015. The preliminary toxicity screen began on Oct.8, 2015, and the testing ended on Oct.30, 2015. This study was completed in the Lab of Shanghai Biomaterials Research & Test Center (SBRTC) and was conducted in accordance with the provisions of the ISO/IEC 17025-2005. MATERIALS The test article provided by the sponsor was identified and handled as follows: Test Article: Non-absorbable Surgical Suture(model:PTFE) < Specification: Z21302-60(USP2-0/EP2.5) > Identification No: 150642 Package Delineation: STERILE Storage Conditions: Room temperature Extraction Vehicles: 0.9% sodium chloride injection (SC) < Lot N2.KI41028E> DMSO< Lot N2.20141118> Test Article Extract: Based the ISO, 6cm2/ml [Surface area of the test article to volume of extraction vehicle],84.8cm2 of the test article was respectively immersed in 14.1m1 of SC and DMSO under sterile conditions for preparing the SC and DMSO test extracts at 37C for 72h.The test extracts were then serially diluted with the same vehicle and resulted in the following concentrations: Test A: 12.5% diluent of the test extraction Test B: 25% diluent of the test extraction Test C: 50% diluent of the test extraction Test D: 100% test extraction according to the requirement The extracts were used immediately after extraction. Page 5 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, No 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: OT " 3 Web site: www.sbrtc.com E-mail: 163.com Lab NQ L 1538 Negative Control: Positive control: Condition of Extracts: SBRTC-2015-1052-6 0.9% SC or DMSO without test material was similarly subjected to the extraction conditions Without S9: TA 100 and TA 102 !VMS TA97 atebrin TA98 P-Nitroquinoline TA1535 Sodium azide With S9: TA97,TA98, TA100 and TA 1535 TA102 1, 8-dihydroxyanthraquinone 2 aminofluorene All the extracts of the test and controls were clear. Identification of Test System: The test system were Salmonella typhimurium tester strains TA97, TA98, TAloo , TA 102 and TA1535 The tester strains were obtained from School of Public Health, FuDan University, China. Historically, Salmonella typhimurium tester strains have been used for test for genotoxicity (Bacterial reverse mutation study) because they demonstrate sensitivity to mutagenic articles. METHODS Test System: Each Salmonella Typhimurium tester strain contains a specific mutation in the histidine operon and other mutations that increase their ability to detect mutagens. These genetically altered strains (TA97, TA98, TA loo , TA 102 and TA1535) cannot grow in the absence of histidine respectively. When they are placed in a histidine-free medium, only those bacterials which mutate spontaneously back to their wild type state (histidine independent by manufacturing their own histidine) are able to form colonies. The spontaneous mutation rate (or reversion rate) for any one strain is relatively constant, but if a mutagen is added to the test system, the mutation rate is significantly increased. The amino-acid requirement for growth, the presence or absence of R-factor plasmids and other phenotypic characteristics of salmonella typhimurium strains TA97, TA98, TA100 ,TA102 and TA1535 were checked for another study in Jul.8, 2015. Tester Strain Mutations S.typhimurium TA97 his-, rfa, AuvrB, R-factor S.typhimurium TA98 his-, rfa, AuvrB, R-factor S.typhimurium TA100 his-, rfa, AuvrB, R-factor S.typhimurium TAio2 his rfa, R-factor, pAQ1 S.typhimurium TA1535 his-, rfa, AuvrB his = histidine-free rfa = cause partial loss of the lipopolysaccharide wall which increases Page 6 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, Na 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: 0( 3 Web site: www.sbrtc.com E-mail: 163.com Lab Ns L 1538 SBRTC-2015-1052-6 A uvrB R-factor pAQ1 permeability of the cell to large molecules (i.e crystal violet inhibition) = deficient DNA excision --repair system (i.e. ultraviolet sensitivity) = contain plasmid PKM101 (i.e. ampicillin resistance) = contain plasmid pAQI (i.e. tetracycline resistance) Metabolic Activation: The S9 homogenate was purchased from School of Public Health, FuDan University, China. Just prior to use, the S9 homogenate was mixed with a buffer containing 0.4M MgCl2/1.65M KCI, 1.0M Glucose-6-phosphate, 0.1M NADP, 0.2M sodium phosphate buffer, and sterile purified water. Preparation of Tester Strains: Cultures of Salmonella Typhimurium, TA97, TA98, TA100, TA ' 02 and TA1535 were inoculated to individual Erlenmeyer flasks containing oxoid broth. The inoculate broth cultures were incubated at 37 C in an incubator shaker operating at 120 rpm for 10 hours. Negative Controls: Negative Controls were tested with each tester strain to determine the spontaneous reversion rate. Each strain was tested with and without S9 activation. These data represented a base rate to which the number of revertant colonies that developed in each test plate was compared to determine whether the test article had significant mutagenic properties. Positive Controls: Positive controls were used to demonstrate that the tester strains were sensitive to mutation to wild type state. Standard Plate Incorporation Assay: Separate tubes containing 2 ml of molten top agar supplemented with histidine-biotin solution for the Salmonella Typhimurium were inoculated with 0.1m1 of culture for each of the five tester strains and 0.1m1 of the test article extract. A 0.5m1 of 10% S9 mix, simulating metabolic activation, was added when necessary. The mixture was poured on to Vogel-Bonner E agar plates in triplicate. Parallel testing was also conducted with a negative control and six positive controls. The plates were incubated at 37C for 48h. Following incubation period, the revertant colonies on each plate were recorded. The mean number of revertants from triplicate testing was determined for the negative controls, the test article extracts and the positive controls. The mean number of revertants of the test plates was compared to the mean number of revertants of the negative control for each of the five tester strains employed. Evaluation and Statistics: For the test article extract to be evaluated as a test failure or "potential mutagen" there must have been a two-fold or greater increase in the number of mean revertants over the means obtained from the negative controls for any or all five tester strains. The mean revertant number of each positive control must have exhibited at least a 3-fold increase over the respective negative control mean of the Salmonella tester strain employed, the negative control results of each tester strain exhibited a characteristic number of spontaneous revertants based on historical data collected at SBRTC. Page 7 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, Ng. 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: Web site: www.sbrtc.com E-mail0: ( 3163.com Lab N2 L 1538 SBRTC-2015-1052-6 100% extract Negative control Positive control x SD 1117 11314 226 282 13611 14612 30218 30923 112 x SD 1068 11511 21+2 263 11816 1356 29225 31030 73 x SD 1123118 118779 41236 77959 1259100 1282145 1382105 1479142 STANDARD PLATE INCORPORATION ASSAY RESULTS x SD: Mean of triplicate plates Standard deviation TABLE III STANDARD PLATE INCORPORATION ASSAY RESULTS 44963 124 93 54067 DMSO 12.5% extract 25% extract 50% extract 100% extract Negative control Positive control x SD -- x SD x SD -- x SD xSD -- x SD TA97 - S9 + S9 1078 11411 11113 1208 11413 11817 1159 11913 10412 11811 1123118 118779 Salmonella typhimurium Tester Strains TA98 TAloo - S9 + S9 - S9 +S9 193 273 1238 14115 TA, 02 - S9 + S9 28621 29027 21+3 255 141+17 14615 29823 32127 285 304 12813 12314 29729 29323 205 173 263 252 11211 11913 12517 28526 f 12115 27621 30127 29626 41236 77959 1259100 1282145 1381+105 1479142 x SD: Mean of triplicate plates Standard deviation TAI 535 - S9 + S9 73 103 72 83 94 114 73 82 63 i 103 44963 54067 Results and conclusions apply only to the test article tested. No further evaluation of these results is made by Shanghai Biomaterials Research & Test Center. CONCLUSION Under the conditions of this assay, the test article extract was considered to be nonmutagenic to Salmonella typhimurium tester strains TA97, TA98, TA100, TA102 and TAI 535. The negative and positive controls performed as anticipated. RECORD STORAGE All raw data pertaining to this study and a copy of the final report are to be stored in the designated archive files at Shanghai Biomaterials Research & Test Center. Page 9 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, No 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: 0 13 Web site: www.sbrtc.com E-mail: 163.com