Document zzd49gjLV6V4Z3eZbLdNjQ3Q0

Lab N2 L 1538 SBRTC-20 15-1052-8 TABLE OF CONTENTS SUMMARY 4 INTRODUCTION 5 MATERIALS 5-6 METHODS 6-7 RESULTS 7-9 CONCLUSION 9 RECORD STORAGE 9 PHOTOGRAPH OF THE TEST ARTICLE 10 1 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 1.111113 Web site: www.sbrtc.com E-mail: ,163.com Lab Na L 1538 SBRTC-2015-1052-8 INTRODUCTION The purpose of this study was to evaluate the mutagenic potential of test article extracts based on quantitation of forward mutations at the thymidine kinase locus of L5178Y/Te" Mouse Lymphoma cells in the presence and absence of a metabolic activation system. The test article was received on Jun.16, 2015. The test began on Sep.28, 2015, and ended on Nov.2, 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 N9: 150642 Package Delineation: STERILE Storage Conditions: Room temperature Extraction Vehicles: RPMI-1640 medium < Lot N2.NAF1408> DMSO< Lot N.2.20141118> Test Article Extract: Based the ISO, 6cm2/ml [Surface area of the test article to volume of extraction vehicle],367.3cm2 test article was covered with 61.2m1 RPMI-1640 and 56.5cm2 test article was covered with 9.4m1 DMSO respectively under sterile conditions for preparing the test article extracts at 37C for 72h . The test extracts were then serially diluted with the same vehicle and resulted in the following concentrations: Test A: 25% diluent of the test extraction Test B: 50% diluent of the test extraction Test C: 100% test extraction according to the requirement The extracts were used immediately after extraction. Negative Control: The extraction vehicle without any test article was subjected to conditions identical to which the test article was subjected during its extraction. Positive control: Methylmethansulfonate (MMS) in the absence of S9 (Sigma, LISA) Page 5 of 10 SHANGHAI BIOMATERIALS RESEARCH & TEST CENTER Add: 201, Building 2, NQ 427 Jumen Road, Shanghai, China Zip Code: 200023 TEL: 0086-21-63034903 FAX: 0( 3 Web site: www.sbrtc.com E-mail: 16 3.com Lab I% L 1538 SBRTC-2015-1052-8 cultured for 2 days. Expression of the Mutant Phenotype: During these 2 days, the cultures were counted and adjusted to 2X 105 cells/ml daily for expression of the mutant phenotype. Cultures with less than 2 X 105 cells/ml were not adjusted. After 2 days, the cultures were adjusted to the concentration of 1 X 104 cells/mL by using R20. Part of the cells were prepared for TFT resistance. First, TFT was added to cultures to a final concentration of 3g /mL in 50 mL of the negative control, 50 mL of the test article and positive controls, respectively. Each TFT-treated culture was dispensed at 200L per well onto 96 well-micro-well plates. The rest of the cells were diluted to 8 cells /mL. 200L was placed into each well of the plates. The plates were incubated at 37C in a humidified incubator with 5% CO2 in air for 12 days. Afterwards the plating efficiency was calculated as PE2 Colony Counting After the incubation period, 96 well micro-well plates were counted for the total number of colonies per plate. The number of wells containing colonies was counted by naked eye. The colonies are scored of small and large colonies. The number of negative wells per 96 well micro-well plates will be counted for the survival (PE0), viability (PE2), and mutation (TFT) plates, and then the plating efficiency in both the survival/ viability plates and mutant selection plates is calculated and the relative total growth (RTG) and the mutant frequency (MF) will be determined. Criteria for a Valid Test The following criteria must be met for the mutagenesis assay to be considered valid: Negative and Positive Controls: The range of plating efficiency for the negative control has to be from 60% to 120% for survival (PE0) andfor viability (PE2) plates. The average spontaneous mutant frequency of negative controls with and without S9 mix should ideally be within the range of 35-140x10-6. For the positive control, it must exhibit mutant frequencies >100 x 10-6 mutants over the negative control for the 4 h exposure, or > 300 x 10-6 mutants over the negative control with 40 % small colonies for 24 h exposure. When the MF of the positive controls increases twofold or more over the negative controls, the experimental sensitivity is acceptable. A statistically significant dose-related increase in NW is required for a positive response. RESULTS According to ISO 10993-3 requirement, the range of plating efficiency was relatively constant to be considered valid. The frequency ranges expected from the laboratory's historical control data. There was no concentration-related increase in mutant frequency. The results of the mutagenesis assay were presented in TABLE I -II. Cloning data and total toxicity data for L5178Y/TK +/-mouse lymphoma cells treated with extracts of the test article were shown as follows. 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: OC 3 Web site: www.sbrtc.com E-mail: 1 163.com