Document 2qpOkYKOQwEZkJ5QodKxZQb7R

Ad<3.>-- noi TOXIKON FINAL REPORT: 01-7019-G1 CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T-6889.7) Author Devaki Sadhu, Ph.D. Final Report Date: March 28,2002 MANAGEMENT OF THE STUDY Performing Laboratory Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730 Sponsor: 3M 3M Center 220-2E-02 Saint Paul, MN 55144 Ni rcc~~r, vroo ac aac, M*3 o ' en CONTAIN NO C 000003 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 Title Page TABLE OF CONTENTS Table o f Contents Study Summary Quality Assurance Statement Study Director Signature and Vrification Dates 1.0 Purpose 2.0 References 3.0 Compliance 4.0 Identification o f the Test and Control Articles 5.0 Identification of the Test System 6.0 Justification of Test System and Route of Administration 7.0 Experimental Design 8.0 Dosage 9.0 Evaluation Criteria 10.0 Results 11.0 Conclusion 12.0 Records 13.0 Confidentiality Agreement T ab let Table H: Table HI: Table IV: Table V: Table VI: Table VU: Parallel Cytotoxicity Assay Parallel Cloning Efficiency (Non-Activated) Parallel Cloning Efficiency (Activated) HGPRT Mutagenesis Assay (Non-Activated) HGPRT Mutagenesis Assay (Activated) Summary o f Mutant Colony Frequency in HGPRT Assay (Non-Activated) Summary o f Mutant Colony Frequency in HGPRT Assay (Activated) s Page 2 of 18 000004 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 STUDY SUMMARY The test article, Ammonium Perfluorooctanoate (FL-143), did not induce mutations at the HGPRT locus as evidenced by the absence of a statistically significant increase in the number o f mutant colonies at the highest analyzable test concentration as compared to the negative controls in the presence or absence o f an exogenous mammalian metabolic activation system. The statistically significant increase in the number of mutant colonies in positive controls in both the activated and non-activated assays verified the proper fimctioning of the test system. Therefore, based on the evaluation criteria of the protocol, the test article is considered non-mutagenic, under the experimental conditions employed. Page 3 of 18 OOOoOS Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 QUALITY ASSURANCE STATEMENT This study was conducted in compliance with U.S. Food and Drug Administration regulations set forth in 21 CFR, Part 58. The sections of the regulations not performed by or under the direction of Toxikon Corporation, exempt from this Good Laboratory Practice Statement, included characterization and stability o f the test article and its mixture with carriers, 21 CFR, Parts 58.105 and 58.113. The Quality Assurance Unit conducted inspections on the following dates. The findings wore reported to the Study Director and to Toxikon's Management. INSPECTIONS DATE OF INSPECTION DATE REPORTED DATE REPORTED STUDY DIRECTOR MANAGEMENT DOSING . 01/02/02 CELL COUNTING 02/07/02 RAW DATA 03/28/02 FINAL REPORT 03/28/02 01/02/02 02//07/02 03/28/02 03/28/02 01/02/02 02/07/02 03/28/02 03/28/02 Mansi Desa^ B.Sc. Quality Assurance 0% Date Page 4 o f 18 000r>06 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES This study met with the technical requirements o f the protocol. The study also met with the requirements of the Good Laboratory Practice Regulations, 21 CFR, Part 58, with the exemptions noted in the QA Statement. Protocol Number: 3MC/VTTRO/003-01/000 Study Director: Devaki Sadhu, Ph.D. Company: Toxikon Corporation 1 !L (aSignature: Date: 0 jttft Study Supervisor: Leigh Waugh-Cohen, B.A. VERTFTCATTQN.DATES: The study dates were as follows: Protocol Effective Date: Test Article ReceiptProject Log Date: Technical Initiation: Technical Completion: Final Report Date: 12/12/01 12/12/01 12/12/01 02/05/02 02/20/02 03/28/02 Page 5 o f 18 0Q007 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 1.0 PURPOSE The CHO/HGPRT Forward Mutation Assay evaluated the mutagenic potential o f a test article via its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. The CHO assay system utilized toxic purine analogs to select for resistant cells that are presumed deficient in the purine salvage enzyme HGPRT. 2.0 REFERENCES The test was conducted based upon the following references: 2.1 Biological Evaluation of Medical Devices - Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity, ANSFAAM3/ISO 10993-3:1993. 2.2 OECD Guidelines for the testing o f Chemicals, "Genetic Toxicology: Tn vitro Mammalian Cell Gene Mutation Test," Test Guideline # 476, current version. 2.3 Hsie, A.W., Casciano, D.A., Couch, D.B., Krahn, D.F., O'Neill, J.P., Whitefield, B.L. "EPA's Gene Tox Program," Mutation Research, 86:193-214 (1981). 3.0 COMPLIANCE The present study conformed to all applicable laws and regulations. Specific regulatory requirements included the current FDA, .21 CFR, Part 58 - Good Laboratory Practice for Nonclinical Laboratory Studies. 4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES The following information was supplied by the Sponsor wherever applicable. Confidential information does not apply. The Sponsor was responsible for all test article characterization data as specified in the GLP regulations. 4.1 Test Article: Ammonium Perfluorooctanoate (FL-143) CAS/Code #: 3825-26-1 / (T-6889.7) Lot/Batch#: Lot 332 Physical State: Solid Color: White / Crystal Density: Not Supplied by Sponsor (N/S) pH: 4 - 7 Stability: Stable Solubility: >lg/mL Storage Conditions: Room Temperature, -20C or 4C Safety Precautions: Standard Toxikon Laboratory Safety Precautions Page 6 o f 18 000008 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 4.2 Control Articles - (Toxikon Supplied) 4.2.1 Negative Control Article Name: Ham's F-12 Complete Medium QC Inventory #: LPR-01-11-001-CC Physical State: Liquid Color: Colorless Storage Conditions: Room Temperature Safety Precautions: Standard Laboratory Safety Precautions 4.2.2 Positive Control Article Name (Non-Activated Assay): Ethylmethanesulfoxide (EMS) QC Inventory #: CSC-01-03-004-CC Physical State: Liquid Color: Clear Storage Conditions: 42C Safety Precautions: Standard Laboratory Safety Precautions 4.2.3 Positive Control Article Name (Activated Assay): Dimethylbenzanthracene (DMBA) QC Inventory #: CSC-97-09-013-CC Physical State: Liquid Color: Clear Storage Conditions: 42C Safety Precautions: Standard Laboratory Safety Precautions 5.0 IDENTIFICATION OF THE TEST SYSTEM Chinese Hamster Ovary (CHO) cells utilized in this assay were obtained from the American Type Culture Collection, Manassas, Virginia. The cells were derived from an ovarian biopsy o f a Chinese hamster. 6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 6.1 This assay evaluated the mutagenic potential o f a test article via its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. This locus is responsible for production of HGPRT, an enzyme that is presort in normal cells and allows cells to salvage hypoxanthine and guanine from the culture medium in order to synthesize DNA. In normal cells, when a toxic substance called 6-thioguanine is included in the growth medium, it is salvaged by the HGPRT enzyme along with hypoxanthine and guanine and incorporated into DNA, thereby causing cell death. Exponentially growing cells, sensitive to die toxic effects o f 6-thioguanine, were exposed to a various concentrations of the test material. If the test material is potentially mutagenic, normal cells (which can utilize hypoxanthine, guanine and 6-thioguanine) mutate to become incapable o f utilizing hypoxanthine, guanine, or 6-thioguanine from the culture medium. However, mutant cells retain their ability to grow as well as normal cells in culture medium because DNA synthesis is made possible by alternate synthetic pathways. Taken together, these findings indicate that the basis for selection of HGPRT mutants is the lack of any ability to utilize the toxic 6-thioguanine. Therefore, cells that grow to form colonies in the presence o f 6-thioguanine are assumed to have undergone mutation either spontaneously or by exposure to the test material. Page 7 of 18 000009 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 The CHO/HGPRT Assay has been used extensively in the detection of mutagenic activity of a wide range of chemical classes. 6.2 The test article was administered in vitro, through a solvent compatible with the test system, per Sponsor's specification. This was the optimal route of administration available in this test system. 7.0 EXPERIMENTAL DESIGN 7.1 Cell Line The CHO-K1 cell line was selected for its high cloning ability and rapid doubling time. It was not necessary to select the cell cultures to reduce the background frequency o fHGPRT mutants. 7.2 Maintenance o f CHO Cells The CHO cells were grown in complete Ham's F-12 medium, buffered with 10 mM HEPES. Complete medium consisted of Ham's F-12 medium containing 10% fetal bovine serum (FBS), 1-2 mM L-glutamine, 100-units/mL penicillin, and 100-pg/mL streptomycin. Incomplete medium was serum-free complete medium. The cells were incubated at 371C, 51% CO2, and saturated humidity. 7.3 Forward Mutation Assay 7.3.1 Metabolic Activation System (S9): . The S9 microsomal fraction was prepared from Sprague Dawley rat livers induced with AroclorR 1254. The S9 rat liver homogenate was purchased from Molecular Toxicology Inc (157 Industrial Park drive, Boone, NC 28607) and stored at -8Q5C until use. A combination o f S9 fraction, isocitric cofactors and incomplete medium was prepared just before exposure and used as the metabolic activation system. The cofactor mixture consisted o f isocitrate (trisodium salt) and NADP (disodium salt) at a final concentration o f 4.5 mg/mL and 2.4 mg/mL, respectively. The S9 fraction was added to each flask at a concentration o f20 pL/mL. 7.3.2 Parallel Cytotoxicity Assay A parallel cytotoxicity assay was performed along with the Mutation assay. The cultures were treated exactly as the mutation plates, incubated for 9 days following the removal o f the test article, fixed, stained, and colonies counted. 7.3.3 Preparation of Test Cultures: Aijproximately 24 hours prior to exposure, duplicate 100 mm plates were seeded at a density o f 1 x 106cells/dish and triplicate parallel cytotoxicity plates were seeded at 200 cells/60 mm dish. 7.3.4 Exposure Periods: The cells were exposed to the test article for 17 hours in the non-activated assay and 5 hours in the activated assay. After exposure, cells were washed twice with PBS and supplemented with complete medium. OOOOlO Page 8 of 18 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 7.3.5 Phenotype Expression: Approximately 48 hours following termination of non-activated exposure period and approximately 64 Vz hours following termination of activated exposure period, cells were trypsinized, counted, and plated at 1 x 106 cells per 100 mm dish. The cells were passed every 48-72 hours to maintain exponential growth during phenotypic expression for 6 days. 7.3.6 Selective Growth: Following the phenotypic expression period, cells were grown in selective medium to select for mutant cells. The selective medium included hypoxanthine-free Ham's F-12 with 10% fetal bovine serum, penicillin-streptomycin (P/S) and 10 pM 6-thioguanine. Ten dishes (five from each duplicate) were used for each test condition. Cell density was 2 x 105/100 mm petri dish. The cultures were incubated for 9 days to allow colonies to develop. 7.3.7 Parallel Cloning Efficiency (PCE): Concurrently, the cloning efficiency was determined by plating cells in selective medium without 6-thioguanine. Six dishes were seeded for each concentration (three from each duplicate) at 200cells/60 mm dish. The cultures were then incubated for 9 days to allow colony formation. 7.3.8 Termination: At the end of the incubation period, plates were rinsed with PBS, fixed in methanol and stained with Giemsa. Only colonies with 50 or more cells were counted. 7.4 Control Articles 7.4.1 Positive Control Article: The positive control for the non-activated system was Ethylmethanesulfoxide (EMS). For the activated system, Dimethylbenzanthracene (DMBA) was used as the positive control. 7.4.2 Negative Control Article: Ham's F-12 Complete Medium served as the negative control article for the non-activated and activated assays. 8.0 DOSAGE 8.1 The test article was dissolved in the culture medium at a concentration of 5 mg/mL as per OECD guidelines. 1:128, 1:256 and 1:512 dilutions o f 5 mg/mL concentration o f the test article were used for dosing. 8.2 Dose Selection 8.2.1 A cytotoxicity preliminary dose-range finding was performed using several dilutions of the 5 mg/mL test article and 1:128, 1:256 and 1:512 dilutions were selected for testing. The positive control for the non-activated assay, EMS, was used at a concentration of 244 pg/mL. The positive control for the non-activated assay, DMBA, was used at a final concentration of 9 pg/mL. Page 9 of 18 0OOO13. Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 9.0 EVALUATION CRITERIA 9.1 Evaluation of Test Results The results o f the CHO/HGPRT Locus Mutation Assay were evaluated based on the number of TG-resistant mutants per 1 x 106 surviving cells. The significance o f the test results were determined by using the statistical program, Tallarida, R.S. and R.B. Murray's Pharmacological Calculations Procedure, ANOVA (analysis o f variance) and Newman- Keuls Test for confirmation This statistical method determined if there was a significant (p < 0.05) increase in the mutation frequency o f the test article compared to the negative control article. The test article was considered to have caused a positive response in the assay if the test article had exhibited a reproducible and statistically significant increase in the number o f mutants per 1 x 106 cells over its concurrent negative control article. 10.0 RESULTS A preliminary Range Finding Assay was conducted to determine the dilutions o f the test article to be used in the Forward Mutation Assay. Exposure to dilutions of the test article resulted in cytotoxicity ranging from 50% cell survival at 1:128 and 80% greater at 1:256 and 1:512. Based on these findings, three dilutions ranging from 1:128 to 1:512 were utilized in the Forward Mutation Assay. The parallel cytotoxicity assay indicated that the positive control article exhibited a cell survival parentage o f 10% and 45% in the non-activated and activated assays respectively (Table I). The dilutions of the test article extract, 1:128,1:256 and 1:512 exhibited cell survival values o f 105, 99 and 102 respectively in the non-activated assay and 94, 104 and 118% respectively in the activated assay. The negative control article exhibited values of 79 and 110% in the non-activated and activated assays respectively. The test article was tested at the three dilutions (1:128, 1:256, 1:512) established in the Range Finding Assay in non-activated assay. None of the three dilutions tested showed a statistically significant increase in the number o f mutants per 1 x 106 surviving cells as compared to the corresponding negative controls in both the non-activated and activated assays (Tables II-VII). The positive controls did show a statistically significant increase in the number of mutants per 1 x 106 surviving cells as compared to the corresponding negative controls. 11.0 CONCLUSION The test article, Ammonium Perfluorooctanoate (FL-143), did not induce mutations at the HGPRT locus as evidenced by the absence o f a statistically significant increase in the number o f mutant colonies at the highest analyzable test concentration as compared to the negative controls in the presence or absence o f an exogenous mammalian metabolic activation system. The statistically significant increase in the number of mutant colonies in positive controls in both the activated and non-activated assays verified the proper functioning of the test system. Therefore, based on the Page 10 o f 18 000012 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 evaluation criteria o f the protocol, the test article is considered non-mutagenic, under the experimental conditions employed. 12.0 RECORDS 12.1 Original raw data is archived at Toxikon Corporation. 12.2 A copy o f the final report and any amendments is archived at Toxikon Corporation. 12.3 The original final report and a copy o f any protocol amendments or deviations is forwarded to the Sponsor. 12.4 All unused test articles shall be returned to the Sponsor by Toxikon, per sponsor's specification. 12.5 Final reports will not be reproduced except in full, without the written authorization/approval from Toxikon. 13.0 CONFIDENTIALITY AGREEMENT Statements of confidentiality were as agreed upon prior to study initiation. Page 11 o f 18 000013 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 Table I: Parallel Cytotoxicity Assay (M utagen Assay) Dilution of E x tra ct Neeative Positive OEMS1 1:128 1:256 1:512 M etabolic Activation Non-Activated Non-Activated Non-Activated Non-Activated Non-Activated T otal# of Foci 147 155 170 10 12 35 232 194 201 187 202 202 200 213 199 Average # Foci Der Plate 157 19 209 197 204 Percentage Survival* 79 10 105 99 102 Negative Positive (DMBA 1:128 1:256 1:512 Activated Activated Activated Activated Activated 222 208 230 95 90 87 182 184 200 194 232 198 232 240 237 220 90 188 208 236 110 45 94 104 118 * Percentage Survival^ Average colonies per plate / number of cells plated (200 cells) x 100 % Page 12 o f 18 000014 ixikon Corporation CHO/HGPRT Forw ard Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 TABLE II PARALLEL CLONING EFFICIENCY NON-ACTIVATED NEGATIVE CONTROL (MEDIUM) # COLONIES/PLATE SURVIVING 1 2 3 4 5 6 FRACTION* 70 80 106 103 117 110 0.488 POSITIVE CONTROL (EMS) 20 19 8 8 5 7 0.056 TEST ARTICLE EXTRACT 79 98 91 89 87 75 (1:128) 0.433 TEST ARTICLE EXTRACT 94 99 112 91 116 102 (1:256) 0.512 TEST ARTICLE EXTRACT 109 84 93 116 117 72 (1:512) 0.493 AVERAGE COLONIES 98 11 87 102 99 * SURVIVING FRACTION" AVERAGE COLONIES PER PLATE/NUMBER OF CELLS PLATED (200 CELLS/PLATE) Page 13 o f 18 000015 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number. 01-7019-G1 TABLE III PARALLEL CLONING EFFICIENCY ACTIVATED NEGATIVE CONTROL (MEDIUM) # COLONIES/PLATE 12 3 45 116 113 91 87 101 SURVIVING 6 FRACTION* 106 0.512 POSITIVE CONTROL (EMS) 6 10 15 12 2 13 0.048 TEST ARTICLE EXTRACT 76 72 71 72 96 93 (1:128) 0.400 TEST ARTICLE EXTRACT 101 (1:256) 95 108 79 77 106 0.472 TEST ARTICLE EXTRACT 108 75 (1:512) 112 76 103 113 0.489 AVERAGE COLONIES 102 10 80 94 98 * SURVIVING FRACTION" AVERAGE COLONIES PER PLATE/NUMBER OF CELLS PLATED (200 CELLS/PLATE) Page 14 of 18 000016 Toxikon Corporation CHO/HGPRT Forward Mutation Assay - ISO (T-6889.7) Project Number: 01-719-G1 TREATMENT NEGATIVE (ACTUAL) NEGATIVE (NORMALIZED)!- POSITIVE (ACTUAL) POSITIVE (NORMALIZED)!- TEST ARTICLE EXTRACT (1:128 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)!- TEST ARTICLE EXTRACT (1:256 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)!- TEST ARTICLE EXTRACT (1:512 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)!- TABLE IV HGPRT MUTAGENESIS ASSAY (NON-ACTIVATED) # MUTANT COLONIES/PLATE 1 2 3 4 5 6 7 8 9 10 1 10 0 0 0 0 1 1 0 2.05 2.05 0.00 0.00 0.00 0.00 0.00 2.05 2.05 0.00 40 39 33 716.42 698.51 591.04 54 967.16 47 12 12 841.79 214.93 214.93 19 14 340.30 250.75 11 197.01 000 0 02 1 10 1 0.00 0.00 0.00 0.00 0.00 4.62 2.31 2.31 0.00 2.31 100 0 110 2 0 0 1.95 0.00 0.00 0.00 1.95 1.95 0.00 3.91 0.00 0.00 0 00 2 0 1 1 0 0 0 0.00 0.00 0.00 4.06 0.00 2.03 2.03 0.00 0.00 0.00 + NORMALIZED MUTATION FREQUENCY" NORMALIZED TO 1 X 10( CELLS PER PLATE BASED UPON CORRESPONDING SURVIVAL FRACTION (SEE TABLE D) Page 15 of 18 000017 Toxikon Corporation CHO/HGPRT Forward Mutation A tiay - ISO (T-6889.7) Project Number: 01-7019-G1 TREATMENT NEGATIVE (ACTUAL) NEGATIVE (NORMALIZED)+ POSITIVE (ACTUAL) POSITIVE (NORMALIZED)* TEST ARTICLE EXTRACT (1:128 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)* TEST ARTICLE EXTRACT (1:256 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)!- TEST ARTICLE EXTRACT (1:512 ACTUAL) TEST ARTICLE EXTRACT (NORMALIZED)!- TABLE V HGPRT MUTAGENESIS ASSAY (ACTIVATED) # MUTANT COLONIES/PLATE 1 2 3 4 5 6 7 8 9 10 00 1 1 110 00 0 0.00 0.00 1.95 1.95 1.95 1.95 0.00 0.00 0.00 0.00 45 32 26 931.03 662.07 537.93 22 455.17 20 18 413.79 372.41 15 36 31 29 310.34 744.83 641.38 600.00 200 2 10 0 30 0 5.00 0.00 0.00 5.00 2.50 0.00 0.00 7.50 0.00 0.00 001 0 20000 2 0.00 0.00 2.12 0.00 4.24 0.00 0.00 0.00 0.00 4.24 1 10 0 0 10 0 1 0 2.04 2.04 0.00 0.00 0.00 2.04 0.00 0.00 2.04 0.00 + NORMALIZED MUTATION FREQUENCY" NORMALIZED TO I X 10* CELLS PER PLATE BASED UPON CORRESPONDING SURVIVAL FRACTION (SEE TABLE III) Page 16 of 18 00Q018 Toxikon Corporation CHO/HGPRT Forward M uta lion Assay - ISO (T-6889.7) Project Number. 01-7019-G1 TABLE VI SUMMARY OF MUTANT COLONY FREQUENCY IN HGPRT ASSAY (NON-ACTIVATED ASSAY) TREATMENT NEGATIVE CONTROL POSITIVE CONTROL TEST ARTICLE (1:8) TEST ARTICLE (1:16) TEST ARTICLE (1:32) TOTAL# TOTAL# PLATES FOCI 10 4 AVERAGE FOCI PER PLATE 0.4 AVERAGE FOCI* PER PLATE (NORMALIZED) 0.82 10 281 28.1 503.28 10 5 0.5 1.16 10 5 0.5 0.98 10 4 0.4 0.81 * NORMALIZED TO 1 X 10* CELLS PER PLATE BASED UPON THE CORRESPONDING SURVIVAL FRACTION (SEE TABLE H) Page 17 o f 18 000019 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 TABLE VH SUMMARY OF MUTANT COLONY FREQUENCY IN HGPRT ASSAY (ACTIVATED ASSAY) TREATMENT NEGATIVE CONTROL TOTAL# TOTAL# PLATES FOCI 10 4 AVERAGE FOCI PER PLATE 0.4 AVERAGE FOCI* PERFLATE (NORMALIZED) 0.78 POSITIVE CONTROL 10 274 27.4 566.90 TEST ARTICLE (1:8) 10 8 0.8 2.00 TEST ARTICLE (1:16) 10 5 0.5, 1.06 TEST ARTICLE (1:32) 10 4 0.4 0.82 * NORMALIZED TO I X 10*CELLS PER PLATE BASED UPON THE CORRESPONDING SURVIVAL FRACTION (SEE TABLE HI) Page 18 of 18 000020 12-2001 MED 12:32 PM TOXI KON FAX NO. 7812711133 P. 02/12 TOXIKON TEST PROTOCOL FDA GLP GUIDELINES CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T-6889.7) TfVXmON PR O TO rm NTHVTRKR13MC/VITRO/003-01/000 p r o t o c o t -d a t Ri i 2/03/01 KFFRCTIVF. DATR- 12/12/01 COMPLIANCE 2 1 CFR, Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies MANACtKMRNT o f t h e s t u d y Performing Laboratory: Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730 Sponsor 3M Company 3M Center Corporate Toxicology Building 0220-02-E-02 S i Paul, MN 55144 000021 DEC-_l_2-201 WED 12:32 PM TOXIKON Toxikon Corporation CHO/HGPRT Forw ard M utation Assay - ISO (T-6889.7) Protocol Num ber: 3M C/VTTRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 FAX HO. 7812711133 PROTOCOL ACCEPTANCE Devaki Sadhu, Ph.D . Study Director Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730 Qj j u Quality Assuz Toxikon Coipotafion 15 Wiggins Avenue Bedford, MA 01730 1 <y Sponsor's Representative 3M Company 3M Center Corporate Toxicology Building 0220-02-E-02 St. Paul, MN 55144 |x | l* .| ) Date Date P. 03/12 * Page 2 of 11 DEC-12-2001 WED 12:32 PM TOXI KON Toxikan Corporation CHO/HGPRT Forward M utation A ssay - ISO (T-6889.7) Protocol N um ber 3MC/VTTRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 FAX NO. 7812711133 TABLE OF CONTENTS Title Page Protocol Acceptance Table of Contents LO Purpose 2.0 References 3.0 Compliance 4.0 Identification of Test and Control Articles 5.0 Identification o f Test System 6.0 Justification o f the Test System and Route o f Administration 7.0 Experimental Design 5.0 Dosage 9.0 Evaluation Criteria 10.0 Records 11.0 Confidentiality Agreement 12.0 Protocol Amendments/Deviations P. 04/12 Page 3 o f11 000023 DEC-12-2001 WED 12_32 PM TOXIKON Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Protocol Number; 3M C/VITRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 1.0 PURPOSE FAX NO. 7812711133 P. 05/12 The CHO/HGPRT Forward Mutation Assay (with Confirmation) evaluates the mutagenic potential o f a test article via its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. The CHO assay system utilizes toxic purine analogs to select for resistant cells that are presumed deficient in the purine salvage enzyme HGPRT. The gene mutations are induced at the HGPRT locus in cultured Chinese hamster ovary (CHO) cells. 2.0 REFERENCES The test will be conducted based upon the following references: 2.1 Biological Evaluation of Medical Devices-Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity, ANSI/AAMI/SO 10993-3: 1993. 2.2 OECD Guidelines for the testing o f Chemicals, "Genetic Toxicology: Tn vitro Mammalian Cell Gene Mutation Test," Test Guideline # 476, current version. 2.3 Hsie, A.W., Casctano, D.A., Couch, D.B., Krahn, D.F., O'Neill, J.P., Whitefield, B.L. "EPA's Gene Tox Program," Mutation Research, 86: 193-214 (1981). 2.4 Extraction procedures, if applicable, will be based upon the standard titled Biological Evaluation o f Medical Devices-Part 12: Sample Preparation and Reference Materials, EN/ISO 10993-12 (1997). 3.0 COMPLIANCE The study will conform to all applicable laws and regulations. Specific regulatory requirements include the current Good Laboratory Practice for Nonclinical Laboratory Studies, FDA, 21 CFR, Part 58. 4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES The following information will be supplied by the Sponsor on a test requisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor will be responsible for all test article characterization data as specified in the GLP regulations. Test and control articles (exclusive o f extracts) that are mixed with carriers require verification of concentration, homogeneity and stability. Samples o f test and control article mixtures will be returned to the Sponsor for characterization and verification, wherever applicable. Page4 of li 000024 DEC-12-2001 WED 12:32 PM TOXIKON FAX NO. 7812711133 Toxikon Corporation CHO/HGPRT Forward M utation A ssay - ISO (T-6889.7) Protocol Num ber: 3M C/VIXRO/002-0I/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 4.1 Test Article; To Be Determined (TBD) Lot/Batch #: CAS/Code#: Physical State: Color: Density: pH: Stability: Solubility: Expiration Date: Storage Conditions: Safety Precautions: 4.2 Control Articles (Toxikon Supplied): 4.2.1 Negative Control Article Name: TBD Toxikon QC #: TBD Physical State: Liquid Color: Colorless Storage: Room Temperature Safety Precautions: Standard Laboratory Safety Precautions 4.2.2 Positive Control Articles: 4.2.2.1 Positive Control Article Name: TBD Toxikon QC #: TBD Physical State: Color: Storage: Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions 4.2.2.2 Positive Control Article Name: TBD Toxikon QC#: TBD Physical State; Color: Storage: Safety Precautions: Known Mutagen. Appropriate Laboratory Safety Precautions P. 06/12 4 Page 5 o f 11 Q000&5 DEC-12-2001 WED 12:33 PH TOXIKON FAX NO. 7812711133 P. 07/12 Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO {T-^889.7) Protocol Number: 3M C/VTRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 5.0 IDENTIFICATION OF THE TEST SYSTEM Chinese Hamster Ovary (CHO) cells utilized in this assay are obtained from the American Type Culture Collection, Rockville, Maryland. The cells are derived from an ovarian biopsy o f a Chinese hamster. 6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 6.1 This assay evaluates the mutagenic potential o f a test article via its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus. This locus is responsible for production ofHGPRT, an enzyme that is present in normal cells and allows cells to salvage hypoxanthine and guanine from the culture medium in order to synthesize DNA. In normal cells, when a toxic substance called 6-thioguanine is included in the growth medium, it is salvaged by the HGPRT enzyme along with hypoxanthine and guanine and incorporated into DNA, thereby causing cell death. Exponentially growing cells, sensitive to the toxic effects o f 6-thioguanine, are exposed to several concentrations of the test material. If the test material is potentially mutagenic, normal cells (which can utilize hypoxanthine, guanine and 6-thioguanine) mutate to become incapable o f utilizing hypoxanthine, guanine, or 6-thioguanine from the culture medium. However, mutant cells retain their ability to grow as well as normal cells in culture medium because DNA synthesis is made possible by alternate synthetic pathways. Taken together, these findings indicate that the basis for selection o fHGPRT mutants is the lack o f any ability to utilize the toxic 6thioguanine. Therefore, cells that grow to form colonies in the presence o f 6-thioguanine are assumed to have undergone mutation either spontaneously or by exposure to the test material. The CHO/HGPRT Assay has been used extensively in the detection of mutagenic activity of a wide range o f chemical classes. 6.2 The test article will be administered in vitro, directly or through a solvent compatible with the test system. These are the only routes o f administration available in this test system. 7.0 EXPERIMENTAL DESIGN 7.1 Cell Line The CHO-K1 cell line is selected for its high cloning ability and rapid doubling time. To reduce the negative control article frequency o f HGPRT' mutants to as low as possible, cell cultures can be selected against this phenotype and returned to normal growth medium for three or more days before use. 7.2 Maintenance o f CHO Cells The CHO cells will be grown in complete Ham's F-12 medium, buffered with 10 mM HEPES. Complete medium will consist o f 10% fetal bovine serum (FBS), 1-2 mM L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin. Incomplete medium is serum-free complete medium. The cells will be incubated at 371C, 51% CO2, and saturated humidity. Page 6 o f 11 pooo?e DEC-12-2001 WED 12:33 PM TOXIKON FAX HO. 7812711133 P. 08/12 Toxikon Corporation CHO/HGPRT Forward M utation A ssay - ISO (T-6S89.7) Protocol Number: 3M C/VITRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 7.3 Cytotoxicity Assay The Cytotoxicity Assay (if necessary) is conducted without metabolic activation, prior to the Mutation Assay. The highest concentration is based on maximum solubility, 10 mg/ml, or Sponsor specifications. Liquid test articles will be tested at 100 ul/ml-0.01 ul/ml. Each test substance concentration, negative control and solvent control (if applicable) is tested in triplicate. Cultures are initiated in 60 mm dishes at 200 cells/plate approximately 24 hours prior to dosing. The cultures are exposed to the test article and controls for 15-17 hours. At the end o f the exposure period, dishes are rinsed with medium or Phosphate Buffered Saline (PBS) and incubated for an additional 6-9 days to allow colonies to develop. At the end o f the incubation period, the dishes are fixed in methanol, stained with Giemsa and colonies counted. Relative survival is obtained by comparing the number of surviving colonies for each dose to that o f the negative or solvent control. 7.4 Forward Mutation Assay 7.4.1 Metabolic Activation System (S9) The S9 microsomal fraction is prepared from Sprague Dawley rat livers induced with Aroclor 1254. The S9 rat liver homogenate is stored at -805C until use. A combination o f S9 fraction, isocitric cofactors and incomplete medium is prepared just prior to exposure and used as the metabolic activation system. The cofactor mixture consists of isocitrate (trisodium salt) and NADP (disodium salt) at a final concentration o f 4.5 mg/ml and 2.4 mg/ml, respectively. The S9 fraction is added to each flask or directly to the cofactor mixture at a concentration of 20 ul/ml. 7.4.2 Parallel Cytotoxicity Assay A parallel cytotoxicity assay is performed along with the Mutation assay. The cultures are treated exactly as the mutation plates, incubated for 6-9 days following the removal of the test article, fixed, stained, and colonies counted. 7.4.3 Preparation of Test Cultures Approximately 24 hours prior to exposure, duplicate 100 mm mutation plates are seeded at a density of 5 x 10* cells/dish and triplicate parallel cytotoxicity plates are seeded at 200 celis/60 mm dish. 7.4.4 Exposure Periods The ceils will be exposed to the test article for 15-17 hours (4-6 hours for highly reactive chemicals) in the non-activated assay and 4-6 hours in the activated assay. After exposure, cells are washed at least once with medium or PBS and supplemented with complete medium. Page 7 of 11 000027 DEC-12-2001 WED 12:33 PM T0XIK0N FAX HO, 7812711133 F. 09/12 Toxikon Corporation CHO/HGPRT Forward Mutation A ssay - ISO (T ^889.7) Protocol Num ber. 3MC/VITRO/02-01/00 Protocol Date: 12/03/01 Effective Date: 12/12/01 7.4.5 Phenotype Expression Approximately 24-48 hours following termination o f exposure period, cells are trypsinized, counted, and plated at 1 x 106 cells per 100 mm dish. The cells are passed every 48-72 hours to maintain exponential growth during phenotypic expression for approximately 7-9 days. 7.4.6 Selective Growth Following the phenotypic expression period, cells are grown in selective medium to select for mutant cells. The selective medium includes hypoxanthine-ffee HAM'S F-12 with 10% dialyzed fetal bovine serum, penicillin-streptomycin (P/S) and 10 p. 6-thioguanine. A total of ten dishes (five from each duplicate) are used for each test condition. Ceil density is 2 x 105/ 100 mm petri dish. The cultures are incubated for 6-9 days to allow colonies to develop. 7.4.7 Parallel Cloning Efficiency (PCE) Concurrently, the cloning efficiency is determined by plating cells in selective medium without 6-thioguanine. Six dishes are seeded for each concentration (three from each duplicate) at 200 cells/60 mm dish. The cultures are then incubated for 6-9 days to allow colony formation. 7.4.8 Termination At the end o f the incubation period, plates are rinsed with PBS, fixed in methanol and stained with Giemsa. Only colonies with 50 or more cells are counted. 7.5 Control Articles 7.5.1 Positive Control Article The positive control for the non- activated system is Ethylmethanesulfoxide (EMS) or 4Nitroquinoline-1-oxide (4NQ), For the activated system, 9,10,- Dimethyl-1,2-benzanthracene (DMBA) or Dimethylnitrosamine (DMN) can be used as the positive control. 7.5.2 Negative Control Article The appropriate solvent, extractant or cell culture medium will serve as the negative control article. 7.6 Confirmatory Assay The results o f the Forward Mutation Assay will be confirmed through an independent Confirmatory Assay. The Confirmatory Assay will be performed as described in Sections 7.0 and 8. 0. Page 8 o f 11 000Z8 DEC-12-2001 WED 12:34 PM TOXIKON Toxikon Corporation CHO/HGPRT Forward M utation Assay - ISO (T-6889.7) Protocol N um ber 3MC/VITRO/002-4>1/00 Protocol Date: 12/03/01 E ffective Date: 12/12/01 FAX HO. 7812711133 P. 10/12 8.0 DOSAGE 8.1 Preparation of Test Article 8.1.1 Medical Devices: The test article will be extracted at ratios specified by EN/ISO 10993-12. Extraction vehicles may be one of the following media: serum-free medium, complete medium, or 0.9% USP Sodium Chloride for Injection, USP (NaCl). Extraction conditions will be as specified by Sponsor: (please check desired condition) 1212C for one hour 702C for 24 hours 502C for 72 hours 371C for 72 hours 371 C for 24 hours per Sponsor's directions (______C for _____ hours). Extracts prepared with medium will be tested at 100% (neat) concentration. Extracts prepared with Sodium Chloride will be diluted with 2X medium and tested at 50% extract concentration (considered neat). Modifications to test article preparation will be as specified by the Sponsor. 8.1.2 Solid/Liquid Test Articles Solid test articles will be dissolved or suspended in a vehicle appropriate for the test system or as specified by the Sponsor. Liquid test articles will be administered as received at predetermined concentrations or as specified by the Sponsor. 8.2 Dose Selection 8.2.1 The concentrations for the Mutation Assay are selected based on toxicity information, maximum solubility o f the test article or Sponsor specifications. If necessary, a Cytotoxicity Assay will be performed to determine toxicity. If toxicity is evident, the doses should include the highest concentration which causes a low level of survival (approximately 10%) and four lower doses, including one dose with no apparent cytotoxicity. If no apparent cytotoxicity is observed, the maximum concentration will be based on the limit o f solubility or 10 mg/ml, whichever is lower (or as specified by the Sponsor). Subsequent doses will decrease in approximate half-log increments. In the absence of toxicity, liquid test articles will be tested at 100 ul/ml-0.01 ul/ml. Dose selection modifications will be as specified by the Sponsor. 8.2.2 Medical device extracts will be analyzed at one concentration only (neat extract). Dilutions o f medical device extracts are not analyzed unless otherwise required, in which case the justification shall be indicated in the final report. An initial Cytotoxicity Assay is not performed for device extracts. Page 9 o f 11 O00029 DEC-12-2001 WED 12:34 PM TOXIKON Toxikon Corporation CHO/HGPRT Forward M utation A ssay - ISO (T-6889.7) Protocol Number: 3M C/VITRO/002-01/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 FAX NO. 7812711133 P. 11/12 9.0 EVALUATION CRITERIA The results o f the CHO/HGPRT Locus Mutation Assay will be evaluated on the basis o f the number of TG-resistant mutants per 1 x 106surviving cells. The significance o f the test results will be determined by either one o f the following methods: 9.1 The test results are analyzed using a statistical program such as Tallarida, R.S. and R.B. Murray's Pharmacological Calculations Procedure, ANOVA (analysis of variance) and NewmanKeuls Test for confirmation o f pairwise comparisons. This statistical method determines ifthere is a significant (p < 0.05) increase in the mutation frequency of the test article compared to the negative control article. The results obtained for the mutation frequency at the various dose levels will be analyzed by the method o f Linear Regression using a program such as "Linear Regression I" by R.J. Tallarida and R.B. Murray, (Manual of Pharmacologic Calculations with Computer Programs, Springer-Verlag, New York, 1986, pp 10-13). This will determine if there is a positive dose response. 9.2 The test article is considered to have caused a positive response if the test article shows a statistically significant positive dose response or at least one test article dose shows a reproducible statistically significant increase in the number o f mutants per 1 x 10fi surviving cells as compared to the corresponding negative control article. 9.3 Confirmation Assay The confirmation assay will validate the reproducibility o fthe mutagenesis assay. 10.0 RECORDS 10.1 Original raw data will be archived at Toxikon Corporation. 10.2 A copy o f the final report and any amendments will be archived at Toxikon Corporation. 10.3 The original final report and a copy o f any protocol amendments or deviations will be forwarded to the Sponsor. 10.4 All unused test article will be handled as specified in the Test Requisition Form. Otherwise, all remaining test article will be discarded. 11.0 CONFIDENTIALITY AGREEMENT Statements o f confidentiality may be agreed upon prior to study initiation. Page 10 of 11 000030 DEC-12-2001 WED 12:34 PH TOXIKON FAX NO. 7812711133 Toxikoit Corporation CHO/HGPRT Forward M utation A ssay - ISO (T-6889.7) Protocol Number: 3MC/VTTRO/002-<)1/000 Protocol Date: 12/03/01 E ffective Date: 12/12/01 12.0 PROTOCOL AMENDMENTS/DEVIATIONS P. 12/12 All changes to the approved protocol and the reason for the changes will be documented in writing, signed by the Study Director, dated, and maintained with the protocol. No protocol amendments will be made without written approval in the form o f a Sponsor Communication Log between the Sponsor and the Study Director which will be generated as closely as possible to the time of the change. Page 11 of 11 000031