Document kDNBG3VqxG7wgZonkVp7E8rwn

7) OECD 473-OPPTS 870.5375, In Vitro Mammalian chromosome aberration (CHO cells), 0623-3110 7 7c18 5 SAN JTlZED DEC 0 9 2003 FINAL REPORT Primedica Redfield Test Article: Potassium Perfluorobutane Sulfonate SITEK Study NO.0623-3110 July 26, 2001 A7. SITEIC R E S E A R CLHABORATORIES 15235 Shady Grove Road, Suite 303, RocltviUe, Maryland 20850 301/9264900 FAX 301/926-8891 FINAL REPORT Studv Title Test for Chemical Induction of Chromosome Aberration in Cultured Chinese Hamster Ovary (CHO) Cells With and Without Metabolic Activation Test Article Potassium Perfluorobutane Sulfonate Author Jing Xu, M.D. Studv Initiation Date July 31, 2000 Studv Completion Date July 26, 2001 Testing Facilitv SITEK Research Laboratories 15235 Shady Grove Road, Suite 303 Rockville, Maryland 20850 Laboratory Project ID Sponsor's Study No. 132-009 SITEK Study NO. 0623-3110 Sponsor Primedica Redfield 100 E. Boone Street Redfield, Arkansas 72132 Page 1 of 87 SITEK Study NO. 0623-3110 GLP COMPLIANCE STATEMENT Study NO. 0623-3110 Sponsor's Test Article I.D. Potassium Perfluorobutane Sulfonate This study was conducted in compliance with the following Good Laboratory Practice standards : United States Food and Drug Administration, Title 21 Code of Federal Regulations Part 58, Revised April 1, 1998. Organisation for Economic Cooperation and Development, The OECD Principles of Good Laboratory Practice, Environment Monograph No. 45 [ENV/MC/CHEM(98)171, Paris 1998, Except for the stability of the test article and control substances under the experimental conditions, which was not determined by SITEK Research Laboratories. The dosing solutions ,j were analyzed by Southern Research Institute, Birmingham, Alabama, however, the analysis was not performed under GLP conditions. Signature Jin#Xu, M.D. Date Study Director 2 SITEK Study NO. 0623-3110 QUALITY ASSURANCE UNIT'S STATEMENT Study NO. 0623-31 10 Sponsor's Test Article I.D. Potassium Perfluorobutane Sulfonate The performance of this study was audited for adherence to the Good Laboratory Practice regulations for nonclinical laboratory studies by the Quality Assurance Unit of SITEK Research Laboratories. In this context, the facilities, equipment, personnel, methods, practices, controls, original data and reports have been inspected as per SITEK's Quality Assurance Unit's Standard Operating Procedures. The information contained within this report accurately reflects the raw data generated from this study. Protocol Review Date: 08/01/00 The following phases were inspected for this study: Inspection c,l Date Phases Inspected Date Findings Reported to Study Director Date Findings Reported to Management 08/22/00 Removal of the Test Article 08/22/00 08/22/00 10/17/00 Workbook Audit 10/ 17/00 10/20/00 10/20/00 Draft Report Audit 10/20/00 10/25/00 7/25/01 Final Report Audit 7/25/01 . 7/25/01 Signature Marcie A . Bauernschub, B.S. Manager, Quality Assurance Unit 3 SITEK Study NO. 0623-3110 STUDY DIRECTOR'S SIGNATURE PAGE This study was performed under the supervision of Jing Xu, M.D., Study Director for In Vitro Cytogenetic Assays, at SITEK Research Laboratories, 15235 Shady Grove Road, Suite 303, Rockville, Maryland 20850. The Final Report on this study was written by the Study Director and released on July 26, 2001. Jing Xu, M.D. Study Director Signature Date 7 b b / 4 SITEK Study NO. 0623-3110 ABSTRACT The test article, Potassium Perfluorobutane Sulfonate, was tested for its potential to induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells with and without exogenous metabolic activation. A Range Finding Test (RFT) was performed to assess the toxicity of Potassium Perfluorobutane Sulfonate to CHO cells in this system. Potassium Perfluorobutane Sulfonate was dissolved in dimethyl sulfoxide (DMSO), therefore, DMSO was used as the solvent. Eight concentrations of the test article were tested ranging from 5.0-5000 pg/mL both with and without metabolic activation. DMSO and water were included in both systems as the solvent and untreated controls, respectively. The treatment time was 3 hours for both the nonactivated and activated systems. Toxicity was assessed by the reduction in the Relative Cell Growth (RCG). Based on the RCG results, Concentrations of 500, 1000, 2500 and 5000 pg/mL were selected in both systems for the definitive Chromosome Aberration Assay (Bl). In the definitive Chromosome Aberration Assay, Potassium Perfluorobutane Sulfonate was tested with concurrent untreated (water only), solvent (DMSO) and positive controls in both systems. Mitomycin-C (MMC), at concentrations of 0.08 and 0.2 pg/mL, was used as the positive ,) control in the non-activated system. Cyclophosphamide (CP), at concentrations of 7.5 and 12.5 pg/mL, was used as the positive control in the activated system. The parallel toxicity was determined by the reduction in the RCG and/or Relative Mitotic Index (RMI). Based on the RCG and RMI results, chromosome aberrations were scored from the three highest concentrations of 1000, 2500 and 5000 pg/mL in both systems. The corresponding untreated, solvent and positive controls (MMC at 0.2 pg/mL and CP at 7.5 pg/mL) were also scored. One hundred (100) metaphases were scored from each of the duplicate cultures at each concentration and the controls. A confirmatory assay (B2) was performed without activation only, since the results from the definitive assay (Bl) were negative in both systems. The procedures followed were the same as in the B1. The concentrations tested were 50, 100, 500, 1000, 2500 and 5000 pg/mL. A continuous treatment up to the harvest time of 1.5 x normal cell cycle time (18 hours) was performed, and the harvest time was 18 hours after the initiation of treatment. The parallel toxicity was determined by reduction in the RCG and/or RMI. Based on the RCG and RMI results, chromosome aberrations from the confirmatory assay were scored from the cells treated with the three concentrations of 500, 1000 and 5000 pg/mL. The corresponding untreated, solvent and positive controls (MMC at 0.2 pg/mL) were also scored. One hundred (100) metaphases were scored from each of the duplicate cultures at each concentration level and the controls. 5 SITEK Study NO. 0623-3110 The data from the definitive and confirmatory assays were statistically analyzed and evaluated. The results indicate that Potassium Perfluorobutane Sulfonate did not induce a statistically significant increase in the percentage of cells with aberrations at any of the concentrations tested, both with and without metabolic activation, in either assay when compared to the solvent controls. Under the conditions of this study and according to the criteria set for evaluating the test results, Potassium Perfluorobutane Sulfonate was negative in the in vitro Chromosome Aberration Assay in CHO cells when tested with and without an exogenous metabolic activation system, and is not considered to be a clastogenic agent. 6 SITEK Study NO. 0623-3110 TABLE OF CONTENTS GLP COMPLIANCE STATEMENT .................................................................... 2 QUALITY ASSURANCE UNIT'S STATEMENT ................................................... 3 STUDY DIRECTOR'S SIGNATURE PAGE .......................................................... 4 ABSTRACT................................................................................................... 5 INTRODUCTION ........................................................................................... 9 MATERIALS ............................................................................................... 10 Test Article Control Substances Indicator Cells Culture Medium Metabolic Activation System ) EXPERIMENTAL PROCEDURES.................................................................... 13 Solubility Test Determination of pH Determination of Osmolality Preparation of Dosing Solutions Preparation of Test Cultures Test System Identification Range Finding Test (A1) Defmitive Chromosome Aberration Assay (B1) Confirmatory Chromosome Aberration Assay (B2) Statistical Analysis CRITERIA FOR A VALID ASSAY ................................................................... 20 EVALUATION OF TEST RESULTS ................................................................. 21 7 SITEK Study NO. 0623-3110 RESULTS .......................................................................................... 22 Solubility Test Determination of pH Osmolality Test Range Finding Test (Al) Definitive Chromosome Aberration Assay (B1) Confirmatory Chromosome Aberration Assay (B2) Statistical Analysis CONCLUSIONS ........................................................................................... 26 ARCHNES ................................................................................................. 26 REFERENCES .......................... ................ ............. 27 APPENDIX I Data Tables ......................................... 29 APPENDIX I1 .................................................. SITEK's Historical Data ......... 38 APPENDIX I11 .................................................................... Study Protocol and Protocol Amendments ......... 47 APPENDIX IV ............................................................................ Dosing Solution Analysis Report ......... 65 8 SITEK Study NO. 0623-3110 INTRODUCTION This study was conducted by Jing Xu, M.D., Keyan Wang, M.D., Dione Washington, B.A. and Weiyu Xie, M.D., at SITEK Research Laboratories from August 9, 2000 to September 25, 2000. The experimental procedures used to perform this study are described in the protocol. The purpose of this study was to evaluate the test article, potassium perfluorobutane salfonate for its potential to cause genetic damage as manifested by induced chromosome aberrations in cultured Chinese hamster ovary (CHO) cells (1-5). 9 SITEK Study NO.0623-3110 MATERIALS TEST ARTICLE 1. Source (Supplier): 2. Name: 3. CAS No.: 4. Lot No.: 5. Physical Appearance: 6. Date Received: 7. Storage Conditions: 8. Purity Information: 9. Expiration Date: Primedica Redfield Laboratories LSponsor) Potassium Perfluorobutane Sulfonate 29420-49-3 White powder June 8, 2000 and September 7, 2000 Room Temperature 100 % 04/06/01 CONTROL SUBSTANCES Positive Controls I Mitomycin-C (MMC), which induces chromosome aberrations in the absence of metabolic activation, was used at 0.08 and 0.2 pg/mL in the non-activated system. Information on the MMC used in this study is provided below: 1. Source: 2. CAS Registry No.: 3. Lot No.: 4. Storage Conditions: 5. Expiration Date: Sigma Chemical Company 50-07-7 48H25 11 1-5C December 10, 2000 Cyclophosphamide (CP), which induces chromosome aberrations in the presence of metabolic activation, was used at 7.5 and 12.5 pg/mL in the activated system. Information on the CP used in this study is provided below: 1. Source: 2. CAS Registry No.: 3. Lot No.: .4. Storage Conditions: 5. Expiration Date: Fluka Biochemika 6055-19-2 29945611 1-5C July 18, 2002 and Jan. 24. 2004 10 SITEK Study NO. 0623-3110 MMC and CP were dissolved in sterile, deionized, distilled water (DDH,O), diluted to 40 pg/mL (MMC) and 1.25 and 1.50 mg/mL (CP), dispensed in small aliquots, and stored at -10 to -20C. One vial of each was thawed just prior to treatment and used in treating the cells. Information on the DDH,O used in this study is provided below: 1. Source: 2. Batch No.: 3. Storage Conditions: 4. Expiration Date: SITEK Research Laboratories 24 1-5C Julv 14, 2001 The stability of MMC and CP, under the experimental conditions, was not determined by SITEK Research Laboratories. However, both substances were used before their expiration dates. Solvent Control DMSO was used to dissolve the test article and to prepare the dosing solutions. Therefore, it was also used as the solvent control. Information on the DMSO used this study is provided below: 1. Source: 2. CAS Registry No.: 3. Lot No.: 4. Storage Conditions: 5. Expiration Date: Fisher 67-68-5 963769 Room Temperature December 12, 2001 INDICATOR CELLS Source The clone CHO-W-B1 of the CHO cell line, used in this study, originated at Litton Bionetics and was obtained by SITEK through the Environmental Health Research and Testing Laboratories, Lexington, Kentucky, in 1988. The doubling time of this cell line is approximately 12 hours, and its modal chromosome number is 21. The karyotype analysis of the cell line is periodically performed and documented at SITEK Research Laboratories. Stock Cultures The CHO cells were propagated in antibiotic-free medium to obtain a sufficient number of cells for freezing a large number of stock ampules. The cells were cryopreserved in McCoy's 5A medium, supplemented with 10%heat-inactivated fetal bovine serum and 8 % DMSO, and stored in liquid nitrogen. Prior to using the stock cultures for the test, 11 SITEK Study NO. 0623-3110 representative ampules were tested for contaminating microorganisms, including mycoplasma, and found free of the above. Cell cultures, initiated from the stock ampules and maintained by subculturing for a maximum of 15 passages, were used in the assays. CULTURE MEDIUM The modified McCoy's 5A medium used in this study was obtained in powder form. The liquid culture medium (SITEK Batch No. 39) was prepared at SITEK Research Laboratories. Information on the medium used in this study is provided below: 1. Source: 2. Lot No.: 3. Storage Conditions: 4. Expiration Date: Gibco BRL 1019960 1-5C April 12. 2001 The fetal bovine serum was obtained from Summit Biotechnology, heat inactivated and used in this study. The antibiotics (penicillin and streptomycin) and the supplement (L- glutamine), used in this study were obtained from Gibco BRL. The lot numbers were 1 recorded in the 0623-3110 study notebook. METABOLIC ACTIVATION SYSTEM The metabolic activation mixture consisted of phenobarbital/ll-naphthoflavone-induced rat liver homogenate (S-9) and the cofactor pool (6). The S-9 was made by SITEK Research Laboratories. Information on S-9 used in this study is provided below: Source S-9 Batch No. Protein Content Expiration Date SITEK 101299 36.8 mg/mL October 12, 2002 Prior to use in the assay, the S-9 was evaluated for its potential to induce an acceptable level of aberrations in CHO cells with CP. Immediately prior to use, the S-9 was thawed at room temperature and mixed with the cofactor pool to form the metabolic activation mixture, which consisted of 4mM NADP, 5 m M glucose-6-phosphate, 30mM KC1, lOmM MgCl,, 50mM sodium phosphate (pH 7.4) and 100 pL/mL of S-9 fraction. This mixture was diluted 1:4 by volume with serum-free medium before refeeding the cultures. 12 SITEK Study NO. 0623-3110 EXPERIMENTAL PROCEDURES SOLUBILITY TEST The test article had previously been tested for its solubility in water (in another study) and was insoluble. Therefore, DMSO was selected as the solvent for the solubility test. One hundred (100) mg of the test article was weighed and DMSO was added in 0.1 mL increments until the test article was dissolved or until 1.5 mL of the solvent had been added. Based on the solubility of the test article in DMSO, the solubility of the test article in culture medium was determined. Twenty five (25) pL of test article at a concentration of 500 pg/mL in DMSO was added to 2.5 mL of culture medium in order to determine the appropriate concentrations to be used for the Range Finding Test. DETERMINATION OF pH To determine the pH of the test article 25 pL of test article at a concentration of 500 mg/mL in DMSO was added to 2.5 mL of complete medium, resulting in a final test article concentration of 5000 pg/mL in medium. If the test article caused a change in color of the (j medium, thereby altering the medium's pH and thus affecting the growth of cells, a buffered medium would have been used in the assay (7,8). DETERMINATION OF OSMOLALITY The osmolalities of the untreated, solvent controls and the highest test article concentration (5000 pglmL) used in treating the cells in the Range Finding Test, were measured using a vapor pressure osmometer. Osmolalities in 500 mOsmol/kg of water and above are considered likely to cause DNA damage and would not be used in the assay (9). PREPARATION OF DOSING SOLUTIONS Dilution schemes were prepared prior to each treatment. The specified amount of test article was measured and dissolved in the required volume of DMSO to reach the highest concentration in the Range Finding Test and Chromosome Aberration Assay. The remaining concentrations were made by subsequent dilution. The dilution of the test article and the preparation of the dosing solutions were done immediately prior to treatment of the cells. The stability of Potassium Perfluorobutane Sulfonate under the experimental conditions and the concentration of the test article dosing solutions were not analytically determined by 13 SITEK Study NO. 0623-3110 SITEK Research Laboratories. However, dosing solution samples (2.0 mL of each concentration) from the definitive and confirmatory Chromosome Aberration Assays (B 1 and B2, respectively) were saved, stored frozen (-10 to -20C) and shipped to the Sponsor on dry ice for analysis. PREPARATION OF TEST CULTURES Stock cultures, growing in T-75 cm2tissue culture flasks in antibiotic-free medium and showing 50-70% confluency, were harvested and used to prepare the test cultures. The culture medium from the T-75 cm2 flasks was discarded, and the cells were washed with Ca++-and Mg++-freephosphate buffered saline (PBS). The cells were then dissociated by incubation at 37 & 1C with 0.05% trypsin. The cells were resuspended in complete culture medium containing 10%HIFBS, 2mM L-glutamine, 50 units/mL of penicillin and 50 pg/mL of streptomycin, the cell suspensions were pooled, and an aliquot of the cell suspension was diluted to the appropriate concentration and counted using a cell counter. Based on the cell counts, a separate cell suspension with lx105 cells/mL was prepared in complete medium. Five (5.0) mL of this suspension was seeded in each T-25 cm2 tissue culture flask to give 5x105cells per flask. These cultures were used in the Range Finding Test and the Chromosome Aberration Assays. The flasks were gassed with a mixture of approxi- 1 mately 5% CO, and 95% air, tightly capped, and incubated for approximately 20-24 hours prior to treatment. TEST SYSTEM IDENTIFICATION All of the test cultures were labeled in indelible ink with the SITEK study number, the activation system, the test article concentrations/controls, code number for the concentrations, A or B for distinguishing between tubes receiving the same treatment, and date of harvest. The slides were labeled with SITEK's study number, the code numbers for the concentrations tested, followed by A or B for the same treatment conditions, and the dates the slides were prepared. RANGE FINDING TEST (Al) In order to determine the toxicity of the test article, a Range Finding Test was performed. Test cultures seeded approximately 24 hours earlier were used in the Range Finding Test. Two replicate cultures were used at each concentration level in both systems. The cells 14 SITEK Study NO. 0623-3110 were treated with concentrations of 5.0, 10, 50, 100, 500, 1000, 2500 and 5000 pg/mL in both the non-activated and activated systems, along with the untreated (water only) and solvent (DMSO) controls. In the non-activated and activated systems, the culture medium was removed from the flasks and 5.0 mL of fresh, complete medium or 5.0 mL of serum-free medium with the S-9 activation mixture were added to each flask, respectively. The cells were exposed to the test article for 3 hours. The medium was then removed, and the cells were rinsed with PBS containing Ca++ and Mg", refed with 5.0 mL of complete medium, and incubated for an additional 15 hours, with 0.1 pg/mL Colcemid present during the final 2 hours. All of the cultures were harvested 18 hours after the initiation of treatment (1.5 x normal cell cycle time). The medium with dividing cells was transferred into labeled centrifuge tubes, and the monolayer of cells was washed with PBS, dissociated with 0.05% trypsin, and resuspended in the collected medium. An aliquot of this cell suspension was counted using an electronic cell counter. The number of cells per flask was calculated for each concentration, and the Relative Cell Growth (RCG) was calculated according to the following formula: RCG = No. Cells in Test Flask X 100 No. Cells in Solvent Flask 1 The cytotoxicity was evaluated on the basis of the reduction in the RCG (10, 11). If possible, a concentration causing greater than 50% reduction in RCG was selected as the highest test concentration for the Chromosome Aberration Assay. In addition, three or more lower concentrations were included in the Assay. If no cytotoxicity was observed at the maximum concentration tested, the Chromosome Aberration Assay was performed at four decreasing concentrations starting with the maximum soluble concentration or one or two concentrations with precipitate. DEFINITIVE CHROMOSOMEABERRATION ASSAY (Bl) The definitive Chromosome Aberration Assay was conducted with a single harvest at 1.5 x normal cell cycle time. Parallel toxicity was assessed by a reduction in the RCG and/or the Relative Mitotic Index (RMI). The slides for the RMI were also used for the determination of chromosome aberrations. The test cultures were prepared as described earlier. Two replicate cultures, seeded with 500,000 cells each approximately 20-24 hours earlier, were treated at each concentration level in the non-activated and activated systems. The cells were treated at concentrations of 500, 1000, 2500 and 5000 pg/mL of the test article in both the non-activated and activated 15 SITEK Study NO. 0623-3110 systems. MMC was used as the positive control at 0.08 and 0.2 pg/mL in the non-activated system, and CP was used at 7.5 and 12.5 pg/mL in the activated system. Untreated and solvent controls were included in each system. In the non-activated and activated systems, the culture medium was removed from the flasks and 5.0 mL of fresh, complete medium or 5.0 mL of serum-free medium with the S-9 activation mixture was added to each flask, respectively. The cells were exposed to the test article for 3 hours. The medium was then removed, and the cells were rinsed with PBS containing Ca++ and Mg", refed with 5.0 mL of complete medium, incubated for an additional 15 hours, with 0.1 pg/mL Colcemid present during the final two hours, and harvested 18 hours after the initiation of the treatment (1.5 x normal cell cycle time). The cells were processed to determine the RCG as described in the Range Finding Test. The remaining cell suspension was processed to determine the RMI as described below. The cells were collected by centrifugation (800 rpm), swelled in hypotonic KC1 (0.075M), and fixed in methano1:glacial acetic acid (3: 1) fixative. The fixed cells were stored at 1-5C. The cells were then collected again by centrifugation, resuspended in a small volume of fresh fixative, and dropped on microslides. The slides were air dried, stained in 5% Giemsa stain, and mounted in Permount using #1 cover glasses. The coded slides were scored for Mitotic Index (MI). A total of 1000 cells were scored from each concentration I (500 from each duplicate flask), and the number of dividing cells were recorded. The MI for each concentration was calculated using the following formula: MI = No. of Dividing Cells from 1000 Cells 10 The RMI was calculated as shown below: RMI = Test Concentration MI X 100 Solvent Control MI Based on the RCG and RMI results, the chromosome aberrations were scored from the 3 highest concentrations of 1000, 2500 and 5000 pg/mL in both the non-activated system and activated system. The corresponding controls were also scored. The concentrations of 0.2 pg/mL for MMC and 7.5 pg/mL for CP were scored from the positive controls in the nonactivated and activated systems, respectively. One hundred (100) metaphases were scored for chromosome aberrations from each of the two duplicate flasks, thus providing 200 metaphases per concentration level. Only cells with 19-23 chromosomes were scored, and the microscope coordinates of each cell with aberrations were recorded. In addition, the number of polyploid and endoreduplicated cells in a total of 100 dividing cells was scored and recorded for each culture. 16 SITEK Study NO. 0623-3110 The types of chromosome aberrations scored and the corresponding abbreviations used are given below (12, 13, 14): 1. Chromatid-type Aberrations Simple: tg - Chromatid gap - an achromatic region occurring along the length of a chromatid in which there is no misalignment. tb - Chromatid break - a discontinuity occurring along the length of either of the two chromatids in which there is a misalignment. isb - Isochromatid break - a discontinuity occurring in both the chromatids at the same locus showing complete rejoining or sister chromatid union at both the broken ends or incomplete rejoining, i.e., only at one of the two broken ends. Complex: qr tr - Quadriradial - chromatid interchanges between chromosomes leading to four-armed configurations. This could be asymmetrical with formation of a dicentric and an acentric chromatid, if union is complete, or symmetrical where there is no formation of a dicentric and an acentric chromatid. Triradial - isochromatid-chromatid exchanges resulting in threearmed configurations and sometimes fragments. The latter should not be scored as an independent aberration. The triradial could be monocentric or dicentric. id ci cr - Interstitial deletion - intra-arm intra-changes resulting in deletion of small fragments which, however, stay in association with the parent chromatid. Chromatid intrachange - exchanges occurring between arms of the same chromosome resulting in asymmetrical (rings) or symmetrical configurations. Complex interchanges - multiarmed configurations resulting from breakage and reunion of two or more chromosomes. 17 2. Chromosome-tvpe Aberrations Simple: sg - Chromosome gap - an achromatic region occurring in both chromatids of the chromosome at the same locus with no misalignment. sb - Complex: d - r - Chromosome break - a discontinuity at the same locus in both chromatids, giving one acentric fragment which may be misaligned and a shortened monocentric chromosome, and where there is no sister chromatid union. Dicentric - an asymmetrical exchange between two chromosomes resulting in a chromosome with two centromeres with or without an accompanying acentric fragment which should not be score as a second aberration. Ring - inter-arm intrachange happening within the chromosome, leading to formation of a centric ring with or without a chromosome fragment. The fragment should not be scored as a second aberration. dm - Double minutes - intra-arm intrachanges leading to tight acentric paired rings. 3. Other Aberrations PU - Pulverized chromosome or chromosomes - shattering of chromatid material resulting in several minute pieces. The identity of the chromosome is not decipherable. Considered as a single aberration. sd - Severely damaged cell - cell with 10 or more aberrations. PP - e - Polyploid cells - metaphases with multiples or approximate multiples of the haploid set of chromosomes. Not scored for structural aberrations. Endoreduplication - metaphases with paired duplicated chromosomes or diplochromosomes; they are not scored for structural aberrations. 18 SITEK Study NO. 0623-3110 The chromosome aberration data from the score sheets were consolidated on a Summary Table. The number of aberrations per cell and the percentage of cells with one or more aberrations for each concentration level were calculated. The data were consolidated separately for the two cultures at each concentration, then pooled and presented together. Chromatid gaps and chromosome gaps were scored, but they were not included in calculating the percentage of cells with aberrations and the number of aberrations per cell. Of the remaining aberrations, each aberration scored was counted as one, except severely damaged cell (sd), which was considered equal to 10 aberrations in calculating the number of aberrations per cell. Endoreduplicated and polyploid cells were recorded separately in percentages. CONFIRMATORYCHROMOSOME ABERRATION ASSAY @2) A confirmatory assay (B2) was performed in the non-activated system only, since the results from the B1 showed a negative response in both systems. The procedures were the same as in the B1, except that the test article treatment period was extended to 18 hours (1.5 x normal cell cycle time). The concentrations tested were 50, 100, 500, 1000, 2500 and 5000 pglmL. The cells were harvested 18 hours (1.5 x normal cell cycle time) after the initiation of treatment. Parallel toxicity was determined by the RCG and RMI. Based on the RCG and RMI results, chromosome aberrations were scored from cells treated with three concentrations of 500, 1000 and 5000 pg/mL. One hundred (100) metaphases were scored from each of the two replicate cultures at each concentration level and the controls. STATISTICAL ANALYSIS The data for the percentage of cells with aberrations for each concentration were compared to the solvent control values using the Chi-square test. A validated statistical package (Epistat) was used to calculate the p values for the Chi-square test. Results were considered significant if p I 0.05. Statistical analysis was not performed if the test concentration value was equal to or less than the concurrent or historical solvent control. If a positive response was indicated by the Chi-square test, the Cochran-Armitage test (trend test) was performed for evidence of a concentration-related response (15). The trend test was considered positive if p 20.05. The trend test was not performed for this study. 19 SITEK Study NO. 0623-3110 CRITERIA FOR A VALID ASSAY 1. In the solvent control, the percentage of cells with aberrations should not have exceeded 4 % . 2. At least 25% of the cells scored in the positive control should have showed one or more chromosome aberrations. 3. At least one of the test concentrations scored should have showed greater than 50% reduction in the RCG and/or RMI. This requirement should not be applied to test articles where no apparent toxicity could be achieved at the maximum soluble concentration or the highest allowable concentration. 20 SITEK Study NO. 0623-3110 EVALUATION OF TEST RESULTS Positive Response The test article was considered to have caused a positive response in this assay if the test article showed a positive dose-response trend and a statistically significant increase over that of the solvent controls in the percentage of cells with aberrations at one or more concentrations. Negative Response The test article was considered to have caused a negative response if none of the test concentrations showed a statistically significant increase in the percentage of aberrant cells. Equivocal Response The test article was considered to have caused an equivocal response if there was a statistically significant increase in the percentage of cells with aberrations without an accompanying positive dose-response trend. 21 SITEK Study NO. 0623-3110 RESULTS SOLUBILITY TEST One hundred (100) mg of the test article was dissolved in 0.2 mL of DMSO yielding a solution with a concentration of 500 mg/mL in DMSO. The test article was soluble in medium at 5000 ,ug/mL. Based on the solubility in the medium, 5000 pg/mL was selected as the highest concentration for the Range Finding Test. DETERMINATION OF pH It was not necessary to determine the pH of the test article, since the color of the pH indicator in the complete medium did not change at the highest test article concentration of 5000 pg/mL. OSMOLALITY TEST The osmolalities of the untreated, solvent and test article-containing medium used in ) treating the cells at 5000 pg/mL in the Range Finding Test are given below: Concentration OSMOLALITY (mOsmol/kg water) Without With Activation Activation Untreated (water) Solvent (DMSO) 5000 pg/mL 277 290 382 334 394 3 14 Even at the highest concentration tested, changes in osmolality did not exceed 500 mOsmol/kg of water, above which DNA damage is considered probable. Therefore, no effect on osmolality was caused by the addition of the test article at the maximum concentration. RANGE FINDING TEST (Al) The results of the Range Finding Test (Al) are summarized and presented in Table 1 (RCG) (Appendix I). The results showed that there was no obvious reduction (more than 50% versus that of the solvent control) in RCG even at the highest concentration of 5000 pg/mL in both the non- 22 SITEK Study NO. 0623-3110 activated and activated systems. The RCGs ranged from 84%-109%and 128-304%, for the non-activated and activated systems, respectively, for the test article concentrations of 5.O5000 pg/mL. DEFINITIVE CHROMOSOME ABERRATION ASSAY (Bl) Based on the toxicity results from the Range Finding Test, the concentrations of 500, 1000, 2500 and 5000 pg/mL were selected for the Chromosome Aberration Assay (Bl) in both the non-activated and activated systems. The parallel toxicity results, as determined by the reduction in the RCG and/or RMI of the treated cells in the non-activated and activated systems, are presented in Tables 2 (RCG) and '3 (RMI) (Appendix I). The RCG results showed that no obvious reduction (more than 50%)was observed at any concentration in either system. The RCGs ranged from 104-133% in the non-activated system, and from 84-110% in the activated system. The RMI data showed that the RMIs ranged from 100-144% in the non-activated system, and from 61-94% in the activated system. Therefore, chromosome aberrations were scored from the 3 highest concentrations of 1000, 2500 and 5000 pg/mL in both the non-activated and activated systems. In addition, the corresponding untreated (water), solvent (DMSO) and positive controls (MMC at 0.2 pg/mL and CP at 7.5 pg/mL) were also scored. One hundred (100) metaphases were scored from each of the two replicate cultures at each concentration and the controls. The results of the Chromosome Aberration Assay (Bl) in the non-activated and activated systems are summarized and presented in Tables 4 and 5 , respectively (Appendix I). The averages of the percentage of cells with aberrations scored from the assay are summarized below: Average Percentage of Cells with Aberrations Treatment Without Activation With Activation Untreated (water) Solvent Control (DMSO) Test Article Concentrations (1000, 2500 and 5000 pg/mL) Positive Control 0.0% 0.0% 0.0% 25.5%" 0.0% 0.0% 0.0% 26.0%" * Statistically significant response using the Chi-square test. 23 SITEK Study NO. 0623-3110 CONFIRMATORY CHROMOSOME ABERRATION ASSAY (B2) The parallel toxicity results of the confirmatory assay (B2) without activation are presented in Tables 6 (RCG) and 7 (RMI) (Appendix I). The results showed that the RCGs ranged from 46-165%. The RMIs ranged from 17-88%. No metaphases were found at the concentration of 2500 pg/mL. Therefore, chromosome aberrations were scored from the three concentrations of 500, 1000 and 5000 pg/mL. In addition, the corresponding untreated, solvent and positive (MMC at 0.2 pg/mL) controls were also scored. One hundred (100) metaphases were scored from each of the two replicate cultures at each concentration and the controls. The results of the confirmatory Chromosome Aberration Assay (B2) are summarized and presented in Table 8 (Appendix I). The averages of the percentage of cells with aberrations scored in this assay are summarized below: Average Percentage of Cells with Aberrations Treatment Without Activation Untreated (water) Solvent Control (DMSO) Test Article Concentrations (500, 1000 and 5000 ,ug/mL) Positive Control (MMC at 0.2 ,ug/mL) 0.0% 0.0% 0.0-0.5 % 29.0%" * Statistically significant response using the Chi-square test STATISTICAL ANALYSIS Statistical analysis showed that, none of the test article concentrations tested in the nonactivated or activated systems, in either assay, were found to have induced a statistically significant increase in the percentage of cells with aberrations over the solvent controls. The percentage of polyploidy (pp) and endoreduplicated cells (e) was in the normal range (0-5.0%and 0-1.O% for pp and e, respectively). All of the criteria for a valid assay were met. 24 SITEK Study NO. 0623-3110 ANALYSIS OF DOSING SOLUTIONS The test article dosing solutions were analyzed by Southern Research Institute and the results are presented in Appendix N .Dosing solution samples ranging in concentration from 5.0 to 500 mg/mL were analyzed from the definitive and confirmatory Chromosome Aberration Assays. All dosing solution samples were found to be in the acceptable range of &lo%of the labeled (targeted) concentration, except two; 100 mg/mL from the definitive assay was 111% of the targeted concentration and 250 mg/mL from the confirmatory was 117%. The latter sample was analyzed a second time and found to have a concentration of 116% of the targeted value. However, this had no effect on the study outcome. i 25 SITEK Study NO. 0623-3110 CONCLUSIONS Test article, Potassium Perfluorobutane Sulfonate, was tested for its potential to induce chromosome aberrations in cultured CHO cells with and without exogenous metabolic activation in the Chromosome Aberration Assay. Under the conditions of this study, and according to the criteria set for evaluating the test results, Potassium Perfluorobutane Sulfonate was negative in the Chromosome Aberration Assay with and without exogenous metabolic activation and is not considered to be a clastogenic agent. ARCHIVES The raw data, documentation, protocol, protocol amendments/deviations and the Final Report along with an electronic files containing data tables and the Final Report of the study will be maintained, for at least 10 years, at SITEK Research Laboratories' archives at 15235 Shady Grove Road, Suite 303, Rockville, Maryland, 20850. After that time, the Sponsor will ,I be notified for further arrangements regarding this study's records. 26 SITEK Study NO. 0623-3110 REFERENCES 1. Evans, H.J. Cytological Methods for Detecting Chemical Mutagens, In: Chemical Mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) Plenum Press, New York and London, pp. 1-29 (1976). 2. Galloway, S. M., et al. Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in the laboratories. Environ. Mutagen., 2:1-51, 1985. 3. Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, G.H. Margolin, M.A Resnick, G. Anderson and E. Zeiger. Chromosome aberration and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environ. molec. Mutagen (suppl. lo), 1-175 (1987). 4. Mutagenicity ("In Vitro" Mammalian Cytogenetic Test) Official Journal of the European Communities, B. 10. L383A, Volume 35, 1992. 5. Galloway, S.M., et al. Report from working group on in vitro tests for chromosomal aberrations. Mut. Res., =:241-261, 1994. 6. Elliot, B.M., et ai. Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity i assays. Mutagenesis, 2:175-177, 1992. 7. Scott, D., S.M. Galloway, R.R., Marshall, M. Ishidate, D. Brusick, Jr., J. Ashby and B.C. Myhr. Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Tast Group 9. Mutation Res., 257: 147-204, 1991. 8. Morita, T., et al. Clastogenicity of low pH to various cultured mammalian cells. Mut. Res., 268: 255-261, 1992. 9. Galloway, S. M., et al. False positive in vitro chromosome aberration tests with nonmutagens at high concentrations and osmolalities. 1985 EMS Abstracts:g. 10. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatorv Genotoxicity Tests for Pharmaceuticals. Recommended for Adoption at Step 4 of the ICH Process on 19 July 1995 by the ICH Steering Committee. 11. OECD Guideline for the Testing of Chemicals. Proposal for Updating Guideline 473. In Vitro Mammalian Chromosome Aberration Test. Draft (Feb. 97). 12. Evans, H. J., and M. L. O'Riordan. Human peripheral blood lymphocytes for the analysis of chromosome aberrations in mutagen tests. Mut. Res., 31:135-148, 1975. 27 SITEK Study NO. 0623-3110 13. Savage, J. R. Classification and relationships of induced chromosomal structural changes. J. Med. Genetics, 13:103-122, 1976. 14. Scott, D., B. J. Dean, N. D. Danford and D. J. Kirkland. Metaphase chromosome aberration assays in vitro. In: Basic Mutagenicity Tests: UKEMS Recommended Procedures. Cambridge University Press, pp. 62-86, 1990. 15. Margolin, B.H., et al. Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environ. Mutagen., 8:183-204, 1986. 28 SITEK Study NO. 0623-3110 APPENDIX I DATA TABLES I 29 SITEK Study NO.0623-3110 TABLE I CHROMOSOME ABERRATION ASSAY IN CHO CELLS RCG - RANGE FINDING TEST TEST ARTICLE: Potassium Peduorobutane Sulfonate SPONSOR: Primedicr Rcdficld SOLVENT DMSO Test Article Conc. (pglmL) WllllOUT ACTIVATION No. of Cells per Flask Mean No. of Cells X 10' RCG' Untreated A 2.34 Untreated B 2.22 2.28 165% SITEK STUDY NO.: 0623-31 IO TIUALNO.: A I Test Article Conc. (pglml) WITH A C n V A l l O N No. of Cells per Flask Mean No. of Cells X 10' Untreated A 2.61 Untreated B 2.37 2.49 RCG' 311Oh Solvent A 1.11 Solvent B 1.64 1.38 100% Solvent A 1.33 Solvent B 0.26 0.80 100% 5.0 A 1.57 5.0 B 1.45 1.51 109% 5.0 A 1.25 5.0 B 1.so 1.58 198% 10 A 0.80 10 A 1.37 i 10 B 1.68 1.24 90% 10 B 1.92 1.65 206% 50 A 1.07 50 B 1.25 1.16 84% 50 A 1.73 50 B 1.64 1.69 211% 100 A 1.45 100 B 1.08 1.27 92% 100 A 1.07 100 B 1.41 1.24 155% 500 A 1.27 500 6 1.11 1.19 86% 500 A 0.82 500 B 1.47 1.15 144% 1000 A 1.48 1000 B 1.15 1.32 96% 1000 A 0.78 1000 6 2.06 1.42 178% 2500 A 1.02 2500 B 0.73 1.28 93% 2500 A 1.37 2500 B 0.67 102 128% 5000 A 1.51 5000 B 1.49 150 109% 5000 A 2.15 5000 B 2.70 2.43 *RCG = Relative Cell Growth = No. of Cells in the Test Flask No. of Cells in the Solvent Flask x 100 30456 30 SITEK Study NO. 0623-3110 TABLE 2 IN WQfXlU RCG - DEFINITIVE ASSAY TEST ARTICLE Potassium Pcduombutanc Sulfonate SPONSOR: Prim& Rodlicld SOLVENT: DMSO SlTEK STUDY NO.: 0623-3110 TFUALNO.: B1 WITHOUT ACTIVATION w(TH ACTIVATION 1 Test Article Mean No. Test Article Mean No. Conc. No. of Cells of Cells Conc. No. of Cells of Cells (pglmL) per Flask x 10' RCG' (pglml) per Flask x loe Untreated A Untreated B 2.54 2.54 2.54 155% Untreated A Untreated B 2.20 2.23 2.22 Solvent A Solvent B 1.40 1.88 1.64 100% Solvent A Solvent B 2.44 1.92 2.18 100% 500 A 2.06 500 A 2.24 500 B 2.29 ' 2.18 133% 500 B 1.41 1.83 84% 1000 A 1.87 1000 B 1.54 1.71 104% 1000 A 2.38 1000 B 2.35 2.37 109% 2500 A 2.03 2500 6 1.95 1.99 121% 2500 A 2.55 2500 E3 2.24 2.40 110% 5000 A 2.02 5000 B 2.1 1 *RCG = Relative Cell Growth = 2.07 126% 5000 5000 No. of Cells in the Test Flask A 2.25 B 2.25 x 100 2.25 103Oh 31 TABLE 3 MITOTIC INDEX - DEFINITIVE ASSAY SPONSOR : Primedica Redfield SOLVENT : DMSO TEST ARTICLE : Potassium Perfluorobutane Sulfonate STUDY NO. : 0623-311 0 TRIALNO.: B1 Tube I Dividing I Mitotic I Mitotic I No. ~Cells/5001Index -I 61% w w 5000 19 A 26 B 33 5.9 107% 5000 60 A 17 MMC 0.08 B 19 1 , 74 A .I12 MMC 0.20 B (6 3.6 45% 1.8 23% CP 7.5 CP 12.5 The positive controls were compared to Untreated since the solvent for MMC and CP was water. +I86% 21 % 18% I M-I = . . . 'Oo0 dlii RMI = lasrslnskMl x 100 10 Solvent Control MI Z 0 TABLE 4 - CHROMOSOME ABERRATIONS DEFINITIVEASSAY TSEKSNTSOARRT: IRCLimE:cdPiduaRtscdimfidPdduorobumc Sulfonate TREA'IMENT TIME: 3 Houn IIARL'EST TIME: IS burr SOLVEKT: DMSO SlTlX STUDY NO.: 0623-3110 TRIAL NO.: ni M l Z A B O J X ACIIVATION: Yes ( ) No (X) w w ca?c * sd I 10 absrrationr in calculations. **Statistical analyses done on the % cells with aberrations. Z? Ia the Chi-square test, MMC was compared to SEEK'Shistorical Untreated control data (0.62%)since the solvent for MMC was water and the concurrent value was 0%. I E T ARTICLE: Pouirium Pduorobutane Sulfonate SPONSOR Primd~mRcdReld SOLVENT. DMSO TABLE 5 l - ' I t X A " T l l h E 3 Houn HARVEST TlME 18 Hwrs SITEK STUDY NO.: 0623-3110 TRIAL NO.: B1 hfETABOUCA C I W A T I O N Yes 0 No ( ) sd - 10 aberrations in calculations. . Z **Statistical analyses done on the % cells wilh aberrations In the Chi-square test, CP was compared to SITEK's historical Untnarcd control data (0.60%) since the solvent for CP was water and the concurrentvalue was 0%. 0 SITEK Study NO.0623-3110 TABLE 6 RCG - CONFIRMATORY IN ASSAY TEST ARTICLE: SPONSOR Poflsriurn Pcrfluombugac SullaU(c Primcdiu Rcdl-icld SOLVENT: DMSO Article Conc. No. of Cells per Flask Mean No. of Cells x lo6 RCG Solvent 1 50 A 50 B 1 100 A 100 8 1 500 A 500 B 2500 3.22 2.46 2.84 100% 3.57 1 1 6.79 4.71 1 1 3.06 2.28 1 1 0.31 4.68 3.89 1.30 165% 137% 46% 2.41 1 1 0.33 1.37 40% I I l:f 1 1:; 2.05 SITEK STUDY NO.: 06213110 TRLALNO.: 82 WlTIi ACTIVATION The Activated System Was not perforned in the 82. 5000 A 5000 B 1.46 1.72 I I *RCG = Relative Cell Growth = No. of Cells in the Test Flask No. of Cells in the Solvent Flask x 100 35 SPONSOR : Primedica Redfield TABLE 7 SOLVENT : DMSO STUDY NO. : 0023-3110 TRIAL NO.: 82 'Ith Activation - Treatment: NIA Hours Harvest: N/A Hours Activated System was not performed in 62 MI- = ... 10 'Oo0 d k - t h e solvent for MMC was water. RMI = x loo Solvent Control MI TABLE 8 CHROMOSOME ABERRATIONS - CONFIRMATORY ASSAY TEST ARTICLE: Paauiurn Pcrfluorotuunc Sulfonate TREATMENT TIME: 18 Hours SPONSOR Primcdiu Rcdficld HARVEST TIME I8 Hwrs SOLVEKT: DMSO TREATMENT CELLS 1 NOT I NUMBER AND TYPE OF ABERRATIONS Chromatid Type Chromosme Type SITEK STUDY NO.: 0623-3110 TRIAL NO.: E2 METABOLIC ACTIVATION: Yu ( ) No ( X ) NO.OP ABS. Z CELLS P-VALUE w 4 * sd - 10 aberrations in calculations. **Statistical~MIYSCS was done on the % cells with aberrations and wiul SITEK'S historical data (0.67%)since the solvent control was OX. In the Chi-square test. MMC was compared to SITEK's historical Untreated control data (0.62%)since the solvent for M M C was water and the concurrent value was 0% Z 0 SITEK Study NO. 0623-3110 APPENDIX I1 SITEK'S HISTORICAL DATA I 38 SITEK Study NO.0623-3110 HISTORICAL DATA FOR NEGATIVE CONTROL (UNTREATED) CHO IN VITRO CHROMOSOME ABERRATION ASSAY NON-ACTIVATED SYSTEM STUDY NUMBER 0212-3112 0212-3112 0212-3112 021 2-31 12 0212-3112 0212-3112 0221-3112 0221-3112 0221-3112 0221-3112 0226-3112 0226-31 12 0226-31 12 0226-31 12 0227-31 12 0227-3112 0227-31 12 0227-31 12 0231 -31 12 0231 -31 12 0231 -31 12 0231 -31 12 0232-31 13 0232-31 13 0232-31 13 0236-31 13 0236-31 13 0242-31 12 0242-31 12 0242-31 12 0242-31 12 0244-31 12 0244-31 12 0244-31 12 0244-31 12 0256-31 14 0256-31 14 0256-31 14 0256-31 14 0250-31 14 0250-31 14 0250-31 14 0265-31 13 0265-31 13 0265-3113 - 0268-31 10 0271 31 1 4 0271 -31 14 0271 -31 14 0273-31 13 0273-31 13 0273-31 13 0276-31 10 0203-31 10 0209-31 14 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 %CELLS WITH ABS. - - 2.00 0.00 0.00 0.00 1 .00 1 .00 0.50 2.00 0.00 0.00 1 .00 0.50 1 .00 1.50 1 .00 0.50 0.50 1 .00 1 .00 0.50 0.50 0.50 1 .oo 0.00 0.50 1 .00 0.50 0.50 0.50 1 .50 0.50 1 .00 0.50 . 0.00 0.00 0.50 0.50 1.50 0.00 0.00 0.00 0.50 0.50 0.50 0.50 0.00 0.50 1 .00 1 .00 0.50 0.50 1 .00 0.50 0.00 0.50 p.1 of 3 39 SITEK Study NO. 0623-3110 HISTORICAL DATA FOR NEGATIVE CONTROL (UNTREATED) CHO IN VITRO C H R O M O S O M E ABERRATION ASSAY NON-ACTIVATED SYSTEM STUDY NUMBER 0289-3114 W09-3114 0327-31 14 0327-31 14 0327-31 14 0334-31 14 0334-3114 0334-31 14 0336-31 10 (3346-31 14 0346-31 14 0346-31 14 0347-31 14 0347-3114 0347-31 14 0351-3114 0351-3114 0351 -31 14 0352-31 14 0352-31 14 0352-31 14 0362-31 14 0362-31 14 0362-31 14 0372-31 14 0372-31 14 0372-31 14 0384-31 14 O384-3114 0384-31 14 0387-3114 0387-31 14 0387-31 1 4 0389-31 14 W9-3114 0309-3114 0389-31 14 0389-31 1 4 0389-31 1 4 03%--3114 0397-31 14 0398-3114 0399-31 14 0402-31 14 0403-3114 0404-3114 0406-31 10 0415-3110 041 5-31 10 0415-3110 0425-31 10 0426-31 10 0426-3110 0428-31 10 0428-31 10 0432-31 10 i?OF METAPHASES SCORED 2- -0-0 200 200 200 200 200 200 200 200 200 2- _0_0 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 XK, 200 200 200 200 200 200 200 200 200 200 200 200 %CELLS WITH ABS. 0.50 0.50 0.00 1 .00 0.50 0.50 1 .00 1.50 1.50 1 .00 0.50 1 .00 1 .00 0.50 0.00 0.00 0.00 0.50 0.50 0.50 1 .00 0.00 0.50 1 .00 0.00 1 .00 1 .00 1 .00 0.50 0.00 1 .00 1.50 0.50 0.50 1 .00 0.50 0.50 2.00 ' 0.50 0.50 2.00 1 .00 0.00 0.50 2.00 1.50 0.00 0.50 1 .00 1.50 0.00 0.00 1 .00 0.00 0.50 0.50 p.2 of 3 40 SITEK Study NO.0623-3110 HISTORICAL DATA FOR 'NEGATIVE CONTROL (UNTREATED) CHO IN VITRO CHROMOSOME ABERRATION ASSAY NON-ACTIVATED SYSTEM STUDY NUMBER 0432-3110 0437-3110 0452-3114 0452-31 14 0460-31 14 0460-31 14 0470-31 10 0470-3110 0470-3110 0475-3110 0475-3110 0490-31 10 0490-31 10 0493-31 10 0493-31 10 0504-31 10 0504-3110 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 RANGE :0.0 - 1.5% I MEAN f. S.D.: JUL 1992 - MAR 1999 %CELLS WITH ABS. 1 .oo 0.00 1 .00 0.50 0.50 0.50 0.00 0.50 1 .00 0.00 0.50 0.00 0.50 0.50 0.50 0.00 0.00 0.62 2 0.51 p.3Of3 41 SITEK Study NO. 0623-3110 HISTORICAL DATA FOR NEGATIVE CONTROL (UNTREATED) CHO IN VITRO CHROMOSOME ABERRATION ASSAY ACTIVATED SYSTEM STUDY NUMBER 0212-31 12 0212-31 12 0212-31 12 0212-31 12 0212-31 12 0221-31 12 0221-31 12 0221-31 12 0221-31 12 0226-31 12 0226-31 12 0226-31 12 0226-3112 0227-31 12 0227-3112 i 0227-31 12 0227-31 12 0231-31 12 0231-31 12 0231-31 12 0231-31 12 0232-31 13 0232-31 13 0232-31 13 0236-31 12 0236 -31 12 0242-31 12 0242-31 12 0242-31 12 0242-31 12 0244-31 12 0244-31 12 0244-31 12 0244-31 12 0256-31 14 0256-31 14 0256-31 14 0256-31 14 0258 -31 14 0258-31 14 0258-31 14 0265-31 13 0265-31 13 0265-31 13 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 42 .- %CELLS WITH ABS. 0.50 0.50 1.oo 0.00 0.50 0.50 1.50 0.50 0.00 1.oo 0.50 0.50 0.50 1S O 1.50 0.50 1.oo 0.50 1.oo 0.00 0.50 0.00 0.50 0.00 1.50 1 .oo 1.oo 0.50 0.50 0.50 0.00 0.50 0.50 0.50 1.oo 1.oo 1.oo 0.50 1.oo 0.00 0.00 0.00 0.50 0.00 SITEK Study NO. 0623-3110 HISTORICAL DATA FOR NEGATIVE CONTROL (UNTREATED) CHO IN VITRO CHROMOSOME ABERRATION ASSAY ACTIVATED SYSTEM STUDY NUMBER 0268-31 10 0271 -31 14 0271 -31 14 0271 -31 14 0273-31 13 0273-31 13 0273-31 13 0276-31 10 0283-31 10 0289-31 14 0289-31 14 0327-31 14 0327-31 14 0327-31 14 0334-31 14 0334-31 14 0334-31 14 0336-31 10 0346-31 14 0346-31 14 0346-31 14 0347-31 14 0347-31 14 0347-31 14 0351 -31 14 0351 -31 14 0351-31 14 0352-31 14 0352-31 14 0352-31 14 0362-31 14 0362-31 14 0362-31 14 0372-31 14 0372-31 14 0372-31 14 0384-31 14 0384-31 14 0384-31 14 0387-31 14 0387-31 14 0387-31 14 0389-31 14 0389-31 14 0389-31 14 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 43 %CELLS WITH ABS. 0.00 0.50 0.50 0.50 1 .oo 0.50 1 .oo 1.50 0.50 0.50 0.50 0.00 1 .oo 1S O 0.50 0.00 0.00 1 .oo 0.50 1 .oo 1 .oo 1.50 0.00 1 .oo 1 .oo 0.50 0.00 1 .oo 1 .oo 1.50 0.50 1 .oo 0.00 0.50 0.00 1 .oo 1.50 0.50 0.50 1 .oo 1.50 1 .oo 1 .oo 0.00 0.50 SITEK Study NO.0623-3110 HISTORICAL DATA FOR NEGATIVE CONTROL (UNTREATED) CHO IN VITRO CHROMOSOME ABERRATION ASSAY ACTIVATED SYSTEM STUDY ~~ NUMBER 0389-31 14 0389-31 14 0396-31 14 0397-31 14 0398-31 14 0399-31 14 0402-31 14 0403-31 14 0406 -31 10 0415-3110 0415-31 10 0415-3110 0425-31 10 0426-3110 0426-31 10 0428-31 10 0432-3110 0437-3110 0452-31 14 0452-31 14 0453-31 14 0453-31 14 0460-31 14 0460-31 14 0470-31 10 0470 -31 10 0470-3110 0475-31 10 0490-31 10 0493-31 10 0504-31 10 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 RANGE : 0.0 - 1.5% %CELtS WITH ABS. 0.00 1.oo 0.00 0.50 0.00 0.00 0.50 0.50 0.00 0.50 0.50 0.50 1.oo 1.oo 0.00 0.50 0.50 0.50 1.oo 1.oo 1.oo 1.oo 0.50 0.00 0.50 0.50 1 .oo 0.50 0.00 0.00 0.00 MEAN -I S.D.: 0.60 +- 0.45 NOV 1992- MAR 1999 44 SITEK Study NO.0623-3110 STUDY NUMBER 0212-31 12 0212-31 12 0212-3112 0212-31 12 0212-31 12 0212-31 12 0221 -31 12 0221 -31 12 0221 -31 12 0221 -31 12 0231 -31 12 0231 -31 12 0231 -31 12 0231 -31 12 0242-31 12 0242-31 12 0242-31 12 0242-31 12 0256-31 14 0256-31 14 0256-31 14 0256-31 14 0268-31 10 0289-31 14 0289-31 14 0289-31 14 0289-31 14 0327-31 14 0327-31 14 0327-31 14 0336-31 10 0346-3114 0346-31 14 0346-31 14 0346-31 14 0346-31 14 0346-31 14 0347-31 14 0347-31 14 0347-31 14 0347-31 14 0347-31 14 0347-31 14 0351 -31 14 0351 -31 14 HISTORICAL DATA FOR DMSO CHO IN VITRO CHROMOSOME ABERRATION ASSAY NON-ACTIVATED SYSTEM 45 SITEK Study NO. 0623-3110 HISTORICAL DATA FOR DMSO CHO IN VITRO CHROMOSOME ABERRATION ASSAY NON-ACTIVATED SYSTEM STUDY NUMBER 0351 -31 14 0352-31 14 0352-31 14 0352-31 14 0404-31 14 0415-31 10 0415-3110 0415-3110 0425-31 10 0426-31 10 0426-31 10 0428-31 10 0428-31 10 0432-31 10 0432-31 10 0437-31 10 0460-31 14 0470-31 10 0470-31 10 0475-31 10 0475-31 10 0493-31 10 0493-31 10 0504-31 10 0504-31 10 # OF METAPHASES SCORED 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 RANGE :0.0 - 1.5% MEAN +- S.D.: - -. %CELLS WITH ABS. 0.50 0.50 1 .oo 0.50 1 .oo 0.50 0.50 0.50 0.00 0.00 1 .oo 0.50 1 .oo 0.50 0.50 0.50 0.50 0.50 1.50 0.00 0.50 0.50 1 .oo 0.50 0.00 0.67 5 0.47 NOV 1992- MAR 1999 46 SITEK Study NO. 0623-3110 APPENDIX I11 STUDY PROTOCOL AND PROTOCOL AMENDMENTS i 47 z ,-SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-3110 t ' r TEST FOR CHEMICAL INDUCTION OF CHROMOSOME ABERRATION IN CULTURED CHINESE HAMSTER OVARY CHO CELLS WITH AND WITHOUT METABOLIC AC IVA ION This protocol .is presented in 80parts. Part One is designed to collect specific information ertaining to the test article and study. Part Two descnbes the study desi n in detail. Pi!ase complete all sections in Part One and sign section 8.0 to approve #e protocol. PART ONE 1.0 SPONSOR 1.1 Name: Prirnedica Redfield 1.2 Address: 100 E. Boone Street Redfield. AR 72132 1.3 Sponsor`s Study Coordinator: John Sena, Ph.D. 2.0 TESTING FACILITY 2.1 Name: SITEK Research Laboratories 2.2 Address: 15235 Shady Grove Road Suite 303 Rockville, Maryland 2085b ) 2.3 Study Director: Jing Xu, M.D. 3.0 STUDY NUMBERS * 3.1 Testing Facility's Study No.: Ob23- -3/0/ 3.2 Sponsor`s Study No.: 132-009 4.0 TEST ARTICLE 4.1 Identification Name: Potassium Perfluorobuntane Sulfonate. CAS #29420-49-3 Batch/Lot No.: Lot #2 4.2 Description Color: White Physical Form: Solid, free flowinq Dowder * lo be completed by the Testing Facility. Protocol No. 311O.AB 111999 Page 1 of 15 48 ~ __~ E 1 SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-3110 4.3 Analvsis Purity Information: 100% pure Does the Sponsor require the use of a correction factor to account for impurity? -Yes X No If yes, what is the correction factor? Deteqination of the test. article characteristics as defined by Good Laborato Practices will be the responsibility of the S onsor. The s ecific GLP references for U . g a encies are: FDA = 21 CFR, 58.105; EPLTSCA = 40 CFR, 792.105 and EPA FIFRA = 48 CFR 160.105. 4.4 Stability Storage Conditions (check one): - X Room Temperature -Refrigerated (1-5C) -Frozen (-10 to -20%) -Other (please specify): Expiration Date: 04-06-01 4.5 Preferred Solvent (check one): -H,O -Culture Medium -Other (please specify): - DMSO - X To be decided by the Testing Facility -Acetone -Ethanol 4.6 Special Handlinq Instructions: Standard precaution (MSDS attached) 5.0 REGULATORYAGENCY SUBMISSION This study will be conducted in compliance with the following Good Laboratory Practice standards: United States Environmental ProtectionA ency, Title 40 Code of Federal Regulations Parts 160 and 792, Revised July 1, 199? United States Food and DN Administration, Title 21 Code of Federal Regulations Part 58, Revised April 1, 1938. Japanese Minist of Agriculture, Forestry and Fisheries 59 Nohsan, Notification No. 3850, Agricurure Production Bureau, August 10, 1984. Protocol No. 31IO.AB I 11999 Page 2 of 15 49 SITEK Study NO. 0623-3110 SITEK RESEARCLHABORATORIES I Ir,I Japanese Ministry of Health and Welfare, Ordinance No. 21, April 1, 1997. Ja anese Ministry of International Trade and Industry, Notification No. 85, Basic InSustries Bureau, March 31, 1984. Organisation for Economic Cooperation and Development, The OECD Principles of Good Laboratory Practice, Environment Monograph No. 45, Paris 1992. Will this study be submitted to a regulatory agency? Yes - No If so, which agency(ies)? OECD 6.0 DOSING SOLUTIONS 3 The Sponsor will be responsible for deterrninin the,stren th and stability. of *e dosing solutions. The U.S. requirements for analysis dosing so%tions are speufied in: FDA = 21 CFR, 58.113; EPA TSCA = 40 CFR, 792.113 and EPA FIFRA = 40 CFR 160.1 13. Does the Sponsor want dosing solution analysis? Yes** - No If yes, please complete the rest of this section. 1 If re uested by the Sponsor, SITEK Research Laboratorieswill determine the stren th and stab!ity.of the dosin solutions. The method of analysis ma be provided by &e Sponsor, or if requested #y the Sponsor, SITEK Research Labora&nes will develop the method of analysis. Alternative1 ,the Sponsor will be responsiblefor determining the strength and stability of the dosing sobions. Dosing solution analysis will be performed by: -SlTEK Research Laboratories - X Sponsor What dosing solutions will be analyzed? From the Range Finding Test? -Yes X No From the Assay? - X Yes -No Which concentration(s)? All 'xp Additional charges will apply. See Special Services price schedule. Protocol No. 311O.AB 111999 Page 3 of 15 50 -7 -SITEK RESEARCHLABORATORIES AL SITEK Study NO.0623-3110 Spdnsor's Authonzed Kepresentative * Quality Assurance Manager bate l o be completed by the Testing Facility. Protocol No. 311O.AB 111999 51 Page 4 of 15 SITEK RESEARCHLABORATORIES 1: STUDY DESIGN SITEK Study NO. 0623-3110 PART TWO 9.0 PURPOSE The purpose of this stud is .to evaluate the test article for its. potential to cause enetic damage as manifeste2by induced chromosome aberrations in cultured Chinese {amster ovary (CHO) cells. 10.0 JUSTIFICATION FOR SELECTION OF TEST SYSTEM Y The CHO cells have been used extensive1 in-theChromosomeAberration Assay and have been demonstrated to be effective in de ecting the clastogenic activity of chemicals from a wide range of chemical classes (1-5). I 1.O ABBREVIATIONS CHO . CP - Chinese Hamster Ovary Cyclophosphamide DMSO - Dimethyl Sulfoxide G-6-P - Glucose-6-phosphate HEPES - N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HIFBS - Heat-Inactivated Fetal Bovine Serum KCI - Potassium Chloride MMC - MI - Mitomycin-C Mitotic Index NADP - Nicotinamide-adenine Dinucleotide Phosphate (Sodium Salt) DPBS - Dulbecco's Phosphate Buffered Saline (with Ca" and Mg") PBS - Phosphate Buffered Saline (without Ca" and Mg") RMI - Relative Mitotic Index - Complete Culture Medium McCoy's 5A medium supplemented with 10% HIFBS, 2mM L-glutamine, 50 units/mL of penicillin and 50 pg/mL of I streptomycin Antibiotic-Free Medium - McCoy's 5A medium supplemented with 10% HIFBS and 2mM L-glutamine Protocol No. 311O.AB 111999 52 Page 5 of 15 151SITEK RESEARCHLABORATORIES SITEK Study NO. 0623-3110 12.0 INDICATOR CELLS 12.1 Source The clone CHO-W-BI of the CHO cell line, used in this stud , ori inated at Litton Y i # Bionetics and was obtained by SITEK through the Environmental ea1 Research and Testing Laboratories, Lexin ton, Kentucky, in 1988. The doublin time of this cell line is a roximately 12 hours, anCYits modal chromosome number is 21. $he ka oty e analysis x g o f i e cell line is periodically performed and documented at SlTEK Researc La oratones. 12.2 Culture Conditions 9 The stock cultures of CHO cells are routinely grown in sterile {lastic tissue culture flasks in antibiotic-free medium. The test cultures are rown in f-.5 cm2 plasbc tissue culture flasks in complete medium. The cultures are kep in a humidified incubator maintained at approximately 37C in an atmosphere of approximately 5% CO, and 95% air. The stock cultures are routinely subcultured before confluency using 0.05% trypsin for dissociating the cells. 12.3 Stock Cultures 13.0 ROUTE OF ADMINISTRATION OF TEST ARTICLE The test article will be administered in vitro directly,or through a .solvent compatible with the test system. This is the only route of administration available in this test system. 14.0 TEST SYSTEM IDENTIFICATION All test cultures will be labeled in indelible ink with the SITEK stud number, the test article concentrations/contrls, the act!vation system, code number for txe concentrations, AothoerrBin/Cforomr aDtiodnesthigant aistipnegrttuinbeenst troectheeivAinsgsathe. same treatment, date of harvest and any Slides will be labeledwith the SITEK study number, the code numbers for the concentraxons tested, followed by A or B/C or D for the same treatment conditions and the date the slides are prepared. 15.0 CONTROL SUBSTANCES 15.1 Positive Controls Mitomycin-C (MMC), which causes chromosome aberrations without metabolic activation, will be dissolved in water and used at 0.08 and/or 0.2 pg/mL in the non-activated system. Cyclophosphamide (CP),which requ,ires metabolic activation, will be dissolved in water and used at 7.5 and/or 12.5 pg/mL in the activated system. Protocol No. 311O.AB 1 11999 Page 6 of 15 53 A:* - SITEK RESEARCLHABORATORIES , SITEK Study NO. 0623-3110 If necessary, other appropriate positive controls can be used with the approval of the Sponsor. 15.2 Solvent Controls The solvents used for dissolying the .test article and positive controls will be used as the solvent controls. Culture medium, deionized distilled water, DMSO CAS #67-68-5), ethanol (CAS #64-174, and acetone (CAS #67-84-1) are some of the so(vlents which are corn atible with this test system. If there is a need to use other solvents, the approval of the Eponsor will be obtained prior to their use. 16.0 DOCUMENTATION Detailed documentation of the procedures, results, and methods used for the analysis of the results of this study will be entered in a study notebook. The study notebook also includes copies of the protocol, protocol amendments and deviations, study reports, and all relevant communications with the Sponsor. 17.0 EXPERIMENTAL PROCEDURE 17.1 Determination of SolubilitvlMiscibility In order to determine the appropriate vehicle for delivering the test article to the test system or to determine the maximum achievable concentration in the solvent requested by the kponsor, a solubilitylmiscibility test will be performed. The test article will be tested for its solubility/miscibility in deionized, distilled water, DMSO, acetone, ethanol andlor other appropnate solvents. Solid and viscous liquid test articles will be tested for solubility in weight per volume, and nonviscous.!iquids will be tested for miscibility in volume or weight per volume. The solubility/mtscibility test will be performed as described below. For solid and viscous liquid test articles, the solubility test will consist of wei hing out 25-100 m aliquots of test article and addiqg solvent in 0.1 mL increments, with?horough mixing beheen additions, until the test article is dissolved or until 1.5 mL of solvent has been added to the vessel. If the test article does not dissolve in 1.5 mL of solvent, more solvent will be added in aliquots of 0.5 mL until 5.0 mL has been added. The volume of solvent re uired for complete dissolution, and any additional observations, will be recorded in the stu& workbook. Test articles that do not dissolve in 5.0 mL of solvent will be recorded as either "not soluble." "Rartially soluble forming a t!omogeneous suspension," or "partially soluble not forming a omogeneous suspension. ty For nonviscous liquid test articles. a miscibili test will be conducted. 0.5 mL of each of the preferred solvents in 0.1 rnL increments wil be added to 0.5 mL aliquots of the test article. If the test article does not dissolve in 1.5 mL of solvent, more solvent will be added in 0.5 mL increments until 5.0 mL has been added. The resulting solution will be thorou hly mixed and observed for miscibility. The test article will be rated as either "not miscibg " "partiall miscible," or "completely miscible" in each of the preferred solvents. The mi&ibility raGng and any additional obseivations will be recorded in the study workbook. The solubility/miscibilitytest need not be performed if adequate information regarding the solvent and maximum soluble concentration is available. The solubility or miscibility of the test article in culture medium will also be checked to determine the appropriate concentrations for the tests. Protocol No. 311O.AB 111999 Page 7 of 15 54 I 1 SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-3110 17.2 PreDaration of Test Cultures The CHO stock cultures grown in antibiotic-free medium and showing approximately 50-70% confluency will be harvested and used to pre are the test cultures for the Assa The culture medium from the flasks will be discarde8, and the cells will be washed w i k hosphate buffered saline (PBS).The cells will then be dissociated b trypsin at 37 f 1.- Y 6.C and resus ended in fresh complete culture medium. An aliquot o the cell suspension will be diluted the appropnate concentration and counted using a cell counter. Base4 on the cell counts, a separate cell suspension in complete culture medium with 1x10 cells/mL will be prepared to seed the test flasks. An appropriate number of T-25 cm2 tissue cullure flasks will be seeded with 5.0 mL of cell suspension to obtain test cultures with 5x10 cells/flask. The cultures to be maintained be ond 48 hours after their initiation, will be seeded with an appropnately reduced number o! cells (250,000400,000 cells per flask in order to avoid over rowth of the monolayer. In the p s e of test article2 which t' 4 reac with plastic, 60 mL sten e lass culture flasks will be used instead of T-25 cm culture flasks. The flasks wl! be incubated for approximately 20-24 hours treatment. 17.3 Preparation of Metabolic Activation Svstem The metabolic activation mixture will consist of henobarbital/n-na hthoflavone induced rat liver homogenate (S-9 fraction) (6) and the coyactor pool. The S-5 fraction will be stored at or below -70C in small aliquots. The S-9 will be validated for acceptable levels of rotein content and metabolic activity. Immediately prior to use, the S-9 will be thawed armom temperature and mixed with the cofactor pool to form the metabolic adivation mixture which will consist of 4mM NADP, 5mM lucose-6-phosphate, 30mM KCI. 1OmM.M CI ,50mM sodium hosphate (pH 7.4) and 1O{ pUmL of S-9 fraction.. This mix- ture will l e diluted 1:4 by vogme with serum-free medium and used in refeeding the cuItures. 17.4 Preparation of Test Article The desired. amount of the test article will be weighed as s ecified .in the dilution scheme which will be prepared pnor to treatment for: either the 8ange Findin Test or Assay. The stock solution of the hi hest concentration will be pre ared b aiding the P E Y appropriate volume of solvent to the. est article just prior to use and t orough y mixin the resulting solution until the desired dissolution is achieved. The remaining stock solu?ions specified in the dilution scheme will be prepared b a subsequent dilution or b dissolving Y the required amount of test article in the solvent a each concentration. In all Yreatments, the amount of solvent delivered to the target cultures will be limited to a level which has no si nificant cytotoxic effect on the cells. If necessary, the test article ma be added B direc y to the culture medium. If the test article is found to alter the pH ofYthe culture medium to an extent that is toxic to the cells (7-8). either HEPES buffered medium will be used during treatment time or necessary adjustmentswill be made to the stock solution(s r' or treatme.nt medium pnor to chemical exposure. A record of the pH measurements wi I be maintained in such cases. 17.5 Ranqe Findinq Test If sufficient information is not available regarding the toxici of the test article, a Y Range Finding Test will be performed in order to determine the tes article concentrations that will produce 0-100% cytotoxicity. The test article will be weighed and a serial dilution will be prepared. If there is no solubilit limitation, prior knowled e of cytotoxicity indicates 4; differently, or the Sponsor specifies di erently, the test article w% be tested at eight to ten concentrations at a maximum concentration of 5000 pg/mL and lower concentrations Protocol No. 31IO.AB 11I 9 9 9 55 Page 8 of 15 I ISITEK RESEARCLHABORATORIES AI SITEK Study No. 0623-3110 covering four log dilutions. A solvent control will also be included in both the non-activated and activated systems. An untreated control (exposed only to water) will be included if a solvent other than water or culture medium is used. If a narrower concentration range or lower concentrations are re uired to determine the desired cytotoxic range, the Range Finding Test will be repeatel. The test cultures seeded approximately 20-24.hours earlier and are in the log phase will be used in the Range Finding Test. Duplicate cultures will be used at each concentration level. B In the non-activated system the culture medium will be removed, and 5 mL of fresh complete medium will be added to each of the culture flasks. The cells will then be ex osed to the test article for 3 hours. After the ex osure period the cells will be washed w i g DPBS, refed with complete medium, allowe to grow for i 5 hours with 0.1 pg/mL Colcemid present durin the final 2 hours, and harvested 18 hours after the initiation of the treatment (1.5 x norma? cell cycle time). 17.5.1 Determination of Relative Cell Growth (RCG) (10-11) Y 9 After the Colcemid ex osure the medium with dividin celjs in each flask will be transferred into labeled cenkfuge tubes, the monola er of ce Is will be washed with PBS, dissociated with 0.05% trypsin and resuspended in he collected medium. An aliquot of this cell sus ension will be counted using an electronic cell counter. The number of cells er flask wifbe calculated for each concentration, and the Relative Cell Growth (RCG) will ge calculated according to the following formula: RCG = No. Cells in Test Flask X 100 No. Cells in Solvent Flask TI The cytotoxicity will be evaluated on the basis of the cell number (RCG). If possible a concentrationcausin greater than 50% reduction in RCG will be selected as the hi hest test concentration for $e Chromosome Aberration Assay. .In.addition, four or more ?ower concentrationswill be included in the Assay. If no cytotoxici is observed at the maximum concentrationtested, the Chromosome AberrationAssay will e perfoved at four decreasing concentrations startin with the maximum soluble concentration or one or two concentrations with precipigte. The actual concentrations for the assay.,once determined, will be added to the protocol in the form of an amendment. from 9 Concentrations exceedin an the Chromosome Aberra ion osmolali of Assay (93: 500 mOsmol/kg of water will be excluded 17.6 Chromosome Aberration Assay The Chromosome Aberration Assay will be performed with a single harvest at 1.5 x normal cell cycle time. Parallel Toxicity will be determined by the RCG of treated cells in comparison with solvent control. The procedure is the same as in the Range Finding Test. Protocol No. 31IO.AB I1I 9 9 9 Page 9 of 15 56 ~ S ITEK RESEARcH LABoRATO RIES SITEK Study NO.0623-3110 The test cultures will be prepared as described in Section 17.2. Dup!icate cultures will be treated and used at each concentration in each system in the evaluation of induced chromosome aberrations, RCG and the Relative Mitotic Index (RMI). The treatmen! procedures for the Chromosome Aberration Assay will be the same as in the Range Finding Test. The cells will be treated with four or more concentrations of the test article, two concentrations each of the two positive controls and the solvent control in both the activated and non-activated systems. Untreated controls (only .exposed to water) will be included in the Assay if a solvent other than water or culture medium is used. c: In the non-activated system, the cells. will be treated in corn lete medjum for three hours. After the exposure penod, the medium will be removed, t e cells will be washed with DPBS, refed with complete medium and incubaJed for 15 hours with 0.1 g/mL Colcemid present during the last 2 hours. The cells will be harvested 18 hours a&r the initiation of treatment (1.5 x normal cell cycle time). Inthe activated system, the cells will be treated in serum-free,. S-9 containingmedium for 3 hours. The removal procedure and incubation and harvest times are the same as in the non-activated system described above. After the Colymid exposure, the cell suspension wil! be rocessed to deteyine the RCG first as descnbed in the Range Finding Test, section 77.5.1.. The remaining cell suspension will be processed to determine the Relative Mitotic Index (RMI).. and chromosome aberrations as described below: f 1 The cells will be collected by centrifugation, swelled in hypotonic KCI 0.075M , and fixed in methanol: lacial acetic acid (3:l) fixative. The fixed cells will be ept at -5C. The cells will then%e collected by centrifugation, resuspended in a small volume of fresh fixative and dropped onto microslides to prepare chromosome spreads. The slides will be air dried, stained in Giemsa stain, and mounted in Permount using # I cover glasses. i i The slides will be scored for Mitotic Index (MI). A total of 1000 cells will be scored from each concentration (500 from each.duplicate flask) and the number of dividin cells recorded. The MI for each concentration level will be calculated using the fol%wing formula: MI = No. of Dividina Cells from 1000 Cells The RMI will be calculated as shown below: RMI = Test Concentration MI Solvent Control MI X 100 1 The same slides will be used to score chromosome aberrations, and scored "blind! in order to avoid bias on the part of the scorer s). A total of three test concentrations, if possible, the highest of which causes more t an 50% reduction in RCG, one positive control concentration, the solvent and untreated controls will be scored from the activated and non-activatedsystems. Whenever possible, 100 meta hases will be scored from each of the two du licate flasks. Consequently, 200 metaplases will be scored for each concentration for chromosome aberrations. Only cells with 19-23 chromosomes will be scored, and the microscope coordinates of each cell with findings will be recorded. In addition, the number of endoreduplicated and polyploid cells in a total of 100 metaphases per culture will be scored and recorded. The types of Chromosome Aberrations scored and the corresponding abbreviations used are given below (12-14): Protocol No. 311O.AB I 11999 Page 10 of 15 57 A: SITEK RESEARCHLABORATORIES ? SITEK Study NO. 0623-3110 tb Chromatid break - a discontinuity occurring alon the length of either of the two chromatids, in which there is a misafgnment. isb - Complex: qr - tr - Y lsochromatid break - a discontinui occurring in both the chromatids at the same locus showing compete rejoining or sister Chromatid union at both the broken ends or incomplete rejoining, i.e., only at one of the two broken ends. - Quadriradial chromatid interchanges .between chromosomes leading to four-armed configurations. This could be asymmetrical with formation of a dicentric and an acentric chromatid if union is complete, or symmetrical where there is no formation of a dicentric and an acentnc chromatid. - Triradial isochrpmatid-chromatid exchanges resulting in three- armed configurations and sometimes fragments. The latter should not be scored as an independent aberration. The triradial could be monocentric or dicentric. id - Interstitial deletion ;intra-arm intrachanges resujting in deletion of small fra ments which, however, stay in association with the parent chromati%. i ci Chromatid intrachange - exchanges occurring between arms of the same chromosome resulting in asymmetrical (rings) or symmetrical configurations. - cr Corn lex interchanges multiarmed configurations resulting from breaEage and reunion of two or more chromosomes. 2. Chromosome-tvpe Aberrations Simple: sg sb Chromosome gap - an achromatic region occurring in both chromatids of the chromosome at the same locus with no mtsalign- ment. - Chromosome break a discontinuity at the same locus in both chromatids, giving one acentric fragment which ma be misaligned and a shortened monocentric chromosome, and wlere there is no sister chromatid union. Complex: d 8 Dicentric - an asymmetrical exchange between two chromosomes resulting in a chromosome with two centromeres with or without an accom anying acentric fragment which should not be scored as a secon aberration. Protocol No. 3110.AB 111999 Page 11 of 15 58 A: ~ SlTEK RESEARCHLABORATORIES r - Rin - inter-arm. intrachange .happening within the chromosome, l e a j n g to formation of a centnc nng with or without a chromosome fragment. The fragment should not be scored as a second aberration. dm Double .minutes - intra-arm intrachanges leading to tight acentric paired rings. 3. Other Aberrations - PU - Pulverized chromosome or chromosomes shattering of chromatid material resulting in several minute pieces. The identity of the chromosome is not decipherable. Considered as a single aberration. sd - Severely damaged cell - cell with ten or more aberrations. - PP Pol ploid cells metaphases with multiples or approximate multi les of &e haploid set of chromosomes. Not scored for struc&ral aberrations. e - Endoreduplicatedcells - metaphaseswith paired duplicatedchrorno- somes or diplochromosornes. They are not scored for structural aberrations. b The chromosome aberration data from the score sheets will be consolidated on a Summary Table. The number of aberrations per cell and the percentage of cells with one or more aberrations will be calculated separately for each duplicate culture and then pooled for each concentration. Chromatid gaps and chromosome gaps will not be included in calculating the percentage of cells with aberrations and the number of aberrations per cell. Of the remainin aberrations, each aberration scored will be counted as one, except a severely damage] cell sd) which will be considered .equal to ten abe,rrations n calculating the number of a errations per cell. Endoreduplicated and polyploid cells will be recorded separately in percentages. 17.7 Statistical Analvsis The data for the percentage of cells with aberrations for each concentration will be compared to the solvent control values using a Chi-square test. The results will be considered significant if p 5 0.05. If the solvent control value isso%,the data will be ana! zed using the historical solvent control values. Statistical analysis will not be performed iythe test concentration value is equal to or less than the concurrent or historical solvent control. Ifa positive response is indicated by the Chi-square test, the Cochran-Armitage test (trend test) will be perfoped for evidence of a dose-related response (15). The trend test will be considered positive if p S 0.05. 17.8 Confirmatow Chromosome Aberration Assay A confirmatory assay will not be performed if the first assay is positive either with and/or without activation. PA confirmatory Chromosome.Aberration Assay without activation will be performed if the results of the first assay without activation produced a ative response. A continuous treatment u . to the harvest time of 1.5 x norma! cell time (18 hours) will be erformed. NegaEve results for the first assay with may require a con irmation on a case by case basis. Protocol No. 311O.AB 111999 Page 12 of 15 59 SITEK RESEARCHLABORATORIES SITEK Study NO. 0623-3110 9 The RCG and RMI determination, harvest and chromosome aberration scorin procedures will be the same as in the definitive assay. Parameters, such as tes concentrations, may be adjusted in the confirmatory assay. A confirmatory chromosome aberration. assay with and without activation will be performed, if the first assay produces an equivocal response. 17.9 Criteria for a Valid Assav 1. In the solvent control, the percentage of cells with aberrations should not exceed 4%. 2. At least 25%.of the cells scored in the positive control should show one or more chromosome aberrations. 3. At least one of the test concentrations scored should show greater than 50% reduction in the RCG. This requirement should not be applied to test articles where no a parent toxicity could be achieved at the maximum soluble concentration or highest aiowable concentration. 17.10 Evaluation of Test Results 17.10.1 Positive Response The test article will be considered to have caused a ositive respon.se in this assay if the test article shows a positive dose-response tren8 and a statistically. significant increase over that of the solvent controls in the percentage of cells with aberrations at one or more concentrations. 17.10.2 Neqative Response The test article will be considered to.have caused a negative response if none of the test concentrations shows a statistically significant increase in the percentage of cells with aberrations. 17.10.3 Equivocal Response The test article will be considered to have caused an equivocal response if one of the test concentrations shows a statistically significant increase in the percentage of cells with aberrations without an accompanying positive dose-response trend. 17.10.4 Other Considerations The above criteria will b e used as guidelines in evaluating the test ,results. However, the Study Director may take other factors info consideration in evaluating the test results. 18.0 PROTOCOL AMENDMENTS AND DEVIATIONS s R If chan es in the approved protocol are necessary, such changes will be documented in the form o? protocol amendments and protocol deviations. Protocol amendments will be generated when chan es in the protocol are made prior to performing a study or part of a study affected by tze changes. In such cases, a verbal agreement to make such changes will be made between the Study Director and the Sponsor. These chan es and the reasons for.themwill be documented and attached to the protocol as an adc?endum. Protocol deviations will be generated when the procedures used to erform the study do not conform to the approved rotocol. The Sponsor will be informecf)ofthese deviations, and as soon as ractical, SUC changes, along with their reasons or explanations, will be documented an kept in the study notebook. Protocol No. 311O.AB I 11999 Page 13 of 15 60 ~~~~ ~~~ E LSlTEK RESEARCLHABORATORIES SITEK Study NO.0623-3110 19.0 REPORT OF RESULTS 19.1 Content i The results.of the stud. will be submitted to the Sponsor in the form of a final re ort. A draft report will be submi ed before the final report is issued. The report will incgde, but not be limited to, the following: 1. Name and address of the testing faci!ity and the dates on which the study was initiated and completed, terminated or discontinued. 2. Objectives and procedures stated in the approved protocol, including any changes in the onginal protocol. 3. Methods used to analyze the data. 4. The test and control substances. 5. Description of the methods used to perform the study. 6. The name of the Study Director and the names of other technical personnel or other professionals who participated in performing the study. P 7. A description of the transformations, calculations or o erations performed on the data, a summary and analysis of the data, and a statement of he conclusions drawn from the analysis. 8. The signed and dated reports of the Study Director or other professionals involved in the study. 9. The location where the raw data and reports are to be stored. 10. A statement from the Quality Assurance Unit. 19.2 Chanses and Corrections to the Final Report All changes to the final report will be in the form of report amendments which will include the reason(s) for the change, and these amendments will be added to the final report as an addendum. 20.0 ARCHIVES The raw data, slides, protocol, documentation, electronic file containin the data tables, and final report of the study will be maintainedin the SlTEK Research La%oratories Archives, 15235 Shady Grove Road, Suite 303, Rockville, Maryland 20850 according to the terms and conditions of the study. 21.O REFERENCES 1. Evans, H.J. Cytolo ical Methods for Detecting Chemical Muta ens, In: Chemical Mutagens, Principles and #lethods Press, New York and London, pp. 1fo-2r 9th(e1ir97D6e)t.ectio.n, Vol. 4, Hollaenzer, A. (ed) Plenum 7 2. Galloway, S . M., et al. Develo ment of a standard protocol for in vitro cytogenetic testing with Chinese hamster ova cets: Comparison of results for 22 compounds in the laboratories. Environ. Mutagen., -:1-51, 1985. Protocol No. 311O.AB 111999 Page 14 of 15 61 A:y ' SITE!( RESEARCLHABORATORIES SITEK Study NO. 0623-3110 8. 3. Gallowa , S.M., M.J. Arrnstron , C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. dakamura, M. Ahmed, Duk, J. Rimpo, G.H. Margolin, M.A Resnick, G. Anderson and E. Zeiger. Chromosome aberration and sister chromatid exchanges in Chinese hamster ova cells: Evaluation of 108 chemicals. Environ. molec. Mutagen 10 (suppl. lo), 1-175 (1937). &! 4. Mutagenici.ty ("In Vitro" Mammalian C o enetic European Communitles, B.10. L383A. Volume ?992. Test) Official Journal of the 5. Galloway, S.M., et al. Report from working group on in vitro tests for chromosomal aberrations. Mut. Res., =:241-261, 1994. 6. Elliot, B.M., e t al. Alternatives to Aroclor 1254-induced S9 in in vitro genotoxicity assays. Mutagenesis, 1:175-177, 1992. 6. 7. Scott, D., S.M. Galloway R.R., Marshall, M. Ishidate, D. Brusick, Jr.. J. Ashby and B.C. M hr. Genotoxicity under gxtreme Culture Conditions. A report from ICPEMC Tast Group Mutation Res., 257: 147-204, 1991. 8. Morita, T. et al. Clastogenicity of low pH to various cultured mammalian cells. Mut. Res., 268: 2!%-261, 1992. 9. Gallowa , S. M;; et al. False positive in vitro chromosome aberration tests with non-mutagens aY high concentrations and osmdams. 1985 EMS Abstracts:s. F I O . . International Conference on Harmonisation of Technical Requjrements for Re istration of Pharmaceuticals for Human Use. ICH Harmonrsed Trr artrte Guideline S21. Guidance on Specific Aspects of Requlatow GenotoxicityTests for harmaceuticals. Federal Kegister 61 (80): 18198-18202, 1996. 11. OECD Guideline for the Testin of Chemicals. Proposal for U dating Guideline 473. In Vitro Mammalian ChromosomeqAberration Iest. Draft (Feb. 97). 12. Evans, H. J., and M. L. O'Riordan; Human en heral blood lymphoc tes for the analysis of chromosome aberrations in mutagen tesk. hut. Res., 3J:135-148, 1975. 13. Sava e J. R. Classification and relationships of induced chromosomal structural changes. J. ded. Genetics, =:103-122, 1976. % 14. Scott, D., B. J. Dean, N. D. Danford and D. J. Kirkland. Meta hase chromosome aberration assays in vitro. In:. Basic Muta enicity Tests: UKEhS Recommended Procedures. Cambridge University Press, pp. 2-86, 1990. 15. Margolin, B.H.,et al. Statistical analysis for in vitro cytogenetic assays using Chinese hamster ovary cells. Environ. Mutagen., 4:18X?w1986. Protocol No. 311O.AB 111999 62 Page 15 of 15 SITEK Study NO. 0623-3110 PROTOCOL AMEh?)MENTS Amendment Nos. : Sponsor: Testing Facility: Sponsor's Study No.: SITEK's Study No.: Test Article I.D.: i Protocol Title: 1-3 Primedica Redfield 100 E. Boone Street Redfield, Arkansas 72132 SITEK Research Laboratories 15235 Shady Grove Road, Suite 303 Rockville, Maryland 20850 132-009 0623-3110 Potassium Perfluorobutane Sulfonate Test for Chemical Induction of Chromosome Aberration in Cultured Chinese Hamster Ovary (CHO) Cells With and Without Metabolic Activation Amendment No. 1: Protocol Page 1, Section 4.1, Identification - The test article name should be Potassium Perfluorobutane Sulfonate instead of Potassium Perfluorobuntane Sulfonate. Reason for Amendment No. 1: To correct a misspelled name. Amendment No. 2: Protocol Page 3, Section 5.0, Remulatow Agencv Submission: The GLP standard for the OECD should be Organisation for Economic Cooperation and Development, The OECD Principles of Good Laboratory Practice, Environment Monograph No. 45 [ENV/MC/CHEM(98)171, Paris 1998. Reason for Amendment No. 2: The OECD GLP standard was updated. 63 SITEK Study NO.0623-3110 Protocol Amendment Nos. 1-3 SITEK Study NO. 0623-3110 Page 2 Amendment No. 3: Protocol Page 9, Section 17.5.1, Determination of Relative Cell Growth mCG) (10-111 - The Chromosome Aberration Assays were performed with the following concentration: Definitive Assay (Bl): 500, 1000, 2500 and 5000 pg/mL in both the non-activated and activated systems. Codmatory Assay (l32): 50, 100, 500, 1O00, 2500 and 5000 pg/mL in the nonactivated system only. Reason for Amendment No. 3: As specified in the protocol, to be added in the form of an amendment once determined. I APPROVAL: Jhg Xu': M.D. Study Director John Seng, Ph.D. Date Sponsor's Study Coordinator 64 SITEK Study NO. 0623-3110 APPENDIX IV DOSING SOLUTION ANALYSIS REPORT i 65 _ _ _ ~ ~~~~ ~~~ - SITEK Study NO. 0623-3110 DOSE FORMULAION ANALYSIS OF PERFLUOROBUTANESULFONATE IN DMSO BY HPLCMS STUDY ID: A239.1 SPONSOR STUDY NO. 132-009 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 66 SITEK Study NO. 0623-3110 1. OBJECTIVE The objective of this study was to determine the dose concentration of PFBS in the supplied dosing solutions received from the study director. 2. SAFETY All necessary procedures to ensure safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by the study director for the test article used in this study. 3. Compliance This work was performed using a validated analytical method (BACG 3533), which along with the validation report is included in the appendix of this report. While this work was not audited in compliance with GLP regulations, it was performed in the spirit of the regulations using calibrated and validated instrumentation. 4. EXPERIMENTAL i 4.1 Analytical Procedures The sample preparation and analysis procedures as described in the analytical method BACG 3533 were employed for all analyses. Each sample was allowed to warm to room temperature and was then vortexed well before an aliquot was taken. Duplicated aliquots were taken from each sample and diluted as described in the method. Two standard curves was prepared over a concentration range of 500 to 10,000 ng/mL and analyzed along with the samples. A single composite curve with a correlation coefficient of 0.9991 was used to quantitative the samples. N o outliers in the calibration standards were noted and subsequently no standards were dropped from either of the calibration curves. 4.2 Results The results of the analysis are presented in the Table I at the end of the report. 5.0 Conclusion A total of 12 dose formulation samples ranging in concentration from 5 to 500 mg/mL were analyzed by BACG 3533. All samples except two, 100 mg/mL B1, 8/22/00 (111%) and 250 mg/mL B2, 9/12/00 (117% of target) were found to be within k 10 % of the reported 67 SITEK Study NO. 0623-3110 concentration. The latter sample was analyzed a second time and found to have a concentration of 116 % of target. 68 SITEK Study NO. 0623-3110 Table I Dose Fomiulation Analysis of PFBS in DMSO Sample Date Prepared Measured Conc. (mg/mL)* o/o of Target ND = not detected NA = not applicable * = average of duplicate analysis ** = sample analyzed twice producing same result 69 SITEK Study NO. 0623-3110 Quality Assurance Statement Final Report On Dose Formulation Analysis of Perfluorobutane Sulfonate (PFBS) I n DMSO A239.1 Sponsor Study No. 132-009 This study was performed using a method validated for accuracy, precision (repeatability), linearity, range and specificity. No audits were performed during the course of the study, either as the analyses were performed, of the resulting data, or the report. C I J w C.L. Mhrsh, Manager, Quality AssuranceIQualityControl 70 SITEK Study NO.0623-3110 SUMMARY A total of 12 fomiulated dose samples including blanks and vehicles ranging in concentration from 5 to 500 mg/mL were analyzed by analytical method BACG 3533 to determine the concentration of perfluorobutanesulfonate (PFBS) in the formulated mixture. All dose formulations were found to be within k 10% of the reported concentration except for two samples, 100 mg/mL B1 dated 8/22/00 and 250 mg/mL B2 dated 9/12/00. This former sample had a formulation concentration of 110.5 % and the latter sample was initially found to be 117 % of target and upon reanalysis it was found to be 116 % of target. 71 -- ~~~ ~ ~ SITEK Study NO. 0623-3110 KEY PERSONNEL James D. Johnson, M.S., MBA Manager Bioanalytical Chemistry Group Gregory S. Gonnan, Ph.D. Staff Chemist Bioanalytical Chemistry Group 72 SITEK Study NO. 0623-3110 6.0 Approvals Staff Chemist Bioanalytical Chemistry Group 1/ Manager v Bioanalytical Chemistry Group ,7-50/ Date I Date 73 SITEK Study NO. 0623-3110 METHOD VALIDATION REPORT VALIDATION OF ANALYTICAL METHOD FOR DOSE FORMULATION ANALYSIS OF PEWLUOROBUTANE SULFONATE (PFBS) I N 1%CARBOXYMETHYL CELLULOSE (CMC) STUDY ID: A098.1 Southern Research Institute 2000 Ninth Avenue South P.O.Box 55537 Birmingham, AL, 35255-5537 74 SITEK Study NO.0623-3110 SUMMARY Southern Research Institute has successfully validated for 3M an analytical method (BACG 3533) entitled "Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC Mass Spectrometry". Calibration standards were prepared by spiking solvent solutions with known amounts of test article, PFBS, and internal standard, perfluoropentanoic acid. The calibration standards were prepared over a concentration range of 500 to 10,000 ng/mL and the carboxymethyl cellulose (CMC) concentration was adjusted to equal that of each diluted sample in each set. Additionally, three standard curves were prepared and analyzed which contained no CMC as controls. A total of 6 calibration curves were generated during the study which produced correlation coefficients ranging from 0.9992 to 0.9999. I 75 KEY PERSONNEL James D. Johnson, M.S. Manager Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Research Chemist 111 Bioanalytical Chemistry Group Lester Williams, B.S. Associate Chemist II Bioanalytical Chemistry Group SITEK Study NO.0623-3 110 76 SITEK Study NO. 0623-3110 1. OBJECTIVE The objective of this study was to provide a validated analytical method for the determination of PFBS concentration in dose formulation samples containing 1YOCMC. 2. SAFETY All necessary procedures to ensure safety of the analysts were based on information contained in the Material Safety and Data Sheets (MSDS), provided by 3M for the test article used in this study. 3. EXPERIMENTAL 3.1 Analytical Procedures . -The sample preparation and analysis procedures as described in the analytical method BACG 3533 were employed for all analyses. For the preparation of the calibration standards, a known volume of a solvent (e.g., 1 mL) containing CMC was spiked with a known amount of test article and internal standard and vortexed briefly to ensure mixing. Each formulation sample was diluted to a final concentration of 3200 n g h L and ah aliquot of this was placed into autosampler vials for analysis. 3.2 Method Validation - Validation for BACG 3533 "Dose Formulation Analysis of PerfluorobutaneSulfonate in 1% Carboxymethyl Cellulose by HPLC Mass Spectrometry" consisted of analyzing three standard curves containing CMC at the concentration present in the diluted 4 mg/mL dose formulation samples (0.001%) and three curves prepared without CMC. The concentration range for both sets ranged from 500 to 10,000 ng/mL. A total of 7 calibration levels in each curve containing CMC and 6 in each of the three without CMC were evaluated. The summaries are given below: Calibration Results 0% CMC A total of three standard curves each containing 6 individual standards encompassing a range of 500 to 10,000 ng/mL were analyzed in duplicate. The correlation coefficient for the composite curve comprising a total of 36 data points was found to be 0.9992. No data points were dropped from the analysis. A statistical summary for the composite curve is shown below: 77 SITEK Study NO. 0623-3110 STANDARD COMPOSITE CURVE PFBS (0% CMC) Concen. (ng/mL) # of Standards Mean% Accur. Std. Deviation 500 6 97.71 19.47 1000 6 102.9 55.85 3000 6 99.73 110.6 5000 6 99.52 187.8 8000 6 10,000 6 I 1 100.1 100.2 288.2 356.4 Calibration Results (CMC = 0.001%) Three standards curves containing a total of 21 single standards encompassing a range of 500 to 10,000 ng/mL were analyzed. The correlation coefficient for the calibration curve was found to be 0.9997. No data points were dropped from the analysis. A statistical summary for the composite Fume is shown below: STANDARD COMPOSITE CURVE PFBS (0.001% CMC) # of Standards Mean% Accur. Std. Deviation 3 98.11 18.19 3 101.5 17.72 3 101 48.33 3 101 49.67 3 98.44 81.61 3 99.31 76.38 -3 100.7 203.4 3.3 Calculations Calculations were performed using TurboQuan (Version 1.0). The amount of analy-te in the diluted dose formulation samples (ng/mL) was back calculated using a calibration curve generated from a set of calibration standards containing the equivalent amount of CMC as in the diluted samples. The calibration curve was generated by a regression analysis to determine the best fit curve (e.g., linear, quadratic etc.) and amount of weighting. A quadratic fit with 1/X weighting was determined to be the best fit : where: y = Peak area response of test article x = Concentration of the test article in standards. a,b, c = Constants derived from the regression analysis. 78 SITEK Study NO. 0623-3110 4.0 Conclusion A quantitative method (BAGC 3533) has been developed and validated for the determination of PFBS in dose formulation solutions containing 1% CMC. Quantitation for this method is based on internal standard which produces standard curves with a correlation coefficients of 0.9992 or greater over a concentration range of 500 to 10,000 ng/mL. 5.0 Approvals -'/" Greg& S. G%o' rman, Ph.D. Staff Chemist Bioanalytical Chemistry Group 7-5==/ Date v Date Manager - Bioanalytical Chemistry Group 79 SITEK Study NO.0623-3110 Page 1 of 8 ANALYTICAL METHOD Method No.: BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) 1 .o PRINCIPLE Dose formulation samples of perfluorobutane sulfonate (PFBS) in 1 % carboxymethyl cellulose (CMC) are diluted down to 3200 ng/mL and analyzed by HPLC/MS. Quantitation is based on an internal standard using back calculated values from a calibration curve encompassing a concentration range from 500 to 10,000 ng/mL and containing the same concentration of CMC as the dose formulations in the diluted form. - 2.0 I REAGENTS AND SOLUTIONS The listed reagents or their equivalents may be used. 2.1 Neat Reagents 2.1.1 Water, deionized and organic free (from in-house purification system; e.g., Ingalls 210N) - 2.1.2 Methanol, HPLC grade 2.1.3 Perfluorobutanesulfonate (analyte), as provided by the client 2.1.4 Perfluoropentanoicacid (internal standard), 97% 2.1.5 Ammonium acetate, HPLC grade 2.2 2.2.1 Prepared Solutions Appropriate changes in the solutions may be made at the discretion of the analyst 5 mh4 Ammonium acetate in organic free water 80 SITEK Study NO. 0623-3110 Page 2 of 8 ANALYTICAL METHOD Method No.: BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLUMS) 2.2.1.1 For example, to prepare 4 liters, measure out ammonium acetate (e.g., 1.542 g) and add in organic-free water (e.g., 4 L). Mix well and filter through HPLC mobile phase filtration apparatus. 3 .O INSTRUMENTS, MATERIALS, AND APPARATUS i 3.1 The following or their equivalents m a y be used. HPLC pump(s), autosampler, and single quadrupole mass spectrometer 3.2 Autosampler vials with inserts 3.3 Vortex mixers (e.g., touch mixer and IKA-Vibrax @ platform mixer) 3.4 HPLC mobile phase filtration apparatus 3.5 Filters for HPLC mobile phase filtration apparatus (e.g.. Nylon-66, 0.20 pm) 3.7 Analytical balance 3.8 Volumetric flasks (e.g., 10 and 25 mL) 3.9 Disposable Pasteur pipets 3.10 Micropipettor(s) with tips 3.11 Culture tubes with teflon-lined caps 3.12 Assorted glassware and syringes 81 SITEK Study NO.0623-3110 Page 3 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) 4.0 4.1 4.1.1 4.2 4.2.1 4.3 4.3.1 PREPARATION OF STOCKS AND WORKING STOCKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. Actual dilutions will be documented on the preparation sheets. Main Stock Solution of PFBS -1000 pg/mL Prepare an - 1000 pg/mL solution of PFBS in mobile phase (e.g., accurately weigh about 10 mg PFBS into a 10-mL volumetric flask). Add mobile phase to dissolve. Dilute to the mark. Alternatively, weigh the compound into an appropriate vessel (e.g., culture tube) and add 10 mL of mobile phase. Mix well. Transfer the solution to a clean vessel if desired. - Spiking Stock Solution of PFBS -50 pg/mL Prepare an -50 pg/mL solution of PFBS in mobile phase (e.g., measure about 500 pL of -1000 pg/mL PFBS into a 10-mL volumetric flask). Add mobile phase to dissolve. Dilute to the mark. Alternatively, weigh the compound into an appropriate vessel (e.g., culture tube) and add 10 mL of mobile phase. Mix well. Transfer the solution to a clean vessel if desired. - Stock Solution of Internal Standard (PFPA), -200 pg/mL Prepare an -200 pg/mL solution of PFPA in deionized organic-free water (e.g., accurately weigh about 10 mg into a 50-mL volumetric flask). Add deionized organicfree water to dissolve and dilute to the mark with deionized organic-free water. Alternatively, weigh the compound into a an appropriate vessel (e.g., culture tube) and add 50 mL of deionized organic-free water. Mix well. Transfer the solution to a clean vessel if desired. 82 SITEK Study NO.0623-3110 Page 4 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) 4.3 4.3.1 Working Stock Solutions of PFBS To prepare working stock solutions, make the proper dilutions as shown in the following table. Prepare in 10-mI, volumetric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. - Working Stock Level Volumeof - (WSL) Internal Volume of PFBS Volume of mobile 1 Approximate Concentration Standard spiking solution phase (ILL) (ng/mL) (PL) (VU Note: The mobile phase used for each standard curve should contain the same concentration of CMC as the samples. For example, the dilution factor of a 4 mg/mL sample is 1000 (prior to addition of IS). The mobile phase for a 4 mg/mL sample prepared in 1%CMC should contain 1/1000%CMC to account for the dilution. 5.0 PREPARATION OF SPIKED STANDARDS AND BLANKS Appropriate changes in the concentrations of the solutions may be made at the discretion of the analyst. 83 SITEK Study NO. 0623-3110 Page 5 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) 5.1 Multiple (e.g., about three) sets of standards blanks (blank + IS) are analyzed with each set of unknown samples. A double blank (blank-IS) may also be analyzed if desired. Standards may be prepared as shown in the table in 4.3.1. 6.0 PREPARATION OF SAMPLES 6.1 i All samples are to be diluted to approximately the middle of the curve (e.g. 3200 ng/mL). Dilutions are to be made with mobile phase. For example, for a 4 mg/mL sample,-take 1mL and dilute with mobile phase in a 10 mL volumetric flask. Then take 100 pL of the diluted sample and dilute that with mobile phase in a 10 mL volumetric flask. Take 800 pL of the second dilution, place in an autosampler vial, add 200 pL of IS, mix, and analyze. 7.0 ANALYSIS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY MASS SPECTROMETRYMASS SPECTROMETRY (IPLC/MS/MS) 7.1 Conditions are to be optimized if necessary. - 7.1.1 HPTC-C . . Analytical Column: none Guard Column: Elution Flow rate: Injection volume: Mobile phase: Fluofix 120E 10 mm x 2 mm 1000 pL/min. 5 PL A: 5mM ammonium acetate buffer B: methanol Elution Profile: Temperature: Isocratic 30% A : 70% B Ambient 7.1.2 PE Sciex API 150EX Single Quadrupole Mass Spectrometer Conditions 84 SITEK Study NO. 0623-3110 Page 6 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) Software: PE Sciex TurboQuan Note: Values listed under "MS/MS Acquisition,Conditions" override parameters in this table. Auxiliary Gas: Air (e.g., Grade 0.1) at 85 pounds per square inch Parameter Is NC TEM OR RNG Qo 1Q 1 ST RO 1 DF CEM NEB CUR CAD QPE POL VCM IPE Yak -5000 0 450 -25 -170 10 - 11 16 11 300 2400 15 8 0 0 0 0 0 85 SITEK Study NO. 0623-3110 Page 7 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) 8.0 1 8.1 Masses requested: -w PFBS: 299.2 200 PFPA (IS) 218.9 200 CALCULATIONS At the end of the analytical run, review each chromatogram to ensure the retention time, peak shape, and peak height and peak area determination of the test article and the IS are acceptable. The data may be smoothed as appropriate. For quantitation, use the ion profiles at the following mass-to-charge ratios: -Analvte PFBS PFPA -. 299.2 218.9 8.2 Plot the peak area response of PFBS divided by the peak area response of the IS (PFPA) from all standards versus the concentration of the test article in the standards. Alternathely, the peak heights may be used instead of peak areas. Obtain the best curve fit of the data (e.g., quadratic fit weighted with Uconcentration of the test article or a quadratic fit). Note: The best curve fit may be dependent on the range of the standard curve and it may be necessary to have more than one standard curve for various concentration ranges using the following: where y = Peak height response of PFBS divided by peak height response of the IS (PFPA) in standards. x = Concentration of the PFBS in standards. 86 SITEK Study NO.0623-3110 Page 8 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) a, b, c = Constants derived from the regression analysis 8.3 Using the standard curve, calculate the level of PFBS in each unknown sample. Correct the results of samples for any dilutions. 9.0 ACCEPTANCE AND REJECTION CRITERIA - 9.1 Refer to SOP SRI 91-3 for acceptance/rejection criteria except acceptable accuracy for I standards is 80-120% of theoretical. 10.0 REPORTING 10.1 Results of all analyses are tabulated, and the raw data, original chromatograms, and reports are to be filed in the appropriate study file. Authors: Gre&KGorman, 7-/ 3-2mo Ph.D., Research Chemist 111 Date - 7- 17- 2 0 0 0 Date 87