Document VJeqOEyNQO6KdVg958Gjqq6Xp

6) OECD 471-OPPTS 870.5100, Bacterial reverse mutation test (Ames test), 0623-2140 SANITIZED DEC 0 9 2003 F"AL REPORT Primedica Redfield Test Article: Potassium Perfluorobutane Sulfonate SITEK Study NO. 0623-2140 July 12, 2001 7A. .SITEIC R E S E A R CLHABORATORIES 15235 Shady Grove Road, Suite 303, Rockville, Maryland 20850 301/9264900 FAY 301/926-8891 Study Title Evaluation of a Test Article in the Salmonella typhimurium/Escherichia coli Plate IncorporatiodPreincubation Mutation Assay in the Presence and Absence of Induced Rat Liver S-9 Test Article I.D. Potassium Perfluorobutane Sulfonate Author Kamala J. Pant, M.S. Performing Laboratory SITEK Research Laboratories 15235 Shady Grove Road, Suite 303 Rockville, Maryland 20850 Laboratory Project ID SITEK Study NO. 0623-2140 Sponsor's Study No. 132-008 Study Initiation Date August 1, 2000 Study Completion Date July 13, 2001 Sponsor Primedica Redfield 100 E. Boone Street Redfield, AR 72132 Page 1 of 92 - ~~ ~ ~~~~ SITEK Study NO. 0623-2140 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS Study NO. 0623-2140 Sponsor's Test Article ID: Potassium Perfluorobutane Sulfonate The study described in this report was conducted in compliance with the following Good Laboratory Practice standard: 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, Paris 1992, except for the strength and stability of the test article dosing solutions and controls under experimental conditions, which were not determined by SITEK Research Laboratories. However, the dosing solutions were analyzed by Southern Research Institute, Birmingham, Alabama. The analysis was not done under GLP conditions. :I Signature Kamala J. Pant, M.S. Study Director 7 -B ? Q \ Date 2 SITEK Study NO. 0623-2140 QUALITY ASSURANCE UNIT'S STATEMENT :.I Study NO. 0623-2140 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: 08-02-00 The following phases were inspected for this study: Inspection Date Phases Inspected Date Findings Reported to Study Director Date Findings Reported to Management 9-25-00 10-04-00 10-04-00 07- 13-01 Confirmation of the Tester Strain Genotvpes Workbook Audit Draft Report Audit Final Report Audit 09-25-00 10-04-00 10-04-00 07-13-01 09-25-00 10-04-00 10-04-00 - 07-13-01 Signature Paul E. k b y , Ph.D. Quality Assurance Unit 7-f-3-01 Date i 3 SITEK Study NO. 0623-2140 STUDY DIRECTOR'S SIGNATURE PAGE This study was performed under the supervision of Kamala J. Pant, M.S., Study Director for Salmonella typhimurium and Escherichia coli Gene Mutation Assays, at SITEK Research Laboratories, 15235 Shady Grove Road, Suite 303, Rockville, Maryland 20850. The Final Report for this study was written by the Study Director and released on July 13, 2001. Signature Kamala J. Pant, M.S. Study Director & Pn.,*-k Date 7- 1'3t01 f 4 SITEK Study NO. 0623-2140 ABSTRACT The test article, Potassium Perfluorobutane Sulfonate, was tested for its potential to cause mutations at the hstidine operon of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and at the tryptophan operon of Escherichia coli strain WP2uvrA. The test article, dissolved in DMSO, was tested for toxicity to strains TAlOO and WP2uvrA in a Range Finding Test (RFT) at test article concentrations ranging from 5.0-5000 pg/plate. The tester strains were exposed to the test article in the absence of exogenous activation and in the presence of induced rat liver S-9 plus cofactors. The toxicity was evaluated based on: 1) reversion frequency, 2) viability, and 3) integrity of the background lawn. The first (definitive) Mutation Assay (B-1), using the plate incorporation method of treatment, was performed with the four Salmonella typhirnurium tester strains and with Escherichia coli strain WP2uvrA. Based on the results of the l2FT (A-1), the test article was tested at the following concentrations in the Mutation Assay: 50, 100, 500, 1000 and 5000 pglplate. The second (confirmatory) Mutation Assay (B-2), using the preincubation method of treatment, was performed to confirm the results of the definitive assay using the same test 1 article concentrations with and without activation. Both negative and positive controls fulfilled the requirements of the test. The results of both Mutation Assays indicate that the test article did not induce any significant increase in the number of revertant colonies for any of the tester strains in the presence or absence of induced rat liver S-9. Under the conditions of this study, Potassium Perfluorobutane Sulfonate was negative in the Salmonella typhimurium/Escherichia coli Plate IncorporatiodPreincubation Mutation Assay with and without metabolic activation. 5 SITEK Study NO. 0623-2140 TABLE OF CONTENTS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS................................................ 2 QUALITY ASSURANCE UNIT'S STATEMENT............................................................................................. 3 STUDY DIRECTOR'S SIGNATURE PAGE..................................................................................................... 4 ABSTRACT ........................................................................................................................................................... 5 INTRODUCTION................................................................................................................................................. 8 MATERIALS....................................................................................................................................................... 10 TEST ARTICLE CONTROL SUBSTANCES INDICATOR CELLS CULTURE CONDITIONS S-9 METABOLIC ACTIVATION SYSTEM EXPERIMENTAL PROCEDURES.................................................................................................................. 13 .- I I DOCUMENTATION TEST SYSTEM IDENTIFICATION SOLUBILITY TEST PREPARATION OF TEST CULTURES PREPARATION OF S-9 METABOLIC ACTIVATION SYSTEM PREPARATION OF TEST ARTICLE DOSING SOLUTIONS RANGE FINDING TEST MUTATION ASSAYS CRITERIA FOR A VALID ASSAY.................................................................................................................. 21 EVALUATION OF TEST RESULTS............................................................................................................... 22 ARCHIVES............................................................................................................... i.......................................... 23 RESULTS ............................................................................................................................................................ 24 SOLUBILITY TEST RANGE FINDING TEST (A-1) MUTATION ASSAYS DOSING SOLUTION ANALYSIS RESULTS CONCLUSIONS ................................................................................................................................................. 26 REFERENCES.................................................................................................................................................... 27 APPENDIX I ....................................................................................................................................................... 28 DATA TABLES 6 SITEK Study NO. 0623-2140 APPENDIX II...................................................................................................................................................... 35 DETAILED PLATE COUNTS AND BACKGROUND LAWN EVALUATION APPENDIX 111.................................................................................................................................................... 48 STUDY PROTOCOL AND PROTOCOL AMENDMENTS APPENDIX IV..................................................................................................................................................... 67 HISTORICAL POSITIVE AND SOLVENT CONTROL DATA APPENDIX V ...................................................................................................................................................... 71 DOSING SOLUTION ANALYSIS t i 7 SITEK Study NO. 0623-2140 INTRODUCTION The purpose of this study was to evaluate the test article, Potassium Perfluorobutane Sulfonate, for its potential to cause mutations in the histidine operon of Salmonella typhimunum strains TA98, TA100, TA1535 and TA1537 and the tryptophan operon of Escherichia coli strain WP2uvrA. The Salmonella typhimurium/Escherichia coli Plate IncorporatiodPreincubation Mutation Assay has been used extensively and has been demonstrated to be effective in detecting mutations caused by compounds from a wide range of chemical classes. The Ames Assay is the most widely used of all methods for determining the mutagenicity of chemicals. Because the bacterial strains used in this assay lack the enzymes necessary for metabolizing promutagens to ultimate mutagens, rat liver S-9 (induced with either Aroclor 1254 or phenobarbital) was added as a substitute for mammalian metabolism. This assay detects point mutations only and measures reverse mutation from acid auxotrophy to prototrophy. In this method, bacterial strains were used which carry base substitution or frame-shift mutations in operons coding for synthesis of specific amino acids. Therefore, these mutants (unlike their wild-type counterparts) cannot synthesize all their required amino acids from inorganic sources of nitrogen, being auxotrophic for the specific amino acid. This assay determines whether the test article can reverse the effect of the pre-existing mutation by . I introducing a second mutation, either at the structural gene or at another site on the bacterial chromosome. The following are the details of mutations in the different strains (4): * TA98 was derived from TA1538 (pKM101 plasmid added). ** TAlOO was derived from TA1535 (pKM101 plasmid added). rfa - Defective lipopolysaccharide coat. More permeable to chemicals. (Sensitive to crystal violet.) uvrB - Reduced error-free repair of some types of DNA damage. (Sensitive to W light.) R Factor (pKh4lOl) - Increases sensitivity by enhancing error-prone DNA repair. (Ampicillin resistant if piasmd present.) uvrA - Less DNA repair. 8 SITEK Study NO. 0623-2140 This study was conducted by Kamala J. Pant, M.S., Sonia Srivastava, M.S., and Jieyu Chen, M.D., from August 4,2000 to September 25, 2000, at SITEK Research Laboratories. The experimental procedures used to perform this study were essentially those of B. N. Ames, et al. (l),D. Maron and B. N. Ames (2), M. H. L. Green and W. J. Muriel (3), and S. Venitt and J. M. Parry (eds.) (4). 9 SITEK Study NO. 0623-2140 MATERIALS TEST ARTICLE 1. Name: 2. Batch/Lot No.: 3. CAS No.: 4. Physical Appearance: 5. Date Received: 6. Storage Conditions: 7. Purity Information: 8. Expiration Date: Potassium Perfluorobutane Sulfonate C4F9S03-WLot 2 29420-49-3 White Powder June 8, 2000 and September 7, 2000 Room Temperature 100% Pure 04-06-01 CONTROL SUBSTANCES Positive Controls The positive control chemicals used for the tester strains in the presence and absence of exogenous metabolic activation are presented below: ! Strain s-9 Chemical Concentration (wg/plate) TA98 - TA98 + + TA 100 - TAlOO TA1535 TA1535 + TA1537 - TA1537 + WP2uvrA - WP2uvrA 2-NF (2-Nitrofluorene) 5.0 2-AA (2-Aminoanthracene) 1.25 NaAz (Sodium Azide) 1.o 2-AA (2-Aminoanthracene) 1.25 NaAz (Sodium Azide) 1 .o 2-AA (2-Aminoanthracene) 1.25 9-AA (9-Aminoacridine) 50 2-AA (2-Aminoanthracene) 1.25 MMS (Methyl Methanesulfonate) 4000 2-AA (2-Aminoanthracene) 10 10 SITEK Study NO. 0623-2140 The following is the information for each of the positive controls used in this assay: Chemical Source CAS No. Lot No. Storage Conditions Expiration Date 2-AA Sigma 613-13-8 39H0945 1-5C 09-1 6-04 9-AA Aldrich 52417-22-8 08326" 1-5C 07- 15-01 2-NF Aldrich 607-57-8 BY01703EV 1-5C 04-06-02 NaAz Sigma 26628-22-8 110H0269 1-5C 04-06-02 MMS Aldrich 66-27-3 08109BU 1-5C 08-11-04 All of the positive control substances, except NaAz and MMS (dissolved in sterile, distilled, deionized water), were dissolved in dimethyl sulfoxide (DMSO). The source, lot number and expiration date of the DMSO are given below: Source: Mallinckrodt Lot No. : 4948N44H11 Storage Conditions: Room Temperature 1 CAS Number: 67-68-5 Expiration Date: April 2003 The source, batch number and expiration date of the sterile, distilled, deionized water are given below: Source: SITEK Research Laboratories Batch Nos.: 23, 24 and 25 Storage Conditions: Room Temperature Expiration Dates: 12-20-00, 01-14-01 and 02-15-01 respectively. Solvent Control The stock solutions of the test article were prepared in DMSO. Therefore, DMSO was used as the solvent control. The source, lot number and expiration date of the DMSO are the same as stated above. 11 - I SITEK Study NO. 0623-2140 INDICATOR CELLS Source The Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were originally obtained from Dr. Bruce N. Ames, University of California, Berkeley. The Escherichia coli strain WP2uvrA was obtained from Ms. Judy Mayo of Pharmacia and Upjohn Co., Kalamazoo, Michigan. CULTURE CONDITIONS The cells were grown in Oxoid Nutrient Broth No. 2 in a shaker incubator rotating at approximately 120 rpm and maintained at a temperature of 37 f 1C. Stock cultures of the tester strains were cryopreserved at SITEK Research Laboratories. Scrapes from the cryopreserved stock were used to initiate the overnight cultures for the test. S-9 METABOLIC ACTIVATION SYSTEM For the activated portion of the Mutation Assays, the cells were exposed to the test article in conjunction with an exogenous metabolic activation system consisting of Aroclor 1254 or 1 phenobarbital-induced rat liver S-9 in 0.15M KC1 plus cofactors (S-9 mix). The components of the standard S-9 mix were 8 m M MgCI,, 33mM KCI, 5 m M glucose-6-phosphate, 4mM NADP, l O O m M sodium phosphate buffer (pH 7.4), and 10% rat Iiver homogenate prepared from Aroclor 1254 or phenobarbital-induced, Sprague-Dawley rats. The S-9 batches used in this study were also evaluated for their ability to metabolically activate a promutagen in the Salmonella typhimurium Plate Incorporation Mutation Assay (Ames Test) in tester strain TAlOO using a single dose of 2-AA. The following is the information pertaining to the S-9 batches used in this study: Source: Inducing Agent: S-9 Batch No. : Protein Content: Storage Conditions: Expiration Date: Molecular Toxicology, Inc. Aroclor 1254 1064, 1092 and 1120 39.9, 46.1, 45.8 mg/mL, respectively. I-7O"C 03-01-02, 04-10-02, 06-27-02 and 10-12-02, respectively. SITEK Research, Labs. Phenobarbita1 101299 36.8 mg/mL 2 -70C 12 SITEK Study NO. 0623-2140 EXPERIMENTAL PROCEDURES DOCUMENTATION The materials, experimental procedures used in the performance of the study, experimental results and methods used in the evaluation of the results were documented in the study workbook. TEST SYSTEM IDENTIFICATION Labeling Plates for the Mutation Assays A sufficient number of Vogel-Bonner agar plates were removed from refrigerated storage and allowed to warm to room temperature. Each plate was then labeled with the following information: SITEK's test article number, experiment phase, presence or absence of rat liver S-9 mixture, dose level code, and strain code. The following strain and dose level codes were used: Strain Codes: 1 = TA98 2 = TAlOO 3 = TA1535 4 = TA1537 5 = WP2uvrA Dose Level Codes: 0 = Solvent for the Test Article 1 = 1st or highest Test Article dose level 2 = 2nd Test Article dose level 3 = 3rd Test Article dose level 4 = 4th Test Article dose level 5 = 5th Test Article dose level or lowest Test Article dose level for the Mutation Assays 6 = 6th Test Article dose level 7 = 7th Test Article dose level or lowest Test Article dose level for the Range Finding Test In addition to the above, Mutation Assay viability plates that contained 1OX histidinebiotin or 1OX tryptophan were designated with the prefix "T". 13 SITEK Study NO. 0623-2140 Labeling: Positive Control Plates Vogel-Bonner agar plates were removed from refrigerated storage and allowed to warm to room temperature. Triplicate sets were labeled with the test article number, identity and dose of the particular positive control, experimental phase, strain code, and the presence or absence of rat exogenous metabolic activation. Labeling: Tester Strain Titer Plates Each tester strain titer plate was labeled with the following information: SITEK test article number, tester strain identity, and experimental phase and the prefix T. LabelinP Tester Strain Characterization Plates Histidine Requirement A single histidine-biotin plate was divided into four zones by drawing horizontal lines on the bottom of the plate with a marking pen and labeling each zone with a different Salmonella tester strain. A biotin-only control plate was labeled in a similar manner. > rfa Mutation Nutrient agar plates were labeled with the Salmonella tester strain identification and "CV" (crystal violet). R-Factor A single ampicillin agar plate was labeled in a similar manner as the histidine-biotin plate. Tryptophan Requirement A tryptophan plate and a Vogel-Bonner agar control plate were labeled with the code for strain WP2uvrA and used for confirmation of the tryptophan requirement. SOLUBILITY TEST Since Sponsor had not provided the information regarding a preferred solvent to be used, a solubility test was performed with water and DMSO. Two 30 mg samples were weighed and each solvent was added in 0.1 mL increments until the test article dissolved or until 5.0 mL had been added. 14 SITEK Study NO. 0623-2140 PREPARATION OF TEST CULTURES The methods used for the cryopreservation and cultivation of the tester strains are modifications of the procedures used by B. N. Ames, et al. (1) and D. Maron and B. N. Ames (2). Inoculation Procedures Frozen ampules of strains TA98, TA100, TA1535, TA1537 and WP2uvrA for the Mutation Assays were removed from liquid nitrogen and placed into crushed dry ice to prevent thawing. Scrapes were made using the tip of a sterile pipet, and these scrapes were transferred to a shaker flask containing approximately 50 mL of sterile Oxoid Nutrient Broth No. 2. The flasks were placed in a shaker incubator, and a timer was set to start the unit at a time which allowed the strains to incubate at approximately 120 rpm and 37 1C for a period of 8-12 hours for the Salmonella strains and 4-6 hours for the E. coli strain before being harvested. Harvestinp Overnight Cultures Before starting the experiment, the cultures were sampled and their Percent Transmittance ) (%T) was determined using a spectrophotometer set to a wavelength of 650 nm. When the desired cell density of 5x108 to lx109 cells/ml (represented by a % T of between 25% and lo%,Optical Density of 0.6-1.0) was achieved, the cultures were placed on wet ice or kept at 1-5C until needed. PREPARATION OF S-9 METABOLIC ACTIVATION SYSTEM The S-9 cofactor mix was prepared as follows: For each mL of S-9 cofactor mix required, 0.335 mL of sterile, deionized, distilled water was combined with 0.5 mL of 0.2M sodium phosphate buffer (pH 7.4), 0.04 mL of a 0.1M NADP solution, 5.0 pL of 1M glucose-6phosphate, and 0.02 mL of a 0.4M MgCIJ1.65M KC1 salt solution. This mixture was maintained on ice until just prior to use, whereupon 0.10 mL of S-9 in 0.15N KC1 was added to the mixture. PREPARATION OF TEST ARTICLE DOSING SOLUTIONS The test article was weighed in glass tubes and solubilized in DMSO prior to its use in the experiment. Further serial dilutions were also prepared in DMSO. 15 SITEK Study NO. 0623-2140 All of the test article and control substance treatments were done under UV-filtered lights to avoid possible problems of photoinactivation. Stability of the test article dosing solutions under experimental conditions was not determined by SITEK Research Laboratories. However, 2.0 mL samples from the Mutation Assays were saved frozen and shipped on dry ice to the Sponsor for analysis. RANGE FINDING TEST In order to determine the toxicity and to select the appropriate test article concentrations for the Mutation Assays, a Range Finding Test (A-1) was performed using strains TAlOO and WP2uvrA. Seven doses of the test article, ranging from 5.0-5000 pg/plate, were evaluated with and without induced rat liver S-9, using one plate per dose. Spontaneous Reversion Frequency Treatment was performed by adding either 500 pL of sterile, deionized, distilled water or 500 pL of S-9 cofactor mix to tubes containing 2.0 mL of top agar supplemented with 1X histidine-biotin or 1X tryptophan solution. Immediately thereafter, 100 p L of TAlOO or WP2uvrA was added, followed by 50 pL of the appropriate test article dose or solvent. The 1 highest concentration of 5000 pg/plate had a slight precipitate in the treatment tube. Each tube was vortexed for 2-3 seconds, and the contents were evenly distributed over a Vogel- Bonner bottom agar plate. Each plate was placed on a level surface until the top agar solidified. The plates were inverted and incubated at 37 &- 1C for approximately 70 hours. Viable Count Determination Treatment and incubation were performed as described in the preceding paragraphs, except that approximately 250-500 cells of TAlOO or WP2uvrA were added to top agar supplemented with 1OX histidine-biotin or 1OX tryptophan solution. After the incubation period was completed, the plates, starting with the highest test article concentration, were observed for the presence of precipitate. Plates having no interfering precipitate were counted using an automatic colony counter (ARTEK Counter, Model 880). Plates with precipitate that interfered with automatic counting were counted by hand. Three counts were taken by rotating the plate on the counter stage and the median count was entered into a validated, Lotus 123 (version 3.4) spreadsheet program (2140A.WK3 for the Range Finding Test). The background lawn was also evaluated. The following notations were used for the precipitate and background lawn evaluation: 16 SITEK Study NO. 0623-2140 Chemical Precipitate: NP = No precipitate present. SP = Slight precipitate - Noticeable compound on the plate; however, no influence on automated plate counting. MP = Moderate precipitate - Marked precipitate requiring hand counting for colony enumeration. HP = Heavy precipitate - Large'amount of compound on the plate rendering hand counting difficult. Background Lawn Evaluation: Normal, healthy microcolony lawn. A noticeable thinning of the microcolony lawn compared to that of the solvent control plates. Marked thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the solvent control plates. Extreme thinning of the microcolony lawn and a large increase in the size of the microcolonies compared to the solvent control plates. Absence of any microcolony bacterial lawn. Obscured by precipitate. Determination of Relative Cloning Efficiencv The corrected viability counts from each dose with and without activation in Salmonella strain TAlOO and in Escherichia coli strain WP2uvrA were compared with the respective solvent control viability counts. The ratio was converted into a percentage, and the data were included in the Range Finding Test results. 17 SITEK Study NO. 0623-2140 MUTATION ASSAYS Definitive Mutation Assav (B-1) Doses for the definitive Mutation Assay (B-1) were selected based on the results of the Range Finding Test (A-1). The definitive Mutation Assay was performed with the four Salmonella typhimurium tester strains (TA98, TA100, TAL535 and TA1537) and Escherichia coli strain WP2uvrA using the plate incorporation method of treatment. The test article was tested with the following concentrations in the Mutation Assay: 50, 100, 500, 1000 and 5000 pg/plate. Treatment was performed by adding either 500 pL of deionized, distilled water or 500 pL of rat S-9 cofactor mix to tubes containing 2.0 mL of top agar supplemented with 1X histidine-biotin or 1X tryptophan solution. Immediately thereafter, 100 pL of strains TA98, TA100, TA1535, TA1537 or WP2uvrA were added, followed by 100 pL of the appropriate test article dose or solvent. The positive controls were treated with 100 pL of the appropriate stock solutions. Each tube was vortexed for 2-3 seconds, and the contents were evenly distributed over a Vogel-Bonner bottom agar plate. Each plate was placed on a level surface \ until the top agar solidified. The plates then were inverted and incubated at 37 f 1C for approximately 70.5 hours. Tester Strain Titer Determination Each tester strain was diluted to determine the approximate number of viable cells delivered to the assay plates. Therefore, approximately 250-500 cells were added to top agar supplemented with 1OX histidine-biotin or 1OX tryptophan solution. .,Each tube was vortexed for 2-3 seconds, and the contents were evenly distributed on bottom agar plates. The plates were incubated at 37 k 1C for approximately 68 hours. Tester Strain Characterization All of the Salmonella typhimurium strains used in the assay were confirmed for the histidine requirement and the rfa mutation. In addition, strains TA98 and TAlOO were tested for the presence of the pKMlOl plasmid. Escherichia coli strain WP2uvrA was confirmed for the tryptophan requirement. 18 SITEK Study NO. 0623-2140 Histidine or Trvptophan Requirement A streak of each tester strain was made by dipping a flamed wire loop into the appropriate undiluted tester strain suspension and drawing it across the surface in the appropriate region of a labeled histidine-biotin or tryptophan plate, as well as control plates. The plates were incubated at 37 1C for approximately 20 hours. rfa Mutation For each of the Salmonella tester strains, a 100 pL aliquot of the undiluted culture was added to a tube containing 2.0 mL of 1X histidine-biotin solution top agar. Each tube was vortexed for 2-3 seconds, and the contents were poured onto an appropriately labeled nutrient agar plate. After allowing the plate to solidify, a sterile disc was aseptically placed in the center of the agar overlay. Ten pL of a 1.0 mg/mL crystal violet solution was then added to the disc. The plates were incubated at 37 k 1C for approximately 20 hours. R-Factor Plasmid A streak of each of the Salmonella tester strains was made by dipping a flamed wire loop into the appropriate suspension and drawing it across the surface in the appropriate region of an ampicillin plate. The plates were incubated at 37 k 1C for approximately 20 hours. Evaluation of Assay Results After the incubation period was completed, the plates, starting with the highest test article concentration, were observed for the presence of precipitate. Plates having no interfering precipitate were counted using an automatic colony counter (ARTEK Counter, Model 880). Plates with precipitate that interfered with automatic counting were counted by hand. Three counts were taken by rotating the plate on the counter stage and the median count was entered into a validated, Lotus 123 (version 3.4) spreadsheet program (2140B.WK3 or 2150B.WK3 for the definitive and the confirmatory Mutation Assays, respectively). The background lawn was also evaluated. Same notations as in the Range Finding Test were used to evaluate the precipitate and background lawn. Evaluation of Tester Strain Characterization The requirement for histidine or tryptophan was demonstrated by the growth of the tester strains on plates supplemented with histidine or tryptophan and the lack of growth on the control plates. 19 SITEK Study NO. 0623-2140 The presence of the rfa mutation was evaluated by measuring the zone of inhibition around the crystal violet disc. A zone about 14 to -15 mm. in diameter was evidence of appropriate inhibition. The presence of the pKMlOl plasmid was demonstrated by the growth of strains TA98 and TAlOO and the lack of growth of strains TA1535 and TA1537 streaked on ampicillin plates. Tabulation of Colony Counts The colony counts provided by the automatic colony counter were raw counts and were not corrected to reflect actual counts. Correction of the counts was performed by computer. The correction factor was determined by comparing a wide range of manual and automatic counts, as described in SITEK's SOP No. 21.0. The relationship was linear, and the counts were corrected by using the following formula: Corrected Count = Raw Counts (1.0571607) + 3.09496 Confirmatory Mutation Assay (B-2) I To c o n f m the results of the definitive assay (B-1), a second (confirmatory) Mutation Assay (B-2) was performed with and without activation using the same test article concentrations. All test article concentrations, including the controls, were tested in triplicate. The preincubation method of treatment was used. For the confirmatory assay, 500 pL of deionized, distilled water or S-9 mix was dispensed into a series of labeled culture tubes, and treatment was performed by adding 100 pL of tester strain and 100 pL of test article or the solvent. The positive control cultures were treated with 100 pL of the appropriate stock solutions. The tubes were vortexed gently and preincubated at 37 & 1C for 20 minutes. The tubes were shaken at a moderate speed during the preincubation. 2.0 mL of top agar containing 1X histidine-biotin or 1X tryptophan was added to each culture tube after the preincubation period. The contents of each tube were mixed by vortexing, poured onto a bottom agar plate, and evenly distributed by gently tilting and rotating the plates. All of the plates were placed on a level surface until solidified. After all treatments had been performed, the plates were inverted and incubated at 37 & 1C for approximately 68 hours. Remainder of the experiment was performed in the similar manner as the .definitive Mutation Assay. 20 SITEK Study NO. 0623-2140 CRITERIA FOR A VALID ASSAY The following criteria were used as guidelines in evaluating the acceptability of the Mutation Assay. Since it is impossible to formulate criteria that would apply to every configuration of data generated by the assay, the Study Director was responsible for the ultimate decision regarding the acceptability of the results. Solvent Control Cultures The mean reversion frequency of the test article solvent control plates for each tester strain should have fallen within the following ranges: TA98 TAlOO TA1535 TA1537 20 & 15 100 & 70 20 k 15 15 f 12 WP2uvrA 15 -t 10 Positive Controls The results for the positive control cultures were considered acceptable if the treated strains had a mean reversion frequency that was three times or more greater than the mean reversion frequency of the solvent control plates. Tester Strain Characterization All of the Salmonella typhimurium strains were confirmed positive for histidine dependence. Escherichia coli strain WP2uvrA was confirmed positive for tryptophan dependence. All of the Salmonella typhimurium strains were confirmed positive for the rfa mutation as evidenced by sensitivity to crystal violet. The R-factor strains, TA98 and TA100, were confirmed positive for the pKMlOl plasmid as evidenced by ampicillin resistance. The titer of the stock cultures for each strain indicated that the stock cultures contained approximately between 5 . 0 ~ 1 0an~d 1.0x109bacteria per mL. 21 ~~~ SITEK Study NO. 0623-2140 EVALUATION OF TEST RESULTS The following criteria were used as guidelines in evaluating the results of the Mutation Assay for a negative, positive or equivocal response. Since it is impossible to write criteria that would apply to every configuration of data generated by the assay, the Study Director was responsible for the ultimate decision concerning the results. Criteria for a Negative Response A response was considered to be negative if all of the strains treated with the test article had mean reversion frequencies that were less than twice that of the mean reversion frequencies of the corresponding solvent control plates in TA98 and TAlOO and less than three times in TA153.5, TA1537 and WP2uvrA, and there was no evidence.of a dose-dependent response. Criteria for a Positive Response A response was considered to be positive if either strain TA98 or TAlOO exhibited a mean reversion frequency that was at least double the mean reversion frequency of the corresponding solvent control in at least one dose, or if either strain TA1535, TA1537 or ) WP2uvrA exhibited a three-fold increase in the mean reversion frequency compared to the solvent control in at least one dose. In addition, the response must have been dose dependent or increasing concentrations of the test article must have showed increasing mean reversion frequencies. In evaluating the results, consideration was given to the degree of toxicity exhibited by the dose causing the two-fold/three-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency. Criteria for an Eauivocal Response A response was considered equivocal if it did not fblfill the criteria of either a negative or a positive response and/or the Study Director did not consider the response to be either positive or negative. 22 SITEK Study NO. 0623-2140 ARCHIVES All of the raw data, documentation, protocol, protocol amendments/deviations, and final report along with an electronic file containing the data tables and final report of the study, will be maintained at SITEK Research Laboratories' Archives at 15235 Shady Grove Road, Suite 303, Rockville, Maryland 20850. i 4 23 SITEK Study NO. 0623-2140 RESULTS SOLUBILITY TEST The test article was tested for solubility in water and DMSO. Two 30 mg samples were weighed and tested for their solubility in water and DMSO. The test article was not soluble in up to 0.5 mL of water, however, 0.1 mL of DMSO was enough to solubilize a 30 mg sample. Thus, the maximum solubility of test article in DMSO was 300 mg/mL. Based on the results of the solubility test, DMSO was used as the solvent for the test article. RANGE FINDING TEST (A-1) Summaries of the results of the Range Finding Test are presented in Tables 1 and 2 and included in Appendix I. The individual plate counts and background lawn evaluation are presented in Appendix 11. TAlOO With and Without Activation: There were no signs of toxicity related with the background lawn or the number of revertants. The Relative Cloning Efficiencies (RCEs) among the test article concentrations of 5.0 to 5000 @plate ranged from 94% to 78% in the non-activated portion and all concentrations were greater than 100% in the activated portion. WP2uvrA With and Without Activation: There were essentially no signs of toxicity related to the test article treatment. The RCEs for the test article concentrations of 5.0 to 5000 pg/plate ranged from 108% to 94% without activation and from 106%to 94% with activation. MUTATION ASSAYS Definitive Mutation Assav 03-1) Summaries of the results of the definitive Mutation Assay are presented in Tables 3-4 included in Appendix I. The individual plate counts and background lawn evaluation are presented in Appendix 11. 24 - SITEK Study NO. 0623-2140 The definitive Mutation Assay, using the plate incorporation method of treatment, was performed with the four Salmonella tester strains (TA98, TA100, TA1535, TA1537) and with E. coli strain WP2uvrA. Based on the results of the Range Finding Test, the following concentrations were tested in the Mutation Assay: 50, 100, 500, 1000 and 5000 pg/plate. All the test article concentrations were nontoxic. In the definitive Mutation Assay (B-1) with and without activation, all the tester stains treated with test article exhibited a mean reversion frequency that was similar to or less than double the corresponding solvent control, and there was no dose-related response. Both the negative and positive controls fulfilled the requirements of the test. Confirmatory Mutation Assay CB-2) Summaries of the results of the confirmatory Mutation (B-2) Assay are presented in Tables 5-6 included in Appendix I. The individual plate counts and background lawn evaluation are presented in Appendix 11. I The confirmatory Mutation Assay (B-2) was performed with the same test article concentrations as the definitive Mutation Assay (B-1) with and without activation, but using the preincubation method of treatment. In the confirmatory Mutation Assay (B-2), as in the definitive Mutation Assay with and without activation, all of the tester strains treated with the test article concentrations exhibited a mean reversion frequency that was similar to or less than double the corresponding solvent control. There was no dose-related response. The test article in both Mutation Assays produced a negative response in the presence and absence of S-9 metabolic activation. Both the positive and negative controls fulfilled the requirements of the test. All of the criteria for a valid assay were met. DOSING SOLUTION ANALYSIS RESULTS The analysis results are presented in Appendix V. All of the test article concentrations from the definitive (B-1) and confirmatory (B-2) Mutation Assays were within 5 % of the targeted values. I 25 ~~~~ ~ SITEK Study NO. 0623-2140 CONCLUSIONS The test article, Potassium Perfluorobutane Sulfonate, was tested in the Salmonella typhimurium/ Escherichia coli Plate IncorporatiodPreincubation Mutation Assay in the presence and absence of induced rat liver S-9. The definitive Mutation Assay (B-1), using the plate incorporation method, was performed with four Salmonella tjphimurium tester strains, TA98, TA100, TA1535 and TA1537, and Escherichia coli strain WP2uvrA. Since the results of the Mutation Assay were negative, a second (confmatory) Mutation Assay (B-2) was performed to confirm the results using the preincubation method of treatment. The results of the Mutation Assays indicated that the test article did not induce any significant increase in the number of revertant colonies for any of the tester strains in the presence or absence of induced rat liver S-9. The positive and negative controls fulfilled the requirements of the test. Under the conditions of this study, Potassium Perfluorobutane Sulfonate was negative in the Salmonella typhimurium/Escherichia coli Plate IncorporatiodPreincubation Mutation \ Assay. 26 SITEK Study NO. 0623-2140 REFERENCES 1. Ames, B. N., J. McCann and E. Yamasaki. Methods for detecting carcinogens and mutagens with the Salmonellalmammalian-microsome mutagenicity test. Mut. Res., - 31:347-367, 1975. 2. Maron, D., and B. N. Ames. Revised methods for the Salmonella mutagenicity test. Mut. Res., m:173-215, 1983. 3. Green, M. H. L., and W. J. Muriel. Mutagen testing using trp+ reversion in Escherichia coli. In: B. J. Kilbey, et al. (eds.), Handbook of Mutagenicity Test Procedures, pp. 65-94, Elsevier North Holland Biomedical Press, Amsterdam, 1977. 4. Venitt, S., and J. M. Parry (eds.). Mutagenicity Testing: A Practical Approach. IRL Press, Oxford, England and Washington, D.C., 1984. 27 SITEK Study NO. 0623-2140 APPENDIX I DATA TABLES 28 -I 1 SITEK RESEARCHLABORATORIES I -1r t TABLE 1 SITEK Study NO. 0623-2140 SALMONELLA TYPHIMURlUM PLATE INCORPORATlON MUTATION ASSAY RANGE FINDING TEST RESULTS WITHOUT ACTIVATION Back- Test No. of ground Article Rever-Chem. Lawn Conc. tantsl PPT. Evalu- pg/plate Plate Eval.* ation** 5.0 10 50 100 500 1000 5000 59 NP NL 62 NP NL 52 NP NL 72 NP NL 55 NP NL 71 NP NL 58 NP NL No. of RelaViable tive Colo- Cloning nies/ EffiPlate ciency WE) 212 78% 244 90% 240 89% 211 78% 254 94% 256 94% 249 92% Test Article Conc. pg/plate 5.0 10 50 100 500 1000 5000 WITH S-9 No. of ReverChem. tants/ PPT. Plate Eval.* 69 NP 70 NP 84 NP 67 NP 59 NP 63 NP 62 NP ACTIVATION Back- No. of ground Viable Lawn ColoEvalu- nies/ ation** Plate NL 540 NL 574 NL 539 NL 545 NL 508 NL 523 NL 541 Relative Cloning Efficiency (RCE) 115% 122% 115% 116% 108% 112% 115% SOLV. CONT. 59 NP NL 271 100% SOLV. CONT. 73 NP NL 469 100% RCE = No. of Colonies in Test Plates x 100 No. of Colonies in Solvent Control Plates Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate; large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 29 - F - SITEK Study NO. 0623-2140 TABLE 2 ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY RANGE FINDING TEST RESULTS SPONSOR: EXPERIMENT NO.: TEST ARTlC LE: Primedica Redifield A-1 Potassium Perfluorobutane SITEK STUDY NO.: SOLVENT: STRAIN: 0623-2140 DMSO WP2uvrA WITH0 UT ACTIVATION WITH S-9 ACTIVATION Test No. of ground Article ReverChem. Lawn Conc. tants/ PPT. Evalu- pg/plate Plate Eval.* ation** 5.0 10 50 100 500 1000 5000 13 NP NL 13 NP NL 10 NP NL 10 NP NL 8 NP NL 15 NP NL 10 NP NL Viable tive Colo- Cloning nies/ EffiPlate ciency WE) 1016 107% 998 1000 887 105% 106% 94% 1018 1006 1000 108% 106% 106% Test No. of ground Article ReverChem. Lawn Conc. tank/ PPT. Evalu- pg/plate Plate Eval.* ation** 5.0 10 50 100 500 1000 5000 18 NP NL 16 NP NL 13 NP NL 12 NP NL 12 NP NL 17 NP NL 22 NP NL Viable tive Colo- Cloning nies/ EffiPlate ciency WE) 1100 96% 1144 100% 1196 104% 1080 94% 1122 98% 1181 103% 1218 106% SOLV. CONT. 17 NP NL 946 100% SOLV. CONT. 20 NP NL 1149 100% RCE = No. of Colonies in Test Plates x 100 No. of Colonies in Solvent Control Plates * Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate; large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 30 SITEK Study NO.0623-2140 TABLE 3 SALMONELLA TYPHIMURIUMIESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY MUTATION ASSAY RESULTS - WITHOUT ACTIVATION S. typhimurium STRAIN: TA98 REVERTANTS DATE PLATED: STD. DEV. D8/15/00 LAWN CELLS SEEDED: 1.692 x 10' PRECIPITATE STRAIN: TA100 REVERTANTS DATE PLATED: STD. DEV. 08ll 5/00 LAWN CELLS SEEDED: 0.964x 10' PRECIPITATE STRAIN: TAl535 REVERTANTS DATE PLATED: STD. D EV. 0811 5/00 1 LAWN 1.798 x 10'1 PRECIPITATE STRAIN: TA1537 I REVERTANTS DATE PLATED: STD. DEV. .. LAWN CELLS SEEDED: 1.128x 10' PRECIPITATE E. coli STRAIN: WP2uvrA DATE PLATED: 0811 5/00 CELLS SEEDED: 2.334x 10' REVERTANTS STD. DEV. LAWN PRECIPITATE Positive Control 740 28 Average No. of Revertants Per Plate Solvent Concentration per plate Control 50 100 500 1000 30 38 37 37 34 '4 6 6 4 11 5000 30 12 NL NL NL NL NL NL NL NP 484 33 NP NP NP NP NP NP 67 83 78 80 78 74 6 8 14 1 1 11 7 NL NL NL NL NL NL NL NP 453 16 NP NP NP NP NP NP 201 17 23 16 16 13 71 3 3 3 2 1 NL NLI NL NL NL NL NL NP 165 56 NP/ NP NP NP NP NP 91 6 5 9 7 7 41 2 1 4 2 2 NL NL NL NL NL NL NL NP NP NP NP NP NP NP Positive Control 466 7 Solvent Control 16 2 Concentration per plate 50 100 500 1000 14 .14 15 16 4 5 3 5 5000 16 4 NL NL NL NL NL NL NL NP NP NP NP NP NP NP 31 SITEK Study NO. 0623-2140 TABLE 4 - SALMONELLA TYPHIMURIUM/ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY MUTATION ASSAY RESULTS WITH S-9 ACTIVATION SPONSOR: EXPERIMENT NO.: TEST ARTICLE: Primedica Redfield 5-1 Potassium Perfluorobutane Sulfonate SITEK STUDY NO.: SOLVENT: CONC. IN: 0623-2140 DMSO pg/plate DATE PLATED: 0811 5/00 LAWN' I CELLS SEEDED: 1.798 x 10' PRECIPITATE STRAIN: TAl537 REVERTANTS DATE PLATED: STD. DEV. 0811 5/00 LAWN CELLS SEEDED: 1.128 x 10' PRECIPITATE NL NP 288 3 NL NP NL NL NL NL NL NL NP NP NP NP NP NP 9 9 11 9 5 7 5 3 4 1 2 1 NL NL NL NL NL NL NP NP NP NP NP NP E. coli STRAIN: WP2uvrA REVERTANTS DATE PLATED: STD. D EV. 0811 5/00 LAWN CELLS SEEDED: 2.334 x 10' PRECIPITATE NL = Normal, healthy microcolony lawn. Positive Control 197 24 Solvent Control 21 3 Concentration per plate 50 100 500 1000 23 20 25 17 3 7 2 2 5000 17 4 NL NL NL NL NL NL NL NP NP NP NP NP NP NP NP = No precipitate. 32 SlTEK RESEARCHLABORATORIES SITEK Study NO. 0623-2140 TABLE 5 SALMONELLA TYPHIMURIUMIESCHERICHIA COLI PRINCUBATION MUTATION ASSAY MUTATION ASSAY RESULTS - WITHOUT ACTIVATION S. typhimurium STRAIN: TA98 DATE PLATED: 09/20/00 CELLS SEEDED: 1. I 60 x 10' STRAIN: TA100 DATE PLATED: 09/20/00 CELLS SEEDED: 2.026 x 10' STRAIN: TA1535 DATE PLATED: 09/20/00 CELLS SEEDED: 2.002 x 10' STRAIN: TA1537 DATE PLATED: 09/20/00 CELLS SEEDED: 0.814 x 10' REVERTANTS STD. DEV. LAWN PRECIPITATE REVERTANTS STD. DEV. LAWN PRECIPITATE REVERTANTS STD. DEV. LAWN PRECIPITATE REVERTANTS STD. DEV. LAWN PRECIPITATE STRAIN: WP2uvrA DATE PLATED: 09/20/00 CELLS SEEDED: 2.360 x 10' REVERTANTS STD. DEV. LAWN PRECIPITATE Positive Control 544 23 Average No. of Revertants Per Plate Solvent Concentration per plate Control 50 100 500 1000 24' 24 24 27 27 7 9 6 6 2 5000 33 3 NL NP 495 30 NL, NL NL NL NL NL NPI NP NP NP NP NP 691 75 76 82 74 83 8! 8 2 6 9 4 NL NL NL NL NL NL NL NP 405 39 NP NP NP NP NP NP 16 19 16 15 17 17 2 4 7 3 4 4 NL NL NL NL NL NL NL NP 290 48 NL NP NP NP NP NP NP NP 133 ~ 3 6 2 7 3 6 2 6 2 NLi NL NL NL NL NL I N P ' NP NP NP NP NP 552 17 NL NP 181 18 ' 2 2 21 21 17 6: 1 7 3 4 8 NLII NL NL NL NL NL N P / NP NP NP NP NP 33 I 1 SITEK RESEARCHLABORATORIES I - . I I SITEK Study NO. 0623-2140 TEST ARTICLE: Potassium Perfluorobutane Sulfonate CONC. IN: pg/plate S. typhirnurium STRAIN: TA98 REVERTANTS DATE PLATED: 09120100 STD. D EV. CELLS SEEDED: LAWN 1.160 x l o 8 PRECIPITATE STRAIN: TA100 REVERTANTS DATE PLATED: STD. DEV. 09/20/00 LAWN CELLS SEEDED: 2.026 x 10' PRECIPITATE STRAIN: TAl535 REVERTANTS DATE PLATED: 09/20/00 STD. DEV. LAWN , CELLS SEEDED: 2.002 x 10' PRECIPITATE STRAIN: TA1537 REVERTANTS DATE PLATED: 09/20/00 STD. DEV. LAWN CELLS SEEDED: 0.814 x 10' PRECIPITATE Positive Control 623 19 Average No. of Revertants Per Plate Solvent Concentration per plate Control 50 100 500 1000 35 38 36 40 39 1 4 6 5 4 5000 38 5 NL NL NL NL NL NL NL NP 495 38 NP NP NP NP NP NP 82 97 88 98 95 88 5 14 10 13 4 8 NL NL NL NL NL NL NL NP 123 10 NP NP NP NP NP NP 17 18 19 16 16 13 2 3 3 2 1 6 NL NL NL NL NL NL NL NP NP NP NP NP NP NP 58 11 6 9 9 10 9 2 3 1 2 1 4 1 NL NL NL NL NL NL NL NP NP NP NP NP NP NP E. coli STRAIN: WP2uvrA REVERTANTS DATE PLATED: 09/20/00 STD. DEV. LAWN CELLS SEEDED: 2.360 x lo8 PRECIPITATE NL = Normal, healthy microcolony lawn. Positive Control 229 17 Solvent Control 19 4 Concentration per plate 50 100 500 1000 23 . 25 24 20 2 6 2 4 5000 19 4 NL NL NL NL NL NL NL NP NP NP NP NP NP NP NP = No precipitate. 34 SITEK Study NO. 0623-2140 APPENDIX I1 DETAILED PLATE COUNTS AND BACKGROUND LAWN EVALUATION i 35 SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-2140 SALMONELLA TYPHlMURlUM PLATE INCORPORATION MUTATION ASSAY RANGE FINDING TEST COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: A-1 SITEK STUDY NO.: TEST ARTICLE: Potassium Petfluorobutane SOLVENT: S ulfo nate STRAIN: 0623-2140 DMSO TAlOO WITHOUT ACTIVATION Test Article Conc. pglplate 5.0 10 50 100 500 1000 5000 No. of Revertants Chem.Background Per Plate PPT. Lawn (raw) (corrected) Eval.* Evaluation** 53 59 NP NL 56 62 NP NL 46 52 NP NL 65 72 NP NL 49 55 NP NL 64 71 NP NL 52 58 NP NL No. of Viable Colonies/Plate (raw) (corrected) 198 21 2 228 244 224 240 197 21 1 237 254 239 256 233 249 R elative Cloning Efficiency (RCE) 78% 90% 89% 78% 94% 94% 92% S 0LVENT CONTROL 53 59 NP NL 253 27 1 100% RCE = No. of Colonies in Test Plates No. of Colonies in Solvent Control Plates x 100 * Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate; large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 1 36 1J- SITEK RESEARCHLABORATORIES I -1r t SITEK Study NO. 0623-2140 SALM0NELLA TYPHIMURI UM PLATE INCOR PORAT10N MUTAT10N AS SAY RANGE FINDING TEST COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: A - 1 SlTEK STUDY NO.: TEST ARTICLE: Potassium Perfluorobutane SOLVENT: Sulfonate STRAIN: 0623 -21 40 DMSO TA100 WITH S-9 ACTIVATION Test Article Conc. pg/plate 5.0 10 50 100 500 1000 5000 No. of Revertants Per Plate (raw) (corrected) 62 69 63 70 77 84 60 67 53 59 57 63 56 62 Chem. Background PPT. Lawn Eval.* Evaluation** NP NL NP NL . NP NL NP NL NP NL NP NL NP NL No. of Viable Co lo nies/Plate (raw) (corrected) 508 540 540 574 507 539 51 3 545 478 508 492 523 509 541 Relative Cloning Efficiency (RCE) 115% 122% 115% 116% 108% 112% 115% SOLVENT CONTROL 66 73 NP NL 44 1 469 100% RCE = No. of Colonies in Test Plates No. of Colonies in Solvent Control Plates x 100 * Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate; large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 37 SITEK Study NO. 0623-2140 ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY RANGE FINDING TEST COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: A-1 TEST ARTlC LE: Potassiurn Perfluor0butane Sulfonate SITEK STUDY NO.: SOLVENT: STRAIN: 0623-2140 DMSO WP2uvrA WITHOUT ACTIVATION Test Article Conc. pg/plate 5.0 10 50 100 500 1000 5000 No. of Revertants Chem.Background Per Plate PPT. Lawn (raw) (corrected) Eval.* Evaluation** 9 13 NP NL 9 13 NP NL 7 10 NP NL 7 10 NP NL 5 8 NP NL 11 15 NP NL 7 10 NP NL No. of Viable Colonies/P late (raw) (corrected) 958 1016 94 1 998 943 1000 836 887 960 1018 949 1006 943 1000 Relative Cloning Efficiency (RCE) 107% 105% 106% 94% 108% 106% 106% SOLVENT CONTROL 13 17 NP NL 892 946 100% i RCE = No. of Colonies in Test Plates x 100 No. of Colonies in Solvent Control Plates * Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate;.large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 38 SITEK Study NO. 0623-2140 ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY RANGE FINDING TEST COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: A-1 SITEK STUDY NO.: TEST ARTICLE: Potassium Perfluorobutane SOLVENT: Sulfonate STRAIN: 0623-2140 DMSO WP2uvrA WITH S-9 ACTIVATION Test Article Conc. pg/plate 5.0 10 50 100 500 1000 5000 No. of Revertants ChemBackground Per Plate PPT. Lawn (raw) (corrected) Eval.* Evaluation** 14 18 NP NL 12 16 NP NL 9 13 NP NL 8 12 NP NL 8 12 NP NL 13 17 NP NL 18 22 NP NL No. of Viable Colonies/Plate (raw) (corrected) 1038 1100 1079 1144 1128 1196 1019 1080 1058 1122 1114 1181 1149 1218 Relative Cloning Efficiency (RCE) 96% 100% 104% ' 94% 98% 103% 106% SOLVENT CONTROL 16 20 NP NL 1084 1149 100% RCE = No. of Colonies in Test Plates x 100 No. of Colonies in Solvent Control Plates * Chemical Precipitate Evaluation NP = No precipitate SP = Slight precipitate; noticeable precipitate on the plate, but no interference with automated plate counting MP = Moderate precipitate; marked precipitate necessitating hand counting for colony enumeration HP = Heavy precipitate: large amount of precipitate rendering hand counting difficult or impossible ** Background Lawn Evaluation NL = Normal, healthy microcolony lawn SR = Noticeable thinning of the microcolony lawn compared to control MR = Marked thinning of the microcolony lawn and increase in size of microcolonies compared to control ER = Extreme thinning of the microcolony lawn and large increase in size of microcolonies compared to control AB = Absence of microcolonies OP = Obscured by precipitate 39 SITEK Study NO. 0623-2140 WITHOUT ACTIVATION S. typhimurium STRAIN: TA98 REVERTANTS PER DATE PLATED: PLATE 08115/00 !AWN CELLS SEEDED: 1.692 x 10' PRECIPITATE STRAIN: TA100 REVERTANTS PER DATE PLATED: PLATE 08115/00 LAWN CELLS SEEDED: 0.964 x 10' PRECIPITATE STRAIN: TA1535 REVERTANTS PER 1 DATE PLATED: PLATE 08115/00 LAWN CELLS SEEDED: 1.798 x 10' PRECIPITATE STRAIN: TA1537 REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 1 . 1 2 8 ~10' PRECIPITATE Positive Control 683 727 680 Solvent Control 21 27 27 NL NL NP NP 423 55 485 61 457 66 NL NL NP NP 408 8 436 21 432 18 NL NL NP NP 108 4 141 9 21 1 3 NL NL NP NP Concentration per plate 50 100 500 1000 34 37 31 17 26 25 28 32 38 35 36 38 5000 20 39 19 NL NL NL NL NL NP NP NP NP NP 80 63 77 70 75 67 63 61 82 65 78 87 81 61 62 NL NL NL NL NL NP NP NP NP NP 16 22 16 13 10 12 17 10 10 8 11 18 11 12 10 NL NL NL NL NL NP NP NP NP NP 1 2 3 4 2 3 3 10 6 6 5 2 4 2 4 NL NL NL NL NL NP 'NP NP NP NP E. coli STRAIN: WP2uvrA REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 2.334 x 10' PRECIPITATE NL = Normal, healthy microcolony lawn. Positive Control 444 438 431 Solvent Control 11 14 11 Concentration per plate 50 100 500 1000 14 9 13 14 7 15 8 7 9 7 12 16 5000 16 12 8 NL NL NL NL NL NL NL NP NP NP NP NP NP NP NP = No precipitate. 40 SITEK Study NO. 0623-2140 SALMONELLA TYPHIM URIUM/ESCHERlCHlA COLI PLATE INCORPORATION MUTATlON ASSAY MUTATION ASSAY CORRECTED COLONY COUNTS AND BACKGROUND LAWN EVALUATIO' N S. typhimurium STRAIN: TA98 REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 1 1.692 x 10'1 PRECIPITATE STRAIN: TA100 REVERTANTS PER DATE PLATED: PLATE 08l15/00 LAWN CELLS SEEDED: 0.964 x 10' PRECIPITATE STRAIN: TA1535 REVERTANTS PER DATE PLATED: PLATE 08115/00 LAWN 1.798 x 10' STRAIN: TA1537 DATE PLATED: 0811 5/00 PRECIPITATE REVERTANTS PER PLATE LAWN STRAIN: WP2uvrA DATE PLATED: 08115/00 1 REVERTANTS PER PLATE Positive Control 725 772 722 NL Solvent Control 25 32 32 NNLLI NP NP I 450 61 I 51 6 68 486 73 NL NLI I I NP NPI 434 12 464 25 460 22 NL NP NP 117 7 152 13 226 6 NL NLI Concentration per plate 50 100 500 1000 39 42 36 21 31 30 33 37 43 40 41 43 5000 24 44 23 NL NL NL NL NL NP NP NP NP NP 88 70 84 77 82 74 70 68 90 72 86 95 89 68 69 NL NL NL NL NL NP NP NP NP NP 20 26 20 17 14 16 21 14 14 12 15 22 15 16 14 NP NP NP NP NP 4 5 6 7 5 6 6 14 9 9 8 5 7 5 7 NL NL NL NL NL 1 Positive Solvent 1 Concentration per plate 1 472 151 18 13 17 18 20 466 18 10 19 12 10 16 459 15 13 10 16 20 12 41 SITEK Study NO. 0623-2140 SALMONELLA TYPHIMURIUMIESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAX MUTATION ASSAY RAW COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: TEST ARTICLE: S. typhimurium B-1 SITEK STUDY NO.: Potassium Perfluorobutane Sulfonate SOLVENT: CONC. IN: WITH S - 9 ACTIVATION 0623-2140 DMSO pglplate 1 Positive Solvent 1 Concentration per plate PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 1.692 x 1O6 PRECIPITATE STRAIN: TA100 REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN 0.964 x 10'1 PRECIPITATE STRAIN: TA1535 I REVERTANTS PER DATE PLATED: PLATE 08/15/00 LAWN 1.798 x 10'1 PRECIPITATE STRAIN: TA1537 REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 1.128 x 10' PRECIPITATE 1- 1472 47 1409 35 I NL NL\ NP NP/ 1098 76 1276 73 1187 77 NL NL/ NP NP/ 175 101 188 6 192 22 NL NL/ I NP NP 267 3 269 3 272 11 NL NL NP NP 1 Positive Solvent 1 35 47 46 40 42 43 58 34 NL NL NP NP NP NP 66 80 70 61 82 84 67 71 53 73 62 73 NL NL NL NL NP NP NP NP 20 7 9 12 7 11 5 8 7 14 7 12 NL NL NL NL NP NP NP NP 5 8 7 2 4 3 5 1 8 10 5 4 NL NL NL NL NP 'NP NP NP Concentration per plate 47 32 NP 57 72 76 NL NP 8 7 7 NL NP 5 3 5 NL NP, 1 DATE PLATED: 0811 5/00 CELLS SEEDED: 2.334 x 10' PER PLATE LAWN PRECIPITATE 200 192 NL NP 15 22 15 20 22 22 NL NL NL NL NL NL NP NP NP NP NP NP 42 SITEK Study NO. 0623-2140 SALMONELLA TYPHIMURIUM/ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY MUTATION ASSAY CORRECTED COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: TEST ARTICLE: B - 1 SITEK STUDY NO.: Potassium Perfluorobutane Sulfonate SOLVENT: CONC. IN: WITH S - 9 ACTIVATION 0623-2140 DMSO pglplate S. tvphirnurium STRAIN: TA98 REVERTANTS PER DATE PLATED: PLATE 08/15/00 LAWN CELLS SEEDED: 1.692 x 10' PRECI PITATE STRAIN: TA100 REVERTANTS PER DATE PLATED: PLATE 08/15/00 LAWN CELLS SEEDED: 0.964 x 10' PRECIPlTATE STRAIN: TA1535 REV ERTA NTS PER DATE PLATED: PLATE 08/15/00 LAWN CELLS SEEDED: 1.798 x 10' STRAIN: TA1537 PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 1.128x l o e PREClPlTATE I Positive Control 1468 1559 1493 Solvent 1 Control 34 53 40 NL NL NP NP 1164 83 1352 80 1258 04 NL NL I NP NP/ 188 14i 202 9 206 26 NL NL Concentration p. er .plate 50 100 500 1000 42 49 52 42 40 53 52 45 47 49 64 39 1 5000 45 53 37 NL NL NL NL NL NP NP NP NP NP 73 88 77 68 63 90 92 74 78 79 59 80 69 80 83 NL NL NL NL NL NP NP NP NP NP 24 10 13 16 12 10 15 8 12 10 10 18 10 16 10 NL NL NL NL NL 287 291 NL NP 6 7 6 8 4 6 15 12 14 8 7 8 NL NL NL NL NL NL NP NP 'NP NP NP NP E. coli STRAIN: WP2uvrA REVERTANTS PER DATE PLATED: PLATE 0811 5/00 LAWN CELLS SEEDED: 2.334 x 10'1 PRECIPITATE Positive Control 170 21 5 206 Solvent Control 24 19 19 NL NL Concentration per plate 50 100 500 1000 20 20 27 16 26 13 23 19 24 26 26 17 5000 17 14 21 NL NL NL NL NL 43 - I 'rr t SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-2140 SALMONELLA TYPHIMURIUM/ESCHERICHIA COLI PRINCUBATION MUTATION ASSAY MUTATION ASSAY RAW COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: TEST ARTICLE: 8-2 SITEK STUDY NO.: Potassium Perfluorobutane Sulfonate SOLVENT: CONC. IN: WITHOUT ACTIVATION 0623-2140 DMSO pglplate S. typhimurium Positive Solvent Concentration per plate Control Control 50 100 500 1000 5000 REVERTANTS 530 13 18 16 23 21 31 PER 51 0 26 13 17 DATE PLATED: PLATE 487 19 29 26 28 09/20/00 LAWN NL NL NL NL NL NL NL CELLS SEEDED: PR ECI PlTATE NP NP NP NP NP NP NP REVERTANTS 470 58 60 71 71 76 73 PER 435 59 68 69 81 59 79 DATE PLATED: PLATE 491 72 75 67 73 60 74 09/20/00 LAWN NL NL NL NL NL NL NL CELLS SEEDED: PRECIPlTATE NP NPI NP NP NP NP NP REVERTANTS 41 7 121 13 19 9 12 12 PER 'i DATE PLATED: PLATE 379 11 13 12 14 10 18 344 14 20 7 10 17 10 09/20/00 LCELLS SEEDED: 2.002 x 10' STRAIN: TA1537 DATE PLATED: 09/20/00 CELLS SEEDED: 0.814 x 10' LAWN P RECl PlTATE REVERTANTS PER PLATE LAWN PRECl PlTATE NL -NL NL NL N L .NL NL I NP 31 1 281 221 N P / NP NP NP NP NP 101 4 4 4 3 3 11 6 1 1 4 1 6 1 4 7 1 4 NL NL NL NL NL NL NL NP NP NP NP NP NP NP STRAIN: WP2uvrA DATE PLATED: 09/20/00 I REVERTANTS PER PLATE LAWN I Positive I Control I 535 519 503 I Solvent Control I 151 20 8 NL NL( Concentration per plate 50 100 500 1000 14 17 19 14 15 25 18 16 13 12 14 21 1 5000 14 6 20 NL NL NL NL NL 44 SlTEK RESEARCLHABORATORIES ' I lir t SITEK Study NO. 0623-2140 SALMON ELLA TYPHIM URIUM/ESCHERICHIA COLI PAINCUBATION MUTATION ASSAY MUTATION ASSAY CORRECTED COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: TEST ARTICLE: 8-2 SITEK STUDY NO.: Potassium Perfluorobutane Sulfonate SOLVENT: CONC. IN: WITHOUT ACTIVATION 0623-2140 DMSO pg/plate S. tvphimurium I Positive Solvent 1 Concentration per plate STRAIN: TA98 I REVERTANTS PER DATE PLATED: PLATE 09/20/00 LAWN CELLS SEEDED: 1.160 x 10' PRECIPITATE STRAIN: TA100 DATE PLATED: I REVERTANTS PER PLATE 09/20/00 LAWN CELLS SEEDED: 2.026 x 10'1 PRECIPITATE STRAIN: TA1535 1 REVERTANTS PER DATE PLATED: PLATE 09/20/00 LAWN CELLS SEEDED: 2.002 x 10' PRECIPITATE STRAIN: TA1537 REV ERTANTS PER DATE PLATED: PLATE 09/20/00 LAWN CELLS SEEDED: 0.814 x IO' PRECIPITATE 563 551 51 8 NL NP I 500 463 522 NL I NP I 444 404 367 NL NP 332 300 237 NL NP 171 22 20 27 25 31 17 21 22 27 23 34 31 33 28 NL NL NL NL NL NP NP NP NP NP 64 67 78 78 83 651 75 76 89 65 79 82 74 a0 75 NL NL NL NL NL N P / NP NP NP NP 161 17 23 13 16 15 17 16 18 14 18 24 10 14 21 NL NL NL NL NL NP NP NP NP NP 14 7 7 7 6 15 9 4 4 7 9 4 7 10 4 NL NL NL NL NL NP NP NP NP NP I 36 34 30 NL NP 80 871 81 NL N 1 22 14 NL NP 6 4 7 NL NP STRAIN: WP2uvrA REVERTANTS PER DATE PLATED: PLATE 09/20/00 LAWN CELLS SEEDED: 2.360 x 1O6 PRECIPITATE Positive Control 569 552 535 Solvent Control 19 24 12 NL NL NP NP Concentration per plate 50 100 500 1000 18 21 23 18 19 30 22 20 17 16 18 25 5000 18 9 24 NL NL NL NL NL NP NP NP NP NP 45 I 1 SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-2140 S.typhimurium STRAIN: TA98 DATE PLATED: 09/20/00 CELLS SEEDED: 1.160x 10' STRAIN: TAlOO DATE PLATED: 09/20/00 CELLS SEEDED: 2.026 x 10' STRAIN: TA1535 DATE PLATED: 09/20/00 CELLS SEEDED: 2.002 x 10' STRAIN: T A I 537 ' DATE PLATED: 09/20/00 CELLS SEEDED: 0.814 x 10' REVERTANTS PER PLATE LAWN PRECIPITATE REVERTANTS PER PLATE LAWN PRECIPITATE REVERTANTS PER PLATE LAWN PRECIPITATE REVERTANTS PER PLATE LAWN PRECIPITATE STRAIN: WP2uvrA DATE PLATED: 09/20/00 CELLS SEEDED: 2.360 x 10' REVERTANTS PER PLATE LAWN PRECIPITATE Positive Control 567 603 588 Solvent Control 30 30 31 NL NL NP NP 445 73 507 79 443 71 NL NL NP NP 104 11 113 14 122 15 NL NL NP NP 52 8 53 4 50 9 NL NL NP NP Positive Control 226 196 21 8 1 C"Ivoenntrtol 19 12 15 NL NL NP NP Concentration per plate 50 100 500 1000 37 29 30 37 30 27 38 36 32 38 38 29 5000 37 33 28 NL NL NL NL NL NP NP NP NP NP 96 78 76 85 76 74 91 97 05 77 96 72 97 92 90 NL NL NL NL NL NP NP NP NP NP 11 15 1 1 13 1 1 16 17 15 12 4 14 12 1 1 12 14 NL NL NL NL NL NP NP NP NP NP 2 7 6 7 5 4 4 7 3 6 3 7 5 9 7 NL NL NL NL NL NP 'NP NP NP NP Concentration per plate 50 100 500 1000 17 15 20 13 21 21 22 21 18 26 19 14 5000 15 19 11 NL NL NL NL NL NP NP NP NP NP 46 1SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-2140 SALMONELLA TYPHIMURIUM/ESCHERICHIA COLI PRINCUBATION MUTATION ASSAY MUTATION ASSAY CORRECTED COLONY COUNTS AND BACKGROUND LAWN EVALUATION EXPERIMENT NO.: TEST ARTICLE: 8-2 SITEK STUDY NO.: Potassium Perfluorobutane Sulfonate SOLVENT: CONC. IN: WITH S-9 ACTIVATION 0623-2140 DMSO pg/plate 3. typhirnuriurn STRAIN: TA98 )ATE PLATED: 29/20/00 I REVERTANTS PER PLATE 1 LAWN Positive Control I 603 641 625 Solvent Control 35 35 36 I NL NLI Concentration per plate 50 100 500 1000 42 34 35 42 35 32 43 41 37 43 43 34 5000 42 38 33 NL NL NL NL NLI 1.160 x 10'1 PRECIPITATE NP NP/ NP NP NP NP NP >ATE PLATED: 39/20/00 PER PLATE LAWN 539 47 1 NL 87 81 99 106 93 84 78 105 79 106 100 98 NLI NL NL NL NL NL 2.026 x 10'1 PRECIPITATE STRAIN: TA1535 REV E RTA NTS PER DATE PLATED: PLATE 09/20/00 LAWN CELLS SEEDED: 2.002 x 10'1 PRECIPITATE STRAIN: TA1537 I REVERTANTS PER DATE PLATED: 09./~20./00 ~ PLATE LAWN CELLS SEEDED: 0.814 x 10' PRECIPITATE IE. coli STRAIN: WP2uvrA I09/20/00 CELLS SEEDED: I REVERTANTS PER PLATE LAWN NP NP/ 113 15 123 18 132 19 NL NL I NP NPI 58 121 7 1 59 56 13' I NL NL NP NP I Positive Solvent 1 242 231 21 0 16 234 19 NL NL NP NP NP NP 15 19 15 17 20 21 19 16 18 16 15 16 NL NL NL NL NP NP NP NP 5 10 9 10 ; 7 10 10 0 6 13 NL NL NL NL NP . N P NP NP Concentration per plate 21 19 24 17 25 25 26 25 22 31 23 10 NL NL NL NL NP 15 7 18 NL NP 8 9 10 NL NP i 19 23 15 NLI 47 SITEK Study NO. 0623-2140 APPENDIX I11 STUDY PROTOCOL AND PROTOCOL AMENDMENTS 48 I Ar, I SITEK Study NO. 0623-2140 EVALUATION OF A TEST ARTICLE IN TH ESCHERICHIA COLI PLATE INCORPORAT A S S A Y I N T H E PRESENCE AND A RAT LIVER S-9 P This protocol is resented in two parts. Part One is designed to colled information ertaining o the test article and study. Part Two describes the studyzs? in detail. P l a s e complete all sections in Part One and sign Section 8.0 to a p p m protocol. PART ONE 1.0 SPONSOR 1.1 Name: Pnmedica Redfield 1.2 Address: 100 E.Boone Street Redfield. AR 72132 1.3 Sponsor's Study Coordinator: John Senci. Ph.0. 2.0 TESTING FACILITY 2.1 Name: SITEK Research Laboratories 2.2 Address: 15235 Shad Grove Road, Suite 303 Rockville. trlatyland 20850 2.3 Study Director. Kamala J. Pant, M.S. 3.0 STUDY NUMBERS * 3.1 Testing Facility's Study No.: dhg.3-2/4f3 3.2 Sponsor's Study No.: 132-008 4.0 TEST ARTICLE 4.1 Identification Name: Potassium Perfluombutane Sulfonate, CAS # 2942049-3 Batch/Lot No.: Lot #2 l o be completed by the Testing Facility. Protocol No. 2140.4 031299 49 Page I of 16 A: ' SITEK RESEARCHLABORATORIES 4.2 Description ' Color: White Physical Form: Solid, free flowins powder 4.3 Analysis Purity Information: 100% Does the Sponsor require the use of a correction factor to account for impurity? - Yes - X No If yes, what is the correction factor? Determination of the test article characteristics as defined by Good Laborato U.g Practices will be the res onsibill of the S onsor. The specific GLP references for a encies are: FDA = 2PCFR. 5x105; EP61 TSCA = 40 CFR, 792.105 and EPA FIFRA = 4a CFR 160.105. 4.4 Stabilitv Storage Conditions (check one): - X Room Temperature -Refrigerated (1-5C) I -Frozen (-10 to -20C) -Other (please specify): Expiration Date: 04-06-01 4.5 Preferred Solvent (check one): -H P -Acetone -DMSO -Ethanol -Other (please specify): X To be decided by the Testing Facility 4.6 Special Handlinq Instructions: Standard precaution (MSDS attached) Protocol No. 2140.4 031299 50 Page 2 of 16 ' ' SlTEK RESEARCHLABORATORIES A: 5.0 REGULATORY AGENCY SUBMISSION PracticeThsitsansdtuadrd(s:will be conducted in compliance with the following Good Laboratory United States Environmental Protection A ency, Title 40 Code of Federal Regulations Parts 160 and 792, Revised July 1, 1999. United States Food and Drug Administration, Title 21 Code of Federal Regulations Part 58, Revised Apnl I,1998. Japanese Ministry of Agriculture, Forestry and Fisheries, 59 Nohsan, Notification No. 3850, Agriculture Production Bureau, August 10, 1984. Japanese Ministry of Health and Welfare, Ordinance No. 21, April 1, 1997. 2 Japanese Minist Industries Bureau, Mar of International 31, 1984. Trade and Industry, Notification No. 85, Basic Organisation for E-mnomic Cooperation and Development, The OECD Principles of Good Laboratory Practice, Environment Monograph No. 45, Pans 1992. Will this study be submitted to a regulatory agency? Yes - No \; Ifso, which agency(ies)? OECD 6.0 TEST ARTICLE/DOSING SOLUTIONS CHARACTERIZATION The U S . re uirements for anal sis of dosing solutions are specified in: FDA = 21 CFR, 58.113; EPACf`SCA = 40 CFR, h2.113; and EPA FIFRA = 40 CFR, 160.113. Does the Sponsor want dosing solution analysis? Yes** - No Ifyes, please complete the rest of this section. If re uested by the Sponsor, SlTEK Research Laboratories will determine the 2 strength an stability of the dosin solutions. The method of analysis may be provided by !i the Sponsor, or if requested by t e Sponsor, SITEK Research Laboratories will develop the method of analysis. R Additional charges Will apply. See Special Services pnce schedule. i Protocol No. 2140.4 031299 51 Page 3 of 16 SlTEK RESEARCHLABORATORIES ~~ ~ ~ ~~~ SITEK Study NO. 0623-2140 Alternatively, the Sponsor will be responsible for determining the strength and stability of the dosing solutions. Dosing solution analysis will be performed by: -SITEK Research Laboratories Sponsor What dosing solutions will be analyzed? From the Range Finding Test? -Yes -& No From the Assay? X Yes -No Which concentration(s)? All What amount of each concentration? 2.0 mL At what temperature should the dosing solutions be stored? -Room Temperature X Frozen (-10 to -20C) -Refrigerated (1-5C) ,! 7.0 STUDYDATES 7.1 Proposed Experimental Start Date: B ldm Defined as the first date the test article is applied to the test system. * 7.2 Anticipated Experimental Completion Date: IO I1I& Defined as the last date on which data are collected directly from the study. * i o be completed by the Testing Facility. I Protocol No. 2140.4 031299 52 Page 4 of 16 SITEK RESEARCLHABORATORIES SITEK Study NO. 0623-2140 8.0 PROTOCOL APPROVAL t Study Director pL4 ** 4244r &&? Sponsor's Authonzed Kepresentative * Quality Assurance Manager io be completed by the Testing Facility. Protocol No. 2140.4 031299 53 Page 5 of 16 1:y SITEK RESEARCLHABORATORIES STUDY DESIGN PART TWO 9.0 PURPOSE 7 h The pu ose of this study is to evaluate the test article for its potential to cause mutations in t e histidine operon of Salmonella himurium strains TA98, TA100, TA1535 and TA1537 and the tryptophan operon ot t s en la strain WP2uvrA. 10.0 JUSTIFICATION FOR SELECTION OF TEST SYSTEM The Salmonella himurium and Escherichia coli strains have been used extensively idnettheectPinlgattehelnmcou' paogr-eaniiocnactuiviatyioonf Assay and haveTeen demonstrated to be effective chemicals from a wide range of classes (1-4). in I 1.O ABBREVIATIONS 2-AA - 2-Aminoanthra&ne 2-NF - 2-Nitrofluorene 9-AA - 9-Aminoacndine DMSO - Dimethyl Sulfoxide 1 MMS - Methyl Methanesulfonate NaAz - Sodium Azide NADP - O.D. - %T - s-9 - Nicotinamide-adenine Dinucleotide Phosphate Optical Density Percent Transmittance Induced Rat Liver Homogenate 12.0 INDICATOR CELLS 12.1 Source 1 C. McCoy, Case Western Protocol No. 2140.4 031299 54 Page 6 of 16 ' ' SITEK RESEARCLHABORATORIES 1; + rK 12.2 Culture Conditions The Salmonella himurium.and Escherichiacoli strains are routinel grown in Oxoid Nutrient Broth No. 2 in a s a er incubator rotating3 approximately 12 rpm and maintaining a temperature of 37 +1"C. 12.3 Stock Cultures The Salmonella himurium and Escherichia coli strains were propa ated to obtain Pn-7-f- a sufficient number o ce s or reezing a lar e nurnEr of stock am ules. ?he cells were c o reserved in Oxoid Nutnent Broth No. fsupplemented with 8-8% dimethyl sulfoxide #MgO and stored in liquid nitro en vapor phase. Scrapes from stock ampules are used b 8 initiate the stock cultures for t e test. 13.0 METABOLIC ACTIVATION 9 cp The standard rat liver Sy9 will be pre ared by inducing male Sprague-Dawley rats with Aroclor-I254 or phenobarbital and/or If)-naphthoflavone. The livers will be aseptically removed, washed, minced, homogenized and centrifu ed at 9OOOx . The su ematant fraction will be pooled, dispensed in ap ropnate aliquo s, and store below - 7 8 C for up to 3 years. Prior to its use in this stud Phe S-9 will be evaluated for acceptable metabolic activity in a standard Salmonella W~&-nuriummutation assay using strain TAIOO and a single dose of 2-AA. 14.0 ROUTE OF ADMINISTRATION OF TEST ARTICLE ) with P The test article will be-administered in vitro djrectly or throu h a the test cultures. This is the only route of administration avai able solvent compatible in this test system. 15.0 TEST SYSTEM IDENTIFICATION All test plates will be labeled using an indelible pen with a code system which clearly identifies the experiment number, the SITEK test a.rticle number, controls, doses, and whether or not the plate was treated in conjunction with an exogenous activation system. The test article will be designated b the unique fourdigit number assigned by SITEK J when the f the letter test article is received A (Range Findin Test eo.gr.b0(M7u4ta).t.ioTnheAsesxapenfmolelonwt epdhabsyeawnilul mbebedredsiegsniatneadtinby (80 the trial number. This will%e followed b the letter N Activation) or S p i t h S;97 w h i d J will be followed b Y by the numbers t2h,e3d, o..s.einadnicdastitnraginthIe entification numbers. highest to the lowest The doses dose. The will be identified strain identifica- tion numbers will be as follows: Salmonella typhimuriurn 1 = TA98 2 = TAIOO 3 = TA1535 4 = TA1537 Escherichia 5 = WP2uvrA Protocol No. 2140.4 031299 55 Page 7 of 16 1:7 ' SlTEK RESEARCHLABORATORIES ' d An example of a plate label follows: 0074B1-S-1-3 0074 B1 - = S - - - 1 3 - SITEK Test Article Number First Mutation Assay With S-9 Highest Test Article Dose Strain TAl535 3< Y In addition to the above, the Range Findin Test and Mutation Assa viability plates that contain 1OX (0.5mM) histidine biotin or 10 (0.5mM) tryptophan wil be designated with the prefix T. 16.0 CONTROL SUBSTANCES 16.1 Positive Controls The positive control chemicals that will be used for the tester strains in the presence and absence of exogenous metabolic activation are presented below. The abbreviations are defined in Section 11.O. Strain - S-9 Chemical Dose bqlplate) Salmonella b phimurrum I TA98 TA98 TA100 TA100 TA1535 TAI 535 TA1537 TA1537 - 2-N F + 2-AA 2.5-7.5 1.25-5.0 - NaAz + 2-AA 0.5-2.0 1.25-5.0 - NaAz + 2-AA 0.5-2.0 1.25-5.0 - 9-AA + 2-AA 25-75 1.25-5.0 Escherichia - COll WP2uvrA - WP2uvrA + MMS 2-AA 2000-4000 10-20 DMSO will be used to solubilize the positive controls 2-AA, 2-NF and 9-AA. H,O will be used to dissolve NaAz and MMS 1 Protocol No. 2140.4 031299 56 Page 8 of 16 K ' SlTEK RESEARCLHABORATORIES If necessary, other appropriate positive controls can be used with the approval of the Sponsor. 16.2 Solvent Control L The solvent used for dissolving the test article will be used as the solvent control. Deionized distilled water, dimethyl sulfoxide (CAS #67-68-5), ethanol (CAS #64-17-5) and acetone (kAS #67-64-1) are some of the solvents which are compatible with this test s stem. If there is a need to use other solvents, the approval of the Sponsor will be o tained prior to their use. 17.0 DOCUMENTATION J All procedures, results, significant observations, and methods used for analysis of results will be documented in a study notebook. The stud notebook will also include copies of the protocol, all protocol amendments and protocol eviations, study reports, and all relevant communications with the Sponsor. 18.0 EXPERIMENTAL PROCEDURE 18.1 In order to determine the optimal vehicle for deliverin the test artide to the test ? system or to determine the maximum achievable concentra ion in the solvent requested by the Sponsor, a solubility/miscibility test will be erformed. The solvents of choice for this P system are water, DMS0,acetone and ethanol: f the test article is not sufficiently soluble ) in any of these solvents, additional solvents will be screened. For solid and viscous test articles, the solubili .test will cpnsist of weighin out 20- ty 9 to 100-mg aliquots of test article and adding solven in. 0.1 mL increments, with horough mixing between additions, until the test article is dissolved as determined b visual Y inspection or until 5.0 mL of solvent has been added to the vessel. The volume o solvent required for complete dissolution and any additional .observations will be recorded in the study notebook. Test articles that do not disso1ve.m 5.0 mL of solvent will be visually inspected and recorded as either "not soluble," "partially soluble forming a homogeneous suspension," or "partially soluble not forming a homogeneous suspension." For liquid test articles a miscibility test will be conducted. 0.5 rnL of solvent wi!l be added to 0.5 mL aliquots.of the test article.. The resultin solution will be thoroughly mixed 9 and observed for miscibility. The test article will be ra ed by visual inspection as either "not miscible," "partiall miscible," or "completely miscible" in each of the four preferred r solvents. The miscibi ity rating and any additional observations will be recorded in the study notebook. + 18.2 Preparation of Test Cultures The strains of Salmonella himurium and Escherichia coli will be re ared from cultures that were started from scra es p aced in Oxoid NutrienfBoth No. &e cultures will be placed on the shaker, and a [mer turns on the incubator approximately 8-12 or 4-6 Protocol No. 2140.4 031299 57 Page 9 of 16 1SITEK RESEARCLHABORATORIES 1v SITEK Study NO. 0623-2140 !F-'-f- E x &lac. hours for Salmonella himurium or Escherichia @ respectively,' nor to sam ling the cultures for arowth de ermina ion. i h e incubator will be set at 12 rpm and 7 Samples fro6 each culture will be checked for Percent Transmittance (%T) at 650 nm. d 1 Only cultures that have a %T of between 25% (O.D. 0.6 ,and 10% OgD. 1.O) will be used. These cultures will have approximately between 50x1 and 1.Ox 0 cells per m L 18.3 Preparation of S-9 Metabolic Activation Mix 18.4 Preparation of Test Article The desired amount of the test article as specified in the dilution scheme will be weighed or measured just pri0.r to use in either the-Range Finding.Test or the Mutation Assay. The dosing solutions will be prepared byadding the appropnate volume of solvent to the test article and thoroughly mixing the resulting solution until the test article goes completely into solution or a homogeneous suspension is achieved.. The remaining doses specified in the dilution scheme will be prepared by either pelformin a serial dilution or by varying the volume delivered fr0.m the stock concentratton to %e cu!tures. In all treatments the amount of solvent delivered to the target cultures will be limited to a level which has no cytotoxic effect on the cells. If necessary, the test article may be added directly to the top agar. 18.5 Ranqe Findina Test In order to determine the test article concentrations that.will roduce from 0-100% toxicity, a Ran e Findin Test will be performed with and withou! S-9 activation usi % tester strains T i 100 and P2uvrA only. The test article wi!l be wet hed or measured, a 3 B a serial dilution will be repared. If there are no solubility/miscibility imitations prior k n o w edge of cytotoxicity inCYicates differently, or the Sponsors ecifies different1 , the treatment sp concentrations for solid and viscous test articles will-be 000, 1000, 500, TOO, 50, 10 and 5.0 pglplate. If the results based on the dosing re imen indicate-that the threshold level f? of complete toxicity is below 5.0 pg/plate an addi ional Range Finding Test will be per- formed. Only one plate per concentration condition will be used. 18.5.1 Treatment 9 been2amddLeadl,iqwuilol tbseofdmisopletennsetodptoagaasre, rtioeswohficchutltruarceetuabmeosumntasi-notfahinisetiddiante4a5n+d lbaioctin.Threaavte- ment will be performed by addin 0.5 mL of s-9 rnix .or 0.5 mL of sterile distilled, deionized water, 0.1 mL of tester s rain TAIOO or WP2uvrA, and 0.1 mL of tesf article to the top agar. Appropriate solvent controls will also be prepared. Protocol No. 2140.4 031299 58 Page I O of 16 1: . dosesInwaitdhdaiti2on.0, ~pl1a0ledsilufotirondeotfetremsitneirnsgtrvaiianbTilAityIOwOillobrechpPre2puavrrAedinb&pplaagtianrgctohnetateinsitnagrt1icOlXe (0.5mM) histidine-biotin or 1OX (0.5mM) tryptophan, respectively. The contents will be mixed by vocexjng the tube, and then the contents will be poured onto a bottom agar plate and evenly distnbuted by gently tilting and rotating the plate. The late will be placed on a flat, level surface until solidified. After all treatment is performed, Phe plates will be inverted and incubated at 37 +1"C for 48-72 hours. 18.5.2 Determination of Toxicity After 48-72 hours of incubation the lates will be removed from the incubator and evaluated or placed in cold storage (1-5'6 until evaluated. Evaluation of test article toxicity on the tester strain will be based on three end points: v 1. Viability of cells plated on .minimal medium plates supplemented with excess histidine-biotin or tryptophan. Toxici wi!l be measured as a decrease in the number of colonies per plate with increasing tes article concentration. P 2. The number of revertant colonies on minimal.medium plates supplemented with trace amounts of histidine-biotin .or trypto han. .Toxicity will be measured as a reduction in the number of revertant colonies per p ate with increasing test article concentration. Y 3. The integrity of the background microcolony lawn. Toxicity will be measured as a thinning or disappearance. of the background lawn usual1 occumng with an increase in the size of the remaining microcolonies relative to the con 1-01plates. 9 The number of revertants per plate and the number of viable colonies per plate will be determined by countin them with an automatic colony counter or by hand as described in Sections 18.6.5.1 and 8.6.5.2. % The counts will be.entered direct1 in the Lotus 123 computer pro ram 2140A.WK3, and the calculations will be performeJ. The computer printouts will e included in the study notebook. 18.6 Mutation Assay positive controls and Protocol No. 2140.4 031299 59 Page I 1 of 16 18.6.1 Test Culture Preparation and Exposure 18.6.1.1 Plate Incorporation Method at 37 +1"C for 48-72 hours. 18.6.1.2 Preincubation Method Cultures of Salmonella hirnurium, TA98, -.TAlOO TAl535 and TA1537, and Escherichia coli WP2uvrA for u h u t a t i o n Assay will be prepared as. described in Section 1&-he test article .will be wei hed or measured, and a sena! dilution will be 4 performed as previously descnbed in Sec ion 18.4. 0.5 rnL of sterile, deionized, distilled water or S-9 mix will be dispensed into a senes of labeled culture tubes, and treatment will be performed by adding 0.1 rnL of tester strain and 0.1 mL of test article. The tubes will be vortexed gent1 and preincubated at 37 +1"Cfor 20 minutes. The tubes are shaken at J ' a moderate spee during the preincubation. ,Appropnafe .solvent and positive controls will also be prepared. 2.0 mL of top agar containing 1X histidine-biotin or 1X tryptophan will be added to each culture tube after the preincubation penod. The contents of each tube will be mixed b vortexin poured onto a bottom agar late, and evenly distributed by J 1 gently tiltin an rotating &e plates. All of the plates will e placed on a flat, level surface until solidifaed. After all treatments have been performed, the plates will be inverted and incubated at 37 k1"C for 48-72 hours. 18.6.2 Confirmation of Tester Strain Genotypes 18.6.3 Tester Strain Viabilitv Determination After the Mutation Assa has been plated, a dilution.of each tester strain will be Y pre ared, and ap roximately 50-500 bactena will be lated in top a ar supplemented with 1 0 2 0.5mM) histdine-biotin or 1OX (0.5mM) tryptoplan. These ?ates will be incubated i P for 4 -72 hours, and then the total number of colonies that deve op will be determined. Protocol No. 2140.4 031299 60 Page 12 of 16 ' SITEK RESEARCLHABORATORIES 1: - 18.6.4 Backqround Lawn Evaluation 3 & The integri of the back round microcolony lawn will be evaluated by viewing each plate with the ai of a 2X to microscope. The lawns will be rated as normal, slightly reduced, markedly reduced, extremely reduced or absent. 18.6.5 Enumeration of Colonies After 48-72 hours of incubation, the plates treated with the hi hest test article ? concentrationwill be observed .for the presence of recipitate. If recipi ate is absent the P P entire assay will be counted. usin an automatic co ony counter. f observation of the high dose plates reveals precipitate %at interferes with accurate automatic counting, those lates will be counted b hand. The procedure will be repeated for each subsequent dose Y Kvel or until no precipi ate is evident. 18.6.5.1 Automatic Colony Countinq ! Each late will be placed on the stage, and three counts are made with the automatic counter. {e plate will be rotated on the stage ap roximately 120"between each count, and the median count will be entered directly into he computer. 18.6.5.2 Hand Countinq the Hand counting of colonies will bottom of the plate. The hand Y be performed b marking a dot over count will be en ered directly into the each colony computer. on The counts will be entered direct1 in the Lotus 123 spread sheet pro ram 2140B or ) 2150B for the Plate Incorporation or l!treincubation method, respectively. ?he computer printouts will be included in the study notebook. 18.7 Confirmatory Mutation Assay Ifthe first Mutation Assay or a portion thereof roduces negative or equivocal results, 4 a confirmatory Mutation Assay will be performed. he test article treatment concentrations may be altered based on the results obtained in the first Mutation Assay. On the other hand, if the results of the first Mutation Assay are clearly ositive, a confirmatory Mutation E Assay may or may not be performed depending on the ponsor's instructions. 18.8 Criteria For a Valid Assay The following criteria will be used as guidelines in determinin the acceptability of the results. Since it IS impossible to. formulate critena to every configuration of data generated by the Mutation Assay, the Study be responsible for the ultimate decision regarding the acceptability of the results. Protocol No. 2140.4 031299 61 Page 13 of 16 11 ' SITEK RESEARCLHABORATORIES ` . 18.8.1 Solvent Control Cultures The mean reversion frequency of the test article solvent control plates for each strain must fall within the range presented below: Salmonella typhimurium TA98 TA100 TA1535 TA1537 20 k15 100 lt70 20 215 15 +12 Escherichia WP2uvrA 15 + l o 18.8.2 Positive Controls The results for the positive control cultures will be considered acceptable if the treated strains have mean reversion frequencies that are three times or greater than the mean reversion frequencies of the test article solvent control plates. 18.8.3 Tester Strain Characterization P*-l-7- 1. All of the Salmonella himurium strains will be confirmed positive for histidine dependence and the tschenc ia s rain for tryptophan dependence. 2. All of the Salmonella himurium strains will be confirmed positive for the rfa mutation as evidenced by sensi IVI o crystal violet. 3. The R-factor strains, TA98 and.TA100, will be confirmed positive for the pKMlO1 1 plasmid as evidenced by ampicillin resistance. 4. The titer of the stock cultures ofFach strain yill indicate that the stock cultures contained approximately between 50x10 and 1.Ox10 bacteria per mL. 18.9 Evaluation of Test Results Y 9 dJ The following criteria will be used as guidelines in evaluating the results of the Mutation Assay for a negative, positive or equivocal response. Since it is impossible to write criteria that would appl to every confi uration of data enerated by the Mutation Assay, the Study Director wil be responsible or the ultimate ecision in the evaluation of the results. The factors considered in making the decision will be discussed in the report. 18.9.1 Criteria for a Neqative Response 1 A response will be considered negative if all of the strains treated with the test article have mean reversion frequencies that are less than v i c e that of the' mean reversion frequencies of the corres onding solvent control plates in TA98 and TAlOO and less than three times in TA1535, T 1537 and WP2uvrA, and there is no evidence of a dosedependent response. I Protocol No. 2140.4 031299 62 Page 14 of 16 I 1 SITEK RESEARCHLABORATORIES 18.9.2 Criteria for' a Positive Response A response will be considered positive-ifeither strain TA98 or TA100 has a dose that produces a mean reversion frequency-that is greater than or equal.to .two times the mean reversion fre uency of the corresponding solvent control plates or if either strain TAI 535, TA1537 or dP2uvrA has a dose roducing a three-fold or greater increase in the mean P reversion frequency compared .to he s.olvent control frequen In additiqn the response 7 must be dose dependent or increasing concentrations of he test article must show increasin mean reversion frequencies. In evaluating the results, consideration will be given to %e degree of toxicity exhibited by the dose causin the two-foldhhree-fold or s reater increase in reversion frequency and the magnitude o the increase in reversion flrequency. 18.9.3 Criteria for an Equivocal ResDonse 4 A response will be considered e uivocal if it-does not fulfill the criteria of either a ne ative or a positive response and/or he Study Director does not consider the response to e! either positive or negative. If an equivocal response is obtained, a repeat assay may be performed at the Sponsor's request. 19.0 PROTOCOL AMENDMENTS AND DEVIATIONS If chan es in the approved protocol are necessary, such changeswill be documented in the form ofprotocol amendments and rotocol deviations. Protocql amendments will be Y generated when chan es in the protoco are made pnor to performing a study or part of i? a study affected by t e 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 them wit1 be documented and attached to the protocol as an ad endum. Protocol deviations will be generated when the procedures used to erform the study do s not conform to the approved protocol. The Sponsor will be informe of these deviations, and as soon as ractical, such changes along with their reasons or explanations will be B documented an kept in the study notebook. 20.0 REPORT OF RESULTS 20.1 Content I; The results of the stud will be submitted to the Spor;lsor in the form of a final re ort. A draft report will be submi ed. before the final report is issued. The report will i n d d e , but not be limited to, the following: 1. Name and address of the testing facijity 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 original protocol. 3. Methods used to analyze the data. 4. The test and control substances. I Protocol No. 2140.4 031299 63 - Page15of16 1 1 SITEK RESEARCHLABORATORIES 5. Description of the methods used to perform the study. 6. The data, mean plate counts, +/- SD, and any observations regarding toxicity and precipitate 7. The name and signature of the Study Director and the names of other technical personnel who participated in performing the study. 8. The location where the raw data and reports are to be stored. 9. A statement from the Quality Assurance Unit. 20.2 Chanqes 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. 21.O ARCHIVES The raw data electronic file confaining the data tables, documentation, protocol and final report of the sfud will be maintained in the SlTEK Research Laboratories Archives, 8 15235 Shad Grove oad, Suite 303, Rockville, Maryland, according to the terms and Y conditions o the study. 22.0 REFERENCES I.Arnes B. N., J. McCann and E. Yamasaki. Methods for detecfing carcino ens and RIP muta ens with the Salmonella/ mammalian-microsome mutagenicity test. ut. Res., 23 - 3113 -367, 1975. 2. Maron, D. and B. N.Ames. Revised methods for the Salmonella mutagenicity test. Mut. Res., &:173-215, 1983. 3. Green, M. H. L., and W. J. Muriel. Mutagen testing usin trp+ reversion in Escherichia coli. .in: B. J. Kilbey, et al. (eds.), Handbook of Mutagenici Test Procedures, PP-65-94 , tlsevier North Holland Biomedical Press, Amsterdam, 4. Venitt, S., and J. M. Parry (eds.). Mutagenicity testing: A practical approach. IRL Press, Oxford, England and Washmgton, D.C., 1984. Protocol No. 2140.4 031299 64 Page 16 of 16 SITEK Study No. 0623-2140 PROTOCOL AMENDMENTS Amendment Nos. : 1-2 Sponsor: Primedica Redfield 100 E. Boone Street Redfield, AR 72132 Testing Facility: SITEK Research Laboratories 15235 Shady Grove Road, Suite 303 Rockville, Maryland 20850 SITEK's Study No.: 0623-2140 Sponsor's Study No.: 132-008 Test Article ID: Potassium Perfluorobutane Sulfonate Protocol Title: Evaluation of a Test Article in the Salmonella typhimurium/Escherichia coli Plate IncorporatiodPreincubation Mutation Assay in the Presence and Absence of Induced Rat Liver S-9 Amendment No. 1: Protocol Page 6, Section 12.1, Source, The Escherichia coli strain, WP2uvrA, that will be used in the assay was received from Ms. Judy Mayo of Pharmacia and Upjohn Co., Kalamazoo, Michigan. Reason for Amendment No. 1: A new strain of Escherichia coli has been obtained and is currently in use at SITEK Research Laboratories. Amendment No. 2: Protocol Page 11, Section 18.6, Mutation Assay, The test article was tested in the Mutation Assays at the following concentrations: 50,100,500,lo00 and 5000 pglplate. Reason for Amendment No. 2: As stated in the protocol, the test article concentrations will be included in the form of an amendment, once determined. i 65 Study Number: 0623-2140 Protocol Amendment Nos. 1 - 2 Page 2 of 2 SITEK Study NO. 0623-2140 APPROVAL: Kamala J. Pant, M.S. 1 JohnSeng, Ph.u. Sponsor's Study Coordinator \ \ e 8,rn Date /Ill Date 66 SITEK Study NO. 0623-2140 APPENDIX IV HISTORICAL POSITIVE AND SOLVENTCONTROLDATA 67 - I SITEK Study NO. 0623-2140 I T E K RESEARCH LABORATORIES t HISTORICAL POSITIVE CONTROL DATA FOR SALMONELLA NPHIMURIUM/ ESCHERICHIA COLI MUTATION ASSAY MUTANTS EXPRESSED IN IO6 SURVIVING CELLS WITH AND WITHOUT ACTNATION WITHOUT ACTIVATION AVERAGE STANGARD DEVIATION (&) MINIMUM VALUE MAXIMUM VALUE N" WKH ACTIVATION AVERAGE STANDARD DEVIATION (k) MINIMUM VALUE MAXIMUM VALUE N* TA9 8 (2W 724 301 189 21 08 I80 TA9 8 (2W 81 1 31 9 151 1699 178 T A 1 00 (Na-w 578 174 272 I433 178 TA1 00 888 302 194 1618 1. 175 TA1535 (NaAz) TA1537 (9AA) 134 141 22 846 I60 TA1535 (24 701 238 187 1410 144 TA1537 179 81 129 48 43 18 1336 349 156 157 E.coli (MMS) 742 391 173 1528 34 E.coli (a) 226 148 19 71 9 145 68 SlTEK Research Laboratories HISTORICAL NEGATIVE CONTROL DATA FOR SALMONELLA TYPHIMURlUM/ ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY MUTANTS EXPRESSED IN IO6 SURVIVING CELLS AVERAGE STANDARD DEVIATION (e) MINIMUM VALUE MAXIMUM VALUE IN* TAI 00 I AVERAGE STANDARD DEVIATION ( 2 ) MINIMUM VALUE MAXIMUM VALUE N* ITA1535 I AVERAGE STANDARD DEVIATION (&) MINIMUM VALUE MAXIMUM VALUE 1 N* ITA1537 AVERAGE STANDARD DEVIATION (&) MlNlMUM VALUE MAXIMUM VALUE I N* [E. COLI AVERAGE STANDARD DEVIATION (2) MINIMUM VALUE MAXIMUM VALUE N" N* = Number of data points. 24.3 3.9 16 33 72 DMSO 89.5 24.1 23 144 73 DMSO 18.3 5.8 10 46 68 DMSO 9.5 - 3.1 3 17 66 DMSO 14.6 3.2 9 24 56 26.4 5.3 11 32 16 ACET 26.0 3.1 21 31 12 CORNOIL 101.6 36.4 26 174 17 ACFT 90.6 16.5 59 118 12 CORNOIL 25.8 3.5 17 33 57 H,O 103.9 22.4 66 159 57 H,O 24.7 5.9 17 35 14 SALINE 91 .I 18.8 70 I32 14 SALINE 19.3 4.6 12 33 16 ACET 16.7 3.8 11 23 12 CORNOIL 18.0 4.0 11 28 40 H,O 16.7 3.6 11 22 14 SALINE 9.6 2.7 6 16 ia ACET 10.6 5.8 3 23 12 CORNOIL 9.1 2.5 5 17 41 H,O 8.0 2.8 4 13 14 SALINE 15.3 4.6 8 25 16 15.3 3.0 10 19 12 17.6 3.5 12 25 40 15.6 3.7 10 25 14 69 SITEK Study NO. 0623-2140 SITEK Research Laboratories HISTORICAL NEGATIVE CONTROL DATA FOR SALMONELLA NPHlMURlUM/ ESCHERICHIA COLI PLATE INCORPORATION MUTATION ASSAY MUTANTS EXPRESSED IN IO6 SURVIVING CELLS WITH S-9 ACTIVATION ITA98 I DMSO ACET CORNOIL H,O SALINE I I N* [TA1535 AVERAGE STANDARD DEVIATION (?I MINIMUM VALUE MAXIMUM VALUE I N* ITA1537 73 DMSO 16.4 4.8 8 311 71 DMSO 16 ACE3 16.4 4.8 8 31 71 ACET 12 CORNOIL 70.8 3.0 15 25 12 CORNOIL 58 14 H,O SALINE 16.7 3.7 8 27 41 H,O 15.4 - 3.6 9 23 14 SALINE AVERAGE STANDARD DEVIATION (2) MINIMUM VALUE MAXIMUM VALUE N* \E.COLI 1 10.2 4.2 3 28 70 DMSO 10.3 2.7 7 14 16 ACET 10.4 . 3.6 6 18 12 CORNOIL 9.8 7.1 2.8 2.2 6 3 17 I1 41 14 H,O SALINE AVERAGE STANDARD DEVlATlON (k) MINIMUM VALUE MAXIMUM VALUE N" N* = Number of data points. 15.2 3.4 0 25 60 16.9 3.4 8 25 60 17.2 5.2 9 26 12 17.5 4.2 10 30 43 16.0 3.5 10 23 14 70 SITEK Study NO. 0623-2140 APPENDIX V DOSING SOLUTION ANALYSIS 71 SITEK Study NO. 0623-2140 DOSE FORMULAION ANALYSIS OF PERFLUOROBUTANESULFONATE IN DMSO BY HPLUMS STUDY ID: A239.1 SPONSOR STUDY NO. 132-008 Southern Reseqrch Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 72 SITEK Study No. 0623-2140 Quality Assurance Statement Final Report On Dose Formulation Analysis of Perfluorobutane Sulfonate (PFBS) In DMSO A239.1 Sponsor Study No. 132-008 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-d%&L C.L. Marsh,'Manager, Quality AssuranceIQuality Control 7/3/& Date 73 SITEK Study NO. 0623-2140 SUMMARY A total of 12 formulated dose samples including blanks ranging in concentration from 0.5 to 50 mg/mL,wereanalyzed 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. I J' 74 James D. Johnson, M.S., MBA Manager Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Staff Chemist Bioanalytical Chemistry Group KEY PERSONNEL SITEK Study NO. 0623-2140 75 SITEK Study NO. 0623-2140 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 ) 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 aliquotwas 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. No 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 0.5 to 500 mg/mL were analyzed by BACG 3533. All samples were found to be within k 10 % of the reported concentration. 76 Table I Dose Formulation Analysis of PFBS in DMSO I I I I I I I Measured I Sample Date Prepared Conc. % of Target 1 (mg/mL)* I 0 mg/mL B2 9/20/00 ND NA 0 mg/mL B1 8/15/00 ND NA 0.5 mg/MI B2 9/20/00 0.49 98.0 0.5 rng/rnL B1 8115/00 0.48 96.0 1 rng/mL B1 811 5/00 1.03 103 1 rng/mL B2 9/20/00 1.03 103 5 mg/mL B1 8/15/00 4.83 96.6 5 mg/mL B2 9/20/00 4.99 99.8 10 mg/mL B2 9/20/00 10.1 101 10 mg/mL B1 8115/00 10.0 100 50 mg/mL B2 9/20/00 49.7 99.4 50 mg/mL B1 8115/00 51.3 103 ND = not detected NA = not applicable * = average of duplicate analysis 77 6.0 Approvals Staff Chemist Bioanalytical Chemistry Group Bioanalytical Chemistry Group SITEK Study No. 0623-2140 -773 - 0/ Date Date 78 ~~~~ - SITEK Study NO. 0623-2140 METHOD VALIDATION REPORT VALIDATION OF ANALYTICAL METHOD FOR DOSE FORMULATION ANALYSIS OF PERFLUOROBUTANE SULFONATE (PFBS) IN 1% CARBOXYMETHYL CELLULOSE (CMC) STUDY ID: A098.1 Southern Research Institute 2000 Ninth Avenue South P.O. Box 55537 Birmingham, AL 35255-5537 79 SITEK Study NO. 0623-2140 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. 80 James D. Johnson, M.S. Manager Bioanalytical Chemistry Group Gregory S. Gorman, Ph.D. Research Chemist I11 Bioanalytical Chemistry Group Lester Williams, B.S. Associate Chemist I1 Bioanalytical Chemistry Group . .. KEY PERSONNEL SITEK Study NO.0623-2140 81 - SITEK Study NO. 0623-2140 I 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 1% CMC. 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 ng/mL and an aliquot of this was placed into autosampler vials for analysis. I. 3.2 Method Validation Validation for BACG 3533 "Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC Mass Spectrometry" consisted of analyzing three standard curves containing CMC at the concentration present in the diluted 4 m g l d 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: 82 SITEK Study NO. 0623-2140 STAMARD COMPOSITE CURVE PFBS (0% CMC) # of Standards Mean% Accur. Std. Deviation 6 97.71 19.47 6 102.9 55.85 6 99.73 110.6 6 99.52 187.8 6 100.1 288.2 6 100.2 356.4 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 Furve is shown below: STANDARD COMPOSITE CURVE PFBS (0.001% CMC) # of Standards Mean % Accur. Std. Deviation 3 98.1 1 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 analyte 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 : y = a x 2 + bx + c 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. 83 SITEK Study NO. 0623-2140 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 Gieg6x-y S. Gorman, Ph.D. Staff Chemist Bioanalytical Chemistry Group 't c/ Manager Bioanalytical Chemistry Group Date '. Date 84 ~~ ~ - SITEK Study NO.0623-2140 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 (HPLCIMS) 1.0 PFUNCIPLE i 2.0 Dose formulation samples of perfluorobutane sulfonate (PFBS) in 1 % carboxymethyl cellulose (CMC) are diluted down to 3200 ng/mL and analyzed by HPLUMS. Quantitation is b&ed on an internal standard using back calculated values from a calibration curve encompassing a concentration range from 500 to 10,000nglmL and containing the same concentration of CMC as the dose formulations in the diluted form. - REAGENTS 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 Perfluoropentanoic acid (internal standa-rd), 97% 2.1.5 Ammonium acetate, HPLC grade 2.2 Prepared Solutio11s Appropriate changes in the solutions may be made at the discretion of the analyst 2.2.1 5 mM Ammonium acetate in organic free water 85 SITEK Study NO. 0623-2140 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 (HPLC/MS) 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. 3.0 3.1 ) 3.2 3.3 3.4 3.5 3.7 3.8 3.9 3.10 3.11 3.12 INSTRUMENTS, MATERIALS, AND APPARATUS The following or their equivalents maybe used. HPLC pump(s), autosampler, and single quadrupole mass spectrometer Autosampler vials with inserts Vortex mixers (e.g., touch mixer and IKA-Vibrax @ platform mixer) HPLC mobile phase filtration apparatus Filters for HPLC mobile phase filtration apparatus (e.g., Nylon-66, 0.20 ,urn) Analytical balance Volumetric flasks (e.g., 10 and 25 mL) Disposable Pasteur pipets Micropipettor(s) with tips Culture tubes with teflon-lined caps Assorted glassware and syringes 86 SITEK Study NO. 0623-2140 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 PREPARATION OF STOCKS AND W O m G 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. , \ 4.1 i Main Stock Solution of PFBS - 1000 p g / d 4.1.1 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. - 4.2 Spiking Stock Solution of PFBS -50 pg/mL 4.2.1 - Prepare an 50 p g / d solution of PFBS in mobile phase (e.g., measure about 500 pL - of lo00 p g / d 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. 4.3 Stock Solution of Internal Standard (PFPA), -200 pg/mL 4.3.1 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 organic- free 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. 87 SITEK Study NO. 0623-2140 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 (HPLCIMS) 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-mL volumetric flasks or other appropriate glassware. If desired a modified dilution scheme can be used and documented in the study records. Working Stock Level Volume of ) (WSL) Internal Volume of PFBS Volume of mobile Approximate Concentration Standard spiking solution phase (PL) (ng/m.L) W) (PL) 500 200 10 790 1000 200 20 780 3000 200 60 740 3200 200 64 736 5000 200 100 700 8000 200 160 640 10000 200 - 200 600 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 m g / d 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. 88 SITEK Study NO.0623-2140 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 (HPLUMS) 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 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 1 mL 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 SPECTROMETRYMSS SPECTROMETRY ("LCMSMS) 7.1 7.1.1 - Conditions are to be optimized if necessary. T.CC o. . w 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 89 SITEK Study NO. 0623-2140 Page 6 of 8 ANALYTICAL Ml3THOD Method No.: BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLUMS) Software: PE Sciex TurboQuan Turboion Spray Source 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 Value IS -5Ooo NC 0 TEM 450 OR -25 RNG -170 QO 10 IQ 1 11 ST 16 RO 1 11 DF 300 CEM 2400 NEB 15 CUR 8 CAD 0 QPE 0 POL 0 VCM I IPE 0 0 90 SITEK Study NO. 0623-2140 ANALYTICAL METHOD Page 7 of 8 Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfonate in 1% Carboxymethyl Cellulose by HPLC/Mass Spectrometry (HPLC/MS) -Masses requested: PFBS: 299.2 200 8.0 1 8.1 8.2 i I 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 JIldkdk 299.2 218.9 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. Alternatively, the peak heights maybe 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: y = axz+ bx + c 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. 91 ~~ - SITEK Study NO. 0623-2140 Page 8 of 8 ANALYTICAL METHOD Method No. : BACG-3533 Title: Dose Formulation Analysis of Perfluorobutane Sulfona t in 1 % C rboxymethy1 Cellulose by HPLC/Mass Spectrometry (HPLCIMS) 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. I' 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&&.Gorman, - 743-JrnO Ph.D., Research Chemist III Date 7-17- 200. Date I i 92