Document YjEdxb47r2G9NX6GmJb90g0dV

A R X -o '33 G-C Final Report Salmonella - Escherichia co/zTMammalian-Microsome Reverse Mutation Assay with PFOS PREPARED FOR: 3M Corporate Toxicology COVANCE STUDY NUMBER: 20784-0-409 001923 C O V A trc > THE DEVELOPMENT SERVICES COMPANY FINAL REPORT SALMONELLA - ESCHERICHIA C0Z//MAMMALIAN-MICROSOME REVERSE MUTATION ASSAY WITH PFOS AUTHOR Michael S. Mecchi, MS PERFORMING LABORATORY Covance Laboratories Inc. (Covance) 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT IDENTIFICATION Covance Study No.: 20784-0-409 3M Study No.: T6295.17 SUBMITTED TO 3M Corporate Toxicology 3M Center Building.220-2E-02 St. Paul, Minnesota 55144-1000 STUDY COMPLETION DATE November 5,1999 1 of 24 001924 QUALITY ASSURANCE STATEMENT Covance 20784-0-409 Salmonella-Escherichia co/i'/Mammalian-Microsome Reverse Mutation Assay with PFOS The report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40 of the U.S. Code of Federal Regulations Part 792. The following inspections were conducted and the findings reported to the Study Director and study director management. Written status reports of inspections and findings are issued to Covance management according to standard operating procedures. Inspection Dates From To 9/9/99 9/9/99 Phase Preparation of S9 Mix Dates Reported to Study Director and Study Director Management 9/9/99 Auditor J. Crouch 10/21/99 10/21/99 Draft Report Review 10/21/99 K. Groeninger 11/04/99 11/04/99 Final Report Review 11/04/99 K. Groeninger C Date -2- C01325 Covance 20784-0-409 STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA - TSCA), Title 40 of the U.S. Code of Federal Regulations Part 792, and with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the Study Director, who certifies that the evaluation of the test article as presented herein represents an appropriate conclusion within the context of the study design and evaluation criteria. All test and control results in this report are supported by an experimental data record and this record has been reviewed by the Study Director. Study Director: - W U i S -yAcC- Michael S. Mecchi, MS Genetic and Cellular Toxicology H -5-91? Study Completion Date -3C 01926 TABLE OF CONTENTS Covance 20784-0-409 Page No. ABSTRACT.................................................................................................................................... 6 STUDY INFORMATION..............................................................................................................7 Sponsor Test Article Assay Information Study Dates Supervisory Personnel OBJECTIVE ................................................................................................................................... 7 TEST SYSTEM RATIONALE.......................................................................................................7 MATERIALS AND METHODS.................................................................................................... 8 Test System Test Article Control Articles S9 Metabolic Activation System Dose Rangefinding Assay Mutagenicity Assay Plating Procedures Scoring the Plates DATA ........................................................................................................................................... 15 Data Presentation Assay Acceptance Criteria Assay Evaluation Criteria RESULTS .......................... , ...................................................................................................... 16 Test Article Handling Dose Rangefinding Assay Mutagenicity Assay CONCLUSION ............................................................................................................................. 17 RECORDS TO BE MAINTAINED ............................................................................................ 18 -4- C01927 REFERENCES DATA TABLES TABLE OF CONTENTS (Continued) Covance 20784-0-409 Page No. ........ 18 19 ABSTRACT Covance 20784-0-409 The objective of this study was to evaluate the test article, PFOS, for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2wvrA. The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding assay using tester strains TA100 and WP2wvrA and ten doses of test article ranging from 6.67 to 5,000 pg per plate, one plate per dose, both in the presence and absence of S9 mix. The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and Escherichia coli tester strain WP2urvA. The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested with the Salmonella tester strains were 5,000, 3,330,1,000, 333, 100, and 33.3 pg per plate in the presence of S9 mix and 5,000,1,000, 333,100, 33.3, 10.0, 3.33, 1.00, and 0.333 pg per plate in the absence of S9 mix. With tester strain WP2uvrA, the doses tested were 5,000, 3,330,1,000, 333, 100, and 33.3 pg per plate both in the presence and absence of S9 mix The results of the Salmonella-Escherichia co/j/Mammalian-Microsome Reverse Mutation Assay indicate that under the conditions of this study, 3M Corporate Toxicology's test article, PFOS, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver (S9). -6- 001929 STUDY INFORMATION Covance 20784-0-409 Sponsor 3M Corporate Toxicology Test Article Sponsor's Identification: PFOS FC-95 Lot 217 Date Received: 08/19/99 Physical Description: white crystalline powder Storage Conditions: room temperature Assay Information Type of Assay: Salmonella-Escherichia co/z'/Mammalian-Microsome Reverse Mutation Assay Protocol No.: 409, Edition 5 Covance Study No.: 20784-0-409 Study Dates Initiation Date: 08/20/99 Experimental Start Date: 08/24/99 Experimental Termination Date: 09/15/99 Supervisory Personnel Study Director: Michael S. Mecchi, MS Laboratory Supervisor: Carlos E. Orantes, BS OBJECTIVE The objective of this study was to evaluate the test article and/or its metabolites for their ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2wvrA. TEST SYSTEM RATIONALE The 5a/wone//a/Mammalian-microsome reverse mutation assay detects point mutations, both ffameshifts and/or base pair substitutions. The strains of Salmonella typhimurium used in this assay are histidine auxotrophs by virtue of conditionally lethal mutations in their histidine operon. When these histidine-dependent cells (his-) are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine) only those cells -7- 001030 Covance 20784-0-409 which revert to histidine independence (his+) are able to form colonies. The trace amount of histidine in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ revertants are readily discemable as colonies against the limited background growth of the his- cells. By utilizing several different tester strains, both base pair substitution mutations and ffameshift mutations can be detected. The Ames Test has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. The Escherichia coli WP2wvrA reverse mutation assay detects point mutations, specifically base pair substitutions. The Escherichia coli tester strain WP2wvrA used in this assay is a tryptophan auxotroph (trp-) by virtue of a conditionally lethal mutation at a site which blocks a step of tryptophan biosynthesis prior to the formation of anthranilic acid. Since the target site for true back mutation is an ochre nonsense mutation, tryptophan-independent revertants (trp+) can arise either by a base change at the site of the original alteration or by suppression by specific suppressor mutations at a second site in tRNA genes (Brusick et al., 1980). When the tryptophan-dependent cells (trp-) are exposed to the test article and grown under selective conditions (minimal media with a trace amount of tryptophan) only those cells which revert to tryptophan independence (trp+) are able to form colonies. The trace amount of tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The trp+ revertants are readily discemable as colonies against the limited background growth of the trp- cells. While the trp reversion system responds to most alkylating agents, base-analog mutagens and certain metals (i.e. soluble chromates), ffameshift mutagens would not be expected to be detected by this system. MATERIALS AND METHODS The experimental materials, methods and procedures are based on those described by Ames et al., (1975) and Green and Muriel (1976). Test System Salmonella typhimurium. The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames et al., (1975). The tester strains in use at Covance were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. The specific genotypes of these strains are shown in the following table. Tester Strain Genotypes Histidine Mutation hisG46 hisC3076 tD 3052 TA1535 TA1537 TA 100 TA98 Additional Mutations LPS Repair R Factor rfa uvrB ______ uvrB + -8- 001031 Covance 20784-0-409 In addition to a mutation in the histidine operon, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall. The second mutation, a deletion of the uvrB gene, results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, all of the tester strains containing this deletion require the vitamin biotin for growth. Strains TA98 and TA100 also contain the PKM101 plasmid which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by ffameshift mutagens. TA1535 is reverted by base substitution mutagens and TA100 is reverted by mutagens which cause both ffameshifts and base substitutions. Escherichia coli. The tester strain used was the tryptophan auxotroph WP2wvrA as described by Green and Muriel (1976). The tester strain in use at Covance was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom). In addition to a mutation in the tryptophan operon, the tester strain contains a uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability since the uvrA repair system would normally act to remove the damaged part of the DNA molecule and accurately repair it afterwards. Tester strain WP2wvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. Frozen Permanent Stocks. Frozen permanent stocks of the tester strains were prepared by growing fresh overnight cultures, adding dimethylsulfoxide (DMSO, 0.09 mL/mL of culture) and freezing small aliquots (0.5-1.5 mL) at -70C. Master Plates. Master plates of the tester strains were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with 1) for -9- 001032 Covance 20784-0-409 Salmonella typhimurium, an excess of histidine, and biotin, and for tester strains TA98 and TA100, ampicillin (25 pg/mL), to ensure the stable maintenance of the pKMIOl plasmid; and 2) for Escherichia coli, an excess of tryptophan. Tester strain master plates were stored at 5 3C. Inoculation of Overnight Cultures. Overnight cultures for use in all testing procedures were inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator which was programmed to begin operation (shaking, 125 25 rpm; incubation, 37 2C) so that the overnight cultures were in log phase or late log phase when turbidity monitoring began. Harvest of Overnight Cultures. To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture turbidity. Cultures were harvested once a predetermined turbidity was reached as determined by a percent transmittance (%T) reading on a spectrophotometer. This target turbidity ensures that cultures have reached a density of at least 0.5 X 109cells per mL and that the cultures have not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when the target %T was reached and were placed at 5 3C. Confirmation of Tester Strain Genotype. Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay. rfa Wall Mutation. For the Salmonella typhimurium tester strain cultures, the presence of the rfa wall mutation was confirmed by demonstration of the sensitivity of the culture to crystal violet. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing 10 pg of crystal violet was added. Sensitivity was demonstrated by inhibition of bacterial growth in a zone immediately surrounding the disk. pKMIOl Plasmid. The presence of the pKMIOl plasmid was confirmed for the appropriate tester strain cultures by demonstration of resistance to ampicillin. An aliquot of an overnight culture of each strain was overlaid onto plates containing selective media and an antibiotic sensitivity disk containing ampicillin was added. Resistance was demonstrated by bacterial growth in the zone immediately surrounding the disk. Characteristic Number of Spontaneous Revertants. The number of spontaneous revertants per plate in the vehicle controls that is characteristic of the respective strains was demonstrated by plating 100 pL aliquots of the culture along with the appropriate vehicle on selective media. - 10- CQ1933 Covance 20784-0-409 Culturing Broth. The broth used to grow overnight cultures of the tester strains was Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder). Agar Plates. Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Overlay Agar for Selection of Revertants. Overlay (top) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and was supplemented with 10 mL of 1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 mL of agar for selection of tryptophan revertants. When S9 mix was required, 2.0 mL of the supplemented top agar was used in the overlay. However, when S9 mix was not required, water was added to the supplemented top agar (0.5 mL of water per 2 mL of supplemented top agar) and the resulting 2.5 mL of diluted supplemented top agar was used for the overlay. This dilution ensured that the final top agar and amino acid supplement concentrations remained the same both in the presence and absence of S9 mix. Test Article The Sponsor was responsible for the determination of the test article stability and the test article characteristics as defined in the GLP regulations. Control Articles Vehicle Controls. Vehicle controls were plated for all tester strains both in the presence and absence of S9 mix. The vehicle control was plated, using a 50 pL aliquot of vehicle (equal to the maximum aliquot of test article dilution plated), along with a 100 pL aliquot of the appropriate tester strain and a 500 pL aliquot of S9 mix (when necessary), on selective agar. Positive Controls. The combinations of positive controls, activation condition and tester strains plated concurrently with the assay are indicated in the following table. -11 - C01934 Positive Controls Covance 20784-0-409 Tester Strain TA98 TA98 TA100 TA100 TA1535 TA1535 TA1537 TA1537 WPluvrA WP2vrA S9 Mix +- - + - + - + - + - Positive Control Cone, per Plate benzo[a]pyrene 2.5 pg 2-nitrofluorene 1.0 pg 2-aminoanthracene 2.5 pg sodium azide 2.0 pg 2-aminoanthracene 2.5 pg sodium azide 2.0 pg 2-aminoanthracene 2.5 pg ICR-191 2.0 pg 2-aminoanthracene 25.0 pg 4-nitroquinoline-N-oxide ____________ 10 ______________ The sources and grades of the positive control articles are as follows: benzo[a]pyrene (CAS #50-32-8), Sigma Chemical Co., purity 98% 2-aminoanthracene (CAS #613-13-8), Sigma Chemical Co., purity 97% 2-nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., 98% sodium azide (CAS #26628-22-8), Sigma Chemical Co., purity 98% ICR-191 (CAS #1707-45-0), Sigma Chemical Co., purity 98% 4-nitroquinoline-N-oxide (CAS #56-57-5), Sigma Chemical Co., purity 99%. Sterility Controls. The most concentrated test article dilution was checked for sterility by plating a 50 pL aliquot (the same volume used in the assay) on selective agar. The S9 mix was checked for sterility by plating 0.5 mL on selective agar. S9 Metabolic Activation System S9 Homogenate. Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Batch 0972 (42.8 mg of protein per mL). The homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with AroclorTM 1254 (200 mg per mL in com oil) at 500 mg/kg as described by Ames et a l, (1975). S9 Mix. The S9 mix was prepared immediately prior to its use in any experimental procedure. The S9 mix contained the components indicated in the following table. - 1 2 - C-01935 S9 Mix Components Covance 20784-0-409 Component h 2o 1M NaH2P 0 4/Na2H P04, pH 7.4 0.25M Glucose-6-phosphate 0.10M N A D P 0.825M KC1/0.2M mgCl2 S9 Homogenate Amount 0.70 mL 0.10 mL 0.02 mL 0.04 mL 0.04 mL 0.10 mL 1.00 mL Dose Rangefinding Assay The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay. Design. The dose rangefinding assay was performed using tester strains TA100 and WP2vrA both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5 mg per plate. Rationale. The cytotoxicity of the test article observed on tester strain TA100 is generally representative of that observed on the other tester strains and because of the comparatively high number of spontaneous revertants per plate observed with this strain, gradations of cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2hwA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 mix may vary greatly from that observed in the absence of S9 mix. Therefore, this would require that different test article dose ranges be tested in the mutagenicity assay based on the presence or absence of the S9 mix. Evaluation of the Dose Rangefinding Assay. Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. Selection of the Maximum Dose for the Mutagenicity Assay. Cytotoxicity was observed in the dose rangefinding study and the highest concentration of test article used in the subsequent mutagenicity assay was a dose which was expected to exhibit a thinning of the bacterial background lawn. Mutagenicity Assay Design. The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2wvrA both in the presence and absence of S9 mix along with the appropriate vehicle and -13- C01936 Covance 20784-0-409 positive controls. The doses of test article were selected based on the results of the dose rangefinding assay. Frequency and Route of Administration. The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et a l, (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, the test article, the tester strain and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of the test article, the vehicle controls and the positive controls were plated in triplicate. Plating Procedures These procedures were used in both the dose rangefinding assay and the mutagenicity assay. Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition and dose level. The S9 mix and dilutions of the test article were prepared immediately prior to their use. When S9 mix was not required, 100 pL of tester strain and 50 pL of vehicle or test article dose were added to 2.5 mL of molten selective top agar (maintained at 45 2C). When S9 mix was required, 500 pL of S9 mix, 100 pL of tester strain and 50 pL of vehicle or test article dose were added to 2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 4 hr at 37 2C. Positive control articles were plated using a 50 pL plating aliquot. Scoring the Plates Plates which were not evaluated immediately following the incubation period were held at 5 3C until such time that colony counting and bacterial background lawn evaluation could take place. Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as 1) normal, 2) slightly reduced, 3) moderately reduced, 4) extremely reduced, 5) absent, or 6) obscured by precipitate. If present on the plates, macroscopic precipitate was scored as slight, moderate or heavy. - 14- C01937 Covance 20784-0-409 Counting Revertant Colonies. The number of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The number of revertant colonies per plate for the positive controls were counted by automated colony counter with the exception of the positive controls for tester strain TA98 in the presence of S9 mix in Experiment 20784-B1, which were counted manually. DATA Data Presentation For all replicate platings, the mean revertants per plate and the standard deviation were calculated- The results of these calculations are presented in tabular form in the Data Tables section of this report. Assay Acceptance Criteria Before assay data were evaluated, the criteria for a valid assay had to be met. The following criteria were used to determine a valid assay: Tester Strain Integrity. rfa Wall Mutation. To demonstrate the presence of the rfa wall mutation, Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet. pKMIOl Plasmid. To demonstrate the presence of the PKM101 plasmid, cultures of the appropriate tester strains exhibited resistance to ampicillin. Characteristic Number of Spontaneous Revertants. To demonstrate the requirement for histidine or tryptophan, the tester strain cultures exhibited a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable ranges for the mean vehicle controls were as follows: TA98 TA100 TA1535 TA1537 WP2wvrA 8 - 60 60 - 240 4 - 45 2 - 25 5 - 40 Tester Strain Culture Density. To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures were greater than or equal to 0.5 x 109bacteria per mL and/or had reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 109bacteria per mL. -15 - C01933 Covance 20784-0-409 Positive Control Values in the Absence of S9 Mix. To demonstrate that the tester strains were capable of identifying a mutagen, the mean value of a positive control for a respective tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain. Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity). To demonstrate that the S9 mix was capable o f metabolizing a promutagen to its mutagenic form(s), the mean value of the positive control for a respective tester strain in the presence of the S9 mix exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain. An acceptable positive control in the presence of S9 mix for a specific strain was evaluated as having demonstrated both the integrity of the S9 mix and the ability of the tester strain to detect a mutagen. Cytotoxicity. A minimum of three non-toxic doses were required to evaluate assay data. Assay Evaluation Criteria Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows: Tester Strains TA98, TA100, and WP2vrA. For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article. RESULTS Test Article Handling The test article formed unworkable, non-homogeneous suspensions in water at concentrations of 500, 399, 300,200, 99.9, and 50.0 mg/mL. In dimethylsulfoxide, the test article formed a non-homogeneous suspension at 501 mg/mL and a homogeneous suspension at 401 mg/mL. For this reason, dimethylsulfoxide (DMSO, CAS# 67-68-5, Acros Organics, Lot No. A012649701) was used as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article formed a translucent white suspension. The -16- 001939 Covance 20784-0-409 test article formed a solution at 2.00 mg/mL and remained a solution in all succeeding dilutions prepared for the mutagenicity assay. Dose Rangefinding Assay Doses tested in the mutagenicity assay were selected based on the results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2vrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5,000 pg per plate, were tested and the results are presented in Tables 1 and 2. These data were generated in Experiment 20784-A1. Indications of cytotoxicity were observed with tester strain TA100 at 10.0 pg per plate and above in the absence of S9 mix as evidenced by the thinning of the bacterial background lawn. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2vrA in either the presence or absence of S9 mix as evidenced by no decrease in the number of revertants per plate and a normal background lawn. Mutagenicity Assay The mutagenicity assay results for PFOS are presented in Tables 3 through 5. These data were generated in Experiment 20784-B1. The data are presented as individual plate counts (Tables 3 and 5) and as mean revertants per plate standard deviation (Tables 4 and 5) for each treatment and control group. The results of the dose rangefinding study were used to select the doses tested in the mutagenicity assay. The doses tested with the Salmonella tester strains were 5,000, 3,330,1,000, 333,100, and 33.3 pg per plate in the presence of S9 mix and 5,000,1,000, 333, 100, 33.3,10.0, 3.33,1.00, and 0.333 pg per plate in the absence of S9 mix. With tester strain WP2wvrA, the doses tested were 5,000,3,330,1,000, 333,100, and 33.3 pg per plate both in the presence and absence of S9 mix. In the mutagenicity assay (Experiment 20784-B1, Tables 3,4 and 5), all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9 mix. All criteria for a valid study were met. CONCLUSION The results of the Salmonella-Escherichia co/i'/Mammalian-Microsome Reverse Mutation Assay indicate that under the conditions of this study, 3M Corporate Toxicology's test article, PFOS, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver (S9). - 17- C01340 RECORDS TO BE MAINTAINED Covance 20784-0-409 All raw data, documentation, records, the protocol, and the final report generated as a result of this study will be archived in the storage facilities of Covance-Vienna for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional period of time or sent to a storage facility designated by the Sponsor. REFERENCES Ames, B.N., McCann, J., and Yamasaki, E., "Methods for detecting carcinogens and mutagens with the Sa/TMOHe/Za/Mammalian-Microsome Mutagenicity Test." Mutation Research, 37:347364(1975). Brusick, D.J., Simmon, V.F., Rosenkranz, H.S., Ray, V.A. and Stafford, R.S., "An evaluation of the Escherichia coli WP2 and WP2vrA reverse mutation assay." Mutation Research, 76:169190(1980). Green, M.H.L. and Muriel, WJ., "Mutagen testing using trp+reversion in Escherichia coli." Mutation Research, 38:3-32 (1976). Maron, D.M. and Ames, B., "Revised methods for the Salmonella Mutagenicity Test." Mutation Research, 773:173-215 (1983). Vogel, H.J. and Bonner, D.M., "Acetylomithinase of E. coli: Partial purification and some properties." J Biol. Chem. 273:97-106 (1956). - 18- C01941 Covance 20784-0-409 DATA TABLES - 19- C0194 Covance 20784-0-409 TABLE 1 : DOSE RANGEFINDING ASSAY RESULTS TEST ARTICLE ID: PFOS EXPERIMENT ID: 20784-A1 VEHICLE: DMSO DATE PLATED: 24-Aug-99 DATE COUNTED: 27-Aug-99 pg/PLATE 0.00 (Vehicle, 50 pL) TA100 REVERTANTS PER PLATE WITH S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* WITHOUT S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* 98 1 86 1 Test Article 6.67 10.0 33.3 66.7 100 333 667 1000 3330 5000 117 1 100 1 96 1 108 1 84 1 93 1 77 1 95 1 76 1 80 1 83 1 73 2 79 2 86 2 97 2 89 2 82 2 87 2 85 2 79 3 * Background Lawn Evaluation Codes: normal 2 = slightly reduced extremely reduced 5 = absent slight precipitate mp = moderate precipitate (requires hand count) 3 moderately reduced 6 obscured by precipitate hp heavy precipitate (requires hand count) -20- 001943 Covance 20784-0-409 TABLE 2 : DOSE RANGEFINDING ASSAY RESULTS TEST ARTICLE ID: PFOS EXPERIMENT ID: 20784-A1 VEHICLE: DMSO DATE PLATED: 24-Aug-99 DATE COUNTED: 27-Aug-99 Hg/PLATE 0.00 (Vehicle, 50 pL) WP2uvrA REVERTANTS PER PLATE WITH S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* WITHOUT S9 REVERTANTS PER PLATE BACKGROUND LAWN EVALUATION* 21 1 18 1 Test Article 6.67 16 1 10.0 14 1 33.3 21 1 66.7 21 1 100 23 1 333 26 1 667 17 1 1000 9 1 3330 20 1 5000 22 1 * Background Lawn Evaluation Codes: 1 = normal 2 = slightly reduced 4 = extremely reduced 5 = absent sp = slight precipitate mp = moderate precipitate (requires hand count) 11 9 18 10 9 10 15 12 16 12 3 = moderately reduced 6 = obscured by precipitate hp = heavy precipitate (requires hand count) 1 1 1 1 1 1 1 1 1 1 -21 - CQ1944 Covance 20784-0-409 TABLE 3 : MUTAGENICITY ASSAY RESULTS - INDIVIDUAL PLATE COUNTS TEST ARTICLE ID: PFOS EXPERIMENT ID: 20784-B1 DATE PLATED: 09-Sep-99 DATE COUNTED: 13-Sep-99, 14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 pL DOSE/PLATE MICROSOMES: RAT LIVER VEHICLE CONTROL TEST ARTICLE 33.3 Pg 100 Pg 333 Pg 1000 Pg 3330 Pg 5000 Pg POSITIVE CONTROL** MICROSOMES: NONE VEHICLE CONTROL TEST ARTICLE 0.333 pg 1.00 Pg 3.33 Pg 10.0 Pg 33.3 Pg 100 Pg 333 Pg 1000 Pg 5000 Pg POSITIVE CONTROL*** TA98 123 23 23 24 17 22 30 20 24 31 17 29 37 14 19 27 20 23 23 13 18 28 365 440 453 12 20 24 12 19 22 12 15 18 19 26 27 20 22 23 17 23 25 14 15 21 13 15 16 8 15 19 6 12 13 186 195 210 REVERTANTS PER PLATE TA100 12 3 TA1535 12 3 95 98 113 10 13 15 75 98 99 86 88 95 111 112 118 64 74 81 87 95 104 90 90 96 8 12 12 11 12 14 6 9 12 6 9 14 4 9 11 7 11 12 835 897 1055 102 117 121 88 94 95 77 88 94 67 77 96 79 87 98 72 81 83 73 78 110 79 84 93 63 69 73 83 83 84 80 88 99 671 716 722 8 9 12 8 8 11 6 7 10 9 13 17 8 14 14 6 14 15 9 13 18 10 10 15 9 13 14 5 7 15 644 688 694 TA1537 123 5 6 10 2 5 12 3 5 12 5 6 10 7 9 10 456 7 89 159 173 183 BACK GROUND LAWN* 1 1 1 1 l 1 1 1 388 458 124 7 9 10 246 258 4 9 11 4 5 12 458 347 443 507 546 1 1 1 1 1 1 1 1 1 2 1 ** TA98 TA100 TA 1535 TA 1537 benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate * Background Lawn Evaluation Codes: 1 = normal 2 = slightly reduced 4 = extremely reduced 5 = absent sp = slight precipitate mp = moderate precipitate (requires hand count) *** TA98 TA100 TA1535 TA 1537 2-nitrofluorene sodium azide sodium azide ICR-191 1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 3 = moderately reduced 6 = obscured by precipitate hp = heavy precipitate (requires hand count) -22- C01945 Covance 20784-0-409 TABLE 4 : MUTAGENICITY ASSAY RESULTS - SUMMARY TEST ARTICLE ID: PFOS EXPERIMENT ID: 20784-B1 DATE PLATED: 09-Sep-99 DATE COUNTED: 13-Sep-99, 14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 pL MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION DOSE/PLATE________ TA98______________TA100_____________TA1535____________ TA1537________ MICROSOMES: RAT LIVER VEHICLE CONTROL MEAN S.D. 23 1 MEAN S.D. 102 10 MEAN S.D. 13 3 MEAN S.D. 73 BACK GROUND LAWN* 1 TEST ARTICLE 33.3 PS 100 Pg 333 Pg 1000 Pg 3330 Pg 5000 Pg 23 7 25 6 28 10 20 7 22 2 20 8 91 14 90 5 114 4 73 9 95 9 92 3 11 2 12 2 93 10 4 84 10 3 65 75 73 92 51 81 1 1 1 1 1 1 POSITIVE CONTROL** 419 48 929 113 113 10 172 12 1 MICROSOMES: NONE VEHICLE CONTROL 19 6 92 4 10 2 63 1 TEST ARTICLE 0.333pg 1.00 Pg 3.33 Pg 10.0 Pg 33.3 Pg 100 Pg 333 Pg 1000 Pg 5000 Pg 18 5 15 3 24 4 22 2 22 4 17 4 15 2 14 6 10 4 86 9 80 15 88 10 79 6 87 20 85 7 68 5 83 1 89 10 92 82 13 4 12 3 12 5 13 5 12 3 12 3 95 62 22 92 42 53 84 74 62 52 1 1 1 1 1 1 1 1 2 POSITIVE CONTROL*** 197 12 703 28 675 27 499 52 1 ** TA98 TA 100 TA 1535 TAI 537 benzo[a]pyrene 2-aminoanthracene 2-aminoanthracene 2-aminoanthracene 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate 2.5 pg/plate * Background Lawn Evaluation Codes: 1 = normal 2 = slightly reduced 4 = extremely reduced 5 = absent sp = slight precipitate mp = moderate precipitate (requires hand count) *** TA98 TA 100 TA1535 TA 1537 2-nitrofluorene sodium azide sodium azide ICR-191 1.0 pg/plate 2.0 pg/plate 2.0 pg/plate 2.0 pg/plate 3 = moderately reduced 6 = obscured by precipitate hp = heavy precipitate (requires hand count) -23 - CO.7.946 Covance 20784-0-409 TABLE 5 : MUTAGENICITY ASSAY RESULTS - INDIVIDUAL PLATE COUNTS AND SUMMARY TEST ARTICLE ID: PFOS EXPERIMENT ID: 20784-B1 DATE PLATED: 09-Sep-99 DATE COUNTED: 13-Sep-99,14-Sep-99 VEHICLE: DMSO PLATING ALIQUOT: 50 pL DOSE/PLATE MICROSOMES: RAT LIVER VEHICLE CONTROL TEST ARTICLE 33.3 Pg 100 Pg 333 Pg 1000 Pg 3330 Pg 5000 Pg POSITIVE CONTROL** MICROSOMES: NONE VEHICLE CONTROL TEST ARTICLE 33.3 Pg 100 Pg 333 Pg 1000 Pg 3330 Pg 5000 Pg POSITIVE CONTROL*** REVERTANTS PER PLATE WP2i/vrA 12 3 9 17 22 10 12 15 14 18 18 18 20 30 17 21 22 11 18 21 14 15 20 213 243 247 18 19 21 9 11 20 16 17 19 10 16 25 12 17 19 11 14 18 9 14 16 157 274 288 MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION WP2uvrA MEAN S.D. 16 7 12 3 17 2 23 6 20 3 17 5 16 3 234 19 BACKGROUND LAWN* 1 1 1 1 1 1 1 1 19 2 13 6 17 2 17 8 16 4 14 4 13 4 240 72 1 1 1 1 1 1 1 1 ' WP2i/vrA 2-aminoanthracene 25.0 gg/plate Background Lawn Evaluation Codes: 1 = normal 2 = slightly reduced 4 = extremely reduced 5 = absent sp = slight precipitate mp = moderate precipitate (requires hand count) *** WP2wv/A 4-nitroquinoline-N-oxide 1.0 pg/plate 3 = moderately reduced 6 = obscured by precipitate hp = heavy precipitate (requires hand count) - 2 4 - C01947 THE DEVELOPMENT SERVICES COMPANY COVANCE STUDY NO.: PROTOCOL 409 E D m O N 5 - ~ ^C? SALMONELLA - ESCHERICHIA COL//MAMMALIAN-MICROSOME REVERSE MUTATION ASSAY Covance Laboratories Inc. (Covance) will conduct this study in compliance with Good Laboratory Practice (GLP) regulations and standards. This protocol, at least one critical phase of the work in progress, and the final report will be audited by Quality Assurance in accordance with SOPs at Covance. This study will be conducted at 9200 Leesburg Pike, Vienna, Virginia 22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 1.0 SPONSOR IDENTIFICATION Company Name: Address: 3M Corporation 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 2.0 TEST ARTICLE IDENTIFICATION PFOS___________________________ _____ 3 fa JfcUj ho. ______________________ 3.0 TEST ARTICLE ANALYSIS Determination of the test article stability and the test article characteristics as defined in the GLP regulations is the responsibility of the Sponsor. 4.0 NOTIFICATION OF REGULATORY SUBMISSION In order to comply with GLP regulations and standards, consulting laboratories must be notified if all or part of a study is intended for regulatory submission. Covance maintains a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the results of this study: Undetermined FDA J 3 EPA-TSCA EPA-FIFRA MAFF MOHW OECD OTHER __________ 12/98 1 of 18 001948 TME DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 5.0 STUDY DATES Proposed Experimental Start Date: __ Proposed Experimental Termination Date: 6.0 APPROVAL OF STUDY PROTOCOL Study Director: 1999 October [SS7 - W U i 1.<5 i M U - _____________ Michael S. Mecchi, M.S. Testing Facility Management: Date: Associate Director Sponsor's Authorized Representative: ~T _________________ __ Marvin T. Case, D.V.M., Ph.D. Date: 12/98 2 of 18 C01919 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 PART 2. STUDY PROTOCOL SALMONELLA - ESCHERICHIA C0L//MAMMALIAN-MICROSOME REVERSE MUTATION ASSAY I. OBJECTIVE The objective of this study is to evaluate the test article and/or its metabolites for their ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2vrA. n. TEST SYSTEM A. Salmonella typhimurium: The Sa/mone/Za/Mammalian-microsome reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains of Salmonella typhimurium used in this assay are histidine auxotrophs by virtue of conditionally lethal mutations in their histidine operon. When these histidine-dependent cells (his-) are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine) only those cells which revert to histidine independence (his+) are able to form colonies. The trace amount of histidine in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ revertants are readily discemable as colonies against the limited background growth of the his- cells. By utilizing several different tester strains, both base pair substitution mutations and frameshift mutations can be detected. The Ames Test has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. B. Escherichia coli: The Escherichia coli WP2vrA reverse mutation assay detects point mutations, specifically base pair substitutions. The Escherichia coli tester strain WP2wvrA used in this assay is a tryptophan auxotroph (trp) by virtue of a conditionally lethal mutation at a site which blocks a step of tryptophan biosynthesis prior to the formation of anthranilic acid. Since the target site for true back mutation is an ochre nonsense mutation, tryptophan-independent revertants (trp*) can arise either by a base change at the site of the original 12/98 3 of 18 C01950 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 alteration or by suppression by specific suppressor mutations at a second site in tRNA genes (Brusick et al, 1980). When the tryptophan-dependent cells (trp-) are exposed to the test article and grown under selective conditions (minimal media with a trace amount of tryptophan) only those cells which revert to tryptophan independence (trp+) are able to form colonies. The trace amount of tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The trp+ revenants are readily discernable as colonies against the limited background growth of the trp- cells. While the trp reversion system responds to most alkylating agents, base-analog mutagens and certain metals (i.e. soluble chromates), frameshift mutagens would not be expected to be detected by this system. 331. MATERIALS A. Tester Strains 1. Salmonella typhimurium: the tester strains to be used will be the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, and TA1537 as described by Ames et al (1975). The specific genotypes of these strains are shown in Table 1. TABLE I. TESTER STRAIN GENOTYPES Histidine Mutation______ Additional Mutations hisGA6 /u`iC3076 WD3052 LPS Repair R Factor TA1535 TA1537 rfa uvrB - TA 100 TA98 rfa vrB +R 12/98 In addition to a mutation in the histidine operon, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e. benzo(a)pyrene) that would otherwise be excluded by a normal intact cell wall. 4 of 18 C01951 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 The second mutation, a deletion of the nvrB gene, results in a deficient DNA excision repair system which greatly enhances the sensitivity of these strains to some mutagens. Since the wvrB deletion extends through the bio gene, all of the tester strains containing this deletion also require the vitamin biotin for growth. Strains TA98 and TA100 also contain the R-factor plasmid, pKMIOl, which further increases the sensitivity of these strains to some mutagens. The mechanism by which this plasmid increases sensitivity to mutagens has been suggested to be by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA1535 is reverted by base substitution mutagens and TA100 is reverted by mutagens which cause both frameshifts and base substitutions. 2. Escherichia coli: the tester strain to be used will be the tryptophan auxotroph WP2uvrA as described by Green and Muriel (1976). In addition to a mutation in the tryptophan operon, the tester strain contains a uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability if the uvrA repair system would normally act to remove the damaged part of the DNA molecule and accurately repair it afterwards. Tester strain WP2wvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. 3. Source of Tester Strains a. Salmonella typhimurium: the tester strains in use at Covance were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. 12/98 5 of 18 C01952 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 b. Escherichia coli: tester strain WP2vrA in use at Covance was received from The National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom). 4. Storage of the Tester Strains a. Frozen Permanent Stocks Frozen permanent stocks will be prepared by growing fresh overnight cultures, adding DMSO (0.09 ml/ml of culture) and freezing away appropriately vialed aliquots. Frozen permanent stocks of the tester strains will be stored at -70C. b. Master Plates Master plates will be prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with either histidine and biotin (and for strains containing the R-factor, ampicillin) or tryptophan. Tester strain master plates will be stored at 5 3C. 5. Preparation of Overnight Cultures a. Inoculation Overnight cultures for use in all testing procedures, will be inoculated by transferring a colony from the appropriate master plate to a flask containing culture medium. Inoculated flasks will be placed in a shaker/incubator which will be programmed to begin operation (shaking, 125 25 rpm; incubation, 37 2C) so that the overnight cultures are in log phase or late log phase when turbidity monitoring begins. b. Harvest To ensure that cultures are harvested in late log phase, the length of incubation will be determined by spectrophotometric monitoring of culture turbidity. Cultures will be harvested once a predetermined turbidity is reached as determined by a percent transmittance (%T) 12/98 6 of 18 CQ1.953 cov/H rc> THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 6. 12/98 reading on a spectrophotometer. This target turbidity ensures that cultures have reached a density of at least 0.5 X 109cells per ml and that the cultures have not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures will be removed from incubation when the target %T is reached and placed at 5 3C. Confirmation of Tester Strain Genotypes a. Salmonella typhimurium: tester strain cultures will be checked for the following genetic markers on the day of their use in the mutagenicity assay: (1) rfa Wall Mutation The presence of the rfa wall mutation will be confirmed by demonstration of the cultures sensitivity to crystal violet. (2) pKMIOl Plasmid The presence of the pKMIOl plasmid will be confirmed for cultures of tester strains TA98 and TA100 by demonstration of resistance to Ampicillin. (3) Spontaneous Reversion The number of spontaneous revertants per plate in the vehicle controls that is characteristic of the respective strains will be demonstrated by plating aliquots of each culture along with the appropriate vehicle on selective medium. b. Escherichia coli: tester strain WP2vrA will be checked for the following genetic marker on the day of its use in the mutagenicity assay: (1) Spontaneous Reversion The number of spontaneous revertants per plate in the vehicle controls that is characteristic of WP2vrA will be 7 of 18 C01954 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 demonstrated by plating aliquots of each culture along with the appropriate vehicle on selective medium. 7. Tester Strain Media a. Culturing Broth The broth used to grow overnight cultures of the tester strains will be Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth #2 (dry powder). b. Minimal Bottom Agar Plates Bottom agar (25 ml per 15 x 100 mm petri dish) will be VogelBonner minimal medium E (Vogel and Bonner, 1956) supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. c. Top Agar for Selection of Revertants Top (overlay) agar will be prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and will be supplemented with 10 ml of 1) 0.5 mM histidine/biotin solution per 100 ml agar for selection of histidine revertants, or 2) 0.5 mM tryptophan solution per 100 ml agar for the selection of tryptophan revertants. When S9 is required, 2.0 ml of the supplemented top agar is used for the overlay. However, when S9 is not required, water is added to the supplemented top agar (0.5 ml of water per 2 ml of supplemented top agar) and 2.5 ml of the diluted supplemented top agar is used for the overlay. This dilution ensures that the final top agar and amino acid supplement concentrations remain the same both in the presence and absence of S9 mix. 12/98 8 of 18 G01355 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 B. Liver Microsomal Enzvme Reaction Mixture (S9 Mix') L S9 Homogenate Liver microsomal enzymes (S9 homogenate) will be purchased commercially and will be prepared from male Sprague-Dawley rats that have been injected (i.p.) with AroclorTM 1254 (200 mg/ml in com oil) at 500 mg/kg as described by Ames et al, 1975. 2. S9 Mix The S9 mix will be prepared immediately prior to its use in any experimental procedure. The S9 mix will contain the components indicated in Table 2. TABLE 2. S9 MIX COMPONENTS h 2o 1M NaH2P 04/Na2H P 04, pH 7.4 0.25M Glucose-6-phosphate 0.10MNADP 0.825M KC1/0.2M MgCl2 S9 Homogenate 0.70 ml 0.10 ml 0.02 ml 0.04 ml 0.04 ml 0.10 ml 1.00 ml C. Controls 1. Vehicle Controls Appropriate vehicle controls will be plated for all strains in the presence and absence of S9 mix. Vehicles compatible with this test system include but will not be limited to: Deionized H20 , dimethylsulfoxide (CAS #67-68-5), ethanol (CAS #64-17-5), and dimethylformamide (CAS # 68- 12-2). 12/98 9 of 18 001956 c o v /h * ^ THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 2. Positive Controls The combinations of positive controls, activation condition and tester strains plated concurrently with the assay are indicated in Table 3. Tester Strain TABLE 3. POSITIVE CONTROLS Cone. S9 Mix Positive Control Der Plate TA98 TA98 TA100 TA100 TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2wvrA + - + - + - + - + - benzo[a]pyrene 2-nitrofluorene 2-aminoanthracene sodium azide 2-aminoanthracene sodium azide 2-aminoanthracene ICR-191 2-aminoanthracene 4-nitroquinoline-N-oxide 2.5 fig 1.0/ig 2.5 fig 2.0 fig 2.5 fig 2.0 fig 2-5 fig 2.0 fig 25.0 iLg 1.0 Mg 3. Sterility Controls a. Test Article The most concentrated test article dilution will be checked for sterility by plating an aliquot of the same volume used in the assay on selective agar. b. S9 Mix The S9 mix will be checked for sterility by plating 0.5 ml on selective agar. 12/98 10 of 18 C01957 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 IV. METHODS A. Dose Rangefinding Study The growth inhibitory effect (cytotoxicity) of the test article to the test system will be determined in order to allow the selection of appropriate doses to be tested in the mutagenicity assay. 1. Design The dose rangefinding study will be performed using tester strains TA100 and WP2vrA both in the presence and absence of S9 mix. A minimum of ten doses of test article will be tested at one plate per dose. Unless otherwise directed by the Sponsor, the test article will be checked for cytotoxicity up to a maximum concentration of 5,000 fig per plate if solubility/miscibility permits. If the test article exhibits limited solubility/miscibility, it will be tested for cytotoxicity up to the maximum workable concentration attainable in the vehicle of choice. a. Rationale The growth inhibitory effect (cytotoxicity) of the test article on tester strain TA100 is generally representative of that observed on the other Salmonella typhimurium tester strains and because of TAlOO's comparatively high number of spontaneous revertants per plate, gradations of cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2wvrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of microsomal enzymes may vary greatly from that observed in the absence of microsomal enzymes. Therefore, this would require that different test article dose ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes. 12/98 11 of 18 C01953 COV/HiC^ THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 2. Evaluation of the Dose Rangefinding Study Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. 3. Selection of the Maximum Dose for the Mutagenicity Assay a. Cytotoxicity Observed When cytotoxicity is observed in the dose rangefinding study, the highest concentration of test article to be used in the subsequent mutagenicity assay will be that which gives a detectable reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn. b. No Cytotoxicity Observed If no cytotoxicity is observed in the dose rangefinding study then the highest dose of test article to be used in the mutagenicity assay will be the same as that tested in the rangefinding study unless 1) the Sponsor specifies an alternate maximum dose, 2) there is limited availability of the test article and/or 3) the test article precipitates heavily in the top agar. B. Mutagenicity Assay 1. Design The assay will be performed using tester strains TA98, TA100, TA1535, TA1537, and WP2vrA, both in the presence and absence of S9 mix. Unless the Sponsor specifies doses to be tested, the doses of test article will be selected based on the results of the dose rangefinding study. If cytotoxicity has been demonstrated in the dose rangefinding study, a minimum of six doses of test article will be tested along with the appropriate vehicle and positive controls. If no cytotoxicity has been demonstrated, a minimum of five doses will be tested. 12/98 12 of 18 C01959 the d evelo pm ent services co m pany PROTOCOL 409 EDITION 5 2. Frequency and Route of Administration The tester strains will be exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, the S9 mix (where appropriate), the tester strain, and the test article are combined in molten agar which is overlaid onto a minimal agar plate. Following incubation at 37 2C for 52 4 hr, revertant colonies will be counted. All doses of test article, vehicle controls and positive controls will be plated in triplicate. C. Plating Procedures These procedures will be used in both the dose rangefinding study and the mutagenicity assay. Each plate will be labeled with a code which identifies the test article, test phase, tester strain, activation condition and dose. The S9 mix and dilutions of the test article will be prepared immediately prior to their use. When S9 mix is not required, 100 pi1of tester strain and 50 pi1of vehicle or test article dose will be added to 2.5 ml of molten selective top agar (maintained at 45 2C). When S9 mix is required, 500 pil of S9 mix, 100 pil of tester strain and 50 pil of vehicle or test article dose will be added to 2.0 ml of molten selective top agar. After the required components have been added, the mixture will be vortexed and overlaid onto the surface of 25 ml of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay has solidified, the plates will be inverted and incubated for 52 4 hr at 37 2C. When necessary, plating aliquots of other than 50 pil of test article/vehicle will be used. Positive control articles will be plated using a 50 ptl plating aliquot. D. Scoring the Plates Plates which are not evaluated immediately following the incubation period will be held at 5 3C until such time that colony counting and bacterial background lawn evaluation can take place. 12/98 13 of 18 G01960 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 1. Bacterial Background Lawn Evaluation The condition of the bacterial background lawn will be evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity will be scored relative to the vehicle control and recorded along with the revertant counts for that dose. 2. Counting Revertant Colonies Revertant colonies for a specific tester strain within a given test article dilution series will be counted either entirely by automated colony counter or entirely by hand (with the exception of the dose rangefinding study). If there is sufficient test article precipitate on the plates at any dose which interferes with automated colony counting, then the plates at all dose levels for that specific strain and activation condition will be counted manually. E. Analysis of Data For all replicate platings, the mean revertants per plate and the standard deviation will be calculated. V. EVALUATION OF TEST RESULTS Before assay data can be evaluated, the criteria for a valid assay must be met. A. Criteria For A Valid Assay The following criteria will be used to determine a valid assay: 1. Tester Strain Integrity: Salmonella typhimurium a. rfa Wall Mutation To demonstrate the presence of the rfa wall mutation, tester strain cultures must exhibit sensitivity to crystal violet. 12/98 14 of 18 0:1961 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 b. pKMIOl Plasmid To demonstrate the presence of the R-factor plasmid, pKMIOl, cultures of tester strains TA98 and TA100 must exhibit resistance to ampicillin. c. Characteristic Number of Spontaneous Revertants To demonstrate the requirement for histidine, the tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable ranges for the mean vehicle controls are as follows: TA98 TA100 TA1535 TA1537 8 - 60 60 - 240 4 - 45 2 - 25 2. Tester Strain Integrity: Escherichia coli a. Characteristic Number of Spontaneous Revertants To demonstrate the requirement for tryptophan, the tester strain culture must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. The acceptable range for the WP2vrA mean vehicle controls is 5 to 40 revertants per plate. 3. Tester Strain Culture Density To demonstrate that appropriate numbers of bacteria are plated, the density of tester strain cultures must be greater than or equal to 0.5 x 109bacteria per ml and/or have reached a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 109bacteria per ml. 12/98 15 of 18 C 010G 2 develo pm ent services company PROTOCOL 409 EDITION 5 4. Positive Control Values a. Positive Control Values in the Absence of S9 Mix To demonstrate that the tester strains are capable of identifying a mutagen, the mean value of a positive control for a respective tester strain must exhibit at least a 3-fold increase over the mean value of the vehicle control for that strain. b. Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity) To demonstrate that the S9 mix is capable of metabolizing a promutagen to its mutagenic form(s), the mean value of the positive control for a respective tester strain in the presence of the S9 mix must exhibit at least a 3-fold increase over the mean value of the vehicle control for that strain. An acceptable positive control in the presence of S9 for a specific strain will be evaluated as having demonstrated both the integrity of the S9 mix and the ability of the tester strain to detect a mutagen. 5. Cvtotoxicitv A minimum of three non-toxic dose levels will be required to evaluate assay data. B. Criteria For A Positive Response Once the criteria for a valid assay have been met, responses observed in the assay are evaluated as follows: 1. Tester Strains TA98. TA100. and WP2vrA For a test article to be considered positive, it must produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be 12/98 16 of 18 C0.13G3 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 accompanied by a dose response to increasing concentrations of the test article. 2. Tester Strains TA1535 and TA1537 For a test article to be considered positive, it must produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article. VI. REPORTING THE RESULTS A report of the results of this study will be prepared by Covance and will accurately describe all methods used for the generation and analysis of the data. Results presented in the report for this assay will include: The results of the dose rangefinding study (if applicable) including the number of revertants per plate and a bacterial background lawn evaluation for each dose the identity of the bacterial tester strains used in the assay dose levels at which the test article was tested individual plate counts for all treated, positive and vehicle control plates calculated mean and standard deviation for all replicate plate counts evaluation of results VH. CHANGES AND REVISIONS Any changes or revisions of this approved protocol will be documented, signed by the Study Director, dated, and maintained with this protocol. The Sponsor will be notified of any changes or revisions. 12/98 17 of 18 C 01964 THE DEVELOPMENT SERVICES COMPANY PROTOCOL 409 EDITION 5 V3. RECORDS TO BE MAINTAINED All raw data, documentation, records, the protocol, and the final report generated as a result of this study will be archived in the storage facilities of Covance-Vienna, for at least one year following submission of the final report to the Sponsor. After the one year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna, for an additional period of time or sent to a storage facility designated by the Sponsor. DC. REFERENCES Ames, B.N., McCann, J. and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Sa/mone/Zo/mammalian-microsome mutagenicity test. Mutation Research 31:347-364. Brusick, D.J., Simmon, V.F., Rosenkranz, H. S., Ray, V.A., and Stafford, R.S. (1980). An evaluation of the Escherichia coli WP2 and WP2vrA reverse mutation assay. Mutation Research, 76:169-190. Green, M.H.L. and Muriel, W.J. (1976). Mutagen testing using trp* reversion in Escherichia coli. Mutation Research 38:3-32. Maron, D.M., and Ames B. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215. Vogel, H J. and Bonner, D.M. (1956). Acetylomithinase of Escherichia coli: Partial purification and some properties. J. Biol Chem. 218:97-106. 12/98 18 of 18