Document gb01QkaVpG6767OmmqVnM1aGQ

CO RN ING Hazleton MUTAGENICITY TEST WITH T-6564 IN THE SALMONELLA - ESCHERICHIA COI//MAMMALIAN-MICROSOME REVERSE MUTATION ASSAY WITH A CONFIRMATORY ASSAY FINAL REPORT AUTHOR Timothy E. Lawlor, M.A. PERFORMING LABORATORY Coming Hazleton Inc. (CHV) 9200 Leesburg Pike Vienna, Virginia 22182 LABORATORY PROJECT ID CHV Study No.: 17750-0-409R SUBMITTED TO 3M Corporation Building 220-2E-02 3M Center St. Paul, MN 55144-1000 STUDY COMPLETION DATE September 13, 1996 CHV Study No.: 17750-0-409R 1 of 34 000232 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT STUDY TITLE: Salmonella - Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay ASSAY NO.: 17750-0-409R PROTOCOL NO.: 409R, Edition 4, Modified for 3M Quality Assurance inspections of the study and review of the final report of the above referenced project were conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to the general requirements of the appropriate Good Laboratory Practice regulations. Findings from the inspections and final report review were reported to management and to the study director on the following dates: Inspection/Date Findings Reported Auditor Scoring Plates - 06/28/96 06/28/96 P. Conley Draft Report Review - 08/29/96 08/30/96 C. Orantes Final Report Review - 09/13/96 09/13/96 C. Orantes Quality Assurance Unit Daie Reieased CHV Study No.: 17750-0-409R 2 000233 C O R N IN G Hazleton STUDY COMPLIANCE AND CERTIFICATION The study was conducted in compliance with the Good Laboratory Practice regulations as set forth by the Food and Drug Administration (FDA) in Title 21 of the U.S. Code of Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) with any applicable amendments. There were no 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. Study Director: Timothy E. Lkiwlor, M.A. Bacterial Mutagenesis Genetic and Cellular Toxicology Study Completion Date CHV Study No.: 17750-0-409R 3 000234 TABLE OF CONTENTS C O R N IN G Hazleton Page No. I. SUMMARY........................................................................................................................ 5 II. STUDY INFORMATION.................................................................................................. 7 III. MATERIALS AND METHODS.................................................... 9 IV. RESULTS AND CONCLUSIONS..............................................................................24 V. DATA TABLES .............................................................................................................. 27 CHV Study No.: 17750-0-409R 4 000235 I CO R N IN G Hazleton > SECTION L SUMMARY 4A INTRODUCTION AND CONCLUSIONS CHV Study No.: 17750-0-409R j 5 000236 CO R N IN G Hazleton SUMMARY A. Introduction At the request of 3M Corporation , Coming Hazleton Inc. investigated T-6564 for mutagenic activity in the Salmonella - Escherichia co/z/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay. This assay evaluated the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains and at the tryptophan locus in an Escherichia coli tester strain both in the presence and absence of an exogenous metabolic activation system of mammalian microsomal enzymes derived from AroclorTM-induced rat liver (S9). The doses tested in the mutagenicity assay were selected based on the results of a dose rangefinding study using tester strains TA100 and WP2wvrA and ten doses of test article ranging from 5,000 to 6.67 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 WP2wwA. The assay was conducted with five doses of test article in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested were 5,000, 3,330, 1,000; 333, and 100 pg per plate in both the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in an independent experiment. B. Conclusions The results of the Salmonella - Escherichia co/z'/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that, under the conditions of this study, 3M Corporation's test article, T-6564, did not cause a positive increase in the number of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver (S9). CHV Study No.: 17750-0-409R 6 000237 C O R N IN G Hazleton SECTION II. STUDY INFORMATION CHV Study No.: 17750-0-409R 000238 C O R N IN G Hazleton STUDY INFORMATION A. Sponsor: 3M Corporation B. Test Article: T-6564 L-13167 FC1015-X 1. Physical Description: clear colorless liquid 2. Date Received: 05/30/96 C. Type of Assay: Salmonella-Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay 1. Protocol Number: CHV Protocol 409R, Edition 4 2. CHV Study Number: 17750-0-409R D. Study Dates 1. Study Initiation Date: 06/04/96 2. Experimental Start Date: 06/12/96 3. Experimental Termination Date: 08/27/96 E. Study Supervisory Personnel Study Director: Timothy E. Lawlor, M.A. Laboratory Supervisor: Michael S. Mecchi, B.S. CHV Study No.: 17750-0-409R 8 000239 CO RN ING Hazleton SECTION III. MATERIALS AND METHODS CHV Study No.: 17750-0-409R 9 G00240 C O R N IN G Hazleton MATERIALS AND METHODS The experimental materials, methods and procedures are based on those described by Ames et a! (1975) and Green and Muriel (1976). MATERIALS A. Tester Strains 1. Salmonella tvphimurium The tester strains used were 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 I. TABLE I. TESTER STRAIN GENOTYPES Histidine Mutation______ ____Additional Mutations hisG46 hisC3076 teD3052 LPS Repair R Factor TA1535 TA1537 rfa uvrB - TA 100 TA98 rfa uvrB +R 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 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. CHV Study No.: 17750-0-409R 10 000241 C O R N IN G Hazleton 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 substitution mutations. 2. Escherichia coli The tester strain used was 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 since the uvrA repair system would normally act to remove the damaged part of the DNA molecule and accurately repair it afterwards. Tester strain WP2uvrA 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 CHV were received directly from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley. b. Escherichia coli The tester strain, WP2wvrA, in use at CHV 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 were prepared by growing fresh overnight cultures, adding DMSO (0.09 ml/ml of culture) and freezing small aliquots (0.5-1.5 ml) at s-70C. CHV Study No.: 17750-0-409R 11 000242 CO R N IN G Hazleton b. Master Plates Master plates were prepared by streaking each tester strain from a frozen permanent stock onto minimal agar appropriately supplemented with 1) for Salmonella typhimurium, an excess of histidine, and biotin, and for tester strains TA98 and TA 100, 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. 5. Preparation of Overnight Cultures a. Inoculation 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. b. Harvest 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 109 cells 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 3 C. 6. Confirmation of Tester Strain Genotypes Tester strain cultures were checked for the following genetic markers on the day of their use in the mutagenicity assay: a. Salmonella typhimurium 1) rfa Wall Mutation 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 CHV Study No.: 17750-0-409R 12 000243 CO R N IN G Hazleton 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. 2) pKMIOl Plasmid R-factor The presence of the pKMIOl plasmid was confirmed for tester strains TA98 and TA100 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 10 pg of ampicillin was added. Resistance was demonstrated by bacterial growth in the zone immediately surrounding the disk. 3) Characteristic Number of Spontaneous Revertants The mean number of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 pi aliquots of the culture along with the appropriate vehicle on selective media. b. Escherichia coli 1) Characteristic Number of Spontaneous Revertants The mean number of spontaneous revertants per plate in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 pi aliquots of the WP2uvrA culture along with the appropriate vehicle on selective media. 7. Tester Strain Media a. 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). b. Agar Plates Bottom agar (25 ml per 15 x 100 mm petri dish) was VogelBonner minimal medium E (Vogel and Bonner, 1956), supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. CHV Study No.: 17750-0-409R 13 000244 C O R N IN G Hazleton c. 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. B. Liver Microsomal Enzyme Reaction Mixture (S9 Mix) 1. S9 Homogenate Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Annapolis, MD 21401, Batch 0642 (39.5 mg of protein per ml) and Batch 0646 (43.4 mg of protein per ml). The homogenate was prepared from male SpragueDawley 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!, 1975. 2. S9 Mix The S9 mix was prepared immediately prior to its use in any experimental procedure. The S9 mix contained the components indicated in Table II. TABLE II. S9 MIX COMPONENTS h 2o 0.70 ml 1M NaH2P04/Na2HP04, pH 7.4 0.10 ml 0.25M Glucose-6-phosphate 0.02 ml 0.10M NADP 0.04 ml 0.825M KC1/0.2M MgCl2 0.04 ml S9 Homogenate 0.10 ml 1.00 ml CHV Study No.: 17750-0-409R 14 000245 CORNING Hazleton C. Controls 1. 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 pi aliquot of vehicle (equal to the maximum aliquot of test article dilution plated), along with a 100 pi aliquot of the appropriate tester strain and a 500 pi aliquot of S9 mix (when necessary), on selective agar. 2. Positive Controls The combinations of positive controls, activation condition and tester strains plated concurrently with the assay are indicated in Table III. TABLE III. POSITIVE CONTROLS Tester Cone Strain S M x Positive Control per piate TA98 + 2-aminoanthracene 2.5 pg TA98 - 2-nitrofluorene 1.0 pg TA 100 + 2-aminoanthracene 2-5 pg TA 100 - sodium azide 2.0 pg TA 1535 + 2-aminoanthracene 2.5 pg TAI 535 - sodium azide 2.0 pg TA1537 + 2-aminoanthracene 2.5 pg TAI 537 - ICR-191 2.0 pg WP2uvrA + 2-aminoanthracene 25.0 pg WP2wvrA - 4-nitroquinoline-N-oxide 1.0 pg a. Source and Grade of Positive Control Articles 2-aminoanthracene (CAS #613-13-8), Sigma Chemical Co., purity ;>97.5%; 2-nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., purity 98%; sodium azide (CAS #26628-22-8), Sigma Chemical Co., purity >98%; ICR-191 (CAS #1707-45-0). Polysciences Inc., purity >95% and Sigma Chemical Co., purity 98%; 4-nitroquinoline-N-oxide (CAS #56-57-5), Sigma Chemical Co., purity >99%. CHV Study No.: 17750-0-409R 15 CO0246 C O R N IN G Hazleton 3. Sterility Controls a. Test Article The most concentrated test article dilution was checked for sterility by plating a 50 pi aliquot (the same volume used in the assay) on selective agar. b. S9 Mix The S9 mix was checked for sterility by plating 0.5 ml on selective agar. METHODS A. Dose Rangefinding Study 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. 1. D esig n The dose rangefinding study was performed using tester strains TA100 and WP2wvrA 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. a. 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 TA 100'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 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. CHV Study No.: 17750-0-409R 16 000247 CO R N IN G Hazleton 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. No Cytotoxicity Observed Since no cytotoxicity was observed in the dose rangefmding study, the highest dose level of test article used in the mutagenicity assay was the same as that tested in the rangefinding study. B. Mutagenicity Assay 1. Design The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2wvrA both in the presence and absence of S9 mix. Five doses of test article were tested along with the appropriate vehicle and positive controls. The doses of test article were selected based on the results of the dose rangefinding study. The results of the initial mutagenicity assay were confirmed in an independent experiment. 2. Frequency and Route of Administration The tester strains were 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 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 at 37 2C for 48 8 hr, revertant colonies were counted. All doses of the test article, the vehicle controls and the positive controls were plated in triplicate. C. Plating Procedures These procedures were used in both the dose rangefinding study and the mutagenicity assay. CHV Study No.: 17750-0-409R 17 000248 CO RN ING Hazleton 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 pi of tester strain and 50 pi of vehicle or test article dose was added to 2.5 ml of molten selective top agar (maintained at 45 2C). When S9 mix was required, 500 pi of S9 mix, 100 pi of tester strain and 50 pi of vehicle or test article dose was 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 48 8 hr at 37 2C. Positive control articles were plated using a 50 pi plating aliquot. D. 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. 1. Bacterial Background Lawn Evaluation The condition of the bacterial background lawn was evaluated for evidence 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 on the data tables using the code system presented at the end of the Materials and Methods Section. 2. 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 WP2uvrA in the absence of S9 mix, in Experiment 17750-B1, which were counted manually. E. Analysis of Data 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. CHV Study No.: 17750-0-409R 18 000249 C O R N IN G Hazleton EVALUATION OF TEST RESULTS Before assay data were evaluated, the criteria for a valid assay had to be met. A. Criteria For A Valid Assay The following criteria were 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 exhibited sensitivity to crystal violet. b. pKMIOl Plasmid To demonstrate the presence of the R-factor plasmid, pKMIOl, cultures of tester strains TA98 and TA100 exhibited resistance to ampicillin. c. Characteristic Number of Spontaneous Revertants To demonstrate the requirement for histidine, 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 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 exhibited a characteristic number of spontaneous revertants per plate when plated along CHV Study No.: 17750-0-409R 19 000250 C O R N IN G Hazleton with the vehicle under selective conditions. The acceptable range for the WP2wvrA mean vehicle controls was 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 were greater than or equal to 0.5 x 109 bacteria 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 109 bacteria per ml. 4. Positive Control Values a. 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. b. Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity) To demonstrate that the S9 mix was 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 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. 5. Cvtotoxicitv A minimum of three non-toxic doses were required to evaluate assay data. B. Criteria For A Positive Response Once the criteria for a valid assay had been met, responses observed in the assay were evaluated as follows: CHV Study No.: 17750-0-409R 20 000251 CO RN ING Hazleton 1. Tester Strains TA98. TA100. and WP2r/vrA 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. 2. 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. 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 Coming Hazleton Inc. 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 Coming Hazleton Inc. for an additional period of time or sent to a storage facility designated by the Sponsor. REFERENCES Ames, B.N., J. McCann, and E. Yamasaki. Methods for detecting carcinogens and mutagens with the Sa/mone/Za/Mammalian-Microsome Mutagenicity Test. Mutation Research 11:347-364 (1975). Brusick, D.J., V.F. Simmon, H.S. Rosenkranz, V.A. Ray, and R.S. Stafford. An evaluation of the Escherichia coli WP2 and WP2uvrA reverse mutation assay. Mutation Research 26:169-190 (1980). Green, M.H.L. and W.J. Muriel. Mutagen testing using trp+reversion in Escherichia coli. Mutation Research H:3-32. (1976). CHV Study No.: 17750-0-409R 21 000252 C O R N IN G Hazleton Maron, D.M., and B. Ames. Revised methods for the Salmonella Mutagenicity Test. Mutation Research 113:173-215 (1983). Vogel, H.J., and D.M. Bonner. Acetylomithinase of E. coli: Partial purification and some properties. J. Biol. Chem. 218:97-106 (1956). CHV Study No.: 17750-0-409R 22 000253 C O RN ING Hazleton BACTERIAL BACKGROUND LAWN EVALUATION CODE The condition of the background bacterial lawn is evaluated both macroscopicallv and microscopically (using a dissecting microscope) for indications of cytotox;:ity and test article precipitate as follows: CODE DEFINITION CHARACTERISTICS OF BACKGROUND LAWN 1 Normal A healthy microcolony lawn. 2 Slightly A noticeable thinning of the microcolony lawn and an Reduced increase in the size of the microcolonies compared to the vehicle control plate. 3 Moderately A marked thinning of the microcolony lawn and an Reduced increase in the size of the microcolonies compared to the vehicle control plate. 4 Extremely An extreme thinning of the microcolony lawn and an Reduced increase in the size of the microcolonies compared to the vehicle control plate. 5 Absent A complete lack of any microcolony lawn. 6 Obscured by The background bacterial lawn cannot be accurately Precipitate evaluated due to microscopic and/or macroscopic test article precipitate. Evidence of macroscopic test article precipitate on the plates is recorded by addition of the following precipitate code to the code number used to evaluate the condition of the background bacterial lawn. sp Slight Noticeable macroscopic precipitate on the plate, Precipitate however, the precipitate does not influence automated counting of the plate. mp Moderate Precipitate The amount of macroscopic precipitate on the plate would interfere with automated counting, thus requiring the plate to be hand counted. hp Heavy The large amount of macroscopic precipitate on the Precipitate plate makes the required hand counting difficult. Example: 4mp would indicate a plate observed to have an extremely reduced background lawn which had to be counted manually due to the marked amount of macroscopic test article precipitate. CHV Study No.: 17750-0-409R 23 000254 CO RN ING Hazleton SECTION IV. RESULTS AND CONCLUSIONS CHV Study No.: 17750-0-409R 24 000255 CORNING Hazleton RESULTS A. Test Article Handling The test article, T-6564, was stored at room temperature. Deionized water (CHV Lots 347, 349, and 352) was used as the vehicle. At 100 mg per ml, which was the most concentrated stock dilution prepared, the test article formed a clear, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay. B. Dose Rangefinding Study Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test article using tester strains TA100 and WP2wvrA in both the presence and absence of S9 mix (one plate per dose). Ten doses of test article, from 5,000 to 6.67 pg per plate, were tested and the results are presented in Tables 1 and 2. These data were generated in Experiment 17750-A1. No cytotoxicity was observed in either the presence or absence of S9 mix as evidenced by a normal background lawn and no decrease in the number of revertants per plate. C. Mutagenicity Assay The mutagenicity assay results for T-6564 are presented in Tables 3 through 7. These data were generated in Experiments 17750-B1, 17750-C1, and 17750-D1. The data are presented as mean revertants per plate standard deviation for each treatment and control group (Tables 4, 6, and 7) and as individual plate counts (Tables 3, 5, and 7). The results of the dose rangefinding study were used to select five doses to be tested in the mutagenicity assay. The doses tested were 5,000, 3,330, 1,000, 333, and 100 pg per plate in both the presence and absence of S9 mix. In the initial mutagenicity assay, Experiment 17750-B1 (Tables 3 and 4), and in the confirmatory assay, Experiment 17750-C1 (Tables 5 and 6), all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of S9 mix. In Experiment 17750-C1, a non-dose responsive 5.0-fold increase which did not meet the criteria for a positive response was observed with tester strain TA1537 in the absence of S9 mix. For this reason tester strain TA1537 was retested in the absence of S9 mix in Experiment 17750-D1. In Experiment 17750-D1 (Table 7), all data were acceptable and and no positive increases in the number of revertants per plate were observed with tester strain TA1537 in the absence of S9 mix. All criteria for a valid study were met. CHV Study No.: 17750-0-409R 25 000256 C O R N IN G Hazleton CONCLUSIONS The results of the Salmonella - Escherichia co///Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that, under the conditions of this study, 3M Corporation's test article, T-6564, did not cause a positive increase in the number of revenants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from AroclorTM-induced rat liver (S9). CHV Study No.: 17750-0-409R 26 000257 CO RN ING Hazleton SECTION V. DATA TABLES CHV Study No.: 17750-0-409R 27 000258 CO RN ING Hazleton TABLE 1 DOSE RANGEFINDING STUDY TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-A1 DATE PLATED: 12-Jun-96 VEHICLE: Deionized water DATE COUNTED: 18-Jun-96 (jg/PLATE 0.00 (Vehicle) (50 pi) Test Article 6.67 10.0 33.3 66.7 100 333 667 1000 3330 5000 TA100 REVERTANTS PER PLATE WITH S9 WITHOUT S9 REVERTANTS BACKGROUND REVERTANTS BACKGROUND PER LAWN PER LAWN PLATE EVALUATION* PLATE EVALUATION* 85 1 78 1 106 1 128 1 120 1 122 1 113 1 126 1 121 1 110 1 112 1 118 1 91 1 96 1 78 1 105 1 84 1 113 1 96 1 86 1 81 1 99 1 * Background Lawn Evaluation Codaa: 1 norial 2 4 m extremely reduced 5 P * alight precipitate P alightly caducad abaant moderata precipitata (raquiraa hand eount) 3 " moderately reduced 6 m obacurad by precipitate hp heavy precipitate (raquiraa hand count) CHV Study No.: 17750-0-409R 28 000259 1 \ C O R N IN G Hazleton TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-Al VEHICLE: Deionized water TABLE 2 DOSE RANGEFINDING STUDY DATE PLATED: DATE COUNTED: 12-Jun-96 18 -Jun-96 pg/PLATE 0.00 (Vehicle) (50 pi) Test Article 6.67 10.0 33.3 66.7 100 333 667 1000 3330 5000 WP2uvrA REVERTANTS PER PLATE WITH S9 . WITHOUT S9 REVERTANTS BACKGROUND REVERTANTS BACKGROUND PER LAWN PER LAWN PLATE EVALUATION* PLATE EVALUATION* 10 1 12 1 13 1 12 1 16 1 91 15 1 16 1 10 1 91 11 1 10 1 14 1 11 1 16 1 22 1 10 1 81 20 1 14 1 61 51 ;j * Background Lawn Evaluation Codaos 1 * normal 2 * alightly raducad 3 " moderately reduced 4 " axtraaaly raducad 3 abaant 6 " obacured by precipitate P " alight precipitata mp * moderate precipitate hp heavy precipitate (require hand count) (requirea hand count) CHV Study No.: 17750-0-409R 29 000260 1 C O R N IN G Hazleton TABLE 3 MUTAGENICITY ASSAY RESULTS INDIVIDUAL PLATE COUNTS TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-B1 DATE PLATED: 20-Jun-96 VEHICLE:: Deionized water DATE COUNTED: 28-Jun-96 PLATING ALIQUOT: 50 (jl REVERTANTS PER PLATE BACKGROUND LAWN* ,,gOPE/PfrAIE________ US!_________ I2_______ U \ m ________TAI_5-3_?_______ V?2wrA_______ 12 3 123 123 123 123 HICROSOMES: Rat Livac VEHICLE CONTROL 25 22 13 118 106 103 15 17 9 9 10 5 15 12 12 1 1 1 TEST ARTICLE 100 Mg 21 29 18 85 109 102 13 13 10 10 11 5 11 10 19 1 333 MS 16 27 25 101 104 96 8 11 15 562 20 24 9 1 S 1000 MS 27 16 29 106 100 101 10 12 13 8 5 12 14 14 14 1 3330 Mg 25 17 19 114 110 125 18 10 13 12 8 10 11 16 8 1 5000 Mg 23 25 25 117 99 109 12 12 10 775 13 13 5 1 POSITIVE CONTROL ** 860 852 970 936 892 810 141 134 124 240 202 190 314 279 270 1 MXCROSOKES: Nona VEHICLE CONTROL TEST ARTICLE 100 MS 333 Mg 1000 Mg 3330 Mg 5000 Mg POSITIVE CONTROL 12 12 12 14 10 13 13 11 4 16 11 12 15 10 2 11 11 13 105 99 86 91 74 85 81 104 83 101 95 86 97 98 92 108 93 75 106 108 104 470 419 460 14 10 6 547 11 9 5 10 13 5 11 10 7 12 10 6 16 14 12 67 1 666 663 451 9 54 460 427 400 1233 918 1110 15 14 10 8 7 13 12 12 11 8 12 9 12 8 11 13 13 9 135 102 85 1 1 1 1 1 1 1 TA98 2 -aainoanthracana 2.5 Mg/plata ** TA98 2-nitrofluorene 1.0 Mg/plata - TA100 2 -aainoanthracana 2.5 Mg/plata TA100 aodiua azide 2.0 Mg/plata j TA1535 2 -aainoanthracana 2.5 Mg/plata TA153 5 aodiua azide 2.0 Mg/plata TA1537 2-aainoanthracana 2.5 Mg/plata TA1537 ICR-191 2.0 Mg/plata VF2uvrA 2 -aainoanthracana 25.0 Mg/plate WP2uvrA 4-nitroquinoline-N-oxida 1.0 Mg/plata Background Lawn Evaluation 1 " noraial 2 " lightly raducad 3 " aodarataly raducad 4 extraaely roducad S * abaant 6 " obaeurad by precipitate ap " alight precipitata ap aodarata precipitata hp " heavy precipitate (require hand count) (require hand count) 11 CHV Study No.: 17750-0-409R 30 3 000261 i \ C O R N IN G Hazleton TABLE 4 MUTAGENICITY ASSAY RESULTS SUMMARY TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-B1 DATE PLATED: 20-Jun-96 VEHICLE: Deionized water DATE COUNTED: 28-Jun-96 PLATING ALIQUOT: 50 pi DOSE/PLATE MICROSOMES: Rat Livar VEHICLE CONTROL TA98 MEAN S.O. 20 6 KEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION TA100 MEAN s . o . ______TAX335____________TA1537______ MEAN S.O. MEAN S . D . 109 8 14 4 83 WP2uvrA MEAN S.O. BACKGROUND LAWN* 13 2 1 TEST ARTICLE 100 MS 333 MS 1000 MS 3330 MS 5000 MS POSITIVE CONTROL .. 23 6 23 6 24 7 20 k 2k 1 89 k 66 99 12 100 k 102 3 116 8 108 9 879 64 12 2 11 4 12 3 14 4 11 1 133 9 93 42 84 10 2 61 211 26 13 5 18 8 14 0 12 4 10 5 288 23 1 1 1 1 1 1 MICROSOMES: Nona VEHICLE CONTROL TEST ARTICLE 100 Hg 333 pg 1000 pg 3330 pg 5000 pg POSITIVE CONTROL *** 12 0 12 2 95 13 3 97 12 1 97 10 83 9 89 13 94 8 96 3 92 17 106 2 450 27 10 4 83 94 92 93 14 2 429 30 52 53 60 61 32 63 1087 159 13 3 93 12 1 10 2 10 2 12 2 107 25 1 1 1 1 1 1 1 TA98 2 -aainoanthracana 2.5 pg/plata TA100 2 -aainoanthracana 2.5 Hg/plata TA1535 2 -aainoanthracana 2-5 Hg/plata TA1537 2 -aainoanthracana 2.5 Hg/plata VF2uvrA 2-aainoanthracana 25.0 Hg/plata TA98 2-nitrofluorana TA100 aodiua acida TA1535 odiua asida TA1537 ICR-191 WP2 uvrA 4-nitroquinolina-N-ozida 1.0 Hg/plata 2-0 Hg/plata 2.0 Hg/plata 2.0 Hg/plata 1.0 Hg/plata * Background Lavn Evaluation Codas: 1 noraal 2 " alightly raducad 4 " axtraaaly raducad 5 abaant ap alight praeipitata ap " aodarata praeipitata (raquiraa hand count) 3 aodarataly raducad 6 m obaeurad by praeipitata hp haavy praeipitata (raquiraa hand count) CHV Study No.: 17750-0-409R 31 000262 C O R N IN G Hazleton TABLE S MUTAGENICITY ASSAY RESULTS INDIVIDUAL PLATE COUNTS TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-C1 DATE PLATED: 25-Jul-96 VEHICLE: Deionized water DATE COUNTED: 30-Jul-96, 31 -Jul-96 PLATING ALIQUOT: 50 Ml DOSE/PLATE____ KICROSOMES: Rat Livor VEHICLE CONTROL TA98 123 23 25 18 REVERTANTS PER PLATE __ TA100____ 123 TA1S35 123 TAI 537____ 123 BACKGROUND LAWN* vrA___________ 123 94 76 103 17 14 17 4 76 12 14 18 1 TEST ARTICLE POSITIVE CONTROL 100 Mg 333 Mg 1000 Mg 3330 MS 5000 Mg 21 21 28 27 33 25 33 26 29 24 30 27 25 33 25 769 624 815 89 99 95 102 105 76 87 115 108 106 96 114 107 87 89 949 1108 1066 8 11 9 16 16 9 12 10 8 9 18 12 8 6 17 146 196 136 11 4 11 8 6 19 7 7 10 844 8 78 89 129 91 14 16 10 10 15 9 24 13 14 24 16 13 21 14 20 151 90 108 1 1 1 1 1 1 MICROSOKES: Nona VEHICLE CONTROL TEST ARTICLE 100 Mg 333 Mg 1000 Mg 3330 Mg 5000 Mg POSITIVE CONTROL *** 13 18 15 16 17 17 21 8 16 20 9 8 21 15 11 10 17 17 207 208 172 79 89 70 71 105 74 89 74 71 78 75 99 80 76 73 88 78 77 548 597 595 16 9 19 16 17 17 14 9 8 99 8 15 14 10 16 13 16 644 619 663 142 65 66 16 16 10 8- 7 4 6 7 3 1 462 477 463 13 16 23 15 10 10 15 IS 19 6 13 19 13 9 6 9 17 10 187 207 160 1 1 1 1 1 1 1 TA98 2 'aainoanthraeene 2.5 pg/plate TA100 2 -aainoanthraeene 2.5 pg/plate TA1535 2-aainoanthraeene 2.5 pg/plate TA1537 2 -aainoanthraeene 2.5 yg/plate VP2uvrA 2 -aainoanthraeene 25.0 pg/plate ** TA98 2 -nitrofluorene TA100 eodiua acide TA1S35 aodiua acide TA1537 ICR-191 WP2uvrA 4 -nitroquinoline-N-oxide 1.0 pg/plate 2.0 pg/plate 2.0 Mg/plete 2.0 Mg/plate 1.0 yg/plete * Background Lavn Evaluation Codoa 1 * noraal 2 4 axtreaely reduced 5 P alight precipitate P lightly radueod abaant odorato precipitata (require hand count) 3 aodarately raducad 6 " obaeurad by precipitata hp haavy precipitate (require# hand count) CHV Study No.: 17750-0-409R 32 000263 ii C O R N IN G Hazleton TABLE 6 MUTAGENICITY ASSAY RESULTS SUMMARY TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-C1 DATE PLATED: 25-Jul-96 VEHICLE: Deionized water DATE COUNTED: 30-Jul-96. 31-Jul-96 PLATING ALIQUOT: 50 pi KEAN UVERTANTS PER PLATE VXTH STANDARD DEVIATION DOSE/PLATE ____ IASS______________TA100_____ _____ TA1335____________TA1337______ KEAN S.D. KEAN S.D. KEAN S.D. MEAN S.D. KICROSOKES: Rat Livar VEHICLE CONTROL 23 4 91 14 16 2 62 VP2uvrA MEAN S.D. BACKGROUND LAWN* 15 3 1 ii TEST ARTICLE 100 pg 23 4 94 5 92 94 13 3 1 < 333 V I 28 4 94 16 14 4 11 7 11 3 1 1000 pg 29 4 103 13 10 2 82 17 6 1 .1 3330 pg 27 3 103 9 13 5 32 18 6 1 3000 pg 28 5 94 11 10 6 81 18 4 1 POSITIVE CONTROL 736 100 1041 82 160 32 103 23 116 31 1 KICROSOKES: Nona VEHICLE CONTROL 15 3 79 10 13 3 22 17 5 1 TEST ARTICLE 100 MS 17 1 84 18 17 1 S1 12 3 1 333 HS 13 7 79 9 10 3 71 16 2 1 1000 Mg 12 7 78 9 91 53 13 7 1 3330 MS 16 5 77 2 13 3 10 7 94 1 3000 MS 15 4 83 12 15 2 5 4 12 4 1 .. .POSITIVE CONTROL 196 21 3B0 28 642 22 467 8 185 24 1 * TA98 2 -aainoanthracana 2.3 MS/pl*t* TA100 2 -aainoanthracana 2.5 M8/pl*t TA1535 2-aainoanthracana 2.5 Mf/plata TA1537 2 'aainoanthracana 2.5 Ms/?l*t+ VP2uvrA 2 -aainoanthracana 25.0 MS/pl** TA98 2 -nitrofluorcna TA100 odiua asida TA1535 aodiua asida TA1537 ICR-191 UP2uvrA 4-nitroquinolina-N-oxida 1.0 MS/pl*t* 2.0 Mg/plata 2.0 MS/pl*** 2.0 Mg/plata 1.0 MS/pl*c* * Background Lawn Evaluation Codaa: 1 normal 2 allghtly raducad 4 axtraaaly raducad 5 abaant ap alight pracipitata np " aodarata pracipitata (raquiraa hand count) 3 ** aodarataly raducad 6 obacurad by pracipitata hp m haavy pracipitata (raquiraa hand count) CHV Study No.: 17750-0-409R ! 33 000264 CO R N IN G Hazleton TABLE 7 MUTAGENICITY ASSAY RESULTS INDIVIDUAL PLATE COUNTS AND SUMMARY TEST ARTICLE ID: T-6564 EXPERIMENT ID: 17750-D1 DATE PLATED: 25-Aug-96 VEHICLE: Deionized water DATE COUNTED: 27-Aug-96 PLATING ALIQUOT: 50 pi REVERTANTS MEAW REVERTANTS PER PLATE PER PIATE VITE STANDARD DEVIATION -DQSE/PLATB___________ T 4M 7________________ TAI5 1 1 123 MEAN S.D. HICROSOMES: None VEHICLE CONTROL 10 2 9 74 TEST ARTICLE 100 pg 333 Mg 1000 pg 3330 pg 5000 pg 965 954 568 6 85 3 19 72 63 62 62 44 POSITIVE CONTROL *** 128 63 60 84 38 BACKGROUND LAWN* 1 1 1 1 1 1 1 ' TA1537 ICR*191 2.0 pg/plate ' Background Lawn Evaluation Codas : 1 " normal 2 slightly raducad 4 " extreeely raducad 5 absent ap slight precipitata mp moderata praeipltata (requires hand eount) 3 * moderately reduced 6 obscured by precipitate hp * heavy precipitate (requires hand eount) CHV Study No.: 17750-0-409R 34 000265