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AR0U'OM3 C O R N IN G Hazleton MUTAGENICITY TEST WITH // ^ '< A \ T-6342 i \* IN THE SALMONELLA - ESCHERICHIA COI//MAMMALIAN-MICRSOME REVERSE MUTATION ASSAY ir-- - ^ 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.: 17073-0-409 SUBMITTED TO 3M Corporation Building 220-2E-02 3M Center St. Paul, MN 55144-1000 STUDY COMPLETION DATE December 14,1995 000353 CHV Study No.: 17073-0-409 1 of 30 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT STUDY TITLE: Salmonella - Escherichia coli/ Mammalian-Microsome Reverse Mutation Assay ASSAY NO.: 17073-0-409 PROTOCOL NO.: 409, Edition 4, Modified for 3M Corporation 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 Plating - 09/19/95 Findings Reported 09/19/95 Auditor M. Murphy Draft Report Review -11/06/95 11/07/95 C. Orantes/ S. Ballenger Final Report Review - 12/14/95 12/14/95 C. Orantes Quality Assurance Unit CHV Study No.: 17073-0-409 000354 2 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 in the Food and Drug Administration (FDA) Title 21 of the U.S. Code of Federal Regulations Part 58, issued December 22, 1978, (effective June 20, 1979) 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. Study Director: Genetic and Cellular Toxicology Study Completion Date CHV Study No.: 17073-0-409 000355 3 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 . . ! ............................................................................23 V. DATA TABLES ..............................................................................................................26 CHV Study No.: 17073-0-409 C0035S 4 C O R N IN G Hazleton SECTION I. SUMMARY INTRODUCTION AND CONCLUSIONS CHV Study No.: 17073-0-409 000357 5 C O R N IN G Hazleton SUMMARY A. Introduction At the request of 3M Corporation, Corning Hazleton Inc. investigated T-6342 for mutagenic activity in the Salmonella-Escherichia co/f'/Mammalian-Microsome Reverse Mutation 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 rangefmding 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 WP2vrA. 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. B. Conclusions The results of the Salmonella - Escherichia co///Mammalian-Microsome Reverse Mutation Assay indicate that, under the conditions of this study, 3M Corporation's test article, T-6342, 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.: 17073-0-409 000358 6 C O R N IN G Hazleton SECTION II. STUDY INFORMATION CHV Study No.: 17073-0-409 000359 7 C O R N IN G Hazleton STUDY INFORMATION A. Sponsor: 3M Corporation B. Test Article: T-6342 1. Physical Description: clear colorless liquid 2. Date Received: 07/21/95 C. Type of Assay: Salmonella-Escherichia co/Z/Mammalian-Microsome Reverse Mutation Assay 1. Protocol Number: CHV Protocol 409, Edition 4 Modified for 3M Corporation 2. CHV Study Number: 17073-0-409 D. Study Dates 1. Study Initiation Date: 08/28/95 2. Experimental Start Date: 09/12/95 3. Experimental Termination Date: 09/22/95 E. Study Supervisory Personnel Study Director: Timothy E. Lawlor, M.A. Laboratory Supervisor: Michael S. Mecchi, B.S. CHV Study No.: 17073-0-409 00036 8 C O R N IN G Hazleton SECTION III. MATERIALS AND METHODS CHV Study No.: 17073-0-409 000361 9 C O R N IN G Hazleton MATERIALS AND METHODS The experimental materials, methods and procedures are based on those described by Ames et al (1975) and Green and Muriel (1976). MATERIALS A. Tester Strains 1. 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 specific genotypes of these strains are shown in Table I. TABLE I- TESTER STRAIN QIVQTYPS Histidine Mutation______ Additional Mutations hisG46 /JWC3076 wD3052 LPS Repair R Factor TA 1535 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 wvrB 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 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. CHV Study No.: 17073-0-409 10 000362 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 substitutions. 2. Escherichia coli The tester strain used was the tryptophan auxotroph WP2vrA 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 WP2nvrA 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, WP2vrA, 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 z- 70C. CHV Study No.: 17073-0-409 11 000363 C O 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 TA 98 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. 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 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 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.: 17073-0-409 12 C003S4 C O 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) pKMl 01 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 WP2wvrA 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.: 17073-0-409 13 00365 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 Enzvme Reaction Mixture iS9 Mixl 1. $9 Homogenate Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Annapolis, MD 21401, Batch 0568 (38.8 mg of protein per ml) and Batch 0569 (38.8 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 al, 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 NaH2P 04/Na2HP04, pH7.4 0.10 ml 0.25M Glucose-6-phosphate 0.02 ml 0.10M NADP 0.04 ml 0.825M KC1/0.2M MgCI2 0.04 ml S9 Homogenate Q-JOml. 1.00 ml CHV Study No.: 17073-0-409 14 0003S6 C O R N IN G 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 TA98 S9 Mix Positive Control + 2-aminoanthracene palpiate 2-5 pg TA98 - 2-nitrofluorene 1.0 pg TA 100 + 2-aminoanthracene 2.5 pg TA 100 - sodium azide 2.0 pg TA1535 2-aminoanthracene 2.5 pg TA1535 - sodium azide 2.0 pg TA 1537 + 2-aminoanthracene 2.5 pg TAI 537 - ICR-191 2.0 pg WP2vrA + 2-aminoanthracene 25.0 pg WP2uvrA - 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%; 4-nitroquinoline-N-oxide (CAS #56-57-5), Sigma Chemical Co., purity >99%. CHV Study No.: 17073-0-409 15 000367 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 inhibitoiy 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. Design The dose rangefinding study was performed using tester strains TA100 and WP2uvrA 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 WP2uvrA 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.: 17073-0-409 16 000368 C O R N IN G Hazleton 2. Evaluation of the Dose Ranpefinding 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 rangefinding study, the highest dose 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 WP2vrA 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. 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. Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition and dose. The S9 mix and dilutions of the test article were prepared immediately prior to their use. CHV Study No.: 17073-0-409 17 000369 C O R N IN G Hazleton 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 3 C 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 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. 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. EVALUATION OF TEST RESULTS Before assay data were evaluated, the criteria for a valid assay had to be met. CHV Study No.: 17073-0-409 18 0003*70 C O R N IN G Hazleton 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 vehicle controls were as follows: TA98 8 - 60 TA100 60 - 240 TA 1535 4 - 45 TA1537 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 with the vehicle under selective conditions. The acceptable range for WP2wvrA 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 109bacteria per ml and/or had reached CHV Study No.: 17073-0-409 19 000373- C O R N IN G Hazleton a target level of turbidity demonstrated to produce cultures with a density greater than or equal to 0.5 x 109bacteria per ml. 4. Positive?Control V riw g 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 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. 2. 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: 1. Tester Strains TA98 .TA100. and WP2nvrA 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. CHV Study No.: 17073-0-409 20 000372 C O R N IN G Hazleton 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 Stf/moHe/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 WP2vrA reverse mutation assay. Mutation Research 7^:169-190 (1980). Green, M.H.L. and W. J. Muriel. Mutagen testing using trp+reversion in Escherichia coli. Mutation Research 38:3-32 (1976). Maron, D.M., and B. Ames. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research H l : 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.: 17073-0-409 21 000373 C O R N IN G Hazleton BACTERIAL BACKGROUND LAWN EVALUATION CODE The condition of the background bacterial lawn is evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate as follows: CODE DEFINITION CHARACTERISTICS OF BACKGROUND LAWN 1 Normal A healthy microcolony lawn. 2 Slightly Reduced A noticeable thinning of the microcolony lawn and an 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 Precipitate The large amount of macroscopic precipitate on the 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.: 17073-0-409 22 000374 C O R N IN G Hazleton SECTION IV. RESULTS AND CONCLUSIONS CHV Study No.: 17073-0-409 23 00375 C O R N IN G Hazleton RESULTS A. T<?$t ArticleJiandlmg The test article, T-6342, was stored at room temperature. Deionized water (CHV Lot 326) 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 WP2vrA 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 17073-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-6342 are presented in Tables 3 and 4. These data were generated in Experiment 17073-B1. The data are presented as mean revertants per plate standard deviation for each treatment and control group (Table 4 ) and as individual plate counts (Table 3). 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 Experiment 17073-B 1 (Tables 3 and 4), 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. All criteria for a valid study were met. CHV Study No.: 17073-0-409 24 000376 C O R N IN G Hazleton CONCLUSIONS The results of the Salmonella-Escherichia co/z/Mammalian-Microsome Reverse Mutation Assay indicate that, under the conditions of this study, 3M Corporation's test article, T-6342, 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.: 17073-0-409 25 000377 C O R N IN G Hazleton SECTION V. DATA TABLES CHV Study No.: 17073-0-409 26 000378 C O R N IN G Hazleton TABLE 1 DOSE RANGEFINDING STUDY TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-A1 DATE PLATED: 12-Sep-95 VEHICLE: Deionized water DATE COUNTED: 15-Sep-95 pg/PLATE 0.00 (Vehicle) (50 pi) Test Article 6.67 10.0 33.3 66.7 100 333 667 1000 3330 5000 TAI00 REVERTANTS PER PLATE WITH S9 _________WITHOUT S9_________ REVERTANTS BACKGROUND REVERTANTS BACKGROUND PER LAWN PER LAWN PLATE EVALUATION* PLATE EVALUATION* 127 1 103 1 100 1 85 1 120 1 112 1 138 107 1 119 1 111 1 118 1 93 1 87 1 81 1 77 1 105 1 98 1 113 1 97 1 79 1 107 1 92 1 * Background Lavn Evaluation Codaa: 1 " normal 4 extremely raducad 2 " lightly raducad 5 m ahaant P alight precipitata P " aoderata precipitata (raquiraa hand eount) 3 TM aodarataly reduced 6 " obacurad by precipitate hp m heavy precipitate (raquiraa hand eount) CHV Study No.: 17073-0-409 27 000379 C O R N IN G Hazleton TABLE 2 DOSE RANGEEINDING STUDY TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-A1 DATE PLATED: 12-Sep-95 VEHICLE: Deionized water DATE COUNTED: 15-Sep-95 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* 16 1 24 1 19 1 11 1 12 1 23 1 20 1 81 14 1 20 1 18 1 20 1 11 1 16 1 11 1 12 1 24 1 23 1 15 1 19 1 21 1 18 1 Background Lawn Evaluation Cod: 1 " norKal k " axtraaaly raducad p m alight praeipitata 2 lightly raducad 5 * abaant ap * aodarata pracipitata (raquiraa hand count) 3 " aodarataly raducad 6 " obacurad by pracipitata hp " haavy pracipitata (raquira hand count) CHV Study No.: 17073-0-409 28 G003S0 C O R N IN G Hazleton TABLE 3 MUTAGENICITY ASSAY RESULTS INDIVIDUAL PLATE COUNTS TEST ARTICLE ID: T-6342 EXPERIMENT ID: 17073-B1 DATE PLATED: 19-Sep-95 VEHICLE: Deionized water DATE COUNTED: 22-Sep-95 PLATING ALIQUOT: 50 Ml DOSE/FLATE MICROSOMES: Rat Livar VEHICLE CONTROL TA98 123 REVERTANTS PER PLATE TA100___ _______TA1535__________ TA1337____ 123 123 12 3 BACKGROUND LAWN- _WP2uvrA 123 il 25 24 117 115 118 13 15 7 10 9 7 20 19 19 1 TEST ARTICLE 100 MS 333 MS 1000 PS 3330 PB 5000 MB 30 36 38 30 27 28 29 34 26 38 31 34 23 19 18 109 115 112 121 114 118 118 114 111 128 123 118 119 108 126 16 13 11 8 il 14 13 17 9 15 14 11 15 10 12 6 12 8 7 5 10 10 3 9 10 11 5 10 7 6 30 14 17 15 21 15 12 14 13 15 16 16 20 29 24 1 1 1 1 1 POSITIVE CONTROL 848 995 810 926 953 889 110 108 122 107 122 108 201 171 163 1 MICROSOMES: Nona VEHICLE CONTROL TEST ARTICLE POSITIVE CONTROL 14 12 13 90 86 86 756 78 1 100 pg 333 pg 1000 pg 3330 h B 5000 pg 19 19 10 11 21 17 12 19 14 10 16 15 20 17 9 97 88 107 103 107 100 104 91 97 82 97 98 92 72 88 585 13 13 19 8 9 17 669 9 16 9 37 7 10 7 13 5 10 4 46 4 12 9 6 129 154 156 841 817 861 582 599 S56 202 224 218 10 15 18 1 26 11 15 18 15 17 27 18 24 9 22 19 10 16 17 1 1 1 1 1 39 45 76 1 TA98 TA100 TA1535 TA1537 WP2uvrA 2-aalnoanthracana 2.5 pg/plata 2-aalnoanthracana 2.5 pg/plata 2-aalnoanthracana 2.5 pg/plata 2 -aalnoanthracana 2.5 pg/plata 2-aalnoanthracana 25.0 pg/plata TA98 2-nitrofluorana TA100 aodiua asida TA1535 aodiua asida TA1537 ICR-191 WP2uvrA 4-nitroquinolina-N-oxida 1.0 pg/plata 2.0 pg/plata 2.0 pg/plata 2.0 pg/plata 1.0 pg/plata - Background Lavn Evaluation Codaa: 1 " noraal 4 " axtraaaly raduead ap alight pracipitata 2 * lightly raduead 5 m abaant ap m aodarata pracipitata (raquirai hand count) 3 " aodarataly raduead 6 " obacurad by praeipitata hp " haavy pracipitata (raquiraa hand count) CHV Study No.: 17073-0-409 29 003S1 C O R N IN G Hazleton TABLE 4t MUTAGENICITY ASSAY RESULTS SUMMARY TEST ARTICLE ID T-6342 EXPERIMENT ID 17073-B1 DATE PLATED 19-Sep-95 VEHICLE: Deionized water DATE COUNTED 22-Sep-95 PLATING ALIQUOT: 50 pi MEAN REVERTANTS PER PLATE WITH STANDARD DEVIATION DOSE/PLATE ____ IA18_____________ TA100_____ _____ TA1335_____ MEAN S.D. MEAN S.D. MEAN S.D. MICROSOMES: Rat Livar VEHICLE CONTROL 20 8 117 2 12 4 TA1537 MEAN S.D. 92 TEST ARTICLE IOO pg 333 pg 1000 pg 3330 pg 5000 pg 35 4 28 2 30 4 34 4 20 3 112 3 118 4 114 4 123 5 118 9 13 3 11 3 13 4 13 2 12 3 93 73 74 93 82 POSITIVE CONTROL ** 884 98 923 32 113 8 112 8 WP2uvrA MEAN S.D. BACKGROUND LAWN* 19 1 1 20 9 17 3 13 1 16 1 24 5 1 1 1 1 1 178 20 1 MICROSOMES: Nona VEHICLE CONTROL 13 1 TEST ARTICLE 100 pg 333 pg 1000 pg 3330 pg 5000 pg 16 5 16 5 15 4 14 3 15 6 POSITIVE CONTROL 146 15 87 2 97 10 103 4 97 7 92 9 84 11 840 22 61 62 15 3 11 5 72 11 4 579 22 54 62 10 3 63 51 93 215 11 14 4 17 8 17 2 23 5 17 7 14 4 53 20 1 1 1 1 1 1 1 TA98 TA100 TA1535 TA1537 WF2UVXA 2-aainoanthracana 2.5 pg/plata 2-aainoanthracana 2.5 pg/plata 2-aainoanthracana 2.5 pg/plata 2-aainoanthracana 2.5 pg/plata 2-aainoanthracana 25.0 pg/plata TA98 2-nitrofluorane TA100 aodiua asida TA1535 aodiua asida TA1537 ICR-191 WP2uvrA 4-nltroquinolina-N-oxlda 1.0 pg/plata 2.0 pg/plata 2.0 pg/plata 2.0 pg/plata 1.0 pg/plata * Background Lawn Evaluation Codaa: 1 * noraal 2 " slightly raduead 4 " axtraaaly raduead 5 " abaant P " alight pracipitata ap " aodarata pracipitata (raqulraJi hand count) 3 " aodarataly raduead 6 * obacurad by pracipitata hp * haavy pracipitata (raquiraa hand count) CHV Study No.: 17073-0-409 30 G00332