Document N2g1vm7yweaBejDjE7G4MRvbD

AfU26-0232 C O RNING Hazleton - '?>' 'VTv1 ^ **, -.V 7w,1 V f N, /' V -J . \ MUTAGENICITY TEST WITH T-6357 V "" J :M , / "<: IN THE SALMONELLA - ESCHERICHIA COI//MAMMALIAN-MICROSOME REVERSE MUTATION 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.: 17387-0-409 SUBMITTED TO 3M Building 220-2E-02 3M Center St. Paul, MN 55144-1000 STUDY COMPLETION DATE April 1,1996 CHV Study No.: 17387-0-409 000573 1 of 30 C O R N IN G Hazleton QUALITY ASSURANCE STATEMENT STUDY TITLE: Salmonella - Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay ASSAY NO.: 17387-0-409 PROTOCOL NO. : 409, Edition 4 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: Inspectipn/Pate FindingsReported Auditor Characterization of Tester Strains - 02/14/96 02/14/96 C. Orantes Draft Report Review - 03/16/96 03/18/96 S. Ballenger Final Report Review-04/01/96 04/01/96 S. Ballenger Quality Assurance Unit ( J CHV Study No.: 17387-0-409 000574 Date Released 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 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: Bacterial Mutagenesis Genetic and Cellular Toxicology Study Completion Date CHV Study No.: 17387-0-409 000575 3 TABLE OF CONTENTS CO RN 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.: 17387-0-409 G0G576 4 C O RN IN G Hazleton SECTION I. SUMMARY INTRODUCTION AND CONCLUSIONS CHV Study No.: 17387-0-409 000577 5 C O R N IN G Hazleton SUMMARY A. Introduction At the request of 3M, Coming Hazleton Inc. investigated T-6357 for mutagenic activity in the Salmonella-Escherichia co/i/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 rangefinding study using tester strains TA100 and WP2uvrA 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/i/Mammalian-Microsome Reverse Mutation Assay indicate that, under the conditions of this study, 3M's test article, T-6357, 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.: 17387-0-409 000578 6 C O R N IN G Hazleton SECTION II. STUDY INFORMATION CHV Study No.: 17387-0-409 000579 7 C O R N IN G Hazleton STUDY INFORMATION A. Sponsor: 3M B. Test Article: T-6357 1. Physical Description: clear amber liquid 2. Date Received: 01/16/96 C. Type of Assay: Salmonella - Escherichia co/i/Mammalian-Microsome Reverse Mutation Assay 1. Protocol Number: CHV Protocol 409, Edition 4 2. CHV Study Number: 17387-0-409 D. Study Dates 1. Study Initiation Date: 01/30/96 2. Experimental Start Date: 02/04/96 3. Experimental Termination Date: 02/21/96 E. Study Supervisory Personnel Study Director: Timothy E. Lawlor, M.A. Laboratory Supervisor: Michael S. Mecchi, B.S. CHV Study No.: 17387-0-409 000530 8 CO R N IN G Hazleton SECTION III. MATERIALS AND METHODS CHV Study No.: 17387-0-409 000581 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 L TESTER STRAIN GENOTYPES Histidine Mutation______ Additional Mutations hisG46 wC3076 hisD3052 LPS Repair R Factor TA1535 TA1537 rfa uvrB - TA100 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 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.: 17387-0-409 000582 10 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 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 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.: 17387-0-409 000533 11 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 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 3C. 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.: 17387-0-409 12 G584 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.: 17387-0-409 000585 13 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 fS9 Mix) 1. S9 Homogenate Liver microsomal enzymes (S9 homogenate) were purchased from Molecular Toxicology, Inc., Annapolis, MD 21401, Batch 0623 (42.4 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 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 H20 0.70 ml 1M NaH2P 0 4/Na2H P04, pH7.4 0.10 ml 0.25M Glucose-6-phosphate 0.02 ml 0.10MNADP 0.04 ml 0.825M KC1/0.2M MgCl2 0.04 ml S9 Homogenate 0.10 ml 1.00 ml 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 CHV Study No.: 17387-0-409 14 000586 C O R N IN G Hazleton 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. TAPEE HE POSITIVE CONTROLS Tester Cone Strain TA98 SSLMix Positive Control + 2-aminoanthracene per plate 2.5 pg TA98 - 2-nitrofluorene 1.0 pg TA100 + 2-aminoanthracene 2.5 pg TA100 - sodium azide 2.0 pg TA1535 + 2-aminoanthracene 2.5 pg TA1535 - sodium azide 2.0 pg TA1537 + 2-aminoanthracene 2.5 pg TA1537 - ICR-191 2.0 pg WP2mrA + 2-aminoanthracene 25.0 pg WP2vrA - 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%. 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. CHV Study No.: 17387-0-409 000587 15 C O R N IN G Hazleton agar. b. S9 Mix The S9 mix was checked for sterility by plating 0.5 ml on selective 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. Design ' 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 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 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. 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. CHV Study No.: 17387-0-409 GGS88 16 C O R N IN G Hazleton 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 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. 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. 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 CHV Study No.: 17387-0-409 000589 17 C O R N IN G Hazleton 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. A. Criteria For A Valid Assay The following criteria were used to determine a valid assay: CHV Study No.: 17387-0-409 000590 18 C O R N IN G Hazleton 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 TA1535 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 the WP2uvrA 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 109bacteria per ml. CHV Study No.: 17387-0-409 000531 19 C O R N IN G Hazleton 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 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: 1. Tester Strains TA98 .TA100. and WP2mvM. 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.: 17387-0-409 000592 20 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 Sa/woe//a/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 WP2wvrA reverse mutation assay. Mutation Research 76:169-190 (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.: 17387-0-409 000593 21 C O R N IN G Hazleton BACTERIAL BACKGROUND LAWN EVALUATION COPE 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 Reduced A marked thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the vehicle control plate. 4 Extremely Reduced An extreme thinning of the microcolony lawn and an 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 Precipitate The background bacterial lawn cannot be accurately 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 Precipitate Noticeable macroscopic precipitate on the plate, 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.: 17387-0-409 G0Gt94 22 C O R N IN G Hazleton SECTION IV. RESULTS AND CONCLUSIONS CHV Study No.: 17387-0-409 000595 23 C O R N IN G Hazleton RESULTS A. Test Article Handling The test article, T-6357, was stored at room temperature. Deionized water (CHV lots 336 and 337) 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 17387-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-6357 are presented in Tables 3 and 4. These data were generated in Experiment 17387-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 17387-B1 (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.: 17387-0-409 000596 24 CO R N IN G Hazleton CONCLUSIONS The results of the Salmonella - Escherichia co///Mammalian-Microsome Reverse Mutation Assay indicate that, under the conditions of this study, 3M's test article, T-6357, 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.: 17387-0-409 000597 25 C O R N IN G Hazleton SECTION V. DATA TABLES CHV Study No.: 17387-0-409 26 C O R N IN G Hazleton TABLE 1 DOSE RANGEFINDING STUDY TEST ARTICLE IDs T-6357 EXPERIMENT ID: 17387-A1 DATE PLATED : 04-Feb-96 VEHICLE: Deionized water DATE COUNTED : 07-Feb-96 pg/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* 97 1 102 1 125 1 87 1 112 1 108 1 103 1 82 1 125 1 136 1 114 1 135 1 85 1 115 1 87 1 88 1 98 1 102 1 86 1 93 1 120 1 105 1 * Background Lawn Evaluation Codaa: 1 ** no m a i 2 "* slightly reduced 4 m extremely reduced 5 m absent sp " slight precipitate np m moderate precipitate (requires hand count) 3 m moderately reduced 6 m obscured by precipitate hp * heavy precipitate (requires hand count) CHV Study No.: 17387-0-409 000599 27 C O R N IN G Hazleton TABLE 2 DOSE RANGEFINDING STUDY TEST ARTICLE ID: T-6357 EXPERIMENT ID: 17387-A1 DATE PLATED: 04-Feb-96 VEHICLE: Deionized water DATE COUNTED: 07-Feb-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* 14 1 11 1 13 1 26 1 24 1 21 1 16 1 20 1 11 1 21 1 19 1 16 1 22 1 19 1 13 1 13 1 20 1 22 1 13 1 11 1 18 1 13 1 Background Lawn Evaluation Codes: 1 m normal 2 " slightly reduced 4 M axtraaaly reduced 5 m absent sp " slight precipitate ap m moderate precipitate (requires hand count) 3 TM moderately reduced 6 " obscured by precipitate hp " heavy precipitate (requires hand count) CHV Study No.: 17387-0-409 000600 28 CO RNING Hazleton TABLE 3 MUTAGENICITY ASSAY RESULTS INDIVIDUAL PLATE COUNTS TEST ARTICLE ID: T-6357 EXPERIMENT ID: 17387-B1 DATE PLATED: 14-Feb-96 VEHICLE: Deionized water DATE COUNTED: 21-Feb-96 PLATING ALIQUOT: 50 Ml KEVERTANTS PER PLATE DOSE/PLATE MICROSOMES: Rat VEHICLE CONTROL u 9 > .HJ TA98 123 29 36 23 TA100 123 130 140 131 TA1535 123 10 9 9 TA1537 i23 478 TEST ARTICLE POSITIVE CONTROL 100 MS 333 MS 1000 Mg 3330 Mg 5000 Mg 30 32 24 26 37 16 15 16 17 26 27 18 19 24 24 135 143 149 113 99 127 122 119 138 134 146 132 139 129 117 1086 1106 1231 1400 1443 1450 12 14 12 12 16 14 11 23 11 12 16 14 10 11 14 227 206 348 10 9 3 10 10 3 5 10 44 8 9 7 2 9 134 101 130 BACKGROUND LAWN* WP2uvrA 123 11 17 6 1 15 7 14 13 13 14 14 11 13 10 20 19 16 15 21 1 1 1 1 1 393 344 372 1 MICROSOMES: None VEHICLE CONTROL TEST ARTICLE 100 Hg 333 Hg 1000 Hg 3330 Hg 5000 Hg POSITIVE CONTROL *** 20 17 8 11 12 9 15 12 11 14 18 12 6 17 9 9 20 11 158 151 162 84 114 79 93 106 91 90 101 77 77 85 84 93 84 80 98 98 86 726 734 779 5 12 11 14 12 9 8 8 10 7 11 13 13 16 10 13 15 10 646 599 701 648 24 79 3 11 35 47 5 5 6 4 5 463 553 742 13 14 12 20 10 14 12 6 12 14 11 9 14 12 13 17 13 7 124 153 119 1 1 1 1 1 1 1 TA96 2 -aainoanthracene 2.5 Hg/plata TA100 2 -aainoanthracene 2.5 Hg/plata TA15 35 2~aainoanthracene 2.5 Hg/plata TA1537 2 -aainoanthracene 2.5 Hg/plata WP2uvrA 2 -aainoanthracene 25.0 Hg/plata TA98 2-nitrofluorene TA100 sodiua azide TA1535 sodium azide TA1537 ICR-191 WP2uvrA 4-nitroquinoline-N-oxide 1.0 pg/plate 2.0 pg/plate 2.0 (jg/plate 2.0 Hg/plata 1.0 Hg/plata Background Lavn Evaluation Codes : 1 ** noraal 2 " slightly reduced 4 ** extreaely reduced 5 m absent sp slight precipitate ap * soderete precipitate (requires hand count) 3 " moderately reduced 6 m obscured by precipitate hp " heavy precipitate (requires hand count) CHV Study No.: 17387-0-409 000801 29 C O R N IN G Hazleton TABLE 4 MUTAGENICITY ASSAY RESULTS SUMMARY TEST ARTICLE ID: T-6357 EXPERIMENT ID: 17387-B1 DATE PLATED: 14-Feb-96 VEHICLE: Deionized water DATE COUNTED: 21-Feb-96 PLATING ALIQUOT: 50 pl MEAN RBVERTANTS PER PLATE WITH STANDARD DEVIATION DOSE/PLATE MICROSOMES: Rat !Liver VEHICLE CONTROL TA9B MEAN S.D. 29 7 ______ TA100_____ _____ TA1535_____ _____ TA1537_____ MEAN S.D. MEAN S.D. MEAN S.D. 134 6 91 62 TEST ARTICLE 100 pg 333 Mg 1000 pg 3330 pg 5000 pg 29 4 26 11 16 1 24 5 22 3 POSITIVE CONTROL .. 1141 79 142 7 113 14 126 10 137 8 128 il 1431 27 13 1 14 2 15 7 14 2 12 2 260 77 91 74 74 64 63 122 18 WP2uvrA MEAN S.D. BACKGROUND LAWN* 11 6 1 12 4 13 1 13 2 16 6 17 3 1 1 1 1 1 370 25 1 MICROSOMES: None VEHICLE CONTROL 15 6 TEST ARTICLE 100 pg 333 pg 1000 pg 3330 pg 5000 pg 11 2 13 2 15 3 11 6 13 6 POSITIVE CONTROL *** 157 6 92 19 94 11 87 6 92 13 87 11 88 5 746 29 94 12 3 91 10 3 13 3 13 3 649 51 62 42 72 74 41 52 586 142 13 1 15 5 10 3 11 3 13 1 12 5 132 18 1 1 1 1 1 1 1 TA98 2-aminoanthracene 2.5 TA100 2-aminoanthracene 2.5 pg/plata TA1535 2-aminoanthracene 2.5 pg/plate TA1537 2 -aminoanthracene 2.5 pg/plate WP2uvrA 2-aminoanthracene 25.0 pg/plata TA98 2-nitrofluorene TA100 sodium azide TAI535 sodium azide TA1537 ICR-191 WP2uvrA 4-nitroquinoline-N-oxide 1.0 pg/plate 2.0 pg/plate 2.0 Mg/plate 2.0 pg/plate 1.0 Mg/plate Background Lavn Evaluation Codes : 1 m normal 2 TM slightly reduced 4 " extremely reduced 5 ** absent sp " slight precipitate mp " moderate precipitate (requires hand count) 3 " moderately reduced 6 " obscured by precipitate hp m heavy precipitate (requires hand count) CHV Study No.: 17387-0-409 30 000602