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IN VITRO MICROBIOLOGICAL MUTAGENICITY OF 3M COMPANY'S COMPOUND T-3727 ASSAYS Final Report arch 1985 m By: Kathleen Moto, Midr6biologist Microbial Genetics Department and Edward S. Ricelo, Assistant Director Microbial Genetics Department Prepared for: 3M Company Medical Department General Offices, 3M Center St. Paul, MN 55144 Attention: Dallas D. Zimmerman Toxicology Specialist SRI Project LSO-3145 Approved by: Kristien E. Mortelmans, Study Director Microbial Gen tsoDrevpartm,ent Get' icrobia Director oxicolo )n B Reidabor J n B. Re1id, Director oxicology Laboratory W. A. Skinner, Vice President Life Sciences Division lnt7enmaUtionLal 333 Ravenswood Ave. o Menlo Park,CA 94025 (415)326-6200 0 TWX: 910-373-2046 o Telex:334-486 SUMMARY SRI International exELmined 3M Company's Compound T-3727 for mutagenic activity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and TA100 of the bacterium Salmonella typhimurium. The assay was performed in the presence and absence of a rat-liver metabolic activation system. All tests were performed in compliance with the United States Food and Drug Administration Good Laboratory Practice Standards. Compound T-3727 was reproducibly to these procedures. nonmutagenic when tested according CONTENTS SUMMARY .....................................i.i..... QUALITY ASSURANCE STATEMENT .....................i.v. INTRODUCTION ..................................1.... MATERIALS .......................0 ........... 3 METHODS .........o .......................... 5 RESULTS AND DISCUSSION .............0.............. 10 TABLES Table 1 .................................11..... Table 2..................................12.. QUALITY ASSURANCE UNIT Final Report Statement SRI Internationalassures the quality and Integrityof this study, Ln-Vitro MicrobiologicalMutagenicity Assays of Compound T-3727, for the 3M Company. The study was inspected on March 12, 1985 during the colony counting phase. The findings of the Quality Assurance Unit inspectionwere reported at the time of the inspectionto the Study Director. SRI management was informed of the inspection results on March 12, 1985. A data audit was performed on March 25, 1985. The Study Director and SRI management were informed of the audit results on March 25, 1985. The final report was audited and reviewed on March 25, 1985. The results of the final report review were communicatedto the Study Director and SRI management on March 25, 1985. The final report accurately describes the methods and standard operating proceduresand reflects the raw data of the study. Any deviations from the approved protocol and standard operating procedures were made with proper authorizationand docu ntation. -7QuaIlty Ar erDirector rt/ Date iv INTRODUCTION SRI International examined 3M Company's Compound T-3727 for mutagenicity in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98, and TAIOO of the bacterium Salmonella typhimurium. An Aroclor 1254-stimulated, rat-liver homogenate metabolic activation system was included in the assay procedure to provide metabolic steps that the microorganisms either are incapable of conducting or do not carry out under the assay conditions. The assay procedure with S. typhimurium has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has about the same reliability in identifying chemicals that are not carcinogenic. However, because the assay systems do not always provide 100% correlatioa with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a chemical is carcinogenic or noncarcinogenic to man. Evaluation of experimental results from the Salmonella assay consists of comparing the number of bistidine-independent colonies on the treated agar plates with the number observed on the control plates. Because all the plated Salmonella indicator organisms undergo a few cell divisions in the presence of the test chemical, the test is semiquantitative in nature. The plate test procedure does not permit quantitative determination of the number of cells surviving the chemical treatment. It is the demonstration of a mutagenic dose-response relationship that is important in establishing mutagenicity. The test chemicals are assayed at several dose levels within a nontoxic dose range--with the exception of the highest dose level, which sometimes.exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial lawn growth, formation of pinpoint colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chemical is considered a mutagen in the Salmonella assay if it elicits a reproducible, dose-related increase in the number of histidine revertants per plate in one or more tester strains. The assays with Compound T-3727 were begun on 20 February 1985 and testing was completed on 27 February 1985. Copies of the final report will be kept in our files (Building M, Room 213) and in SRI's Records Center. The raw data will be retained In Building 205, Room 13, for one year after the laboratory notebook has been filled and then will be stored in SRI's Records Center. All that remains of Compound T-3727 will be kept for six months in our chemical storage room (Building M, Room 217) and then returned to 3M Company. 2 MATERIALS o Test Article - Name: T-3727 - Date Received: 19 February 1985 - Description: Light-amber waxy solid - Storage Conditions: Stored at room temperature in a secondary container - Special Testing Conditions: None - Stability: Assured by Sponsor o Indicator Organisms - Species - Strains: - Source: Salmonella typhimurium LT2 TA1535, TA1537, TA1538, TA98, and TA100 for typhimurium Dr. Bruce Ames, University of California, Berkeley a Metabolic Activation Aroclor 1254-induced, rat liver S-9; SRI Batch F-4; 26.5 mg/ml protein 0 Negative (Solvent) Control Material Acetone, CAS No. 67-64-1 Date Opened: 14 December 1984 Expiration Date: 14 December 1985 Manufacturer: American Scientific Products, McGraw Park, IL Purity: 99.7% Lot No.: KTEA 3 o Positive Control ChemieRls 9-Aminoacridine, CAS No. 90-45-9 Manufacturer: Pfaltz and Bauer, Stamford, CT 2-Anthramiue, CAS No. 613-13-8 Manufacturer: Sigma Chemical Co., St. Louis, MO 2-Nitrofluorene,'CAS No. 607-57-8 Manufacturer: Aldrich Chemical Co..,Milwaukee, WI Sodium Azide, CAS No. 26628-22-8 Manufacturer: Difco Laboratories, Detroit, MI o Counters Used - New Brunswick Scientific BioTran 1110Automated Colony Counter, Model Clll, SRI No. 0030 6126 00 - New Brunswick Scientific BactronicO Colony Counter, Model C110, SRI No. 0012 3108 00 4 METHODS Salmonella typhimurium Strains TA1535, TA1537, TA1538, TA98, and TA100 The Salmonella typhimurium strains used at SRI are all histidine auxotrophe by virtue of mutations in the histidine operon. When these histidine-dependent calls are grown on minimal medium agar plates containing a trace of histidine, only those cells that revert to histidine independence (his+) are able to form colonies, The small amount of histidine allows all the plated bacteria to undergo a few divisions; in many cases, this growth is essential for autag"enesisto occur. The his+ revertants are easily visible as colonies against the slight background growth. The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar, the mutation frequency is increased, usually-in a dose-related manner. We obtained our S. typhimurium strains from Dr. Bruce Ames of the University+of California at Berkeley. In addition to having mutations in the histidine operon, all the indicator strains have a mutation (rfa) that leads to a defective lip@opolysaccharidecoat; they also have a deletion that covers genes involved in the synthesis of the vitamin biotin (bio) and in the repair of ultraviolet (uv)-inducedDNA damage (uvrB). The rfa mutation makes the strains more permeable to many large molecules, t@ereby increasing the autagenic effect of these molecules. The uvrb mutation renders the bacteria unable to use the accurate excision repair mechanism to remove certain chemically or physically induced DNA lesions and thereby enhances the strains' sensitivity to some autagenic agents. Strain TA1535 is reverted to + his by many mutagens that cause base@-pairsubstitutions. Strain TAIOO is derived from Tkl535 by the introduction of the resistance transfer factor, plasmid pKM101. This plasmid is believed to cause an increase in error-prone DNA repair that leads to many more mutations for 5 a given dose of most autagens. In addition, plasmid pKI4101confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasmid in the cell. The presence of this plasmid also makes strain TAIGO sensitive to some frameshift mutagens [e.g., ICR191, benzo(a)pyrene, aflatoxin Bl, and 7,12-dimethylbeuz(a)anthracenel. Strains TA1537 and TA1538 are reverted by many frameshift mutageus. Strain TA98 is derived from TA1538 by the addition of the plasmid pKM101, which makes it more sensitive to some mutageuic agents. All indicator strains are kept frozen in nutrient broth supplemented 9 with 10% sterile glycerol at -80*C in 1-ml aliquoto containing about 10 cells. New frozen stock cultures are made every three months from single colony isolates that have been checked for.their genotypic characteristics @his,.Lfa,2arB, Lio) and for the presence of the plasmid. For each experiment, the frozen 1--mlcell cultures are allowed to thaw at room temperature before inoculation in 50 ml of glucose minimal liquid medium supplemented with an excess of biotin and histidine. The cultures are grown at 37*C, unshaken for 4 hours, then gently shaken (100 rpm) for 11 to 14 hours. All strains are genetically analyzed whenever experiments are performed. Aroclor 1254-Stimulated Metaboliz Activation System Some carcinogenic chemicals (e.g., of the aromatic amine type or the polycyclic hydrocarbon type) are inactive unless they are metabolized to active forms. In--animalsand man, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large number of these chemicals to carcinogens. Some of these intermediate metabolites are very potent mutagens In the S. typhimurium test. Ames has described. the liver metabolic activation system that we use. In brief, adult male Sprague-Dawley rats (200 to 250 g) are given a single 500-mg/kg intraperitoneal injection of Aroclor 1254 (a mixture of pblychlorinatedbiphenyls). This treatment enhances the synthesis of enzymes involved in the metabolic conversion of chemicals. Four days after the injection, the animals' food 6 is removed but drinking water is provided ad libitum. On the fifth day, the rats are killed and the liver homogenate is prepared as follows. The livers are removed aseptically and placed in a preweighed, sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted in an ice bath. The livers are washed with an equal volume of cold, sterile 0.15 M KC1, minced with sterile surgical cissors in three volumes of 0.15 H KC1 (3 ml/g of wet organ), and homoenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged : for 10 minutes at 9000 x jL, and the supernatant, referred to as the S-9 fraction, is quickly frozen on dry ice and stored at -80*C. The metabolic activation mixture for each experiment consists of, for 50 ml: 9 5.0 ml of S-9 fraction 0 1.0 al of Mgcl2 (0.4 M) and KC1 (1.65 M) o 0.25 al of glucose-6-phosphate (1 M) 9 2.0 ml of NADP (0.1 M) . 9 25.0 ml of sodium phosphate buffer (0.2 M, pH 7.4), a 16.75 al of sterile B2 The amount of S-9 fraction delivered to each plate is 50 iLl. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an ini- tial stock. In some cases, a preliminary experiment is conducted to find I a suitable dose range for testing. The artiele is usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/ plate unless solubility, autagenicity, or toxicity dictates a lower upper limit. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 pl/plate. When liquids are tested, occasionally the sample is not diluted and various aliquots are used. All assays are repeated at least once on a separate day. The plate incorporation assay is performed in the following way. To a sterile 13 x 100--amtest tube placed in a 43*C beating block we add: 7 (1) 2.00 ml of 0.6% agar containing 0.6% NaCl, 0.05 mM biotin, and 0.05 mM histidine (2) 0.05 ml of indicator organisms (about 108 bacteria) (3) 0.05 al of a solutLon of the test article (4) 0.50 ml of metabolic activation mixture (if appropriate). This mixture is stirred gently and then poured on plates containing about 25 ml of minimal glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37*C. The number of his+ revertant colonies is counted using a BioTran 11 automated colony counter when possible. When accurate counts cannot be obtained (e.g., because of precipitate), the plates are counted manually using an electric probe colony counter. Concurrent sterility,negative (solvent),and positive controls are run with every experiment. Sterility.controls include plating out separately steps (3) and (4). For negative controls,we use steps (1), (2), (4), and 0.05 al of the solvent used for the test article, if appropriate. For positive controls, we test each-bacterial culture using the steps (1), (2), (3), and (4) with the followingmutagens: Sodium azide for the base-pair substitution mutants TA1535 and TA100 9-Aminoacridine for the frameshift mutant TA1537 2-Nitrofluorene for the frameshift mutants TA1538 and TA98 2-Anthraminefor all tester strains, in the presence of metabolic activation. Statistical Analysis No statistical analysis is performed. Results are a tabulationof the number of colonies appearing on the plates. Criteria for Interpretation Positive. A test article in considered a mutagen when it produces a reproducible, dose-related increase in the number of revertants in one or more strains. This increase must occur for at least three dose levels. Negative. A test article is considered a nonmutagen when no doserelated increase in the number of revertants is observed in at least two independent experiments. The maximum dose level tested for nontoxic compouuds.is 10 mg/plate (unless dictated otberwise by solubility problems). For toxic compounds, only the highest dose level tested should show evidence of toxicity. Inconclusive. When a test article cannot be identified clearly as a autagen or nonmutagen in the standard plate assay, the results are classified as inconclusive. References Ames, B. N., E. G. Gurney, J. A. Miller, and R. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Natl. Acad. Sci. USA i9, 3128-3132 (1972). Ames, B. N., W. E. Dur3ton, E. Yamasaki, and P. D. Lee.. Carcinogens are autagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Natl. Acad. Sci. USA 10, 2281-2285 (1973). Ames, B. N., J. McCann, and E. Yamasaki. Methods for detecting careinogeus and mutagens with the Salmone3.la/mammalian-microsome mutageuicity test. Mutat. Res. .11,347-364 (1975). McCann, J., E. ChoL, E. Yamasaki, and B. N. Ames. Detection of carcinogens as autagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. USA Z2, 5135-5139 (1975). McCaim, J., and B. N. Ames. Detection of carcinogens as mutagens in the Salmone' lla/microsome test: Assay of 300 chemicals: Discussion. Proc. Tatl. Acad. Sci. USA 73, 950-954 (1976). Mortelmans,'K. E., and B.A.D. Stocker. Segregation of the autator property of plasaid R46 from its ultraviolet-protectingproperty. Mol. Gen. Genet. 167, 317-327 (1979). 9 RESULTS AND DISCUSSION 3H Company's Compound T-3727 was screened for mutagenic activity in the Ames Salmonella/microsome in vitro mutagenicity assay using the five standard strains of Salmonella typhimurium. TA1535, TA1537, TA1538, TA98, and TA100. The assays were performed in duplicate, both in the presence and absence of a rat-liver metabolic activation system. Three plates per dose level were tested. Acetone was used as the solvent. The microbial mutagenicity testing of this sample was performed on 20 and 27 February 1985. Dose levels ranging from 10 to 5000 jig/plate were used for both assays (Tables 1 and 2). No dose-related increases in the number of histidine-independent revertants were observed in either assay. A black precipitate was noted at 5000 pg/plate; therefore, these plates were hand-counted. We conclude that Compound T-3727 was reproducibly norlmutagenic when tested according to these procedures. 10 Table I IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3727 Experiment Date: 20 February 1985 Compound Compound Metabolic Added Activation per Plate- TA1535 Negative Control Acetone + 50 lil 20 22 32 50 17 8 8 Histidine Revertants per Plate TA1537 TA1538 TA98 3 10 3 17 18 17 18 26 11 7 6 28 25 21 41 38 Positive Controls Sodium Azide - 9-AminoacrLdine - 2-Nitrofluorene - 2-Anthramine - + + Compound T-3727 - + + + + + + I pg 50 5 1 1 2.5 2.5 10 pg 50 100 500 1000 5000* 10 50 100 500 1000 5000* 690 661 752 37 37 27 405 347 338 25 15 25 30 21 26 28 32 20 23 21 24 28 16 29 25 23 17 13 15 18 10 15 12 16 16 12 5 11 9 12 10 6 12 17 8 520 662 787 1440 1244 1552 942 891 23 34 27 37 36 182 177 164 187 16( 8 11 6 87 89 95 10 7 5 19 19 17 30 l@ 9 6 5 '14 13 17 29 2 886 8 16 10 22 2 8 7 8 14 10 12 23 1 5 7 7 13 16 17 29 1 6 12 6 11 12 7 23 2 15 9 9 5 10 8 6 13 13 5 15 9 11 19 4 5 9 11 21 25 22 53 2 12 24 17 37 4 25 27 23 25 2 25 24 22 28 2, 24 20 21 41 3 21 17 19 31 21 Precipitated at this dose level; hand-counted. Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM COMPOUND T-3727 Experiment Date: 27 February 1985 Compound Compound Metabolic Added Activation per Plate TA1535 Histidine Revertants per Plate TA1537 TA1538 TA98 Negative Control Acetone + 50 tLi 10 22 19 7 6 10 56 8 if 7 16 6 8 8 11 11 19 21 8 18 19 18 26 Positive Controls Sodium Azide - 9-Aminoacridine - 2-Nitrofluorene - 2-Anthramine - + + Compound T-3727 - - - - I pg 50 5 1 1 2.5 2.5 10 M 50 100 500 1000 5000* 464 492 477 24 21 20 215 212 220 21 26 16 20 24 17 23 14 22 20 13 17 23 25 20 19 13 22 111 293 346 1253 1240 1102 783 689 15 22 40 21 193 .158 151 141 168 8 10 6 68 7.2 68 337 16 20 23 19 8 7 18 14 9 28 19 6 ' 3 8 11 9 12 21 19 4 9 8 18 .11 13 32 26 7 46 7 16 10 30 12 7 4 9 10 8 4- 30 23 + 10 11 10 8 16 12 20 11 11 11 32 24 + 50 10 8 8 11 8 6 15 15 9 26 34 + 100 10 .15 17 9 10 11 17 21 12 26 27 + 500 12 11 14 6 12 9 16 9 15 24 25 + 1000 687 7 12 13 16 20 12 18 16 + 5000* 10 7 11 11 9 9 20 13 17 32 26 Precipitated at this dose level; hand-counted.