Document RJQdX8OaV0bD9pda4O2YQOdma

13 ym momma wer ay:Atlan Dba a MwiacrrodblaS.l RGiecnceitoi,csAsDseipsatratnmtenDtirector T acomerS s ne -- C--O SKI Project LSG-3145 Wibaw Oroiinees TTT on 1WG Bess STMMARY SRI International examined 34 Company's Compound T-3727 for muta= genic activity in the standard Anes Salmonella/microsome assay with strains TAIS35, TALS, TALS3S, TA98, and TALOD of the bacterium Salmonella typhimurius. The assay was performed fa the presence and absence of a rat-liver metabolic activation system. ALL tests were performed in coupliance with the United States Food and Drug Adafnistration Good Laboratory Practice Standards. Compound T-3727 was reproducibly nonautagenic when tested according to these procedures. un 01232 coNTENTS SUMMARY. co euvvvvrnesssnsnnneeessssnnnnnnnennnnes 41 QUALITY ASSURANCE STATEMENT.......sversessesesnne 10 INTR+vO sesDssnU nnseC sassT ssseInesO asssN nnnn. nns 1 METHODS veveernnnnesesssnneneesssssnnnnssnsinns 5 RESULTS AND DISCUSSION. ousevvnveeessssnnnnnesssnes 10 TABLES Table Leveereersnrnreensssssneneeeessnnnnes 1 Table Zeereeerunneerinsiinneaesessinneneneee 12 fn 01233 FQiUnAaLlITYReApSoSrUtRASNtCaEtemUNeInTt SRI International assures the quality and integrity of this study, In-Vitro Microbiological Mutagenicity Assays of Compound 1-3727, for the 3M Company. The study was inspected on March 12, 1985 during the colony counting phase. The findings of the Quality Assurance Unit fnspection were reported at the time of the inspection to 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 communicated tothe Study Director and SRI management on March 25, 1985. The final report accurately describes the methods and standard operating procedures and reflects the raw data of the study. Any deviations from the approved protocol and standard operating procedures were made with proper authorization and docur ntation. A AR L Tho eru e 2at 5s (h J9S05 w 01234 INTRODUCTION SRI International examined 3M Company's Compound T-3727 for mutagenicity in the standard Anes Salmonella/microsome assay with strains TAIS35, TALS37, TALS38, TAS, and TALOO of the bacterium Salmonella typhimurium, An Aroclor 1254-stimulated, rat-liver homogenate metabolic activation systen was included in the assay procedure to provide metabolic steps that the alcroorganisms either are incapable of conducting or do mot carry out under the assay conditions. The assay procedure with S. typhimurtun has proven to be 80 to 90% reliable tn detecting carcinogens as mutagens, and it has about the same reliability fn identifying chemicals that are mot carcinogenic. However, because the assay systems do not always provide 100% correlation with carctnogenteity investigations in animals, neither a positive mor a negative response conclusively proves that a chemical is carcinogenic or noncarctuogenic to man. Evaluation of experimental results from the Salmonella assay consists of comparing the number of histidine-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 fn the presence of the test chemical, the test {s semiquantitative in nature. The plate test procedure does not permit quantitative deternination 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 cheatcals are assayed at several dose levels within a non toxic dose range--vith the exception of the highest dose level, which sometines exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial lawn growth, formation of pinpoint 1 01235 colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chenical 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 vas coapleted on 27 February 1985. Copies of the final report will be kept in our files (Building , Room 213) and in SRI's Records Center. The raw data vill be retained in Building 205, Room 13, for ome year after the laboratory notebook has been filled and then will be stored in SI'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 01236 MATERIALS Zest Article - Name: T3727 - Date Received: 19 February 1985 - Description: Light-amber vaxy solid - Storage Conditions: iStnoaredseactondraoroya ctoenmtpaeirnaetrure - Spectal Testing Conditions: Nome - Stability: Assured by Sponsor Indicator Organtsms - Species: Salmonella typhimurium LT2 - Stratns: 5T.AISt3y5p,htmTuArItSu3n7', TALS, TAY, and TALOO for - Source: Dr. Bruce Ames, University of California, Berkeley o Metabolic Activation ~Ar2o6c.l5ormg1/2a5l4-tpnrdoutceeidn, rat liver 5-9; SKI Batch F-; Negative (Solvent) Control Matertal DAacteetonOep,eneCdA:S No1.4 D6e7c-e64m-b1er 1984 EMaxnpuifraacttiuornerD:ate:Amer1i4caDneceSmcbieernti1f9i85c Products, McGraw Park, IL PLuortitNyo.:: 9K9T.E7A1 3 01237  Positive Control Chemicals M9a-nAunftancotaucrreird:ineP,falCtAsS Naou.d B9a0u-e4r5,-9Stamford, CT M2a-nAunftahcrtausrtenre:, CSAiSgmaNo.Che6m1i3c-a1l3-8Co., St. Louis, KO M2a-nNuiftarcotfulrueorr:eneA,ldr`CiAcShNoC.hem6i0c7a-l57-Co8., Milwaukee, WI Sodtun Azide, Manufacturer: CDAiSfcoNo.Lab2o6r6a2t8o-r2i2e-s8, Detroit, KI Counters Used ~ NCeouwntBerru,nsHwoidckelScCiLeLn,tifSiAcI BNoi.oTr00a3n0I6I126Au0t0omated Colony - CN1e1w0,BrSuRnIswNioc.k 0S0c1i2ent3i10f8ic00Bactronic Colony Couater, Model 4 01238 HETHODS Salmonella typhimeiun Strains TAIS3S, TT ALS3A 7, ISTAS3 S,an8dTA,LOO The Salmnella typhimrive strains used at SRI are all histidine auxotzophs by virtue of mutations In the histidine operon. When these histidtoe-dependent cells are grown on minimal medtus agar plates con- tatntog a trace of histidine, only those cells that revert to histidine independence (his*) are able t4dine allovs all the plated to form colonies. The small amount of his bacteria to undergo a few divisions; in say cases, this growth is essential for mutagenesis to occur. The his revertants are essily visible as colontes against the slight background grouth, The spontaneous mutation frequency of each strain is relatively constant, but when a mutagen is added to the agar, the mutation frequency is fncressed, usually in a dose-related mmer. We obtatned our S. typhiavriua strains from Dr. Bruce Ames of the Uatversity of California at Berkeley. In addition to having mutations in the histidine operon, all the fndicator strains have a mutation (cfs) that leads to a defective Lipopolysaccharide coat; they also have a deletion that covers genes fnvolved in the syathests of the vitamin biotin (bio) and ta the repair of ultraviolet (uv)-induced DNA damage (urB). The rfa mutation sakes the strains more pernesble to many large molecules, thereby increastag the mutagenic effect of these molecules. The uved mutation renders the bacteria unable to use the accurate excision repair mechanism to remove certain cheatcally or physically induced QNA lesions and thereby enbances the strains' sensitivity to Lo revertad to hs by many mutagens some that mutagenic agents. Strain TAISHS cause base-pair substitutions. Strata TALOO 1s derived from TAIS3S by the introduction of the resistance transfer factor, plasmid pRAIOL. This plasaid is believed to cause an increase in error-prone DEA repair that leads to many more mutations for 5 01239  given dose of most mutagens. In addition, plasmid pRIOL confers resis tance to the antibiotic ampicillin, which is a conventent marker to detect the presence of the plasmid in the cell. The presence of this plasmid also makes stratn TAIO0 sensitive to some frameshift mutagens [e.g., ICR 191, benzo(a)pycene, aflatoxin By, and 7,12-dinethyIbenz(a)anthracene]. Stratas TAIS37 and TAISIB are reverted by many frameshift mutagens. Stratn TASS is derived from TALS3S by the addition of the plasaid KRNIOL, Which makes Lt more sensitive to sone mutagenic agents. ALL tndicator strains are kept frozen in nutrient broth supplemented With 10% sterile glycerol at ~B0C 1a 1-al aliquots contatning 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, fa, wed, blo) and for the presence of the plasaid. For each experinent, the frozen 1-al cell cultures are allowed to thaw at room temperature before inoculation fa 50 al of glucose ainiesl liquid mediva supplencated with an excess of biotin and histidine. The cultures are grown at 37C, ushaken for & hours, then gently shaken (100 rpm) for 11 to 16 hours. ALL strains are genetically analyzed whenever experiments are perforaed. Aroclor 1254-5timlated Hetaboll: Activation Systen Some carcinogenic chentcals (e.g., of the aromatic amine type or the polyeyelic hydrocarbon type) are inactive unless they are metabolized to active foras. In`animals and man, an enzyse system in the liver or other organs (e.g., lug or kidney) is capable of metabolizing a large number of these chemicals to carcinogens. Some of these internediate metabolites are very potent mutagens in the S. fyphimriua test. Ames has described the liver oetabolic activation system that ve use. Iu brief, adult mle Sprague-Davley rats (200 to 250 g) are given a single 500-ag/kg intraperi= toneal fajection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes favolved in the metabolic conversion of chemicals. Four days after the injection, the animals' food 6 oi1240 1s removed but drinking water is providedad libitum. On the fifth day, the rats are killed and the liver homogenate is prepared as follos. The livers are removed aseptically and placed in a preveighed, sterile glass besker. 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 KCl, minced with sterile surgical scissors in three volumes of 0.15 M KCl (3 ml/g of wet organ), and homo genized with a Potter-Elvehjen apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the S-9 fraction, is quickly frozen on dry ice and stored at -80C. The metabolic activation mixture for each experiment consists of, for 50 ml: 5.0 ml of $=9 fraction 1.0 ml of MgCl, (0.4 M) and KCl (1.65 ) 0.25 ml of glucose-6-phosphate (1 M) 2.0 ml of NADP (0.1 H) 25.0 al of sodiun phosphate buffer (0.2 M, pH 7.4) 16.75 ml of sterile HO. The amount of 5-9 fraction delivered to each plate is 50 ul. 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 a suitable dose range for testing. The article {s usually tested over a minimum of six dose levels, the highest montoxic dose level being 10 mg/ plate unless solubility, mutagenicity, or toxicity dictates a lover upper limit. When extracts are made, various wadiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 yl/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 fncorporation assay is performed in the following way. To a sterile 13 x 100-am test tube placed in a 43C heating block we add: 7 oi241 (1) a2n.d000.&015ofwt0.h6i%stiadgianrecontaining 0.6% NaCl, 0.05 m4 biotin, (2) 0.05 ml of ndicator organtsms (about 103 bacterta) (3) 0.05 ml of a solution of the test article (4) 0.50 nl of metabolic activation mixture (if appropriate). This mixture is stirred geatly aud then poured on plates contatning about 25 ul of minim] glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37C. The number of his* revertant colonies is counted ustag a BioTran II automated colony counter when possible. When accurate counts cannot be obtained (s.g., because of prectpitate), the plates are counted manually using an electric probe colony counter. Concurrent sterility, negative (solvent), and positive controls are Tun ith every expertacat. Sterility controls include plating out sepa rately steps (3) and (4). For negative controls, we use steps (1), (2), (4), and 0.05 m1 of the solvent used for the test article, if appropriate. Tor positive controls, ve test each bacterial culture using the steps (1), (2), (3), and (4) vith the following mutagens: Sodium aztde and TA100 for the base-pair substitution mutants TAIS3S 9-Amtnoacridine for the frameshift mutant TALS37 2-Nitrofluorene for the frameshift mutants TA538 and TASS sZe-tAaubtohlrtaesinaectifvoartiaolnl. tester strains, in the presence of Statistical Analysts Wo statistical analysis is performed. Results are a tabulation of the nuaber of colonies appearing on the plates. Ceiterta for Interpretation Positive. A test article is considered a mutagen uhen it produces a reproductble, dose-related increase in the number of revertants in one or more stratns. This increase must occur for at least three dose levels. 8 coL242 Negative. A test article is considered a monmutagen when no doserelated tncrease {a the nusber of revertants is observed fn at least tuo independent experiments. The maximum dose level tested for nontoxic compounds 1s 10 mg/plate (unless dictated otherwise by solubility prob lems). For toxic compounds, only the highest dose level tested should show evidence of toxteity. Inconclusive. When a test article cannot be identified clearly 82s mutagen or noumutagen fa the standard plate assay, the results are classified as inconclusive. References aAsnesf,raBm.esNh.i,ftE.mitG.ageGnusr:ney,MetJ.aboA.litMeisllaern,d daenrdivH.atiBvaerstscohf.2-aCcaertcyilnaomgetnnsoUfSlAuo6r9e,ne3a1n2d8-3o1t3h2er(a19r7o2m)a.tic amine carcinogens. Proc. Natl. Acad. Sci. Amauetsa,genBs.:N.,A Ws.impE.leDutressttons,ysEt.emYacmoamsbaiknii,nganldiveF.r Dh.omLoegee.nateCsarfcoirnoagceatsivaatrieon a(n1d973b)a.cterta for detection. Proc. Natl. Acad. Sci. USA70, 2281-2283 Agmeenss,`aBn.d Nm.u,taJg.enscCwaintnh, tahendSaE.lmoYannealslsak/im.ammMaeltihaond-saicfroorsodneetemcuttianggeactaerictiyno test. Mutat. Res. 31, 347-34(1975). MgceCnasnna,s Jm.u,tagE.ensCho{ta, tEh.e YSaamlamsoankeil,ln/amndicrB.osoN.neAnteess.t: DAestseacytioonf o30f0ccahrecimno icals. Proc. Natl. Acad. Sci. USA72, 5135-5139 (1975). HSeaClammonn,elJl.a,/aiacnrdosBo.neN. tAesute:s. AsDseatyectofion300ofchceamriccianlosg:ensDisacsusmsuitoang.ensPrionc.the Fatl. Acad. Sci.USA73, 950-954 (1576). MPorrotpeelrstaynso,f Kp.laEs.a,idanR4d6B.fAr.oDm. iSttsocukletrr.avioSleegtr-epgraottieocntiofngtphreopmeurttayt.or Mol. Gen. Genet. 167, 317-327 (1979). 9 01243 RESULTS AND DISCUSSION 3M Company's Compound T-3727 was screened for mutagenic activity fn the Anes Salmonella/nicrosone in vitro mutagenicity assay using the five standard stratns of Salmonella typhimurium: TAIS3S, TALS37, TALS, TA9S, and TAIOO. The assays were performed in duplicate, both in the preseace and absence of a rat-liver metabolic activation system. Three plates per dose level were tested. Acetone vas used as the solvent. The microbial mutagentclty testing of this sample was performed on 20 and 27 February 1985. Dose levels ranging from 10 to 5000 pg/plate vere used for both assays (Tables 1 and 2). No dose-related increases in the nuaber of histidine-independent revertants were observed fn elther assay. A black precipitate vas noted at 5000 yg/plate; therefore, these plates were hand-couated. Ve conclude that Compound T-3727 was reproducibly nonmutagenic when tested accordtag to these procedures. 10 CoLl244 Compound. Negative control Aeotone Table 1 IN VITRO ASSAYS ITH SALMONELLA TYPRINRION neon 1-727 Expectaant Date: 2February 1985 AHcetciavbmotlitoen pCoormhpaoPdulenaddte TT HISE I HistidinT e ReveceantspeR r PlaHtIe E E mm :- w20ST w mom ow 0370 3mwmowowoas o% oBR okmosw Postetve ontals SSFhointtticunonotssicietoidrdetunes --== T-tatheasine <:+ : Compound 73727 -:z -:: 1+ +1 1+ 1s s 0 ee 732 so e620 i21 s 0m wom som os 23 ws ow om wm ow 9s Wo2w w m%Bom ow2 omoxm a805 786 s65 w5o0 nomm ol2e om 85 77 87 Se 3 U6 2 6 Sooo dBo os1s how s1 o9w a9 W 500 o5k00 129 6511339 SWOe 1122 Wwos6 um5 Bsou4 9s ser 402412408 1%552 9452 89"5 R1Uk as as am W207 6 ww 20 av ae in M8 o11 plm ooBH B%WB% W m1M0mn% w1ok 2UmB Boy oBdoiY n imm oa Mou 7D oA om oe amam mG2omom 7om %om 4om o3A I1n06I1a L1s6 B3 oWa DmoBm ouBoDz om unw Aom DwosoA onooH a% Mueasaom om PEAT Trreco tpiiates d at ts his dose Level; hand-counted. & Tobie 2 IN VITO ASSAYS WITH SAMOVELLA TYPADRRII comzono 3727 Bxpactasat Date: 27February 1985 Compound Negative control Acetone Hetabolte CoAmpioduendd lle rae E MisttdiosRBeveE m stantsper Plaetre mm +- m3 w lSmF o7 67 66 08 88 mWooWw owBooA% w1o%w 9RNWH RostetveControls SSioaditonncaAcsrsidoetoe uitoiiorsms =-- 5 hnchianine -i : : Compound 12 -: -z -: +:1 & 11 1 T 3i RE moo ue 3B2soaswormoamon ose momoa o2% w Ww mBBooomx mowoa63 335 787 w$o6 Woe BoBm BBBm oRpm7T4 AL49 68 Soo% Ww 1mdo ots8oosw8 w1 om8 sow oomm6 1F S0o0 EaT 2 4ETF1 76 12 TE onE9E 00 su se 1232 3 1200%1102 193 18 151 7T8e3 10 6Am 168 7327 138 mw om . Bz we Gwu6m ow13 om3 omon mom owBBoooDwluaswr m9 l8 17 1om ows lo1 oom ouw m1 4woamoa a8m 0% ow93 om o2w oo1mA1omom192 owmosomo%pooIm o1 10 8mi0n 116 w3 o1o5 sau EE 3wowa ouusmoat10d31 E&SFrractpiated at this done level; hand-counted.