Document JrMOd3RRkyve6517NdQJjD1qK

[ c ARA26G-0316TM =~ IN VITRO a, MICROBIOLOGICAL MUTAGENICITY ASSAYS : a September 1984 : sw _ Dela Lone. ite) G LL 2 E-- C-- 3M Company em Saal attentions 3031 tomate Yea. ti... Teta semen serscmsens LT flios fe SoB n flue Kei we.AP. SRI SEA 333 Ravenswood Ave. + Menlo Park, CA 94025 005563 SmataRY SRI International examined 34 Company's Compound T-3610 for mutagenic activity in the standard Ames Salmonella/microsome assay with strains TALS35, TALS37, TALS38, TA9S, and TAL0O of the bacterium Salmonella typhimurium. Compound T-3610 was also screened for recombimogenic activity in the yeast Saccharomyces cerevisiae D3 assay. Both assays were performed in the presence and absence of a rat-liver metabolic activation systea. Compound T-3610 was reproducibly nonmutagenic and monrecombimogenic when tested according to these procedures. 1 005564 CONTENTS INTRvvvO vvevDereU ssesC ssseT ssnnIannO nnnnN nann. nes 1 RESULTS AND DISCUSSION..vveverresssssssunnnnnnneee 11 TABLES Table Leesusennnnnnnnnennnnnnnneeennnnnnnnnns 12 Table 2.nnnnnnnnnnnninnnnniiereeanaennnnnen 13 FE C3 FP 1 G05565 INTRODUCTION SRI International exsained 34 Company's Compound T-3610 for mutagenicity in the standard Ames Salmonella/icrosome assay with strains TALS3S, TALS37, TALS3S, TA9S, and TALOO of the bacterium Salmonella typhimriua. Compound T-3610 was also tested for recombinogenic activity in the yeast Saccharomyces cerevisiae D3 assay. Aa Aroclor 1254stimlated, rat-liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the microorganisms efther are facapable of conducting or do not carry out under the assay conditions. The assay procedure with S. typhimiriua 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. The assay procedure with S. cerevisiae is about 60% reliable in detecting carcinogens as sgeats that increase mitotic recombination. However, because the assay systems do not always provide 100% correlation with carcinogenicity investigations in animals, neither a positive nor a negative response conclusively proves that a cheatcal is carcinogenic or noncarcinogenic to man. Evaluation of experimental results froa the Salmonella assay consists of comparing the number of histidine-independent colonies on the treated agar plates vith the number observed on the control plates. Because all the plated Salmonella indicator organisas undergo a few cell divisions in the presence of the test chemical, the test ls semiquantitative in nature. The plate test procedure does not permit. quantitative deteratnation of the musber of cells surviving the chemical treatment. It is the demonstration of a mutagenic dose-response relationship that is faportant in establishing mutagenicity. 1 005566 The test chemtcals are assayed at several dose levels within a nontoxic dose range----vith the exception of the highest dose level, which sometimes exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial growth lava, formation of pimpoint colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chemical 1s considered a mutagen in the Salmonella assay if it elicits a reproductble, dose-related increase in the number of histidine revertants per plate in one or more tester strains. The yeast Saccharomyces cerevisiae D3 is a eukaryotic alcroorganisn capable of detecting mitotic recombination, as expressed through a mutation leading to a defective enzyme in the adenine-metabolizing pathway, resulting in a red-pigmented colony. In this assay, the yeast cells are exposed to several concentrations of the test chemical, usually ranging from a concentration that results fa no killing to one that causes 50% killing. Any concentration that induces 90% killing is considered toxic. When the number of genetically altered colonies per milliliter (yield) and the ratio of altered colonies to survivors (frequency) from the treated cells are unequivocally larger than those of the solvent treated controls, we conclude that the exposure of the cells to the compound induces mitotic recombination. If this event is dose-related, the observation fs termed a positive response. 2 005567 MATERIALS o Test Article - Name: T-3610 - Date Recetved: 19 July 1984 - Description: White, wa-like material - Storage Conditions: Room temperature - Spectal Testing Conditions: Nome - Stability: Assured by Sponsor * Indicator Organtsms - Spectes: Salmonella Eyphimurivm LT2; Saccharomyces cerevisise - Stratus: TALS35, TALS37, TALS3S, TASS, and TALOD for S: typhimurius; D3 for S. cerevisiae - Source: Dtrh.e BSraulcmeoneAlmleas}, UDrn.iveF.raiK.tyZoifmeCraalsiufno,rniWa.,GeBremrakneyl,ey,forfor the yeast Metabolic Activaton A~r2o2c.l0orsg1/25a6l-ipnrdoutceeidn, rat liver S-95 SKI Batch B-3; Negative (Solvent) Control Material ADcaetteonOepened: 13 December 1983 EMaxnpuifraacttiuornerD:ate:Amer1i3cDaencSecsibeenrti1f9i8c4 Products, McGraw Park, IL Positive Control Chemicals M9a-nAunftaucotaucrreird:ine,PfaClAtSsNoa.nd9B0a-v4e5r,9 Stamford, CT 2Ma-nAunftahcrtauartenre!, CASSigmNoa.Ch6e1m3i-c1a3l-8Co., St. Louts, HO 3 005568 2-Nitrofluorene, CAS No. 607-57-8 Manufacturer: Aldrich Chemical Co., Milvaukee, WI Sodtun Azide, CAS No. 26628-22-8 Manufacturer! Difco Laboratories, Detroit, MI M1a,n2u,f3a,c4t-uDrieerp:oxybPuftaalntez,anCAdSBaNuoe.r,14S6t4a-m5f3o-r5d, CT MSatneurfiagcutautroecry:stinC,albCiAoSchNeom., 1L0a04J8o-l1l3a-,2 CA Counters Used ~ NCoeuwntBerru,nsMwoidceklScCiLeLn,tifSRiIc Nboi.oT0r0a3n0I0I151Aut0o0mated Colony ~ CN1e1w0,BruSRnIswNioc.k 0S0c1i3en0t7i8f8ic00Bactronic Colony Couater, Model 4 005569 METHODS Salmonella typhisuriwm Strains TALS3S TALS37, TAIS38, TA98, end TALOO The Salmonella typhimurium strains used at SRI are all histidine auxotrophs by virtue of mutations in the histidine operon. When these histidine-dependent cells are grown on minimal medium agar plates containing a trace of histidine, only those cells that revert to histidine independence (his*) are able to fora colonies. The small amovat of histidine allows all the plated bacteria to undergo a few divisions; in many cases, this grovth is essentisl for mutagenesis to 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 1s increased, ususlly in a dose-related manner. : We obtatned 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 (cfs) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes lavolved in the synthesis of the vitaatn biotin (bio) and tn the repair of ultraviolet (uwv)-induced DNA damage (uveB). The rfa mutation makes the strains more permesble to many large molecules, thereby increasing the mitagentc effect of these molecules. The uiB mutation renders the bacteria unsble to use the accurate excision repair mechanism to remove certain chemically or physically induced DNA lesions and thereby enhances the stratos' sensitivity to some mutagenic agents. Strata TALS3S 1s reverted to his* by many mutagens that cause base-pair substitutions. Strain TALOD is derived from TASIS by the introduction of the resistance transfer factor, plasaid pRAOL. This plasuid is believed to cause an increase in error-prone DNA repair that leads to many more mutations for a 5 005570 given dose of most mtagens. In addition, plassid PRHLOL confers resistance to the antibiotic ampicillin, which is a convenient marker to detect the presence of the plasaid in the cell. The presence of this Plasatd also askes strain TALOO sensitive to some frameshift mutagens {e-g., 108-191, benzo(a)pyrene, aflatoxin B), and 7,12-dizethylbenz (a)anthracene]. Stratus TAISI and TALS3S are reverted by many frameshift mutagens. Strain TAS is derived from TASIB by the addition of the plasatd pRAIOL, which makes it more sensitive to some mutagenic agents. ALL tndtcator scratns are kept frozen in nutrient broth supplemented with 107 sterile glycerol at ~80C fn 1-al aliquots containing about 10 cells. New frozen stock cultures are made every 3 months from single colony fsolates that have been checked for their genotypic characteristics (hts, rfa, web, bio) and for the presence of the plasmid. For each _ expertneat, the 1-al frozen cell cultures are alloved to thaw at room temperature before inoculation fn 50 al of glucose mintmal quid medium supplemented with an excess of biotin and histidine. The cultures are grown at 37C, unshaken for 4 hours, then gently shaken (100 rpm) for 11 0 14 hours. ALL stratus are genetically analyzed whenever experiments are perforned. Aroclor 1254-Stinulated Metabolic Activation Syste Some carctaogenic cheaicals (e.g., of the aromatic amine type or the polycyclic hydrocarbon type) ate inactive unless they are metabolized to active foras. In aniaals aod man, an enzyme systea in the liver or other organs (e.g., lung or kidney) 1s capable of metabolizing a lerge 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 systea that we use. In brief, adult male Sprague-Davley rats (200 to 250 g) are given a single 500-ag/kg intra- . peritoneal fnjection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes involved in the metabolic conversion of chemicals. Four days after the fnjection, the animals' food is removed but drinking water is provided ad lbitus. On the fifth day, the rats are killed and the liver homogenate Ls prepared as follovs. 6 605571 The livers are removed aseptically and placed in a preveighed sterile glass beaker. The organ weight is determined, and all subsequent operations are conducted in aa ice bath. The livers are vashed with an equal volume of cold, sterile 0.15 KCl, minced with sterile surgical scissors in three volumes of 0.15 XC1 (3 ml/g of wet organ), and homogenized vith a PotterElvehjen apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the 5-9 fraction, is quickly frozen on dry ice and stored at -80C. The metabolic activation atxture for each experiment consists of, for 50 al: 5.0 ml of 5-9 fraction 1.0 ul of gC1, (0.4 M) and KCL (1.65 ) 0.25 al of glucose-6-phosphate (1 ) 2.0 al of NADP (0.1 ) 25.0 51 of sodium phosphate buffer (0.2 M, pH 7.4) 16.75 al of sterile 5,0. The amount of S-9 fraction delivered to each plate is 50 pl. Plate Incorporation Assay Prior to testing, the test article is serially diluted from an initial stock. The article fs usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/plate unless solubility, mutagenicity, or toxicity dictates a lover upper limit. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 p1/plate. Occasionally, liquids are tested; 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-m test tube placed fn a 43C heating block we add: (1) 2bi.o0t0ina,l aonfd00..605 amgfarhicsotnitdaiinneing 0.6% NaCl, 0.05 mM (2) 0.05 al of indicator organisms (about 10o bacteria) 7 005572 (3) 0.05 al of a solution 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 al of minimsl glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37C. The mumber of his* revertant colonies is counted using a BioTran II 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 ml of the solvent used for the test article. For positive controls, we test each bacterial culture with the following mutagens using steps (1), (2), (3), and (4): Sodium TAL00. azide for the base-pair substitution mutants TALSIS and 9-Aninoacridine for the frameshift mutant TALS37. . 2-Nitrofluorene for the frameshift mutants TAL538 and TASS. 2-Anthrasioe for all tester metabolic activation. strains, in the presence of Saccharomyces cerevisiae D3 The yeast S. cerevisiae D3 1s a diploid microorganism heterozygous for a mutation leading to a defective enzyme in the sdenine-metabolizing pathway. When grown on medius containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated from the heterozygotes by mitotic recombination. The frequency of this recombinational event may be {ncreased by incubating the organisms with various carcinogenic or recombinogenic agents. The recombimogenic activity of a compound or its metabolite 1s deterained from the number of red-piguented colonies appearing on test plates. : A stock culture of S. cerevisiae is stored at 4C. For each experiment, broth containing 0.05% MgSO,, 0.15% Ki,PO;, 0.45% (NH,),80,, 0.35% peptone, 8 005573 0.5% yeast extract, and 2% dextrose 1s inoculated with a loopful of the stock culture and incubated overnight at 30C with shaking. The in vitro yeast mitotic recombination assay in suspension is conducted as follows. The overnight culture 1s centrifuged and the cells are resuspended at a concentration of 10% cells/al tn 67 u phosphate buffer (pH 7.4). Toa stertle test tube are added: 1.00 nl of the resuspended culture 0.50 al of either the metabolic activation mixture or buffer 0.20 ml of the test chemical 0.30 ul of buffer. Several doses of the test chemical are tested in each experiment, and appropriate controls are included. The suspension mixture is incubated at 30C for 4 hours on a roller drum. The sample fs then diluted serially in sterile physiologic saline, and 0.2 ml of the 107 and 107 dilutions ts spread on plates contatntng the same ingredients as the broth plus 2.0% agar; five plates are spread with the 1073 dilution and three plates are spread with the 10 dilution. The plates are incubated for 3 days at 30C, followed by 1 day at 4C to enhance the development of the red pigment indicative of adenine-deficient homozygosity. Plates containing the 107 dilution are scanned with a dissecting microscope + at 10x magnification, and the number of mitotic recombinsnts (red colonfes or Ted sectors) is recorded. The surviving fraction of organisms is deternined from the total mmber of colonies appearing on the plates of the 10-5 atlution. Statistical Analysts No statistical analysis vas performed for any of the assays. The results Of the plate incorporation assay are a tabulation of the number of colonies appearing on the plates. A positive response is indicated by a reproducible, dose-related increase fn the number of histidine-independent colonies per plate. The results of the S. cerevistae D3 assay are tabulated by calculating the nusber of mitotic recombinants per 10% survivors. A positive response in 9 005574 this assay 1s indicated by absolute number of aitotic a dose-related increase of more than I-fold in recombinants per atlliliter as vell as fn the the relative mmber of mitotic recombinants per 10% survivors. References Afmreasn,cehBi.feN.,sutEa.geGn.s:GurMneety,aboJl.itAe.sMainldlerd,eriavnadti.vesBaorftsIch-.acetCyolrecntinnosgieinosonaesne snd other aromatic emine carcinogens. Proc. Nat. Acad. Sci. USA 69, 3128-3132 1972). ASumteasg,enBs.: bacteria N.,4 for .sdiemtpeE.lcetiDoutner.ssttonPs,ryosctE.e.m NYaactmo.amsbaiAknciia,ndg anlSediivseFr. UShD.AomToL0ge.een.a2t2e0Cs1a-r3cf3oi8rn5ogsee(nrisiovearsr)ieon and Afhmeoearsd,.thSeBc.id.Ne.t,OeschtP.i1o0Dn,. aL7ned6e2,c7l8aa6nsds(iWf1.i9c7Ea9.t)iDounrsotfonm.itagAnensiaapnrdovecdarbcaicntoegretnasl: tPesrtec.sysMtere:m Ames, 3. N., J. McCann, and E. Yamasaki. Methods for detecting carcinogens Hata. Res. 31, 347-368 (TTS) and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Btreucshiaclkq,lesD.viJt.,h yaenadstV.. .EuvMiaryoenr.. HeNaelwthdePveerlsoppemcetn:tsin83m-u8t6age(n1i9c7i3t)y screening Kciiegra,retLt.eD.s,mokEe. Ycaomnadseanksia,tesa.nd P3r.ocN.. NAante.s.AcaDde.tecSetii.onUSoAf 7m1u,ta1ge5n9tc16sc3elv(i1t5y74)f.n eCana, J., E. Got, E. Yamasaki, and 3. N. Ames. Detection of carcinogens as mTuatt.ageenasd.inSctih.e U SalmTS oZnelA l3a79/-m9i8c3ros(o1m9e75)t.est: Assay of 300 chemicals. Proc. NSacatCl.ammomAn,cealdJl..a,/aSciainc.droUsB.SoAneN1.3t,Aenaet9s:5.0-5A3Ds4esatye(c19to7if6o)n3.00ofcchaeraciicanlosg:ensDaisscumsustisogne.ns Pinroct.he MP5ol1ra7ts3ea2lia7damRas4s,5sKfy.r.onB.,iteanldtrB.aAv.iDo.leStt-opcrkoecre.ctiSneggrpergoapteirocny.of Mtohle.muGetmastorGenpsreorper1t6y3 of nZIinttmrsSoeaurcsacahaaancr,iodm,yT.ceIs-Xa.,eccehraeynvldi-s3R.i-aneiS.ccrhovH-aoltl-e.rn.itGreInon.sdougGcuetamienotinsdionf10e0m.aintdot3i-o7ct6hegreCn1ea3l6kc7yo)lnavteirnsgiosnewnittsh 10 005575 RESULTS AND DISCUSSION 34 Company's Compound T-3610 was screened for mutagenic activity fn the Ames Salmonella/microsome in vitro mutagenicity assay using the five standard strains of Salmonella typhimurium: TALSIS, TALS37, TALS3S, TASS, and TAL00. The assays vere performed in duplicate, both in the presence and absence of a rat-liver metabolic activation system. Acetone vas used 48 the solvent. The atcroblal mutagenicity testing of this sample vas performed on 26 July and 3 August 1986. Dose levels ranging from 10 to 5000 sg/plate were used for both assays (Tables 1 and 2). Wo dose-related increases in the number of histidine-independent revertants were observed in either assay. A precipitate was noted at 5000 pg/plate, and these plates were hand counted. Coapound T-3610 was also tested for: recombinogentc activity in the yeast Saccharomyces cerevisiae D3 asssy for mitotic recombination. This assay vas perforsed on 27 July and 10 August 1984, both with and without metabolic activation. A dose range of 0.05 to 5% was used for both assays (Tables 3 and 4). No dose-related increases in the misber of mitotic recombinants per 10 survivors were observed in either assay. In conclusion, Compound T-3610 vas reproducibly nonmtageatc and nonrecosbinogentc vhen tested according to these procedures. u 005576 I31m Assavs viTmasbua1pose even Eepectn[ en Da--tes 2--6 July 1984 Comes WAeertaibmoetiteen peCrohmaPpgioesustned T BisctgH toe_gRevoIstaEstspohT rpolrate mo ` NegaacttovneeControl I- E HE EE R 0EoEwEBEEEAENE 5 PoFsDSirotasiitivumeenostkCcorhrniteiertonlres -Dhntrraates : S1 ar i 6 as 100s 305 367 533 a as Rh Rm we . ::: Bisoomoal a1 sog aos wm i Compound 73600 :: : DRwRmB onow 63OsS4 LmbEooRww Rm oBos Som on oEx RwboEmis on on in om diee . : wE o. EWEI S 8 wou bom o% in g2z i3. : W0BooossTshR 0 sow ooin swmonoAwoboomnwomoN okwhiaiage liem 8S oR RE Bis 9 z&4 :i WBOt. B0nB o5w dBombomom oona aiine is MN Frecipteated at this dose level; hand-counteds "od i 0 110 Ass Tabl2a wi SawoNELA TrPnDORIN Experiaecaotmdaacneo: 13-3A10ug 1984 cospoun [Motrivaetion pr-- per ieee TESTMiscItoton Rovathanteapoee piace mmo Negeaeotve Control I- oSwTO3REM8da7 wsown oobw mooHmm wo : PonSCtohediiivumemcaCrocinttiarniolee - 0Le sm om oa DTnthireenrinee s :3 2 : Sii 10W1L9o10Ee74 D69B6 oS5m87 awwon Isom ow ou ow : Boda nob Comma 00 :: I LewnonwowhoTwNS7BRRBoas iw , ::: EWOO BmoBw BomE osos I wBoNn Bom ookmenia . i+i WSooommEEow5 EwhonwEEooR nEhnEon EoRBEg e ii : SWooe nmoSu Wo lBnw oNnBBn3 howw owoowa uoou ymnaAan &a o Freecipp ttatedr a hin dons level; hand-eoated: a "1 i ante 3 IN VITRO ASSAYS WITH SACCUBONICES CEREVISIAE 03 omzonm 17-3610 Expectasat Date: 27 July 198 Percent Surviving ReMcoemobrltaetats Recwoineboitnsaeges Compound MAeoctiavbaoclitocn Conce(unltyration Geils1p6eg sl SIurevivors bEelro)a surevirvo1r0s5', , NeKgeaectiovne Goatrol - + W0o2 100 s5 1B PoS1stiett,tteg4aac2Dtolnoeecp,cyooarlcysi3bnuc,ane -= 00..002058 aPe] 1000 aso 19050.3 + 0/003 pet 100 158 30) BE Compount 13610 --: 0oo.sr05 waWsss 111000000 8e 4 13 5.3 -: 31 ape 101000 37 1i5 i ++1 o0o.ns05 piwweaos 10100 100 i9 zn5.9 5 1 ` ++ 15 wao1 11000 i. u5w.8 Tealcutactons are expressed uatng two sigaticant figuces. 2&s & s i Table 4 IN VITRO ASSAYS WITH SucomBONCES carmytsiae 03 coeonm 1-2610 Expertaeat Dace: 10 August 1984 FegatiCvoempCoounntdrol Acltoigvghaetlitoen ComPceracmetnitaion io GSaulclveivsienfg'al 16%) Survi3vors RecMoimeboirntaenta. Recmoimcboritneage Gpo1r05}) _seprevirvo1r0%s,' Rencone +- 3397 100 3 11 ` Pos1t,e2t,v3e,4CDolnetproorlybucass Stetigmatocyscia -3 000/.000020s5 a0r 110000 Eni 200100 Coupound T-3610 -- oa 100 150 310 o0.r05 5503 101000 65 116 . --- o51is p35sr5 1100000 875 It1]3 5.1 ' +i+ + 0oo.ns05 pwis 53 11010000 1 10 1s30 25o.l3i ` + 5 53 100 75 094 "Calculations ace expressed ustog two sigateicant figures. a& a23 IT : i