Document o9z0XLv0jw79meY4Z0bnZpg3R

18 VITvE VeICR.OBTOCLOoGErCAoL M1I-T3A60C5BVLCITY ASSATS ac-- September 1984 oO, THiTteraueiaeGrefnietelic,siiDetpbarc(meeT5notR)iet ant a | A FTeaH rrrainY SGeencettollircseeReDseeptoacretnatsnDt TFcEe ------ e3e4 ceeoupapfnyaarceens,ntoH Center pen Sel Acteation: TBmLlcoHenCoonraSicpkectatss -- SKI Project 150-3145 Approved by: teoomn 3GeonerrtieelsaaDnesp,usiDaierencttor [so7fn 9o.lReitao, DfirrectSorT Ton sna Laboratory 4i. A.soSkkiemnmeenrs,DViivciesiPornesident SFr TX NS 203 AavenswAover. + Mano Park, CA 94025 00 $275 smouzy SRI International examined 3M Company's Compound T-3609 for mutagenic TaAcLtSi3vSi,tyTAinLSt3h,e TsAtLaSn3dSa,rdTAAmSSe,s SaanldmoTnAeLlOlDa/omfictrhoesobmaectaesrsiauys wSiatlhsonsetlrlasins typhimurium. Compound T-3609 was also screened for recosbinogenic sctivity ta the yeast Saccharomyces cerevisiae D3 assay. Both assays vere perforned in the presence aad absence of a rat-liver metabolic activation systea. Compound T-3609 vas reproducibly monmutagenic and nonrecosbinogenic When tested according to these procedures. i 005276 CONTENTS METHODS. +evvvvevenseeesensnsssssnnnnneeeeesessnes 5 RESULTS AND DISCUSSION...ssssssssrssnsereeeeesnnes 11 TABLES Table Luveseerrrrrrrrrnssnnsnensnnnnsssisnns 12 Table Zuvererrrrrrrrrnessenesssssrinnnnnenees 13 Table 3uvvrrrrrnrrrrreereeeesnnnnnninnninnes 16 Table Auvrrerrrrrrrrnnrariesensnnnnsssinenes 15 005277 111 INTRODUCTION SRI International examined 3M Company's Compound T-3609 for mutagenicity in the standard Ames Salmonella/microsome assay with stratus TALS35, TALS37, TALS38, TASS, and TALOD of the bacterium Salmonella typhimuriua. Cospound T-3609 was also tested for recombinogenic activity 4n the yeast Saccharonyces cerevisiae D3 assay. An Aroclor 1254stimulated, rat-liver homogenate metabolic activation system was included in the assay procedures to provide metabolic steps that the microorganisms etther are incapable of conducting oc do not carry out under the assay conditions. The assay procedure with 5. typhimurium has proven to be 80 to 90% reliable fn detecting carcinogens as mutagens, and it has about the same Teltabtlity in identifying chemicals that are not carcimogenic. The assay Procedure with 5. cerevisiae is about 60% reliable in detecting carcinogens as agents that increase mitotic recombination. However, because the assay systems do uot alvays provide 100% correlation with carcinogenicity lavestigations in animals, neither a positive mor a negative response conclusively proves that a chemical is carcinogenic or noncarcinogentc to man. Evaluation of experimental results from the Salmonella assay consists of comparing the number of histidine-tndependent colonies on the treated agar plates with the maber observed ou the control plates. Because all the plated Salmonella indicator organisus undergo a few cell divisions in the presence of the test chemical, the test is semiquantitative fn nature. The plate test procedure does not permit quantitative deterataation of the nusber of cells surviving the chemical treatment. It 1s the demonstration of a mutagenic dose-response relationship that is iuportant in establishing mutagenicity. 1 a 005278 The test cheateals are assayed at several dose levels within a nontoxic dose range--vith the exception of the highest dose level, which sometines exhibits toxicity. Toxicity ia evidenced by several phenomena: clearing of the background bacterial growth lava, foraation of plupotat colonies consisting of surviving cells, and a decrease in the muaber of cevertaat colontes below the spontaneous background. A cheatcal 1s considered a mutagen in the Saluonella assay if it elicits a reproducible, dose-related increase in the nusber of histidine cevertants per plate fn one or mre tester strains. Ihe yeast Saccharonyces cerevisiae D3 is a eukaryotic alcroorgantsn capable of detecting mitotic recombination, as expressed through a autation leading to a defective enzyne in the adentne-aetaboliziag pathvay, resulting in a red-pignented colony. cells are exposed to several concentrations of In this assay, the the test cheatcal, yeast usually cangiag fron a concentration that results fa mo killing to one that cavees 5t0o%xick.illiWnhge.n tAhneynucaobnecrentofratgieonnettihcaatllyindaulcfeesre9d0%cokliolnliiesngpeirs caoinlsliidleirteedr (yield) and the rato 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 {nduces mitotic recombination. If this event ls dose-related, the observation is termed a positive response. 2 005279 MATERIALS o Test Article - Name: T1-3609 - Date Received: 19 July 198 - Description: Yellow, glue-like liquid - Storage Conditions: Room temperature - Spectal Testing Conditions: None - Stability: Assured by Sponsor * Indfeator Organtsms - Spectes: Salmonella typhimurium LT2; Saccharomyces cerevisiae - Strains: ST.ASD3ySp,hiTmArLiSuIn7;, TDA3LSf3oSr,S.TA9cSe,reavnidsiaTeALOO for ~ Source: Dtrh.e SBraulcmeoneAlmleas;, DUrn.iveF.rsiX.tyZiofaseCraalaiafao,rniWa., GBeerrmkaneyl,ey,forfor the yeast Metabolic Activaton ~Ar2o2c.l0orug1/2a5l4-pinrdoutceetda, rat liver 5-9; SKI Batch -3; Negative (Diluent) Control Material ADacteetonOepened: 13 December 1983 EMaxnpuifraacttiuornerD:ate:Amer1i3caDenceSmcbieernti1f9i8c Products, McGraw Park, IL Positive Control Chemicals M9a-nAunftaacotaucrreird:ine,PaClAtSzNoa.nd90Ba-u4e5r-,9 Stamford, CT M2a-nAunftahcrtauarteare:, CASSigmXoa.Ch6e1a3t-c1a3l-8Co., St. Louls, M0 005280 3 r 2Nteroe fluorens,ydCrAiSchNo.Che6n07t-c5a7l-8Co., Milwaukee, WT SSoodetutneAzrid:e, GA"DSifNcoo.La2b6o6r2a8t-o2r2i-e8s, Detrott, HI e 12,3,4n -DiepoxybPuecaalness, aGnAdSBNaou.er,14S6t4a-n5f3o5rd, CT Seteeriugraaetorcy:stia,CalCbAiSocNhoe.s,1L0a04J8o-l1l3a-,2 CA Counters Used eeweBreumewvaidcekrScCLiie1n,tiSfRiIc Nboi.oTr00a3n0II01e51Aut0o0mated Colony -- ieworBruSRnEswNioc.k 0S0e1t3en0t7i8f8ic00Bactronice Colony Couster, Nodel 005281 ` wETHODS Salmonellstyphimurium StrainsTALS33, TALS37, TAI538, TAGG, and TAIO0 The Salmonella typhimuriua strains used at SRI are all histidine auxotrophs by virtue of mutations in the histidine operon. When these histidtne-dependent cells are grown on minimal medium agar plates contatning 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 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. Eyphimurtum strains from Dr. Bruce Ames of the University of California at Berkeley. In addition to having mtations in the histidine operon, all the indicator strains have a mutation (rfa) that Leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synthesis of the vitamin biotin (bio) and in the repair of ultraviolet (uv)-induced DNA damage (urd). The rfa autation makes the strains more permeable to many large molecules, thereby increasing the mtagenic effect of these molecules. The wd mutation renders the bacteria unable to use the accurate excision repair mechanisn to reove certatn chemically or physically induced DNA lesions and thereby enhances the strains' sensitivity to some mutagenic agents. Strata TALSS 1s reverted to his* by many mitagens that cause base-pair substitutions. Strata TALOD ts derived from TALS3S by the Introduction of the resistance transfer factor, plasaid pRAIOL. This plasaid is believed to cause an increase ta error-prone DNA repair that leads to many more mutations for a 5 005282 fpievsetnotdaonsce toof atuhestsnmcciabgieonrsi.e smIpnlcatdidiiitni,on,Whipclhass1std&RcIoOmeLntceonnttearascker 0 PdleatseacldtaheisprseaskeenscesiorfsitnheTHpIlOaDsmisdensfiotitvhee tcoells.omeThfersmpersehsiefnccemuoifagcehniss [e.g., ICR-191, benzo(a)pyrene, aflatoxin B,, and 7,12-dimethylbenz- (a)anthracene]. Strains TA1537 and TA1538 are reverted by many frameshift mPultaasgaeinds.pRHLSOtLr,aiwnhiTcAh98saiksesder15ivseodrefrsomensTiAtLi5v3e8 tboy stohmeeamdudtiatgieonnicofagtehnete: ALL tottccor craton sre kept feosen fn mien broth spplementsd wCcieotllhlosn.10%tNesoeltwaecrfeirsloesetgnhaltyscteohrcaokvlecbautelet-nu8r0ecshCecakrieendls-afmdolre taelvhieeqrruyotg3sesSooctnoycnhpteiacinficrnhugmasaasbciotsugetlreta1t0ics e(Bxlpae,risntean,,wetrhke,iHoos)farnotzenfoceltlhcuplrteusreensceacoef atlhleowpeldasmtiod.thewFo8rceaochn ccuepppelreantsuarteedbveiftohresatssosccuelssscioofn b1iaot5i0naa1ndofbgalcuiciosnee,sinTihaeslcullitquuciedssesdrseim agxreow14npohraottuorr3sa7.etC.,AuLnshsatkraeinnsfoarxe4 ghoeunrssc,icatlhleyn agneanltylsyedshwahkeennev(e1r00exrppme)rtsfeonrts11 Aroclor 1350-3stautated vassboltc hettvation system potyeySeotnieccsaprdcstascoapsebnotnc htyapnei)eaales (dnea5c.e,iveofuntlheessarotmhaeyticaceamsiunceabtoylpieseodF <t0he tecrttitvnes Ctooruupse. duTnngaonxiasKildeaesyn)d aien,ca4p3ableensoyfsesestysatbeomlefinngche lLaicvgeer soufsboetrherof atrheesevecyhepntoetaelns mtuotacgaerncstsofgnentsh.e 5:SomEeyphoifsutrhteusme tienstte.rseAdmieantebassetsdbeanlcietiedsed the ver metabolic sciivacion yutem that ve ase. In brief, sdulc sale Sprague-Dawley rats (200 to 250 g) are given a single 500-mg/kg intraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated abwsinipiaahaeblnosyl'lisc)f.osoodmTah1rsiesiroetnmroevaoetfdmecnbhtuetmiecndahclaisna.ckeissTotuvheratesdrayynstfahesstpieserrofotheevnszidiynmfeeidscbtieiiconunvdm,o.lvtehdeOninthethe t1o6lclhond.ay, the sate axe Willed and the ver homogenate fs prepeced os 005283 The Livers are removed aseptically and placed in a preveighed sterile 8lase beaker. The organ veight is deterained, and all subsequeat operations are conducted fn an ice bath. The livers are vashed vith an equal volume of cold, sterile 0.15 KC, minced with sterile surgical scissors in three Volumes of 0.15 M XC1 (3 ml/g of wet organ), and hoaogentzed vith a Potter Elvehjen apparatus. The houogenate fs centrifuged for 10 minutes at 9000 x 5, and the supernatant, referred to as the 5-9 fraction, is quickly frozen on dry tee and stored at -80C. The metabolic activation mixture for each experiment consists of, for 50 a1: 5.0al of 59 fraction 1.0 al of MgCl, (0.4 1) and KGL (1.65 ) 0.25 a of glucose-6-phosphate (1 4) 2.0 a1 of MDP (0.1%) 25.0 al of sodiun phosphate buffer (0.2 , pi 7.4) 16.75 al of sterile 8,0. The amount of 5-9 fraction delivered to each plate 1s 50 pl. Plate Incorporation Asssy Prior to testing, the test article fs serially diluted from an tniclal stock. The article fs usually tested over a minimum of six dose levels, the highest nontoxic dose level being 10 mg/plate unless solubility, mutageatetty, or toxicity dictates a lover upper lint. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 1/plate. Occastonslly, liquids are tested; the sample Ls moc diluted, and various aliquots are used. All assays are repeated at least once on a separate day. The plate facorporation assay is perforsed in the following vay. To a sterile 13 x 100-m test tube placed fn a 43C heating block ve add: (13 2bl.o0t0iaa,1 aonfd00..6075 a=garhicsotnitdaiinneing 0.6% NaCl, 0.05 ut (2) 0.05 al of indicator organisms (about 10 bacteria) 7 005284 (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 thea poured on plates containing about 25 81 of minimal glucose agar. After the top agar has set, the plates are incubated for 48 hours at 37C. The number of his' revertant colonies 1s counted using a BioTran II automated colony couater 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 ul of the solvent used for the test article. For positive controls, we test each bacterial culture with the following mutagens using steps (1), (2), @), and (4): MSo1d0i0u.s azide for the base-pair substitution mutants TALSIS and 9-Antnoacridine for the frameshift mutant TALS37. 2-Nitrofluorene for the frameshift mutants TALSIS aod TAS. m2e-tAanbtohlriacadancetifvoartiaolnl. tester strains, in the presence of Saccharomyces cerevistae D3 The yeast S. cerevisiae D3 is a diploid microorganisa heterozygous for a mutation leading to a defective enzyme in the adenine-metabolizing pathway. When grown on medfun containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated fron the heterozygotes by mitotic recombination. The frequency of this recoabinational event may be increased by incubating the organisms with various catcinogenic of recoabinogenic agents. The recombinogentc activity of a cospound or its metabolite is deternined frou 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% MgS0,, 0.15%KH PO, 0.45% (NH,),S0,, 0.35% peptone, . 005285 0.5% yeast extract, and 2% dextrose 1s fnoculated 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 45 follows. The overnight culture Ls centrifuged and the cells are resuspended 7.4). Toa at a concentration of sterile test tube are 108 cells/al added: tn 67 m4 phosphate buffer (pi 1.00 al of the resuspended culture 0.50 al of either the metabolic activation mixture or buffer 0.20 ul of the test chenteal 0.30 al of butter. Several doses of the test cheatcal are tested fn each experiacnt, and appropriate controls are included. The suspension mixture is tacubated at 30C for 4 hours on a roller drus. The sample is then diluted serially fn sterile physiologic saline, and 0.2 ul of the 10% and 102 dilutions is spread on plates contatntng the same ingredients as the broth plus 2.0% agar; five plates are spread with the 10 dtlution and three plates are spread with'the 1075 dilution. The plates are tacubated for 3 days at 30C, followed by 1 day at 4C to enhance the development of the red pignent indicative of adenine-deficient homozygosity. Plates contatning the 107% dilution are scanned with a dissecting microscope at 10 magnification, and the number of mitotic recombinants (red colonies or red sectors) is recorded. The surviving fraction of organisms is determined from the total mumber of colontes appearing on the plates of the 1075 dtlutton. Statistical nalyats No statistical analysis vas perforned for any of the assays. The results of the plate ncorporation assay are a tabulation of the nuaber of colonies appeartag on the plates. A positive response is indicated by a reproducible, dose-related increase fn the nuaber of histidine-independent colonies pec plate. The results of the S. cerevisiae D3 assay are tabulated by calculating the number of mitotic recombinants per 10% survivors. A positive response in 9 005286 tahbissoluatsesanyumibserinodficmaitteodtibcy raecdoomsbei-nraenltastedperinmcirlelaisleitoefr maosrevetlhlanas3-ffonldthein the relative mmber of mitotic recombinants per 107 survivors. References Aoftmrheaesnr,t.ahBs.irtoeWu.a,mtuitcEa.gmeGin.ns:eGurcnMaeerytc,aibnooJlg.ietnAesds.MialnPdlreorcd,.eriaNvantad.tiHv.AecsBaadorftaSccIhi-..scUescCayalrec3si,insogg3te1ln2us0o5ta1es3n2e and 1972). sSuetsa,genBs.: bacteria N.,A for Wsd.iemtpeElc.etiDoutnre.ssttonsP,ryosctE..emNYaactuo.amsbsiAknciia,ndg. anLSdciiv.eFr. USDh.Aos7oL0ge,een.a2t2eC6se1-r2cf3olr6u5oagce(tnLis9v7a3a)tr:ieon and Amoern,thBe.deN.t,ecFt.ioDn. aLnede,claansdsi.ficaE.tiDounrsotfonm.tegAennsimapnrdovecdarbcaicntoegreinasl. tPesrtoc.sysNtate:a Acad. Sci. USA70, 782-786 (1973). Ames, B. N., J. NeGann, and E. Yamasaki. Methods for detecting carcinogens Hantdatm.utaRgeesn.s31w,ith34t7h-e36S8a(lAm9o7n5e)l.la/mammalian-microsome mutagenicity test. Btreucshincikq,uesD.wiJt.,h ayneadstV.. W.EavMiaryoenr,. HeNaelwthdePveerlaoppemcetn.ts,in63m-t8a6ge(n1i9c7i3t).y screening Kciiegra,tecLt.eD.s,aokEe. Ycaoansdseanksia,tesa.nd P3r.ocN.. AMamte.s.AcaDde.tecSctii.onUSoAf 7m1u,ta4g1e5n9i-c41a6c3tiv(i1t9y74)f.n WeGann, Ju, E. Chot, E. Tasasaki, and D. N. Ames. Detection of carcinogens as mNautt.ageAncsad.inSetih.e USSaAlm7o2n,el9l7a9/-m9i8c3ros(o1m9e75)t.est: Assay of 300 chemicals. Proc. MNSacatlC.aaowRn,eealdlJ.a./Saciainc,droUsB.SoAmeN1.3t,Aeset9s:5.0-9A5sD4estaey(c1t9o7if6o)n3.0o0f cchaeraiccianlosgsensDiasscumsustiaogne.ns iPnrotche NPolrutsenliadanRsU,S Kf.romE.,itsandultBr.Aa.vDi.oleSttoopcrkoetre.ctiSneggrpergoapteirotny.of Mtohle.muGetma:torGepnreotper1t6y7 of 317-327 (1979). 2ninim'tnrSeoarucsachaaancr,iods,y.ceIs-K.a,ecterhaeynvdli-s3Ri.-aneiS.tcrhovHa-otlle-.rn.itGreIonn.sdougGcuetsnineotin.dionf1e00B,iantdo6t3o-tt7c6hegr(e1ea9l6k7c)yo-lnavteirnsgioangevnittsh 10 005287 RESULTS AND DISCUSSION 3K Conpany's Compound T-3609 was screened for mtagentc activity in the Anes Salmonella/aicrosome in vitro mutagenicity assay using the five standard strains of Salmonella typhimurium: TAIS35, TALS, TAIS3S, TASS, and TAI00. The assays were performed in duplicate, both in the presence and sbaeace of a rat-liver metabolic activation system. Acetone was used as the diluent. The microbial mitagentcity testing of this sample was performed on 26 July and 3 August 1986. Dose levels ranging from 10 to 5000 pg/plate were used for both assays (Tables 1 and 2). No dose-related increases in the number of histidine-independent revertants vere observed in either assay. Compound T-3609 was also tested for recosbiuogenic activity fa the yeast Saccharomyces cerevisiae D3 assay for mitotic recombination. This assay vas perforaed on 27 July and 10 August 1984, both with aad without metabolic activation. A dose raage of 0.05 to 5% vas used for both assays (Tables 3 and 4). No dose-related tacreases in the mumber of mitotic recosbinants per 105 survivors were observed in either assay. In conclusion, Compound T-3609 was reproducibly monmutagentc and nonrecoabluogentc when tested according to these procedures. 005288 n Tabt1e IN VETO AssaYs ITH sAusOWELLA TYPHINRIDY anon 1-3609 Bxpectacat Dace: 26July 1984 Compan MAcettiavbaotlitoen peCrAodmPdpleoaducned EATS HiTAsItSiYdine RIevAerStasta TpoOr EPlate EOI ` HeKgaetttovneeControl +- EFou mslosow 64 moBoos 4poBu o2w FoSsotdeiivueACeotndeeeols Shminoscridioe 0Lie 97 do 236 25 was ' Fo 2kuaitcheontihnoere : 1s + i 10m0W5oou5e0s5 561%7 25383 132 u16ss :+ 2d5 omonos w7 w7 i Compound 1-3609 2 -: - o0 e 10 W3omoowmoes5 ou17 wBmoo3sw9 oooNmw w1Bomam uo1m0s ` >Fg :Swe500 EE] 3moma Zoom ow s3 omoau oBa o1wme 1lme 76 sou on wom oa % ++ 510 : 100 BBTooosuwso9 wsouos ommwo om1B%3 n3a0m ue 1wommm P i w500 swe oTonm sou o8s ouowow 1do3aMu wom ox Bo7aoonuss oml1o i | Tube 2 | TT[c --o r-- aemm | IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM coment wAeestaibvoatstieon poprbripgiean--ce HIST BRoAScHtatne SRovBertEancsSporhPleatem STeipsnies Control Smoak mTohoon8 od7 0sonw dow miadm om arCrtehemCtoyncoent - Lie wom 4m oa 5 Daiiwetaiwre p: Sii 1W0B1oU0e7R4 anR3eoaB507 wde w ' :i BBs odwoaw mnoowe CompoundT-3609. - To 1o10w 1g %BBBooEA2ho%o8 h88od 1oo6sNnRE1o6b 2NoR7N Ru2oN9 Em167 163 .!! g2 DIome BBAATTA3 wSowERox RonEe a 2 r Iz ooR wB3ow5s4oh05o5d%nRBoBDnWnYEGEmue TD Loommk wP I hoRELE od E R bonRoEwREoboEay EoRERm] Co i | . ote 3 IN veri ASSAYS ITH sucousonces crrevisIAR 3 coro 1-3609 Expertaens Date: 27 July 1984 Metabollc ConPceerncternattion CeSluleavtvpteogal Survivors RecMoponerbritone]aate Rewciocmoitntaegts bec 105, cospousa Rettcion 0 ahd 8 El sufvivos ' NeHgeaattioven Contzol :- pi4)2 10100 5s n3u Po1Ss,tt2,eitDvielgemctpaonocepmreobttuisncane == 00.0002s5 uwsr 1100 wi we5.3 . : 503 +] 100 wm Compound 1-3609 >z -:z 0oo.sr05 : 3 poiss 101000 10 ue 100 `7 11 7i 35 i 8 :5 us 1% `1 | 88 + 3 0oo.rs05 awwte 1110000 `"5 1n13 :: 15 piots 1100 5 15) Fealculations are expressed ustog tvo stgatttcant figures. i Tate 4 0 IRD asst wim saconmsowtcas coro 1-3609 cearvistay 0) Saperiaent Date: 10 upc 1984 conpouns MMetabe olic ContreoOmrscNeenaecion CesluiravEivhespeal Survivors Recwopemeobreisneaate Reccooomrrpitn1e0a5ges Fie sian . NeTgeartkiovneeConceota :- 327s i10w soonu FoS1s1tt2ee.t3rvetcioCaoinneccpeoootaaeitane T=C 0o2osn : aon piwiro 111000 5woDTam . Conpouna 13609 :-: 0o0.5t05 z? aJiss] 11010000 is 100 iFo E w5a ou i :5 ii [I } +:: 0o50F8 iae 110i00o0 sPFI oomu :: 5v iie 1oo0 Si oomos 8 8a Fonlculacions are expressed tag to stpateicanc Figucen. 8 i