Document Z4o3q0q1LJzRVBOVR9BYkyjqO

harlan STUDY NUMBER 1305602 CELL MUTATION ASSAY AT THE THYMIDINE KINASE LOCUS (TK *) IN MOUSE LYMPHOMA L5178Y CELLS WITH <cC604 AMMONIUM SALT REPORT STUDY COMPLETION DATE: JULY 14, 2010 SCt6ud0y4Naummmboenri1u3m0s5a6t02 Rego Page of 44 1 COPYOF GLP CERTIFICATE ] [------ `GLP-Besichemiinogigu1n09 og.f/GSLCtPhaCotemeplbmieaanncte = HESSEN B Seo Ee GL npekiponeue truOeomcoahaEgrenng Assesmem nt of cnofoordshe s y wihGo SLPHsEcsEi = 8 coesnsy] ror ese Tr B [ES ------ [I ---- DtJi ekTwit0o2m0oe2fn0t8ceio feoOE LY Ccbar sshd dP , desorGLtCoemp eps dls RE ------ e ane foRetetrem.hwiC resoaspeno,tidenren30e.e)war 2000(3 AYouN %) 3,X Nw W BeS (amend Arse GLO nesnd ssfGL. orig Aut SCt6ud0y4Naummmbeorni1u3m05s6al0t2 Report 2 CONTENTS 1 COPY OF GLP CERTIFICATE 2 CONTENTS 3 PREFACE 31 General 32 Responsibilties 33 Schedule 34 Project Staff Signatures 35 Good Laboratory Practice 36 Guidelines 37 Awchiving 3:8 Deviations from the Study Plan 4 STATEMENT OF COMPLIANCE 5 QUALITY ASSURANCE UNIT STATEMENT 6 SUMMARY 7 OBJECTIVE 70 Aimsof the Study 72 RelevofathneTceestSystem 8 MATERIALS AND METHODS 81 Testhem 82 Controls 82.1 Solvent Controls 822 Posilive Control Substances 83 TestSystem 83.1 Reasons for the Choice of the Cell Line L5176Y 84 Cel Cultures 85 Mammalian Microsomal Fraction 9 Mix 85.1 9 (Preparation by Harlan CCR) 852 S9Mix 86 Pre-Teston Toxiclty 87 Dose selection 88 Experimental Performance 88.1 Complete Culture Medium 882 Selective Medium 883 Saline G Solution 89 Size Distribution of the Colonies 810 Data Recording 8.11 Calculation and Processing of the Data 8.12 Acosptabilty of the Assay 8.13 Evaluation of Results 8.14 Statistical Analysis 9 RESULTS AND DISCUSSION 9.1 Conclusion 10 REFERENCES 11 DISTRIBUTION OF THE REPORT Page 3of44 2 3 5 5 5 5 6 6 6 7 7 8 9 0 2 12 2 3 1i4 14 14 15 15 15 6 16 16 6 7 18 18 18 18 19 19 20 21 21 2 23 2 2 2 C`S6tu0dy4Naummmboenri1u3m05s6a0t2 Report CONTENTS CONTINUED 12 ANNEX|: TABLES OF RESULTS 12 Pre-Experiment 122 Main Experiment 12.21 Experiment, Culture | 12.22 Experiment, Culture I 12.2.3 Graphical summary of experiment | Culture | 12.2.4 Graphical summary of experiment | Culture I 12.2.5 Experiment I, Culture| 12.26 Experiment I, Culture I 12.2.7 Graphical summary of experiment l, Culture | 12.2.8 Graphical summaroyfexperiment , Culture I 13 ANNEX II: HISTORICAL DATA Pagedofas 2 2 228 31 Ea 3 36 39 a2 a3 a 3 PREFACE THe:31 General Sponsor Stay Monitor Test Fast -- iaHttohesCTeAhyLmLiydAmipnreMoKmiOanaLNseE1L7So8AcYLusTco(lTsK) VS2i0Ol0Le2V1LAoBYmoSbatOrLedEi1XaI)2S0 SpA or aa arianCyttet Cell Research GH (innHsaorenloanRLCooCpmpRe)aerinsissen 19 3.2 Responsibilities Sty Doctor EE Deputy Stuy Director J Management J---- Head of Qualify Assurance Unit JETER-- 3.3 Schedule Experiment Sarg Dae Experimental Completion Dat Janay 05,2010 February 22, 2010 3.4 PrSotaj ffSe ignc atut res Study iector ------ 3.5 Good Laboratory Practice `Chemikaliengesetz* (Chemicals Act) of the Federal Republic of Germany, "Anhan1g 5002, and arena erin ted dy02,2000 (BGI . 1146) OECD Princes of Good Laborstary Pract, a eid n 1997 (COT BB Final] 3.6 Guidelines c`SCt8ud0y4Naummbmeorni1u3m05s6al0t2 Report 3.7 Archiving Harlan CCR will archive the following data for 15 years: Raw data, study plan, report, and a sample of the test item. No data will be discarded without the sponsors consent. 3.8 Deviations from the Study Plan `There were no deviations to the study plan. Page7 of 44 `Study Number 1305602 Report C604 ammonium salt Page 8of 44 4 STATEMENT OF COMPLIANCE Study Number Test tem: 1305602 C504 AMMONIUM SALT Study Director: Tite: EE Cell Mutation Assay at the Thymidine Kinase Locus (TK) in Mouse Lymphoma L5178Y Calls with cC604 AMMONIUM SALT This study performed in the test facility of Harlan CCR was conducted in compliance with Good Laboratory Practice Regulations: `Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1* (Annex1), dated July 25, 1994 (`BGBI. | 1994", pp. 1703), last revisiondated June 27, 2002. and amended version dated July 02, 2008 (`BGBI.", p. 1146), `OECD Principles of Good Laboratory Practice", as revised in 1997 [C(97)186/Final]. There were no circumstances that may have affected the quality or integrity of the study. Study Director Harlan CCR S5t5ud0y4Nuammbmeorni1u3m056s0a2t Report Pagedot 44 5 QUALITY ASSURANCE UNIT STATEMENT Study Number Test tom 1a0s602 504 AMMONIUM SALT Study Director Tite: RR] CaaltlheMuTthaytmioindiAnsesKaiynase Locus TK") iinhMo6u0s5e0L4yAmpMhMoOmNaILU5M17S8AYLTCells Trehseultgsenaerrealrepfoarctielditso tahndreaspcontsiibleofpeHrasrolnananCdCtRhe aMraenaignespmeecntted periodically and the bSytutdhye pQruoacletdyurAesssuwrearneceinUsnpiet.ctTehdepedraitoedsicaarle.giTvheensbteuldoywplan and hs report were audited g1 QPAShUaseIsnsapencdtioDnasteAsudits DSattuedsMyaofDniRaregepcetomorertnsatntdo t1h0e Stuy Pan November 18,2008 | November 18,2009 1% Amendment o StudyPlan:| November 25, 2009 November 25, 2000 2TM Amendment to Study Plan: December 07, 2009 December 07, 2009 3 Amendment to Study Plan: July 08, 2010 Process Inspection ApPprleipcaartatiioonn for Application and sanary27,2010 Preparationfor Applicaton: |_ Februaty 17, 2010 Report Maayrc1h43,1,20210010 July 09, 2010 JFaenburaurayry2717,, 22001100 Vsayren141,,20210010 This statement is confi that ths present report rfiecs the raw data Head of Qualiy Assurance T= ootQED SCt6ud0y4Naummbmeorni1u3m05s6a0lt2 Report Page 100f44 6 SUMMARY The study was performed to investigate the potential of cCS04 AMMONIUM SALT to induce mutations at the mouse lymphoma thymidine Kinase locus using the cel line L5178Y. `The assay was performed in two independent experiments, using two parallel cultures `each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. "The highest concentration (3640 g/mL) applied in the pre-experiment was chosen with regard to the molecular weigh of the test item corresponding to a molar concentration of `about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and precipitaotfithoen test fem. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentraotfitohen test tem Appropriate reference mutagens were used as positive controls and showeda distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. "The tested concentrations are described in table 2 (page 17). The evaluated experimental points and the results are summarised in table 1 (page 11). Conclusion in conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell ine LS178Y in the absence and presence of metabolic activation. Therefore, cC604 AMMONIUM SALT is considered to be non-mutageric in this mouse lymphoma assay. SCtu6d0y4Naummmboenri1u3m0s5a60l2 Report Page 11of 4 Tabl1e: Summofarersulyts -- we rd em | ma - Cemsa omeana rshod| |oaoats || ccleess|| red [EC xpotmanii ahesiment ||-- weO r S O A wer | poent.tcoonrowini|i|s wtol5{ | w [aeenr asm s | 7a |+ me | a0 | mm0o HoTetesittooonnn || aasm0ss0o ||| assetei|nvas3wr0eotonasa"um | wco |utrw womtcer|m enam frestton | isso] | conn vorconeuns' | eurokcoo nieds' socomr| ST aT Jrw peoss..ca coomtraowicoAr|a 3o0f mwes [mwer lwowee |[awayT|omems 1s | [ofotsftoton | aasl.l|mesos iew awme||e we ||r mmwe |sonm f[feeatrteeon stie1m| wT_o_oogpv)o +|| esftaosss | Hvitem | ;woesw ||[wwwaea||| mw m |ww awzw [[fErSepttectioommrentiiatirrmesnso || Tcooenrsseottco| nnT"m [cwoetevawsrTeomtscrn"Tw [PCorcront--i | wa0l) -|| r 22 a sw mme | mmea || m em wa fresttom | seal | wre wwe | mr a | am fofslotstttoonn ~ 1 vmasse | vmeee mvew masw || mrs |m|maemewomn fefetstttoonm | sowosfl | ceur asretw coia | ciiai|ssvwtcs orii ad [[SEocpeiomerniatheora || eronwtm | Tw [we me[mws| = Jpoornccoomuosmtncuoann|c|on assolf || "mo iase |mo0|| mwie || wsre |woam fl[osstttioomn ~~" 1 Teenoll | woees wr w |ae| w[eesy |w aw |mwm [[ffoosstt eomm Womoopls+|| wwrs aamm woew || wmar || wm es |mom [ffaotsttteomm [e50705005]) ++|| ccuuttie vsartosccoonnttdr" cw ornvm as otninom `Threshol=d numobfmuetarnt coloniesper 10 ceolfelacsh solvent controlplus 126. # culture was not continued due to exceedingly severe cylotoxi efects (5) precipitation istotothe unaided eye Thevaluesprintedinbolda judgedas invalid, sincetheacceptance crlera (age 21) are not met (RTG < 10% in both parall cultures). `2S0t6ud0y4Naummmboenri1u3m05s0al0t2 Report Page 120144 7 OBJECTIVE 7.4 Aims of the Study The assay was performed to assess the potential of the test item to induce mutations by tmheeacnesll olfintewLo5i1n7dYe.penIndetnhte mfrosut seexpleyrimmpehnotmtahthtyrmeiadtimneentkidnuraasteiolnocwuassex4pehroiumresnwtisthusainndg without metabolic activation. Experiment II was performed with a treatment time of 24 hours in the absence and 4 hoursinthe presence of metabolic activation. 7.2 Relevanceofthe Test System Invitro methods are valuable to accurately control the concentration and exposure (me of cells to the test item under study. However, due to the limited capacity for metabolic `activation of potential mutagens an exogenous metabolic activation system is necessary. This in vitro test is an assay for the detectionof forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TKTM cells to TK* mutants. In addi- tdiaonm, aegveidteonctheehTaKs bloeceuns oabntdaiandejdactehantt sgmeanlelsTK(1*). cGoelonneieasnmdacyhrreosmulotsformoem mcuhtraotmioonssomaarle considered as an initial step in the carcinogenic process (2). The L5178Y/TK" Gels are exposed to the test tem with and without exogenous metabolic `maoctniivtatoiroen.d fFoorltlhoewilnogsanofefxupnrcetsisoniaolnTtKimeentzhyemdee.scendants of the treated cell population are TK catalyses the conversion of TFT (Trfuorothymidine) to is cytostatic and cytotoxic trfluorothymidine-monophosphate derivative. Therefore, cels deficient in TK due to a forward mutation are resistant to TFT. These cells are able to prolfrate in the presence of TFT whereas the non-mutated cels die. However, the mulant phenotype requires a certain period of time before i is compllaly expressed. The phenotypic expression is achieved by allowing exponential growh of the cel for 48 hours. The mutant frequency is determined by cloning known numbersofcells in 96-vell plates containing the selective agen to detect mutant cells, and in 96-well petes wihout Seleciive agent to determine the surviving cells (cloning efficiency). Afer a suitable period cofoltoinmieesthceorcroelcotneidesfoarrceeclousnutrveidv.alM(u4t,a5t.ion rates are calculated from the number of mutant In order to establish a dose effect relationship at leat four concentrations of the test fem are tested. These concentrations should yield a dose related toxic effect. The highest concentration level should produce 2 low evel (10 20 %) of cell cuits growth. Reference mutagens are tested in parallel to the test fem in order to demonstrate the sensitivity of the test system. SCt8ud0y4Naommmboerni1u3m05s6a0l2 Report Page 130144 8 MATERIALS AND METHODS 81 Testitem Internal Test item Number: S1069711 The test item and the information concerning the test item were provided by the sponsor. enti: Batch No. Molecular Weight: Purity: Stabilty in solvent: Storage: Expiration Date: <C504 ammonium salt (dry salt) 156139 357 gimol 975% Not indicated by the sponsor In the refrigerator (+2 t0 +6 C) December 31, 2020 On the day of the experiment (immediately before treatment), the test item was dissolved in defonised water. The final concentration of deionised water in the culture medium was 109% viv. "The osmolarity and the pH-valus were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation | Solvent control T| C604poavmmonium salt Osmolarity mOsm | pH-value 212 I 2% 763 | 762 S0t5u0y4Naummmboenri1u3m05s6al0t2 Report Page 14of44 82 Controls 8.24 Solvent Controls. Concurrent solvent controls (deionised water) were performed. 8.22 Positive Control Substances 8.22.1 Without metabolic activation Name: Supplier: Charge: Expiration Date: Puy Dissolved in: Final Concentration: Methyl Methane Sulfonate (MMS) Sigma, 82024 Taufkichen, Germany 76296) May 2010 > 99% Nutrient medium 1139.50 ppgg/mmLL == 00..1182 mmMM ((eexxppeerriimmeenntt IIl "The solutions were prepared on the day of experiment. 8.2.22 With metabolic activation Name: Supplier Charge: Expiration Date: purty: Dissolved in Final Concentration Cyclophosphamide (CPA) Aldrich Chemie, 89555 Steinheim, Germany 097K1311 March 2011 298% 09% saline 30 gimL = 107 pM 45pg=1m6.1LuM "The dilutions of the stock solutions were prepared on the day of experimen. The stability of both postive control substances in solution is proven by the response inthe expected range. mutagenic StCu6d0y4Naummmbearni1u3m05s6a0t2 Report Page 150f4 83 Test System 8.3.1 Reasons for the Choice of the Cell Line L5178Y TLh5e17L85Y17c8olYscaalrlenceharhaacstesruicsceedssbfuyllayhbiegehnpruoslaifderiantiionniartoee(xdpoeurbilminegnttsifmoer 1m0a-ny12yeahrsi.n `stock cultures) and cloning efficiencies of untreated cells of usually more than 50 % both necessaryforthe appropriate performanceofthe study. Thecells have a stable karyotype `with a near diploid (40 + 2) chromosome number (2). AGelllowcumletturaebsoliacs amctaivniytyocfhceemlilcsalusnddeervienvliotproaconmduittaigoennsicis paotdeinstaiadlvaonntlaygeafotferasmseatyasbowliitch `laecatsitvatpiaorntibayllythebemaamcmhaileiveadn obrygansiuspmp.leHmoewnteivnegr,tmheetacbeolliccualcttuirveastiowniotfh clhievemricmailcsrcoasnomaet preparations (S9 mix). 84 Cell Cultures Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cellsforone day in RPMI 1640-HAT medium supplemented with: hypoxanthine 1.0x10M* atmhiynmoipdtienrein 12..60xx11007MM TRhPeMIin1c6ub4a0timoendoifumthecocnatalisniinngHAT-medium is followed by a recovery period of 2 days in hthyypmoixdainntehine 11..604<1100MM (Alctoemrpltehtse. ciunlctuurbeatmieodniutmh)e, LS178Y cals are retumed fo nomal RPMI 1640 medium Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank eofxpeHrairmleannts.CCBRefoarlelowfrienegzitngh,e eraecpheabteeldchusweasofsctrheeenseadmfeorcmelylcocuplltausremabactocnhtamininamtainoyn arenmdaicnhescikmieadrfboerckaaursyeootfypteh straebplryo.ducCiobnesecqhuaerancttleyr.isttihoes poafrthaemectoelrss of the experiments GTehramwaendy)stoicnk RcuPlMtIures16a4r0e pcroomppalgeatleedcuilntuprleastmicedfilausmks ((sGreeeinepra,ge721683).2 FTrhieckecnehlalsuseanr,e subcultured two times prior to treatment, The call cultures are incubated at 37 + 15C na humidified atmosphere with 4.5% carbon dioxide and 95.5 % ambient air. St0u6d0y4Naummbmeorri1u3m05sa0l0t2 Report Page 16.0144 85 Mammalian Microsomal Fraction S9 Mix 8.5.1 S9 (Preparation by Harlan CCR) Phenabarbital-naphihaflavone induced rat fiver S9 was used as the metabolic activation system. The S9 is prepared from - 12 weeks oid male Wistar HdCpb:WU rats (Harlan Laboratories GmbH, 33178 Borchen, Germany). weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. phenobarbital i.p. (Desitin; 22335 Hamburg, Germany) and pda-ynsa.phTthhoefilaivveornsewep.roe. p(rAledprairche,d 82945h5o5urSsteaifnthreitmh,e Gleasrtmraenayi)meenatc.hTohne tShOreferaccotniosnescuwteirvee produced by dition of the iver homogenate wilh a 150 mM KCI solution (1+3) followed by centrifugation at 9000 g. Alquots of the supematan! were frozen and stored in awemepko.ules at -80 C. Small numbers of the ampoules were kept at -20C for up to one The protein concentration of the S9 preparation was 31.7 mg/mL (Lot. No.: 021009) in the pre-experiment and 32.7 mgimL (Lot. No.: 210808) in experiment | and 35.0 mg/mL (Lot. No.: 070110) in experiment Il. 8.5.2 S9 Mix An appropriate quantiy of S9 supematant was thewed and mixed with S9 cofactor solution to ive a fina protein concentratoifon 0.75 mg/mL in the cultures. Cofaciors were added fo the S9 mix to reach the following concentrations: 8mM MgCl, 33m KCI 5mM glucose-6-phosphate 4mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored In an ie bath. Tthheefi$n0almtiexstpmreepdairuamtiwonaswa5s%pe(vr/fv)o.rmed according to Ames et al. (3). The concentration in 8.6 Pre-Test on Toxicity A protest was performed in order to determine the concentration range of the mutagenicily experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test em and i the solvent control without metabolic activation. 1x10" cals (3x10 cals at the beginning of 24 h treatment) were exposed to each concentration of the test fem for 4 and 24 hours without and 4 hours wilh metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treetment the call were washed twice by centriugation (4259, 10 min) and resuspended in "saline G. Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growin period. The cell density was determined immediately afler treatment and a each day of the growin period and adjusted to 3cxu1l0turceesllwsa/smLc,alicfulnaetceedssaatryt.heTehned roeflatthievegrsouwstphenpseiroinodgarcocwotrhdi(nRgStGo)thoef mtheethtordeaotfedCicetlel and Spector (1), St0u5d0y4Naummmboenri1u9m05s6a0l2 Report Page 17of44 87 Dose selection fAocrctohredimnugtattoitohneerxepseurlitmsoenftsth.e pre-esta least four adequate concentrations were chosen b"yThtehheigshoelsutbictoyncoenttraotixonoifshtochueltdesbtet1e0m.mM, but not higher than 5 mg/mL, unless limited eRxSpGeri(mReelna)tibvleoSwu5s0p%ensairoencoGnrsoiwdtehr)edotroriRc.TIGnc(aRseeloaftitvoexiTcoetfailecGs,rotwhtehh)igvhaelsutetsest(mtaeimn acopnpcreonxtirmaattieolny 1of0-t2h0e %.main experiment should reduce the RSG or RTG valus to 2T4hehptrree-aetxmpeenrt)imoefntmewtaasboplecrfaoctrimveatdioinn.thTeesptrefseemncceon(c4ehnttrraetaitomnesnbt)etawnedenab2s8e.n4cpeg(/4mhL aanndd 3640 pg/mL wero used. The highest concentration n the pre-experiment was chosen with treogcardeftfoectthse pwuerrtey o(9b7s.e5rv%e)danatd t1h8e20molyegciumlLarawneidghatbo(3v5e7 ign/tmh)e oafbtsehncteestolfemm.elSatbroolnigc eafctfievcattsiooncc(u4rhretdreaattm4e5n5t)pIgnitmhLe parnedsenabcoeveo.f meFtoalbloolwiincgacctoivnattiinouno(u4shtorueastmternetatm(e2n4t)hotuorxisc): strong toxi effects were determined at 227.5 pgimL and above. 2T4hehotuesrts)mebedfiourme twhaestecshtectkeemdwfaorsprreemciopvteadt.ionAfaetrth4eheonudrsoftreeaacthmetnrteaptrmeecnitiipaeiroinodof(4thoer mteesttabiotleimc awcatisvatoibosn.erFvoeldlowaitng9c1o0ntpigniumoLus2tnrdeatambeonvtenoInpretchiepiptarteisoennwcaesannotded.absence of Tmhaexriemwuamsconnocernetlreavtainotnsohfitthoefptrhee-epxHpevrailmueentor the osmolarity of the medium even at the The dose range of the main experiments was limited by the occurrence of toxic and mpreetcaibpoiltiatci.on.actiTvhaetionindaivnidduatlhecosnceecnoinrdatieoxnpseroifmetnhte wfirhstouetxpmeerliambeolnitc.wiatchtiavantdionwitwheoruet generally spaced by a factor of 20. A narrower spacing was used in the upper concentration range to cover the mit of solubility or cytotoxic effects more closely sTtoarotevderwictohmmeoprreotbhlaenmsfowuirtchonpcoesnstirbaletiodnevsiations in toxicity both main experiments were Table 2: Doses applied in the Mouse Lymphoma Assay with cC04 AMMONIUM SALT - concentaloEnxpseirinmepntgi| ml [[wvihsiooumntsromi | s60 | 1v1a3s8 |[a22m7s5 || 4a5w5 ||o sto ||o n1e36r5 | Experiment I without S9 mix | 28.1 563 | 1125 | 2250 | 3375 | as0 | *4h treatment; 24 h treatment F1o3l8l5owpign/gmLt.he(eprxipnrteesdsiionnboplhdasleettoefrs4)8ihnoeuxrpsetrhiemecnutltu| rweisthaoutthemectoanbcoelnitcraatcitoinvsaotifon91a0ndanadt SCt5u0dy4Naummmboerni1u3m05s6a0l2 Report Page 180144 1365 pg/mL with metabolic activation were not analysed due to exceedingly severe toxic aefnfdect6s7.5Ipngt/hmeLs(eccuolntudreex1)paernidme9n0t0 tphge/mcLu.ltwuirtehs maetta4b5o0lipcga/cmiLvawtiitohnouwtermeetnaobtolciocntaicntiuveadtifoonr the same reason 88 Experimental Performance In the mutation experiment 1x10" (3x10 during 24 h exposure) cells/flask (80 cm" flasks) suspended in 10 ml RPMI medium with 3% horse serum (15 % horse serum during 24 h oerxpaobssuernec)ewoefremaelxapbooslecdatcoivvaatriioonu.sAcfoenrce4nthra(t2i4ohnsinofthtehesetecsotnditeemxpeeirtihmeernitn)tthheeptreestsefnocme w"saaslinreemG*o.veSdubbsyscqeunetnrluygatthieocnal(l4s2w5erxeg,re1s0usmpine)ndaendditnh3e0cemlilscowmeprleetweaschuletdurtewimceediwiutmh and incubatedfo an expression and growth periodof43h. TThhee creelllatdievenssiutyspweanssiodnetgerromwitnhed(ReSacGh) dofaythaenrdeaadtjeudstceeldl tcoul3txu1re0s wcealslsicmall,ciufanteecdesbsyartyh.e dacacyor1difnogldo-tnhcreeamseethiondcoafl invumebaenrdSmupletciptloired(1b)y the day 2 fokdncrease in call number After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4Gxe1r0m'anyc)e.llsThien vsiealbielcittiyve(comneidniguemffi(csieenecy)bewlaosw)dewtietrhmiTnFedT b(ySesrevead,in6g9a0b4o2utH2eicdaellbierpge,r w1.e5l iCntoinmi4.c5ro%tfeCr OpJi9as5.(%s5amweatmeredsiautumrawtietdhoauitr TfFoTr).10T-h15pldaatyess.weTrheenintchuebaptleadteasl w3e7re evaluated 8.8.1 Complete Culture Medium RinPvMiIrog1e6n4)0 m(e3d%iumHS(GIdBuCrOi,ngin4vrohgoeunr) sturpepaltememnetn,ted1w%ihof15 1%00horUsMeOsDarupmgi(HmSl, GPIeBrCiOc,i in/Streptomycin, 220 ygiml. Sodium-Pyruvate, and 0.5 0.75 % Amphotericin used as antifungal (6). 8.8.2 Selective Medium RPMI 1640 (complete culture medium)byaddition of5 ug/mL TFT. 8.8.3 Saline G Solution The "saline G* solution was composad as folows (per lve): KNealCl 840000mmgg Glucose 100mg KNHaHPPOO,X7H,0 pH: 72 219500mmgg. SCt5ud0y4Naummbmeorni1u2m05s6a0t2 Report Page 190144 89 Size Distributionof the Colonies Colonies were counted manualy. In accordance with thei size the colonies were catlaaslslifcioendceinnttorattwioongsrooufpst.h Ttheestcotlamo.nyCsritzeerdiiastfroibduetitoenrwmaisn dceotleornmyisneidz iwnetrhoe tchoentarbolssolauntde csoilzeoonfiesthe(tchoeloopntyic(amlordeentshiatny 1o/f3 tohfea swmeallllfocrollaorngieescoilsongieense)raalnldy thhiegohpetrictahladnentshietyoopftitchale density of the large ones). 810 Data Recording "fTohrem,daitnaclguedninegraetxepderwiemreentraelcogrrdoeudpisntwihterhatwhedatteas.t Ttheme,renseuglattsiwvee,resoplrveesnet,nteadndinptoasbiutilvaer control. The survival ate and vibilty were determined based on the Poisson distribution mmuettahtoido.n TfhreeqzueernocytewramsofdetrhievePdoifsrsoomn tdhiestrcilboutniionng, e[fPf(i0c)]ienmceythuondd,erwassslaucsteivde (7c,on8d).itiTohnes compared fo the corresponding viabilty under non-selective conditions. psmns _-- 8.11 Calculation and Processingof the Data I Bi RThe data listedinthetables (Annex I) are calculated and processedasdescribed below. (24 htreatment): [Wainer ir (100000) (cell nuamt4b 8h/eifcrelnuam t24b h>e 300r 000 7 (24 h treatment): (100000) (cell number at 48 h if ell number at 24 h > 300000 | :>corresponding control In(mean number ofemptywelsperpl/a96)t/ceells seeded cloning efficiencxy 1/0clon0ing effiofccoirreespnoncdiyng per | | | mutant colonie/s 10 cells: (small mutant colonies (column 14) + large mutant colonies (column 15)) `small mutant colonie/s 10 cells: |(-In(mean numobfweelrs not containing small colonies col. 10/ | 1 96 /ce) lls survived Table "Mutagenicity datalpart I. col. 11) x 10 | large mutant colonies/ 10cells: | (-In{mean numobfweelrls not containing large colonies col. 9/ 96) / cells survived Table "Mutagenicity datalpart I", col. 11) x 10 [roses] `number of mutant colonies per 10 cels of each solvent control cloning effcency determined after he expression period fo | CSt6u0dy4Naummmboerni1u3m05s6a0t2 Report Page 2144 8.12 Acceptability of the Assay rAecmuotmamteinodnataisosnasy oifs tchoensIiWdGerTe(drela.cc9e,p1t0a,bl1e1,if12i,t m1e3)etasretchoensfiodlleorweidn)g: creria (the current 1. All plates, from either the doning efficiency or the TFT resistance-testing portion of the experiment are analysable. 2. The absolute cloning efficiency at the time of mutant selection (CE) of the solvent controls s 65-120 %. 3. The total suspension growth of the solvent control calculated by the day 1 fold- increase in call number muliplied by the da2y fold-ncrease in cell number is 8 - 32. Following 24 h treatment the total suspension growth is 32 ~ 180 (re. 13). 4. The range of the solvent control mutant frequency is in the range of 50-170x 10 cols 5. The positive controls (MMS and CPA) should yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF IMF) of at least 300 x 10 cals. At least 40 % of the induced mutation frequency (IMF) should be reflected in the small colony MF. Altamaively, the positive controls should induce atleast 150 small colonies 6. The upper mit of cytotoxicty observed in the postive control culture should be the same as for the experimental culures (Le. the relative total growth ~ RTG- should be greater than 10 % of the concurrent selective control group). 7. The highest concentration of th test fem should be 10 mM or 5000 g/mL, unless limited by toxicty or solubilty of the test tem. If toxicty occurred, the highest concentration shouid lower the relative total growth to approximately 10 0 20 % of survival. If precipitation is noted, the highest analysed concentration should be the lowest concentration where precipitationis observedby the naked eye. 8.13 Evaluationof Results Aexcteesetdsiteam tihsrecslhaoslsidfioefd a1s26muctolaogneineisc piefrth1e0inedlucsedabmoutvaetitohne fcroemqeusepncoyndrienpgrodsuoclivbelnyt control, Arelevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered lo be reproducible if it occurs in both parallel cultures. However, in the evaluation of the test results the historical variably of the mutation rates in the solvent controls of this study are taken into consideration. Results of test groups are general rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fuflled S itTi ro inn oT alOeSa all,s Solisod45sSSeSour Sle hin Sm RSs 8.14 Statistical Analysis A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT*11 (SYSTAT Software, Inc., 501, Canal Sy Lr Dh m Imsbepi by Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant common considered together. SrcSTITT om] CEA oR Ae Sn De experiment I, culture | with S9 mix | experiment I, culture Il with S9 mix = our Et oun | 0.081 | 0.103 SCt6u0dy4Naummbmeorni1u3m05s6al0t2 Report Page 230144 9 RESULTS AND DISCUSSION The study was performed to investigate the potential of cCB04 AMMONIUM SALT to Linsd1u7c8eY.mutations at the mouse lymphoma thymidine Kinase locus using the cell line `The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without fiver microsomal activation anda treatment period of 4 h. The second experiment was performed in the `absence of metabolic activation with a treatment period of 24 hours and in the presence of `metabolic activation with a treatment period of4 hours. `The main experiments were evaluated at the folowing concentrations: Experiment: without $9 mix: with S9 mix: Experiment I 56.9; 113.6; 227.5; and 455 pginL. 56.9; 113.8; 227.5; 455; and 910 pgimL. without $9 mix: 20.1; 56.3; 112.5; 225; and 337.5 pgimL. with $9 mix: 56.3; 112.5; 225; 450; and 675 pgimL. Precipitation of the test tem visible 0 the naked eye was noted at 455 ug/L and above in the first experiment with metabolic activation and at 450 ygimL. and above in the second experiment with metabolic activation. Relevant toxic effects indicated by a relative total growth of less than 50 % of relative total growth were observed in experiment | starting at 227.5 pg/mL in the presence and absence of metabolic activation. In the second experiment toxic effects as described `above occurred at 225 g/mL and above with and without metabolic activation. The data `generated in the second experiment without metabolic activation at 337.5 pg/mL were not judged as acceptable since the RTG dropped far below the 10% limit in both parallel cultures. The same was true for the data generated in the second experiment with metabolic activation at 675 g/mL. The RTG of the second culture was only 26, the first culture was not analysable at ll due to exceedingly severe cyoloxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments up to the maximum concentration with and without `metabolic activation. The mutation frequency exceeded the threshold of 126 above each Solvent control in the first culture of the frst experiment without metabolic activation at 227.5 and 455.0 pgm. In the second cullure of the fist experiment with metabolic aciivation the threshold was exceeded al precipialing concentrations of 450 and 910 pgimL. However, this increase was not reproduced in the parallel culture under identical conditions and consequently judged as artefact. In the second experiment the threshold of 126 above each solvent control count was again exceeded in one of the parallel cultures at a precipitating concentration of 450 g/L with metabolic activation. `Again, no comparable increase was noted in the parallel culture under identical conditions So this isolated increase was judged as precipitation artefact. Precipitation i a problem in the mouse lymphoma assay since L5T78Y cells grow in suspension. The test item is removed by centrifugation at the end of treatment. At precipitating concanirations the test siteedmimceanntsnottocgoemtphelretweiltyh bteherecemlolvs eudpoatn tcheentirnitfeugnadteidone.ndArobfittrraeraytamemnotunsitnsceofthteheprteecsitpiittaetme are carried over 1 the next steps of the assay leading to imeproducibl artefacts. SCt6ud0y4Naummmbeorri1u3m05s6a0l2 Report Page 24of44 A near regression analyss (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. A significant dose dependent rend of the mutation frequency indicated by a probabilty value of <0.05 was detected in the frst Gulure of the first experiment without metabolic aTchieisvaetiaopnpaarnedntintrtehnedssweecroendcocnuslitduereredofirtehleevfiarnstt esixnpceeritmheenttrewnidthwamsetnaobtolriecpraoctdiuvcaetidoni.n the second culture without metabolic activation under identical experimental conditions. In the second culture with metabolic activation the rend was based on precipitation artefacts as discussed above. In this study the rangeof the solvent controls was from 124 up o 196 mutant colonies per 10 cals; the range of the groups treated with the test tem was from 124 up to 764 mutant colonies per 10 cel. The solvent controls exceeded the recommended upper mit of 170 fpoelrlo1w0ingGellosngsotmeermwhtarteaitnmtehnet pfroes2e4ncheouorfs.meHtoawbeolviecr,acttihveatdiaotnaaanrdeijnuodngeedofasboatchcceuplttaubrlees since the WGT recommendations published in 2003 (11) stated an upper limit of 200 McoMloSnie(s19p.e5rp1g0/mcLellins aenxpdearlimseonltve|ntancdont1r3o.l0s rgeimmaLi.neidn ewxitpheirnimtheenthilslt)oraincdal CcoPnAtro(l3.r0anagen.d 4.5 gin) were used as posiive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity ith at least one of the concentrations of the controls. Conclusion n conclusion t can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L178Y in the absence and presence of metabolic activation. SCt6ud0y4Naummbmeorri1u3m05sa6l0t2 Report Page 25 of44 10 REFERENCES 1. D. Cive, JF.S. Spector Laboratory procedureforassessing specific locus mutation at the TK locus in cultured L5178Y mouse lymphoma cells Mutation Research 31, 17-29, 1975 2. P. Howard-Flanders Mutagenesis in mammalian cells Mutation Research 86, 307-327, 1981 3. B.N. Ames, J. McCann, and E. Yamasaki Methods for detecting carcinogens and mutagens with theSalmoneliaimammalian microsome mutagenicity test In: B.J. Kilbey et al. (Eds) "Handbookf Mutagenicity Test Procedures", Elsevier, Amsterdam, 1-17, 1977 4. D. Clive, R. McCuen, J.F.S. Spector, C. Piper and KH. Mavournin `Specific gene mutations in L5178Y cells in culture: A report of the U.S. Environmental Protection Agency Genetox Program. Mutation Research 115, 225-251, 1983 5. DI.nLs.itSupaenndcesrusapnednWs.iJo.n pCraostpocaorlys for chemically-induced mutationatthte locus in L5178Y MOLY cels: dose response and colony size distribution Mutation Research 322, 291-300, 1994 6. EReRc.oNmemsetnmadnend,pRrLo.tocBorlislibngaesre,dJ.oPn.Wa.suGrivlemayno,fCc.uJr.reRnutdpdr,acatnidceSi.nHg.eHn.oStwoixiecrteyngteasting laboratories: 11 Mutation in Chinese hamster lung and L5178Y mouse lymphoma cells Mutation Research 246, 265-284, 1991 7. A4.Ccoolmep,aCr.iFs.onArloefttt,heJ aHgLa.rGcrloeneinn,gJa. nLdomwiecraontdiaWt.ioMnurtieeclhniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells Mutation Research 111, 371-386, 1983 8. KSuHr.vivKalraofmehru,maH.nL.lyWmaptheorbsl,asatnodidJKc.elsBuacfthearnDaNnA damage measuredbygrowth in microtiter wells Mutation Research 72, 285-294, 1980 9. MM. Moore, M. Honma, J. Clements, T. Awogi, G. BolcsfoldiJ. Cole, B. Gollapudi, K. Harringlon-Brock, A. Mitvchell, W. Muster, B. Myhr, M. O'Donavan, M.C. Ouldelhkim, R. San, H. Shimada, LF. Stankowski J. Mouse lymphoma thymidine kinase locus gene mutation assay: Intemational Workshop on Genotoxicty Test Procedures Workgroup Report. Environ Mol Mutagen. 35(3);185-90, 2000 SCt8ud0y4Naummbmeorri1u3m05s6al0t2 Report Page260144 10. MM. Moore, M. Honma,J. Clements, K. Harringlon-Brock, T. Awagi, G. Bolcsfoid, M. Gifone, D. Collard, M. Fellows, K. Flanders, B. Gollapudi, P. Jenkinson, P. Kirby, . Kirchner, J. Kraycer, S. McEnaney, W. Muster, B. My, M. O'Donavan, J. Over, M.C. Ouidelnkim, K. Pan, R. Preston, C. Riach, R. San, H. Shimada, and LF. Stankowski Jr. Mouse lymphoma thymidine kinase gene mutation assay: follow up ntematinal workshop on genotosicity test procedures, New Orleans, Louisiana, April 2000 Environmental and Molecuiar Mutagenesis 40, 292-299, 2002 1. MM. Moore, M. Honma, J. ClementsG,. Bolosfod, M. GifoneR,. Delongchamp, M, Fellows, B. Gollapudi,P. Jenkinson, P. Kity,S. Kirchner, W. Muster, B. Mynr, M. ODonavanJ,.OlverT,.Omori, M.C. Ouldelrkim, K._Pan, R. PrestonC,. Riach, R. San, LF. StankowskiJr.A. Thakur, S. Wakuri, and|. Yoshimura Mouse lymphoma thymidine kinase gene mutation assay: Intemational workshop on genotoxicity tests workgroupreportPlymouth, UK 2002 Mutat Res. 540(2), 127-40, 2003 12. MM. Moore, M. Honma, J. Clements, G. Bolcsfad, 8. Burinson, M. Cifon, J. Clarke, R. Delongehamp, R. Durward, M. Flows, B. Gollapu, S. Hou, P. Jenkinson, M. LoydJ,.ajeska, B. Myhr, M. OonavanT,. Omori, C. Riach, R. San,LF. Stankowski JrA. Thakur, F. Van Goethe,S. Waku, and|. Yoshimura Mouse lymphoma thymicine kinase gene mutation assay: Follow-up meeting of the international workshonp Genotoxily testing ~ Aberdeen, Scotland 2003 ~Assay acceptance criteria, posiive contols and data evaluation EnvMiorlMuotange.n. 47, 1-5, 2006 13. MM. MooreM,. HonmaJ,. Clements, G. Bolcsfld,8. Burinson, M. CioneJ,. Clarke, P. Clay, R. Doppalapuci, M. Fellows, B. Gollapu,S. Hou, P. Jenkinson, W. Muster, K. Pant, D.A. Kidd, E. LorgeM,. Loyd, B. Myhr, M. O'Donavan, C. Riach, R. San, LF. Stankowski Jr.A,. ThakurF,.Van Goethern Mouse lymphoma thymidine kinase gene mutation assay: Meetingof the intemational workshop on genotoxicity testing, San Francisco, 2005, recommendations for 24 treatment Mutat Res. 627, 36-40, 2007 11 DISTRIBUTION OF THE REPORT Sponsor Study Director x copy of report x electronic copy Txoriginal report es 0 WLR esE iE preEtTTeen mTITene[]1 i:Bn ee ili 8 | MO ' tl | J seal | ) Ai AVAt AAVNiR lelsleisizm a2 Bed 8 : ir CEH { - E EE AEEYE FEEREEEETfS FdE ed g filEasEag; sagan: Sy i 5S :3 FPFI] EiEtReES ||] EHiiBE] E3L38 E8= 3|32g% g TE #8 Bzg |B 1iiil " 25 : EiEe..| BERNER: i fF i EsEagss1sEiegncse fpraaRmdsaanfsolyd |bkig | a S8858888 888883838 [37 g sBEp ggLzsLELs | i ELloelgaler sd1 | gTBI Er HH frees ese OTE AR gsr2asfBfEsseids|||| ucpaeeesadilit| i8 2 Pale ER Hom EE f Rares |fagncen [2211 [plefefeleleelele TLLLET 1]Te H |e wend bli gana | ag dL faxes flares11!g Yssssses | sssan| { in2llzz282 | gER2 2 I n PT yp E | | i :8 * EEfRiEiRERTE iGeeg 2g8s [FFEzsaEsslsLeEee:sr33EREaLg;k gi : HHz iS i: sHEE: 5E0 Eg |2 is A TTT lL. 52 2 $zg8: pelleLyLo) L ET 8 gi 13 [lesssonze Blesnaan Bligel,| 5 Bskflilzlainlalzialelz 3HHHH2 xlsleazlal EBHsEEiZHE3 5 g SZIz llsawcasllszsraalsess H2HH2eeasausaelssl| 2 =: 2 eszssslesss ||a||8sszezsszsssal ABRREENEY - soslgsase | | BREE BH taal alas snzasaf 2 | | 23] szslzeee | | zslzzes WH ls/8)ziv/ale|a |sizl2lz/s/g] ll I ii 1 TTTT EE] | 8kza:s Hd alelabslzlulelal || lg sk hapsases T|T| T3]8ces iSE i HEE i |bday #3| ||| sEasmza e- bEi R b Fy HL IES |e | lgslelaale E2fe i  AfiRsiEseRseRs E sasEnaE ed EEE E |B s: [ : EEsgEegrE7 |] 5E85E 2o3F% EH E|Lg i ik HEE [+ i TTT Tk g [Elellpll giI hl| vzsi ssssy sn eazssy eat gf: ZEEE eed BiEEzEifRaEiteEziER sfeiE zddsEizEgipsEaialEREiEF%EE) s|slssias || lngzs1gTa7e lz I" HiaaieiHf eaBl: lal 1 T ge T H I. gE I. 2d Fla 2233338 223833333 |E EEeCEEC E j|iEreCeEeeEdERe grTzpL zEL sd TsL zanagdeedl { i 2 Ea i HrEemLit ann|aapvae,s [8| Fl ee {presreel | b uN esen.gonad| 1HEEi fezsxne (hannsne1r| | Benzzzens fnzzreacItn|g & HE | gg 3 irs es|sseeeeas] ese||esaseneel]sDH aii)i a ess]|| peertlle eed] | peel | 42 2 * + CBEEEEEEREIEEEE EBEEE EEEIERRTLH 8% [gEasErees sanaEEeie iE2E([[]]]FFrE3Es B Pre8s: 8 | eed | | E5 g5 s8 5= |5i T |:3 LL i | 853 BBReEovEeBal. B2z2s8v0a2e8s2 [(hPls | 2 {5Aa0cogac aRaaaaaaIsCN i Elesisianess fElesases Eg i : 2 282s HLL ES i leche Elesacesf [3 ese sche neces Bite Loadedeagfttil | leases | Bassa BEE |le||e ssneas lf 2 11 523] ca|s|seaoseaBs2EE 2 || =mzzes BFE LL Boss || gRaaael 1877 Buleslesal |||| Bse EZEe sarRRETiEseE die||ssezeeeenfl 832 ly|| 34] |I 3 2 LLL 2/T2T5T8]&) I =55|4 BEagsze || sgszaslEc g | 22 sla] = 5 S2t0u5d0y4Naummmboenriu1m30s5a60t2 Report 12.2.3 Graphical summaryof experiment | / Culture | TT. ol - en | Tol . . i | Page34of44 1| iPosld g BiEo | [IR cons L000 -- i Fig. 1: Summary of experiment |, cuture | without metabolic acivaton me Foe' | oo oem || | Fall oer ij=T 1 || NB hE i | JL f M0 d OB as || Pan [ 1 Fig. 2: Summary of experiment |, culture | with metabolic activation StCu0dy4Naummmboenr1i3u0m5s6a0h2 Report Pagesso44 12.2.4 Graphical sumom fexa perr imey nt | / Culture Il - -- | i1 = ---- ml Dobe | fel 3 po fF 8 18 | tH | . --S-- T -------------------------------- Fi. 3 Sumomfexaperrimeynt |, culture l wihoutmetabolic activation | ---- - -| fred | W | . | {ovmmwe | AN PH Fig. 4 Summary of experiment, clue with metabolic activation - ETT EEE HHH HEH | ut zn |sz {H iE E i Sl EO he 8 [BEBRe..|| (BEBEsxE Ts : Flee es pin i: i slgzles g pss : n i ili eld fe BERNE iii : i oe] Lonel Bt nl {=t=1+ seee]]Leasloi 5 |a i 8: FETEEERRY EEE gla prs Hi z| sz E 00IU BN[F7g8L 0 I| I]E feEB R] $E| iikf }|[brrbleeebelE eegnR ealE nl3 || hood 1 ed Modell) |1 IARI RARAR [|zsee! a||seoepmaseoH es1:8 |ngz||anaesnsaar lst so || nse 4 TTat { i*H fOnEY f ReRo || 8iaitf iS:i g3i58 [I1 TBEER 5E5E: 1000 [PE] Pel l]0 8 5] I TTC, = Hidde: iF 3 BE sess | 2a 23 ite { HC i g 33 g82288 f1smseze farses 38223832 Es EY HIAARRAREJARRAR1AD: IE ||siss||sslsslslizlslaslslsB= s sagss |se2s2s2ss 2sallHie0]g [nes oon til | ea||s lgese saars idsd |sgi||ggazzacaerhsd | |de|| n zone al | 4 :8 3 | | EEEEEE EEEEEE gIgLa LL TLEE Ea PH HLL 5 [le a ; ii EL a sn | | jeoe|fnonodnlny Hfooted irernettfl El isnd | 8 : JT 1 ULIEg NnLLLe Ho Eee li E ERRRRERRTTIETEErEreh 2 isn her pJaNssRiNdAsNiNsR|RIgNaNaRnNnRsRaC] :ER13 : Siigun beck | Blk iE| | 4 ee ||oeacr kee 1 sps|z|aeesnaensa HeD i ocoll | 3 LoHs d[iLeEE FEENEY iii 18 3i 4 patie.| sucess B01 Pell] eae | El is | ||[T8 | | 8 388 Tk g floognogoooAsana aaaans an 2: hassel | L Heel | ifi tk ulsl] e| {fbaekaec hEiFee 23 ! IJ AAn UA] MEIaRoIeARA1FR2ERSBE [el ||ee elss ehj cle|se] mana HEE |se|| sa nesecl zEdad | sak|e|sapzeeeds| [A [AAA g2i3RkE AAR2/388) INARRANARRAAARAAC:, i 3 SStyEamRmbeerr313035s8a02 f-- 12.2.7 Graphical summaryof experiment Il, Culture | Page 2018 i-su-l :[I| ii | -- | -- = | | | = NB | Pog * ot | LFiF H e co vom e]{ Fig 5: Summary ofexpercurie imcetnmetta,l actvaion -ml | dm | Ral HR P | bi byn" CURR ER m -- -- | -- Lo imm| | nom BE | CtIdEd-Af+ | PHY a | Fig. 6: Summary of experiment Il, culture | with metabolic activation CS0t5u0y4Naummmboenri1u3m05s6a02 Report Page daar Graphical summary of experiment I, Culture II - | LE oo | | wl || .tooL Hd --I [ oo EE | | i | Pod fog 8 io cnn Fig. 7: Summary of experiment Il, culture Il without metabolic activation " fe] Eoi=- | ie|r | I~ TT - ove | | | 8 "I| SN| Pdr 8 Py a Fig. 8: SuomfexmperimaentIr,cutyure Ilvith metabolic action T Stylee ase porn J 13 ANNEX II: HISTORICAL DATA "These values representthehistorical controldatafrom 2006 to 2008 [ene Swami P mm 0150woa es ctr|Eteoi-- Number of mutant colonies per 10 cells | wow |mos | | wa| ---- Fs Tee [|(medium, em DMSO, water, ethanol, acetone, [mes THF) (CPA) | [Doommsosaomassn| |w TTT m |w0 || ream sergsenrne|TtoHi [ohoiwre |w mw w