Document VDQxLnE23oMdOj6g6rOLpX4p

STUDY NUMBER 1305601 CHROMOSOME ABERRATION ASSAY IN BONE MARROW CELLS OF THE RAT WITH C604 AMMONIUM SALT REPORT Study Completion Date: July 13, 2010 S0tu5d0y4NAuMmbMeOrNI13U0M5S50A1LT Report . Page 20138 1 COPYOFGLP CERTIFICATE m GuteLabrprsisGond Loran Paci Bn GLP-BescheinE igung/Statementoae fGLhP Cmomplsiance = HESSEN ww C Ei GL apktis on rrOberi gdc Ebaby A SuI scomsityR mnhGnLPcI oinfg 0 rote Ciminose = frood =m (UoverecuabaeBeehesngodAbe Uossivos insd es) tog egie eEs py es ATT eat Outendeer T ni--tsof pein ffeectoesatfeweirnsgtarnadiehmmia TShsoenibseet treid tyccs eed tt Coss pnt vente cs ts ob cos, SE TOA Gr 6 Soir oee pi To Oran kinderieeesmriehonrceRo =HPe) a 2 en mtr[ dGLOE er s SeoDsyab WisO tsPos siAbt St0u8d0y4NAumMbMeOrN1I3U0M56S01ALT Report 2 CONTENTS 1 COPY OF GLP CERTIFICATE 2 CONTENTS 3 PREFACE 31 General 32 Responsibiities 33 Schedule 3354 PGrooojedctLaSbtoarffatSoirgynaPtruarcetisce 36 Guidelines 3378 _ ADrecvhiiavtiinogns from the Study Plan 4 STATEMENT OF COMPLIANCE 5 STATEMENT OF QUALITY ASSURANCE UNIT 6 SUMMARY 6.1 Conclusion 7 OBJECTIVE 7.1 Amsof the Study 7.2 Reasons for the Study 8 MATERIALS AND METHODS 8812 TCoensttriotlesm 83 Test System 8845 EDxaptearRiemecnotradlinPgerformance 86 Acceptance Criteria 87 Historical Data 88 _ Evaluation of Results 9 9.1RESPUreL-TExSperiments on Toxicity 92 Toxic Symptoms in the Main Experiment 93 Mutagenicity Experiment 9.4 Summary of Results 10 BIOMETRY 11 DISCUSSION 11.1 CONCLUSION 12 REFERENCES 13 DISTRIBUTION OF THE REPORT 14 ANNEX 1 14.1 Tables of Results 15 ANNEX 2 1155.21 CCeerrttiiffiiccaattee ooff ATnaipmWalatFeorod 153 Certificate of Animal Bedding Page 30f38 2 3 4 4 4 4 55 5 66 7 8 9 5 10 10 10 11 1112 13 1145 16 16 18 177 19 20 20 21 22 2 23 23 24 2 30 2304 3 nT 3 PREFACE 3.1 General Study Monitor ay 300at Sor i GD 3.2 Responsibilities Quality Assurance Unit 3.3 Schedule GD `Study Number 1305601 CCBO4AMMONIUMSALT 3.4 ProjectStaff Signatures Study Director Report ~~~ ------ Page 5 of 38 Management oo QED EE Br) 3.5 Good Laboratory Practice The staywit be performed in complance wit (CehenmoikaliconogteeStuz (2C5,he1m05i4sCABcOtBLof1t9h0F4edeprp.al1R7e00puebtofeGvernmaneyw,es"Aurhnasng271, 2O00E2,CDanPdriancmiepnledseodf GveorosidonLadbaotreadtoJruylyr0a2c,ic2'0,08 as reve in 1687 ("BGBI.", p. 1146). C7)180Fnal) 3.6 Guidelines This study will follow the procedures indicated by the following internationally accepted TEanovrairoonmmenatalSoPnrsaHcaiornoACgrenocsy,osoHmesaAbfeerctisn TTeosstt GuiEdPlAine1s2O2P5P0T.S2267A0u5g36e3n iCoevpaEsvoarpaeimnomCraoL tonEaNst.coCoosmy amigsa3an0e0qu8ation (56) No -- 4353008,-- Once-- Jour SctCuBd0y4NAumMbMeOrN1I3U0M550S1ALT Report Pagers 3.7 Archiving A - =v th folowing data for 15 years: Raw data, study plan, report, ansamdple of the tes em Mictoscopi sides wil be archived for atleast 12 years. No data ill be discarded without the sponsor's writen consent 3.8 Deviations from the Study Plan During the cours ofthe study there were the folowing deviations from th sty lan Du-e-- to mariage the sumame of the study director changedfrom[JE + the frst pe-experient the 30 hour observation interval was mised out + rDeulraitnigve htuhmeidaictcyirmaatnigoen bpehtawseeenof33the65an%imfaolsa fpoerritohdeoffrasptprporxei-meaxtpeelryim&enhtourhse These deviations however do not affect he val of th study Study Number 1305601 cC6O4 AMMONIUM SALT Report 44 SSTTAATTEEMMEENNTT OOFF CCOOMMPPLLIIAANNCCEE Page 7 of 38 Study Number: 1305601 Test Item: cC6O4 AMMONIUM SALT Study Director: Title: Chromosome Aberration Assay in Bone Marrow Cells of the Rat with cC6O4 AMMONIUM SALT This study performed in the test facility of was conducted in compliance with Good Laboratory Practice Regulations: "Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" (Annex 1), dated July 25, 1994 ("BGBI. 1 1994", pp. 1703), last revision dated 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. StSutduydyDDirireeccttoorr | Doaatte:: (HED Study Number 1305601 COSO4AMMONIUMSALT Report EE Page 80138 5 STATEMENT OF QUALITY ASSURANCE UNIT Study Number Test em: 1305601 C504 AMMONIUM SALT TiSttleu:dy Director: RiChromosomei Aberr|alion Assay in Bone Marrow Call of th Rat with cCBO4 AMMONIUM SALT TpehreiogdiecnaelrlaylafnadcitthieosreasndusacatrievirteepsoroftIed 10 he responsible person and the m-a-nagseemcenitcd aSutduidtyedpbryoctehdeuQrueaslitwyerAessuinrsapnecceteUdnitpeTrihoedidcaatle.s aTshegisvteundbyelpoiwa.n and this report were Phases and Dates of QAU Inspections! Audis Study Plan NNoovveembmebre227r0,, 22000009 1 Amendmentto Study Plan: | __ May 14,2010 27 Amendment to Study Plan: | __ July 09, 2010 eStsutdyPeIrnfsoprecmtainocne: || December 16, 2000 Report pr 09 + 12,2010 DDiarteecstoorfaRnedpotrotMsatnoatgheemSetnutdy NNoovveemmbbeerr 2270,, 22000008 May 14,2010 July 09,2010 December 16, 2009 hori 12,2010 This statement i to confi that the present report reflects the raw deta CSt5ud0y4NAumMbMeOrN1I3U05M60S1ALT Report Page 9.0f38 6 SUMMARY Tihnidsucsetcudhyrowmaossopmeerfaobremrerdatitoonsinivnesbtoingaetematrhreopwotceenltsiaolf otfheCr5at.04 AMMONIUM SALT to The test item was formulated in sterile water. The volume administered orally was 10 dmiolskeg gbrooduyp)weaiftgehrtt(hbe.w.t)r.eaTthmeentantihmealbsowneermeatrrreoatwedceollnscew.er2e4 cholalnedcte4d8fhor(conhlryotmhoeshoimgeh aberration analysis. cytogenetic damage. 7 males per test group were evaluated Per animal 100 well spread metaphases wfoerrethsecoorcecdurfroerngcaepso,f dbirseianktse,gratfiroangs.ments, deletions, multiple aberrations, exchanges, and chromosomal The test tem was investigated in ths cytogenetic assay at the folowing doses: Males: 3125, 625, and 1250 mglkg bw. Ttohxiecimtya.x1i2m5u0mmgd/oksgesb.fwo.r twhaesceystotgiemnaetteidcaassstoaybewecrleosdeettoertmhienmedaxiin maupmret-oelexrpaetreidmednostesfo.r a`cTthieviatyn,imrauflfsletdrfeuarteadndwihthuntchhisbadcokseposshtourweedindcliicnaitcianlgsbiigonasvaliikleabaitryedoufctthieonteisnt sfpleomn.taneous iNtoemr,elienvdaincattrinegdutchtaitonthoef ttehset miitteomticatintdhieceisndciocualtdedbecoonbcseenrtrvaetdioanfstewratsreanottmecnyttowtiotxhicthienetshte bone marrow. Ntroeasttmaetnisttiwciatlhlythseigensifticiatnetm ainsccroeamspeariendtthoethferevqeuheicnlceycoonftroalb,errant cells ocurred after Asnhoawpepdroaprdiiasttiencrtefaenrdensctaetimstuitcaalglyensig(nciyfciclaonpthionscprheaamsiedeof) iwnadsucuesdeadbearsraptoisointifvreecqounetnrcoyl.and 6.1 Conclusion In conclusion, it can be stated that during the mutagenicity test described and under the `deextpeerrmiimennetdalbycotnhdeictihornosmroespoormteeda,betrhreatteisotntteesmt wdiitdhnbootneindmuacrerocwhrcoelmlosofomtheemrautt.ations as Tchherroefmoorseo,mecCab8e0rr4atiAoMnMaOssNaIyUiMn vivSo.ALT is considered to be non-mutagenic in this `Study Number 1305601 Report Page 10of 38 S-- AON A-- Y ---- 7 OBJECTIVE 7.4 Aimsof the Study `This in vivo experiment was performed to assess the mutagenic propertiesof the test item by means of the chromosome aberration test in bone marrow cells of the rat. 7.2 Reasons for the Study cTabyheterorgbaetonineoteniscm.adrTarhmoeawsgeceytaiobngeersnroeamttaiitcointcsesctalriess afinsreaaqsnuseanitnyldiyfcoraltetothrheaoldfeftaoecpttohiteoenntdoiafalmsat0rguecitdnudruaclclesc.hmroorHmoeowsseuovbmetarll,e gcohrrmoimnoalsocmeellsdammaaygeleathdattoishecroimtpaabtlieblceytwoigtehnecteilcl daibvnisoimona.itiSeimsi.larHedraimabalgee ciyntdougecneedtiicn aebvneonrtmsaloirtbiiersthadreefekctnso.wn to have deleterious effects in man, e.g. induction of neoplastic iRnattesrvaalrse. eCxhporsoemdosoomthee tpersetpatreamtiboynsanaraeppmroapdrieatferoromutbeoannedmaarrersoawcrfcailcse.d aTthseeqsuteanitnieadl preparations are examined and metaphase cel are scored fo chromosomal aberrations. The bone marrow i the tissue of choice because it offers several advantages: 1. Low incidence of spontaneous chromosome aberrations. 32.. HTeicghhnipcroaplosrtiiomnpolfimocefittathpyehapsreepacarlaltsioonr. analysis. Chromosomal aberrations are generally evaluated in first post-rsatment mitoses. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but `chromosome-type aberrations also occur. "The time at which the aberration frequency s at a maximum varies from agent fo agent. nBmeaecrcerasouswsaercyedliltfofeprpoerpnuetlcasctehileoymnicdiaesfli_snaeshyantvcheehreorfnefoseupcostns,sea.mtuldtDiifpulfsee.relnpotomspatirrotelsactomfdeetnlhtaey scoealrmlpmcleyetcalbetoimlaeinsc.d aatnrhdee cpohnasrimdaecroakbilnyetdieclaysefdfects the. appearance of the frst post-reatment mitosis can be SFoormetheinadiscsaetsiosnmoefntcyolftcolxaisctoyg(epnairctiaacltinvhitby itohen moafmxiitomsuims) twolaesrataepdpdieodseonorcethaantdprsoadmupcliendg aatdd2i4tonhalanddos4e8lehvealfstewretrheettersstaetdmeanctc.ordTiongleys.iabish a dose response fect at least two pTaoraldleemloonstihreatteetthteemsensiiviy of the test system, a reference mutagen was tested in `Study Number 1305601 C604 AMMONIUM SALT Report 8 MATERIALS AND METHODS Page 110f 38 8.1 Test item Internal Test Item Number: S1060711 The test item and the information concerning the test item were provided by the sponsor. Identity: Batch No. Purity: Stability in solvent Storage: Expiration Date: 0604 ammonium salt 156/39 975% Not indicated by the sponsor In the refrigerator (+2 to +8 C) December 31, 2020 On the day of the experiment, the test item was formulated in sterile water. The vehicle wstaasndcahrdosveonlutmoe its of relative 10 milkg non-toxicity b.w. orally. for the animals. All animals received a single Study Number 1305601 Report C804 AMMONIUM SALT Page 120138 82 Controls 82.1 Vehicle Control `The vehicle of th test item was used as vehicle control Name: CSuaptpallioerg:ue no. Route and Frequency of Administration: Volume Administered: sterile water 68.72B4r0a8u2n.0M0e.i0s0ungen AG orally, once 10 milkg bw. 8.2.2 Positive Control Name: Supplier: CDiastsaollovgeudeinn:o. Dosing: oRfouAdtmeinainsdtrFatrieoqnu:ency Volume Administered: CPA; Cyclophosphamide Sigma-Aldrich Vertriebs-GmbH 82041 Deisenhofen, Germany sctoerrileeswater 15 mglkg baw. orally, once 10 mig b.w. `Solution prepared on day of administration "The staboiflCtPyA at room temperature is good. At 25C only 3.5 % of is potency is lost after 24 hours. CSt5u0dy4NAumMbMeOrN1I3U0M56S01ALT Report oT Page 130138 83 Test System 8.3.1 Reasons for the Choice of the Experimental Animal Species TInhieatgaotnsaatnsanmivamaatwiahtiohmsh:as bdeein ousneIfoosr mokanyoyeeaersarsaebleSexopeerimweontealraniomal Phyaiiogcal, phamasologieal and tosogcn hades. TIbheneeUrrsepoouattraetfoomrnatohnfeyFdsedusalitlgasn afavonamltathbheoepCefyrtooimgaemsnauenctch,oaiosnfsvafeys.etibcgaahtloiaomnfosrsowmohmisceuhcahmboaexynpaebrtomnehnteetlspstfualsoin mtahye stan: at stan) - DNoumber of animals: [ a days RIgneaolbohyowemiighe 5 13wets atsiatof veatment males: 1802455 aAncicmoarldsinugntdoertwheenstuapcpceirmsataosnsuirnatnhceeatnheaalnihmoaulssewoefrAa EinEheEatRhyEocorndaiomni.niTmhuemaorfiiTneg ore Dut i paid mls ut nt et 2 or Stor mor mTheeanimalas were ditiuted no the test groups a random and ienied by her cage 83.2 Husbandry aE poratryT comin CouotinTgee: Bedng: Food Water [r-- --T-- ------------ gWaokwtoon Type IV, with wire mesh top CT(bEaiHtbrRaEoRTmonGasmombnbH8ot,riSgeo7nm9C3rm0ea2orGEmimmamsie)ndiCnoge.nK,G.Germany) aprailaensLsaibmodredrodridessGmabdHt, u3m9178 Brchen, Germany) op water, 23 ium (eTGmaepmtoeeaiinPdueurwmeeir2dky2e,332064-036850%RoBdorf, Germany) rica lg 6.00 am 600 pm SCt6u0dy4NAumMbMeOrN1I3U0M56S01ALT Report Page 140136 8.4 Experimental Performance 8.4.1 Pre-Experiments for Toxicity SgAtrropauirnep;livcmeaihcnihacreyU;nsrdtoeuurdtyei,dofennrteiaqccuaueltneccyo,tnodxaiinclidotnyvsowlaaussmeipneorftfhaoedrmmimneuidtstawrgiatethnioitnlw.yo astnuidmyalcsonpceerrnsienxg:anadnitmeaslt sTyhmepatnoimmsalast wientreervatlrseaotfed1ho.nc2e.4ohr,alGlhy,it2h4h,th30tehst(efxecemptanfdoretxhaemfirnstedprfeo-rexapceurtiemetnotx)i,c and 48 h afer sach administrationof th tes fem 8.4.2 Dose Selection citains gbeenfeorramlulraetecdomamnedndademdintiosutsereetdheremparoxdiumciubmlytoolrer2a0t0ed0dmosaekoratshethheiguhpespterdofsmetthfaotr non-toxic test ems. rTehaectmioanxsiwmiuthmouttolhearvaitnegd edfofesctsleovnelssuwnevraeldweittheirnmi4n8ehdoutrosbeafttehrethdeosterseattmhaetntc.aused toxic STwahamrspeiteianakdgeenqianuttaet4rev8alhly 2as4tpeahrceradeftadetormsteerneltaetvmeelnst,.spFaocredthbeyahigfhaecsttorodofs2e, lweevrele aapnplaideddiiaotntahle scaenmtprlael 8T`Se.es4vt.e3GnrmoSautlpuesds:yarPeraoscseidgunredefo each test group. "The animals are identified by their cage number and tail ags as shown below inthe table. est group 2Hourspostreatment4 Negaveconro 7 Low dose 8-14 Medium dose 15-21 High dose 2-2 8-42 Positive control 29-35 CSt5ud0y4NAumMbMeOrN1I3U0M56S01ALT Report Page 15038 8.4.4 Treatment: Abte tahdemibneigsitnenriendgwoafsthaedjtursetaetdmetnottthheeaanniimmaal'lss bwoedryewweeiigghth.edThaendantihmeailnsdirveicdeuailvevdotlhuemetetsot aicteumteontocxei.c sSyemvpetnommaslaersowunedre1 th,re2a-t4edh,p6erh,do2s4ehgarnodup4.8Thhaeftaenriemaaltsewner.e examined for Pinrhiiobrit(o2r:c5otloce3mhiodu(r2s.)0tmogs/akcgrifbi.cwe.,),atnoimaarlresstwecreleliinnjemcetteadpihnatsrea.peritoneally with the spindle 84.5 Preparationofthe Animals: Tephiephaynsiemsawlserweecruet osfafcarinfdictehde bmyarcreorwviwcaalsdfilsulsochaetdioonu.t wTihtheafpeprmooxriamawteerley 5rmelmohvyepdo,tontihce spuostpaesnssiiuomncwhalosritdheensiolnuctuiboante(d0.f5o6r 2%0 mwiinv,atp3r7ewCa.rmTehde c1e0lls37werCe).seTdhimeenhtyepdotobnyica.bcreelfl cweansriffiuxgedatwiiotnh (31+0100abrspoml)u,tetmheethhyapnootion+igcacsiulpeacmeattiacnatciwdafsixadtiisvecafrodre6d0amnind. tThehecnellthpeelcleeltl speildleets wtahesfgiexnattilvye rweassuscpheanndgeeddwaitnhdfeixnaotuivgehafnidxatsitvoerewdasoveardndiegdhttaotm4akC.e Pariroerlattoivmelaykithnign cell suspension. The fixalive-cell suspension was spreadbyflame-drying and stained with OGineemsoar.moCroevesrlidsleispswewreeremamdoeunftreodm ewiatchhEbUoKnIeTmTa(rKrIoNwDsLaEmRp,le.79110 Freiburg, Germany). 8.456 Analysis of Metaphase Cells: Eivmamleurastiioonn oobfjectthievess.idGeasps,wabsreapkesr,fforramgemdentuss,indgeleNtIioKnOs,N exmcihcaronsgceospeasndwcihthrom1o0s0xomaoli dsipsrienatdegmraettiaopnshawseersepreercaonridmeadlawsersetrsuccoturreadl fcorhrcyotmoogsenoemteicadbearmraatgieonos.n cAotdleedasstid1e0s.0 Twehlel number of chromosome aberrations per metaphase was determined. Only metaphases wdietshcrtihbee cahacryatcotteorxiisctiecffcechtrtohmeomsiotomteicniunmdbexer(%ofce4l2ls=in2miwteorsies;in1c0l0u0decdelilns tahree asncaolryesids).wTaos determined. All animals per test group were evaluated as described. 85 Data Recording `tTahbeuldaartfaorgme,neirnactlueddinwgerexepreericmoerndteadlignrtohuepsl,abvoerhaitcolrey,parnotdocpools.itTivheecronetsruollt,s are presented in C`S5tu0dy4NAumMbMeOrN1I3U0M56S01ALT Report Page 16of38 8.6 Acceptance Criteria Tpohseititveestciosntarcoclepsthaobwlse sainstcaetiastticlaelalsyt s5igenivfailcuaanbtlreeasnpiomnasles apnedr tghreouapbewrerarteioanvarialtaebloe,f tthhee `vehicle control (excl. gaps) is below 2 %. 8.7 Historical Data Number Dose Mean number of cells with |studoifes `mbgw/.kg stabaenrdraartidondsevi(aextcilo.n gaps) + |27 [veice0s| | males temates 062041 range0-2 [1[opal25| moesttomaes | 302 | [7Tora] 20 [| maesstomaes | 174144 [os [opal ts |maes| [eo [opal 1s | tomas [14831:27040 [2[opal 12s| mas | 42:03 125 females 93:41 [1 fTeeal 10 females [o [opal 7s |mies[| [+Tea5l| maesaromaes CPA = Cyclophosphamide S9e 6 sree 8:8 Evaluation of Results AnutmebsetrteofmsitruccltausrsailficehdraosmomsuotmaaglenaibcerIrfaitiionndsuacensd eaitrheeprroadudcoisbel-eresltaattiesdtiicanlclryesaisgeniifnictahnet positive response for atleast one of he fst paints. SAotmesat aibaemrrpartioodnucnionrganesittahteirstaicdaolssei-grneilfaitcaendtianncdreraseeprinotdhuecneumpobseirivoef rstersupcolnursaelactharnoymoo-f {he test points is considered non-mutagenic in tis system Tris can be confirmed by means of tha non-parametric Mann-Whitney test. However, both biological and statistical significance should be considered together. `Study Number 1305601 C604 AMMONIUM SALT Report Page 17of 38 9 RESULTS 94 Pre-Experiments on Toxicity In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg `administered bw. was TE rm cC604 AMMONIUM SALT formulated in sterile 10 ml/kg b.w.. water. The volume reactions on [sonmaole / oefemale son fi e Jo| [we[me]+|e] & rr men a Se motes foetas rto0o0| a0[|2a2|[22a2[[va2 || ffTorormwa lwlwTo]o[woo[]on[w20][son]0] i on as beC604 AMMONIUM SALT ts re 1750 mg/kg bw. cC604 AMMONIUM SALT formulated in sterile water. The volume administered was 10 ml/kg b.w.. "The treated animals expressed toxic reactions as shown in the table below: Ce fi mosmeensan| 2a|[a2[[oan[|a[|ve2 a|| oman |wo[oo[22[2 2[ we Twlw]wlw]w]w] toxic hours post-treatment footsomormnssom[|oonv|[2222[2[| 22|[2|2e2]| rons [ooToo[a[so [on]wo] en `Study Number 1305601 Report Page 19 of 38 9.2 Toxic Symptoms in the Main Experiment of 1250 mg /kg b.w. cvs cC604 AMMONIUM SALT formulated in sterile rin water. The volume = -- Reactions fCmooLrsermezrn[] ow1h oe[244[h|[on6wh o[[24|h|o748h]|" For the mid dose group 7 males received orally a single dose of 625 mg /kg b.w. cC604 Ce ormeceension[a0[|2a [e3l|o7]| T owolls 1s] `Study Number 1308601 C604 AMMONIUM SALT 9.3 Mutagenicity Experiment 9.4 Summary of Results Report Page 200f 38 Experimental group dose | preparation [number| % aberrant cells | mean mgkg| hourspost | ofcells| incl. excl mitotic. bw. | administration | scored| gap: gaps | index (%) # coat emmonium si[sas | a0 Jon[oo[an[oar| comps emmontum si[en| Ju]or [or[om | cosas ammonium so [rm | a [wo[or [or[om| Dapp BERERERE a[mo] @Ju]vo]ee] 9.4.1 Analysis of aberration types fmaagn:||tmaognt: daloon |abmoortae- darsotm oot ganar [sJofofefofef[sfolofolo] [+JofoftJoJa[vfolofolol] * MpPohrreeniihaaninoSnabortions xcuing gap 1oocel changes (ut 10othr abaatviersoenrse)d CSt6ud0y4NAumMbMeOrN1I3U0M56S01ALT Report Page 210f38 10 BIOMETRY nStoant-isptaircaalmestirgincifMiacannnceWhatitthneeyfilveespter cent level (p < 0.05) was evaluated by means of the Vehivceirscuosntrol Tost group (aberrant cells excl. Gaps) Significance a2C46h0p4osAtMtMreOaNtmIeUntM SALT, 312.5 mg/kg b.w., C604 AMMONIUM SALT, 625 mg/kg bw. C245h0p4osAtMrMeOatNmIeUntM SALT, 1250 mghkg bw. |[own| F; or Cyclophosphamide, 10 mg/kg b.w. + 224C6h0p4osAtMtMreOaNtmIeUntM SALT, 1250 mgikg bw. 0.0003 48h Apo=st trneoaltmeesntted, as Te mean abarallon rate was nol above he coresponding ="= cnoonttsrioglnviafliucea.nt Sionficant CSt5u0dy4NAumMbMeOrN1I3U0M56S01ALT Report Page 220138 11 DISCUSSION cThhirsosmtousdoymweasabeprerraftoiromnesdintoboansesemsasrtrhoewpcoetlelnstoifaltohfe craCt.504 AMMONIUM SALT to induce TThhee vteosltuimteemadwmaisnifsotremrueldaotreadlliynwsatse1r0wmaitekrgwbhoidcyh wweaisghatls(ob:u4s.e.d26ashtahnedve4h8ichle(ocnolnytrtohle high dose group) afte a single adminisiraton ofthefest tem the bone marrow cells were cweollllecatsedvefhoiclteheacnhdropomsoistiovmeecoanbterrorlatwieonreaneavlyasliusa.teSdevfeorntmhaeleosccpuarrretnecset tofemcygtrooguepn,etaisc dfraamgamgeent.s,Pdeerletainoinmsa,lmul1t0i0plewealblerrsaptrieoansd, mexeclhaapnhgaesse,sanwdercehroscmoorseodmaflordisgianptse,gratbiroenask.s, Trehseposntsuedy(awbaersrataicocneplraatbeleexacsludtihneg pgoaspisivewacsonl7r.o3l %s)hoawnedd tahestaabteirsrtiactailolyn sfirgenqifuiecnacnyt exclusive gaps of the vehicle control was below 2%. 3T1h2e.5te,s6t2t5,emanwdas12i5n0vemsgti/gkagtebdwi.n this cytogenetic assay at the following doses: Tbeheclhoisgehetsot dthoesemaofxi1m25u0m mtgo/lekrgatbedwdowseers.e dTehteermaniinmeadlsin tarepartee-dexwpiethritmheinstdoorsetosxhiocwefdo ciinndiiccaaltinsgigbnisolaivkeaialraebdoiucfltitihtoeny Itntspfoenmtaneous actvy, ruffed fur and hunchback posture STAhLeT,mitiontdiiccaitnidnigcotshtwetrhee treostt rietleemvacnitdlnyortehdauvceedcyatfotetroxtireeaftfmecetnstiwnitthheCb5on0e4maArMroMwO.NIUM Nfor_esqtuaetnicsitecsalolcycursriegndif(iacbaenrtratoironbiroalloegsicoaflltyheretleestvanftlemetnrheaantecdemaennimtalosf ratnhegedabbeerrtawteieonn 0fr.e0q-u1e.n1c%i)esasobsceormvpeadreadftertotrtehaetmevnethiwciethcconCt6ro0l4 vAaMluMeON(0I.4U%M). SATLheT wmeeraencoanbseirsrtaetnitolny cboenltorowl2v%aluae;bersreaentaccaclespleaxnccleuscirvietegriaap).s T(hgeirveen waesrethealuspopenro mrieltevoafnta dtoolseerabdleepevnehdiecnlte increases in the aberration rates of the treated animals `iCnycrcelaospehsosopfhianmdiudceedwaabserruasteiodn afrseqpueonsciiievse. control and showed statistically significant 11.1CONCLUSION einxpceornicmleunstiaoln,cotndciatniobnes sretpaotretdedth,atcCdu6r0i4ngAtMhMe OmuNtIagUeMniSciAtLyTtedsitddnestcriinbdeudceancdhruonmdoesrotmhee mthuetartaitoinnsviavso datermined by the chromosome aberration test with bone marrow cells of `CSCtBudOy4NAuMmbMeOrN1I3U0M56S01ALT Report Page 23.0138 12 REFERENCES 1. MBeencdhearn,iMsmAs.ofGrcihgrgos,moHs.oG.m,alBeadbfeorrrda,tiJoSn.pr(o1d9u7c4t)ion lMlutaCthieomnicRaelsseaarncdhi2o3n,izi1n9g7-ra2d1i2ation 2. GReoleattzi,onPs.hiSprabme,twRe.eJ.n,eDxophenrailmoevnata,lJ.re(s1u9lt7s5)in mammals and man. Cytogenetic aMnuatlaytsiiosnoRfebsoenaercmha3t1r,ow24i7n-ju2r5y4inducedby a single doseofcyclophosphamide 3. F0rovhibveorcgy,toHg,enSecthiucliznev-esStcihgeanticoinnsgn, Mb.on(e19m7a5r)row cals of rats, Chinese hamster `and mice reated with 6-mercaptopurineAvchiv fir Toxikologie 33, 209-224 4. AHlolua,sTo.fC.ma(m1m9a67l)ian chromosomes: Rattus norvegicus Vol. 1, Folio 18 5. `SAucguityeacmyat,ogTe.n,etGoitcoe,ffKe.,ctUoefn2a-k(a2,-fHu.ry(l1)9-735-)(-niro-2-uny-acrylamide (AF-2, food MpurteasteironvaRteisveoean)rcrhat3b1o,n2e41m-a2r4r6ow cls n vivo 6. S`Supgeiciyfaimcaw,inTe.ra(b1i9l7t1y) of the largest tslocentric chromosome of at bone marrow cJoaulrsntaol7o,f1t2h-ediNamteitohnyableCnza(nac)earntIhnsrtaicteuneerange 471, 1267-1275 7. KLorcaaultlhy, mJ.os(t19p7o1w)erful tied rank test in a Wicoxon situation `Annals of Mathematical Statistics, 42, 1949 -- 1956 5. GAaml.elJi.,HJoFs.p.(1Ph9a6r7m)., 24, 425 Gited from: The Extra Pharmacopoea (29" edition) 13 DISTRIBUTION OF THE REPORT Sponsor 1xx ecloepcytroofnircepcoorpty Study Director txoriginal report StayNum 120501 Report C604 AMMONIUM SALT Page 240138 14 ANNEX 1 14.1Tables of Results Table 1: Chromosome aberrations in bone marrow cellsofratsscoring 24 hours EtEERE Moy ol after treatment; Experimental group 1: Vehicle; Sterile water culmbseorores|| madgose | iegaos | ext gap C Cr 2T Twn[www|| CC oT1o o 0|| CI eTn 0| CC o1e o2 || C Cs sT Tm n[[wwoo|] C--o--T--o--| CI C EN melCeoeT|oea || TE EEEE EE Analysisofaberration types in each 100 cells aon| "| |:| om ment [soto crn | ato: : gration |+ Jofofofoofo oJ olool] laloloflofofJoJolJoooJ]o [sJofolo ozo]oo oo] [JoJoloTolololoolTao] [sfoJololoJolololoJolol] [seo ooo ooo] [7Jefe oJolololooool] [om oo] f o vof loo ]olf o] LrLemtnr startsxn co nestrostarrtoh SCt5ud0y4NAumMbMeOrN1I3U0M56S01ALT Report Page 250138 [r=]= 12g ff wp jon Taafbtlere2tr:eatCmhenrto;mEoxspoemriemaebnetrarlatgiroonuspi2n:bToenset mitaermr;ocwC6ce0l4lsaomfrmaotnsisucmorsianltg;23412h.o5urs mg/kg bw. animal no. cenlulmsbsecroroefd|| mg/dkogsebw. | incl. gaps | excl gaps [ee | m [ 0 |] [C T o o 1 [eo | m [ 10 | [e | o oo |] [10 Tm | 100 | [oT oo | [a 1 om [ 10 | CoTo] [2 | m [ 100 | [C | o o | [8Tm | 100 | [o0 T| [1a T m | 10 | [o0 T mem o | 0 ET EEEEE EE Analysisofaberration types in each 100 cells nmaol gop break aber:rat, |change|dgirsaitnitoen- [lofoloToolTo oloool] [e]ofoflofoJJooToolloo] [lofofoloolo ol lolooll] ["]ofefoTooJoooTl]ooll] [Zlofolooolo oT ool ] [lolofofololololololo] [JoJo oJoJololooo To ] LomofoloJoo fo oJ olosl] * Moro than 5 aberrations excluing gaps in one cel: exchanges (ut no her aboratons) wer recorded * Pleo `Study Number 1305601 C604 AMMONIUM SALT Report Page26of 38. Table 3: Chromosome aberrations in bone marrowcellsofratsscoring 24 hours after treatment; Experimentalgroup 2: Test item; cC604 ammonium salt; 625 mg/kg b.w. [oan[oo Jaen[ me |oT | [5 m | 100 | [8 [ m | 100 | [7 m0 | [8m | 100 | [rem[100 | [20 Tm | 100 | [2Tm[0 | Analysisofaberration types in each 100 cells 2 TT 2 I I rT7 ] [o0 T7] [oT oo | [oT oo | mean] or | oz | E : E TE HEREEEEEE 2 [[ofo[ofoolo2][o+ [o]] [floTol2Toovo o[o]o] [PooloJolololo[ofo]o] [l oTo ooll rolo o[ol ]o] [[oToloTolololo of[o]o] [2]ofofoJolofolo[olo]o] [2]oo oJolo oo ooo] Low]oof2To 2s[ofofo]o] o Henaon C`S5tu0dy4NAumMbMeOrN1I3U0M56S01ALT Report Page 27of38. [==]= lam pasate) Table 4: Chromosome aberrations in bone marrow cells of rats scoring 24 hours after treatment; Experimentalgroup2: Test item; cC604 ammonium salt; 1250 mg/kg b.w. crousmescroorted|| [C2z 2 [ Tm mTw 10| | [2Tm T 0 | [2Tm | 0 | [26 Tm T 0| [zz [mT 0| [C2Ca Tm 1 0 | mdgohsgebw| inc gaps | excl gap [ I--C T--o o --| [o [ o oo | [2[2 | [oT oo | [oT oo | mem[ er [1 01]| EEE FEE Analysisof aberration types in each 100 cells break mfernagt-| tion |aberr-at.| change| dgirsaitnitoen- [Z]oJolofo oflooolo o] l [Z oTo l lao ]0 o oof o] [2]oooololo oo oo] [=]oololol02o]oo ol ] [2]oolololoofo loool ] [Z ooooo looo oo] [2]ool +Tol olo ol ooo ] from[0Jo [of ooa lon o] +. oa ro an 5 obraton xchain ap cn al xchange (kno hr sores) wor Foard SE84 Amon SALT `Study Number 1305601 Report Page 280f 38 Table 5: Chromosome aberrations in bone marrow cellsofrats scoring 24 hours 1 =Tala wmine after treatment; Experimental group 3: Positive control; cyclophosphamide number of dose. C Co oT Tnw [cew llws socor|| ed g/kg b.w. incl. Gaps | excl. Gaps Cs15 1 CI orT--m ----0r] C1e 6| I IE E Ce| ICE C EN melI ws [-- ws--] Analysis of boraton typos nesch 100 cel aon|" [| ms| i:| om [<p|shane|tim: ment - gration [#lofofeJolrTolol+]alol] [#fofolsToole]l[ool] [lololefolz]olo ole 0] [l zo o fl rloo l+]l To] [BlofofaJolrfololo]zl0o] [#lofol7Tole+]Telo0ll] [lololafololalo oo] ow]o [owolefalolen]o] ELS La, F ------ after treatment; Experimental group 4: Test item; cC604 ammonium salt; 1250 mg/kg number of EE EE i pi [es Tm | 100 | [or Tm 100 | [ee Tm[100 | [oe [ wm | 10 | [aw [ mw | 100 | [et Tm | 100 | [Tm[10 | 2 2| Cs 5 |] [o0 T| [o0 T| [oT| [2[2 | [+[1 | wea| 11 | 11] Analysisofaberration types in each 100 cells Ella [TofofloJoJoJe]o ooo] [7]ofofofofJeo lvoTo0T] [#lololooloTolo ooo] [*lol lolo oll olo oTT o0o] [#lofoloTolololooo] [#lofol+TolvTololololol] [2]ofolooo oo oo] low]oJo[ +Jofa oo loaoT ] leer So`Sbtuday NAummbmeorn13056S0A1LT Report 15 ANNE2 X 15.1 Certificateof Tap Water Page 30.0f38 non GeafortiuserSra 118 fy zentrallobor isa GRD =U B=h ike = sawn t=on men] so | n |vss owe] [Coenen lT ens oe E-- E ----a -- [Comcast CT NT I | TTwT =r Jee TT we 1] [=p=r Jee TeTee JT es W Tesves] fr=a=gs [msperiezre C=ny F=n Tomrmem T ww ||| TE fEoEre = er = a Tw sw = w] ] Study Number 1305601 C504AMMONIUMSALT Report Page 310138 J ~ pi wr fain E [m r Tc wescosu mors |e rows||e torome isTameow|e] PC os -- wes TTO oS w |C ] eR I EW C[-- oT a fee -- TwI [ma |-- fom esa|= e[me | PProoemsms foe Twwa fT os ws E Te[om |] 0 CT | C C-- --O O EE E -- T -- EBe E-- besms TI w oom[--7) CC E ----O 2 --| ffomm eewwwwe eeww[vWes |m C--O -- | ) EC e ---- --O| [BC oomme -- feweswO e| |r|| oooesm0 |o0os BT o S ewww ow | oom0[wom | T0I 3 EC C e-- -- -- O O| be Peewee ow| oe [ow] Scien108Pe TE,Se 4 S ST RE pos pass --_ ~ Gl /hesvwaYsossesrer Ras [rumenTT eosatom |Groat| ras arnwen| [oe Teemm ee| om |] [00. ute ouncevorwit [ome | eo| om | | [Ott eee |epioz| or | 8 | TTSN TI rn mre [aw ai] pox een Tw oan [0cwan Teme w|@r 3) [eee Te Tw oer [onewra | wrl| [oo Teme Tw| @2 |os | [Bontbornaten Towers wt| wr [Crostos leme Tat [oom le Ter [aan Tome wr ar [il eee ee |ow ar [Peroman Jen TwTes [fondo Towers wt|r [ow 3 Dc fmmers Tw or | [Someta Towers wr| aa |w | [Be ee Tw oar 7] [famomeama wen fmm wr wz ea [ope fms Tw or [Eaves fmm Tw ar == fms TwTe TT] |Co II CTT | [Bao sare [San I C3 [Pesotum emer Twn | oom || |ST J-- [pwotisenpe ewes | wn | oom || [SomeCPO oY wen | wn| ae ar | fo ee Tw|e ar | CS I SC| CE -- jr) w |e|@ or | [oss eee| | we | ar | CT | [l ooops ee | w om | a1 | CS II CT | [Bo IIIT [wn | wm or] Study Number 1305601 cC6O4 AMMONIUM SALT Report Page 33 of 38 Analysen-Nr.: 200927278 Probe: VOM: 07.10.2009 FE171h:essesnewnwaasssseerr zentrallabor Versorgt sein. Sicher sein. Parameter gamme-hlexachlorcyclohexan Heicazinon Isoproturon MCPA MeCoprop (MCPP) Metazachlor Metbabenrthimuron Metobromwon Kenyon Parathion-Ethyl Propazin EeSebuthylazin emSimeon Terbuthylazin feSumme PBSM Methode/Norm Einheit Ergebnis Grenzwert ow EN 030 ices rmi KM <0,02 0.1 011EN 12.0106SS FM DIN En so 1136111(F12] pen <0,02 0,1 WO <0,02 0.1 Nerarreseeer (Lens) Pan <0,02 0.1 reeperneteree PrAten re <0,02 0,1 Del EN ISO IOUS (FM man <0,02 0,1 Pii an no ii.F.) inn <0,02 0,1 WIEN ISO 11303 eel DiN EN MO 11300 FM ygry <0,02 0,1 PO <0,02 0,1 DIN ENISO 10025 (FP DIN EN WC fl ee Frell heen [oe Lem I OMENISO 106e6Pler CT OPEN MO Kne MP ON EN ISO Mee! (Flp Eee yeerscheeelene Ire <0,03 0,1 Pan <0,02 0.1 pgA <0,02 0,1 nil <0,02 0.1 ugn <0,02 0,1 PO 0,02 0.5 nicht bestimmbar. d.h Gehalt ist kleiner ab die derzeitige Bestimmungsgrenze Beurteilung. Die Beschaffenbert der Probe entspricht lensichtlih r n nn1,1 r m r en Anforderungen EYDAC0H LIACALreOrt-Pe es ANNeNtlart nach DIN EN ISOJIEC 17025 mm ---- Pre erelereatries earner ner eur umeneceren eneaen Per Valeerrecrhunnne Veerreesop,ep par Perbertene rred c. ece'. nr MIEneMOI, SSW en. a...geese Yemen, rn some.. f dean >evertor. imager sceptre:nee Geelererereell noarysenbencia vom 06 11 2099 fur Probe 200927278 Seile 4 von 4 SCO amONO SALT `Study Number 1305601 Report 15.2 CertifoifcAnaitmael Food Page34of 38 ToldGataGe 165 2018 Pr Resor & otS m J e eee TEE CoocfAnasys F = Y 53m 1% 1% g 5 E: ffara ih 4i3z ) E2 22 Ni im il bem, - -_ -- BT iz Tet wevan 3 == Ta Th ted EEnT airra 01148 0600 `Study Number 1305601 C604 AMMONIUMSALT Report Page 35 0f 38 Tekdad Certifed Giobal 18% Protein Rodent Diet 2018C [] MutiResidueSoroen PsEheSe ACO 1OOMA ourTEm r 5 w = m=ao5 2 =C=x BE FnoeeE = HE i - ar na cul foPe ooaft E w=i :20 mnt E=na oiiel] fePoagorama-s nSa 3iom] pebaesunt = =zaz ows uo po bE 22 Feoa erss 2u5l5 B ET} LDoreea 2= a55 f= IE pee ERpER EE roto 2 ol 2 Si fre--n feine =3n=e oao]n = femme 223oor]r E8iige 2% harlan- 83 opyoudioresn earchbgeter rfpaotees E: =3oo oo = m2 neree ote eben PCBon 420 MaWId SOTH,AZ +050 483 E31 wembancn apres cStCu5d0y4NAumMbMeOrN1I3U0M86S01ALT Report Page 36 of 38 15.3 Certificate of Animal Bedding EERE EEiEd meni tad [2pmut lemesann.ex TSeasrtiRersohiRts P-- Tra ---- yy pre-- EEE =i `Study Number 1305601 C604 AMMONIUM SALT Ce Report Page 37of38 =iatorE gronee Y [-- wm = gon Fi .# EEE Es mwa S51 3 5 Er RIE bas T BE riam= -- SEoc pa = Study Number 1305601 cC6O4 AMMONIUM SALT Report EE em LUFA-ITL ACROLAS lageP054Wsee .91. 4 I >4.M Xs 7.9.44 .44-44u:me -4a.r.o. tera,se .941 dennehildle444 _--Sample No. 584525 l.4,1 Garon4444 __ Fanfmn 1.199/94 e/sathileno . 1/19444Non i'eankinee,;, Pm0ION441144 DIMON:497e erniehowernle Preencras Gala Teo 'DAP i rlSO 91.4 ,, , D try ta 4940 8238 9 I"0,4,0 ', Ole :ergeg Other Analyses 41101 '7 OlO Am& 4,040 _ 4.14 4),N4 9:4I0 91.011) 4.01D .14:19_ 4014 4.040 4010 .. ame eak. 7 DWO Cate" no Page3013 26 112008 21261 r),m444 4..4 MOM Om ....: Ilelirlf91 _ OH .4 ?: .::ri/ :GIre ia.:l ON 'in ea Wil478 L03941 chr ma ;a ye JetMae CAI . 44ii 8..2titicii4. OW ..4 So 44401`41144 ow . aa . 46.:rei.0.sem oh 4cm:9nit...Uhl. ou .4 e *Oa UM.. OS .men18440 taxes TO.swine&owner ea e'Sin er greedMe.4 fl edge/M.. leellbleeft 0 liwItInt AM. et. 14^', ... 0 ....."9.,....' 31." ,,...NW Ta anavearmub et regd nitietzernesers n .4444:: vaarreor ,Ruin mg swede ofurrmen IIIUM ten>, hp exedra eel. aurae Page 38 of 38 =n