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MIN315022356
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'PERFLUOROOCTANESULFONYL FLUORIDE (POSF)
T-1e6L3>
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES
Sponsor IBMiGCnoerngper00TsSiselesy SWrrSais
CONTA NO gy
THEY
1%
Resarct Laboratory HSNooenoritnyrgoLuif Sciences id HCfeomismmgeonts RoLAND Reprised 2 eer 202
000044
CONTENTS
MIN 315/022356
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Page
(COMPLIANCEWITHGOODLABORATORYPRACTICESTANDARDS ..
wenn
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QUALITY ASSURANCESTATEMENT...
5
ASSEOSFRSESMULTESvoNc T
15
ansin
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090845
"TABLES
CONTENTS (continued)
MIN 3157022356 Page
3. Metanaalyspis hta-arsse t6stosonncomommoosericrcnes 22 5. Metanaalysipsdatha- 6a000s8 e 68onmrreererncrins 26
APPENDICES 21. FHistioricarlpeooigsreaCOkOtMTOciltvh eeemssmgommmmmrreonres 238 5 Apparatusforvapourgashsexposeofclivaiedmammalian cel... 30
4 EyeResearchCentre GLP Compliance Statement,2001 ...mssmsennes 31
sid
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020048
MIN 315022356
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(COMPLIANCE WITH GOOD LABORATORY FRACTICE STANDARDS
hPeacsiyieads,rwiitnh i5 arpepirownsscionlcove,n5coCmpolenwtihtehihse GollaoviengdGoeodaL.abor The UK Good Lavorstony Pie Regan 1599 (Sistry nent No 5106)
[ECCommission Directive 1999/11/EC of 8 March 1999 (Official Journal No. L 77/8). OECD Principles of Good Laboratory Practice (as revised in 1997), 'ENV/MC/CHEM(98)17.
GWientvoith norma prc ini typeof she sy, he protocol dds rie ans of he The cpl ate hs tes substance vas the sesponslyof Spon
iS. suidnDiAcT,pDEAlToe, DEsSSPar ViFre Depurmenof Gees axel.
`Huntingdon Life Sciences Ltd.
C Bue lr 2 2.
"
006047
QUALITY ASSURANCE STATEMENT `The following have been inspected or audited in relation to ths study.
MIN 3150022356
Study Phase
Date of Inspection
Date of Reporting
Protocol Audit
11 January 2002
11 January 2002
Study Based Inspection Culture treatment
17 January 2002
17 January 2002
Process Based Inspections.
Formulation Culture Establishment `Harvesting and slide preparation Slide scoring 5:9 preparation
14 January 2002 11 February 2002 7 February 2002 5 March 2002 15 January 2002
14 January 2002 11 February 2002 8 February 2002 5 March 2002 15 January 2002
Report Audit
15 April 2002
15 April 2002
`DPirroetcotcoorlanAdudCiotm:pAannyauMdaitnaogfetmheenptroatsoicnodlicfaotredthaibsovset.udy was conducted and reported to the Study.
Study Study
DBiarsecetdorInasnpdecCtoiomnp:anAynMiannspaegcteimoennoafas
phaseof indicated
this study above.
was
conducted
and
reported
to
the
ParnodcreespsetBitaisveedprIoncsepdeucrtieosnse:mpAltoyoerdaobonutthitshetytpiemeofthsistusdtyudwyerweascarinriperdoogurte.ssTihnesspeectwieornesocfornoduutcitneed.
and reported to appropriate Company Management as indicated above.
`cRoenpdourctteAdudaintd:rTehpiosrtreedpotrotthhaesSbteuednyaDuidrietcetdorbayntdheCQoumaplaitnyyAMsasnuraagnecmeeDnetpaartimnednitc.ateTdhiasboavued.it was
`The methods, procedures and observations were found to be accurately described and the reported
result to reflect the raw data.
G`ArnogueplaMJaencakgienrgs,, B.Sc. MSc., Fil, MRQA., DepartmentofQuality Assurance, Huntingdon Life Sciences Lid.
:
Lae
is:
Date
000048
RESPONSIBLE PERSONNEL
Linda Allis, DEA Tox., DESS Pharm. Vet, France, Study Director, DepartmentofGenetic Toxicology. Lincoln Pritchard, B.Se., Study Supervisor, `DepartmentofGenetic Toxicology.
MIN 3151022356
0960: 3 oR CERT
SUMMARY
MIN 315/022356
A study was Chromosomal
performed aberrations
to in
assess human
the ability of Perfluorooctancsulfonyl lymphocytes cultured in vitro.
Fluoride
(POSF)
to
induce
Hphuymtaohnsemlaygmgplhuotciyntiens,,andin
whole exposed
blood to the
tesctulstuubrse,tanwceerbeothsiimnutlhaetepdresteoncediavniddeabsbeynceaddoift$io9n
of mix
deenrdoivfetdhferoimncaubaltiivoerns.perSiooldv,encetllanddivpiossiiotnivweacsonatrrroelstceudltuusriensgwCeolrceamlsiodp,rtepharcede.llsThwaorvheosutresd baenfdorselitdhees
prepasroetdha,t metaphase cells couldbeexaminedforchromosomal damage.
In order to assess the toxicity of Perfluorooctanesulfonyl Fluoride (POSF) to cultured human Sloylmvpehnotcyctoentsr,olt.heOmnitotthiecbiasnidseoxftwhaesscealdcautlaa,tetdheffoorlallolwciunlgtucroenscetnrteraatteidownistwhetrhee steelsetcstuedbsftoarncmeetaanpdhatshee. analysis:
Firsttest
`Withandwithout $9mix - 3 hourstreatment, 17hoursrecovery: 125,2.5and 5% vvatmosphere.
Second test
`Without S9mix -20 hours continuous treatment: 08, 1and29%viv atmosphere.
`Wi59tmihx - 3hourstreatment, 17hoursrecovery:2, 5 and7.5% vv atmosphere.
In both the sutisically
saibgsneinficceanatndincprreeasseencienofthe9prmoipso,rtiPoenrflouformooecttaapnheassuelfofniyg]urFelsuocroindteai(niPnOgSFc)hrcoamuosseodmanlo
aberrations atanydoselevel,whencomparedwiththesolventcontrol,ineither test.
`Aqdousaentlievteal.tiNvoe asntaatliysstiiscaflolry psioglnyipfliocaindtyiwnacsremaasdeseinintchueltpurroepsortrteiaotneodfpwoiltyhptlhoeindegcaeltlisvweecroentsreoeln.and highest
aAblelrrpaonstitcievlels,codnetmroolnsctormapottuinnhdgssecnasuistievditylaorfgtehestteastitsstyicsatlelymasingdniftihceanetffiicnaccryoeafstesheiSn 9thmeixp.roportion of
ofisclcaosntcolguedneicd tahcettivtihtey tienst sthuibsstianncveitPreorfclyutoorgoeoncettaincestuelsftonsyylstFelmu,oruinddee(rPtOhSe)exhpaesrismheonwtnalnocoenvdiidteinocnes described.
3 paaeun
:7:
090050
INTRODUCTION
MIN 3157022356
t"oTchiasurseepocrhtrdeosmcorsiobmeaslaasbteurdraytdioenssiginnehdtuomaasnselsymspthhoeacbyitleistycoufPlerufilnrcrieoroodc.tanesulfonyl Fluoride (POSF) The std was conductedin compliancewithth following guidlines:
`OMEaCrmDaliGuaindCelhirnoemofsoormetheAbeTrersattiinogn Toefst,ChGeumiidceallisn.e 4(7139.97) Genetic Toxicology: In Vitro
UChSroEmPoAso(m1e99A8b)errHaetailotnh TEesftf.ectEsPTAes7t12G-uCi-d9e8l:i2n2e3s.. OPPTS 870.5375 In Vitro Mammalian H19u7m5a, nScloyttm,phDoecaynt,esDahnafvoerdbeaennduKsierkilnantdhi19t9y0p)e.ofTshteuydyarfeocaulnturued minobfvyiteeraorrbsut(EdvoannsotandidviOd'eRiuonrldeasns simulatedtodoso. Thisis achibeyavddeindg phytohaemaglutinin (PHA) totheculture that resuisn
`ahigh mitotic yield (Nowell 1960).
icultuhirsesmteuddiy,umb.looTdhetackuelntufrresomweheealitnhcyubmaatleed ninont-hesmporkeisnegncdeoonforPsHwAasbepfooorelebdeianngdtrdeialtuetdedwiwtihththiesseuset substance, Following treatment the cells were amested at metaphase using the mitotic inhibitor, Clorlocmemoisdom.e abCehrrraotimoonss.omTehsebiensttheesset.imamteetoafpthahseeshceelrlastiwoenrferetqhueenncyexsaatmintehdefifsotr etlhle dipvriesseinocnea.ftoefr ithniistilataiobn oofrtatrhetaeotcmreelnyt csyicnlcee tciemrteafionrthyupmeaonfldyammpahogceytmeasyinbewhloolstedbulroiondgcsuultbusreequisenatppcreolxidmiavtiesiloyns1.3-1I4n hours.
Tbohtehstthuedyprweassenpcee ranfdothorenmatbewsdoencseepaorfat$e9ocmciaxs.ionsI.n tIhne tsheecfoinrsdt tteest., aatchorneetihnouuorustrteraetamtemnetnwtawsausseudseidn withoutS9mi, ndthetestwith $9mixwas repeatofthefisttest. Abbeeernraetxicolnusdewderferoscmortehde aqcuacnotridtiantgiotno otfhecchlraosmiofscoamieonsboefmtahteioInsS.CNS(o1m98e5)g.apTsr,adhiocwoenvaelrl,y hgaavpes hbaeveen s1h98o1w).n tIonbethirseasltduidsycotnhteintuoittalieasminbeDrNAof(cHeleldsdlceonatnadinBinogdyacboertreat1i9o7n0s,SbaotthyawPirtahkaansdh,wiHtohuouatndgaPpasthhaaks beencaleulaied. Msyasntyemssutbhsattaanrceesnotdofonuontd einxecrutltaurmedutcaegleln.icTheefrfeefcotreuntthiel ctuhletyurehsaavnedbteeestnsmuebtsatbanocleisweedrebyinecunbzaytmeed
in both the absence and presence of a supplemented liver fraction (S9 mix) prepared from rats.
(preMvioausalnrydtAormeaentsed w1i9t8h3,aNsautbasrtaajnacneet(aAlr.oc1l9o7r6)1.254) known to induce a high level of enzymic activity
Lone
5:
000051
MIN 3157022356,
"oTnhe31prAoutogcuosltw2a0s01pranodvbeydthbeySHtuundtyiDnigrdeocntoLrifoenS1c0ieJnacneusarMya2n0a0g2e.ment on 18 July 2001, by te Sponsor `he study was conducted at Huntingdon Life SiencesLid.Eye, Suffolk, P23 7PX, England. Trehsepeecxtipveerliym.ental sart and completion datesofthe study were 15 January 2002 and 15 March 2002,
REET
29:
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000052
entity:
CAS No:
|
Appearance:
Storage conditions:
Batch number:
Expiry:
Purity Date received:
TEST SUBSTANCE
MIN 315/022356
'
Perflcroocianesulfonyl Fluoride (POSE)
307-357
Clearliqid Room temperature os0227 Sduproantsioonrsofrtehseposntsuidbyilty: assumed sable for the 955% 16 June 2001
Ltn .
10
0005;5
EXPERIMENTAL PROCEDURE
MIN 3150022356
(CULTURE OF LYMPHOCYTES
RHuEmMaIn1b6l4o0odtwisassueccoulletcutreedmaesedpituicmalsluypfprloemmehnetaeldhyw,intohn-1s0m%okfionetgalmlcaelfdosneorrusm,,po1oulendianvd Hdielpuatreidn,wi2t0h mLedUium:pe0n.1iimlllp2h0yohuga/emmlagsgsleupilnoimny)coifnthaendce2l0sumspMensgilountawmeirnee.plaAcelduiontis r(l04unmilverbslaolodco:nt4a.i5nemrls athnedcienllc.ubated at 37Cfor approximatly 48 hous. Thecultures were gently shaken daily to resuspend
POSITIVE CONTROLS In the absence of $9 mix. SuTppelnie:r: BApaptecahrnauncmeb:er: Solvent:
Final concentration:
In the presence of$9 mix SuHpepnliiieyr:: Appearance: SBoaltvcenhtn:umber: Final concentrations
MSiitgommayCchienmCical CoLud B3l1uKe2p5o0w0der Serle purified water 00.1.2 ppgg//mmll c(3ohnotuirntureoatrmeenatm)en)
CAsytcaloMpehdoiscpahaLmidide White powder (StOeNr4il6eSp(uTreisfite1d)awantder1H485(Test2) 10 pgm
PREPARATION OF $9 FRACTION SSpeexc:ies SoSutracien:: AWgeeisght
MRaatle `SCphraarlgeuseR-iDvaewrleUyKderived <7-80weeks
: fen
1: 000054
MIN 315/022356 l5i9vefrrsacwteiroen swtaismulparteepdarbeydAfrroocmloar g1r2o54u,poafdmcian.ist1e0reandaimsalas.sinMgliexeindvafpuenrcittioonneaolxiidnafseectisoynstienmcs oin otiheartata draotssawgeeroefKSil0l0edmga/ndkgthbiordylwieveirgshats.epOincahlleyirtemhovdeady.after injection, following an overnight starvation, the "0T.h1e5 fMolKloCwIin(g3 smtlepKsCIw:ere1 cgalrveed)obuefaotr0be-i4ngCtruanndsefreraesdepttoiacnchoonmdiotgieonniss.erT.heFollivleorwsiwnegrperepplaarcateidoni,n dtihespheonmsoegdeinnaotaelsiqwueortescaenndtisfourgeedaatt-08000Cog fborel1o0wmuinnulterse.quiTrheed.supermatant fraction (9 faction) was
PREPARATION OF $9 MIX 9pHm7i.4x(c1o0nt0aminMe)d,: $gh9cfosaec-i6o-nph(o1s0p%haVt1e)(,5MmgVCDl, N(8AmDMP),(4KmCMI).(33AmlMt)h, scoofdaicutmorosrwtehorpehfolseprh-astteeibluifsfeedr before use
TREATMENT OF CELLS WITHTEST SUBSTANCE - FIRST TEST Acufltteurreapmperdoixuimma.teAltym4o8sphhoeurres,sotfhePecruflltuuroersoowcearneescuelnftorniyf!ugFelduaorniddeth(PcOaSlFls)wweerree reesstaubslpiesnhdeeddininsefarleesdh g2l0a,s4s0baontds70(%16v0mvlatimnoesrpnhaelrev.olAuimre)wawsitwhitshedprtauwmncfaprsom0eagcihvebfoilnael acnodnctehnetnraatnioanpsproofpr1i.a2t5,e 2v5o,l5um,e1o0,f Ptehrefsleuportouomctcaanp.esuAlfftoenrye!vFalpuoorraitdieon(oPfOSPFe)flvwoarsooienttarnoedsuuclefdonuys|inFglauosryirdien(gePOaSnFd)naeneddlee,quiinlsiebrrtaetdiotnhorfoutghhe atmhoespihnceurbeastetd o3n7thCe,rtshiedelsyamtph3o7cytCeicualtruorlelserweaprpeariantjeucste(dceinaopptehndibxil3:e.AppTahreatgulsasfsorbovtalepsouwerrse spphparsoeimeaxtpeolsyu.re Tohfecullytmipvhaotcedytmeasmcmoastlitahn icneslilsd)eo, fwhtihcehborodteatsesantdewbeorteleismmoenrcseedevieryculctguhrte mmienduitoems tohnecereevvoelruytiornv.olTuhoensoalnvdenetxpcoosnetdrodlir(eAtrl)y twoaPserefsltuaobrloioschteadncisnuldfuopnlyilcaFtleuorciudere(PsOSaEn)d fcoornttahinreeds aonf aatnmdaolspshoecroentoafiinre.d aMniattommoyscpihnerCe, oaftaai.final concentration of 0.2 ig/ml, was added to duplicate cultures cIumlmteudrieataenldydibsecfaorrdeedt.reaTthmiesntowafsthreplsaecceodndvisteht o1fmcluolfuSes9, m1ixm.lTohfemceudlituurmeswwaesreretmhoevneadddferdom10eatchhe acopnptrrooplriactueltuglraesss wbeorleeegsitvaibnligsthheed suanmdeersearinesaomfosfpinhaelrceoncoefntairr.atioCnyacloapbhoovsep.hTahmeiddeupwlaicsataedsdoeldven(t duplicate culturesa a inal concentration of 10 g/ml that were contained ina atmosphereofsir "aTnhdrereeshuosupresnadefderindofsriensgh, mtehdicuulmtuurnesdewrearne actenmiorsipfhuegreedoaft a5i00ing ufnoirversmailnuctoenst.ainTehres.ceTlhlseywewreereritnhseend incubated fora further 17 hours.
hin
2
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HARVESTING AND FIXATION
MIN 3151022356
T(Swiogmhao)urtos abecfhorceultthuerecelalts8wefirnealhcaornvcesetnetdr,atmiiotnotoifc 0a.c1tiviigt/ymlw.asAfartreerst2edhobuyrsaddiinctuiboantioofn,Coclaecehmicedll
suspension was transferred to@ centrifuge tube and centrifuged for minutes at 50g0.Thecell pellets
`hwyeproetotnreiacteidnwciutbhsiaohnypatot3o7niCc,sotlhuetisounsp(e0n.s0i7o5nMs KwCelepcreenwtarirfmuegdedatat37SC0)0. Afforte5rami1n0umtiensuatnepderthieodcoelfl p"Tehelfsstfiixveedwbaysardedpiltaiocneodffurftrheesrhlmypersepanriedlcobedcfaimxeaticvoelo(u3rplaesrss.methanol :1 part glacial acetic sed)
SLIDE PREPARATION Tsmhaellpevlloeltusmweeorfe frreessuhspfiexnadteidve,.thAenfceewntdrriofupgsedofstth50c0elgl. sfuosrpenmsiinountewseraenddrfoinpaplelyd roenstuospperned-ecldeainned8 pmircerpoasrceopien bsluifdfeesrewdhiwactherwe(r6eHt6e.8n).alAlfotweerdritnosiainrg-dirny.bufTehreedsiwdaetserwetrhee stlhiednesstwaeirneedleifnt 010a%ir-Gdireymsaan,d tchoemnplmeoucndted in DPX. The remsining cultures i fixative were sored at 4C unt slide analysis was
MICROSCOPIC EXAMINATION "mThietporepcaelrlesdpselride1s0w0e0rcelesxaimnineeadchbycuiltguhremwicarsosrceocporyduesdinegxcaelpotwfporowpoessitoibvjeecctoivnet.roTlhteaptreodpocurlturieosn.f FSorlovemntthceosnetroelsuvlatlsuethoe,fdotsheelreevewlacsaunsoindgecredaesc,rtehee mianxmiimtumoaichnideevabolfeacpopnrcoexnitmaattieolny w5a0s%uoefdthae6 tshleecheidgh.est dose level for the metaphase analysis, The imermediste and low dose levels were alo d"Tohseedcounncleenstsraptrioenliomfienaacrhypsocsaintiovfemceonttroalpcohmgapuosrueendiselnecdtedifaoncrinaasnuafltfyisecisiwednatsletvheelolfowsesbt ceonrceaenltrnsa.tion e"Txhaemsienleedctesdt s2idmeasgnwiefriceatthieonnocofdexd.100M0etuaspihngasaenceollls iwmemreerisdieonntifoibejdeucstiinveg. alOonwepohwunedrreodbjmecettiavpehaasned sfihgouwriesngwearehiegxhamleivneel,ofwhaebreerrapnotssicbellels,. frComhrcoamcohscoumleturaeb.errTahtiisonsnuwmebreer swcaosredredauccceodrdiinngc(ultuhrees elnadsoiriecdautpiloicnaotfetdhceeIllSsCwNer(e19n8o5i)d. Ownhleyncseelelns,itThhe4ve-r4n8iecrhrreoamdionsgosmoefsawleresbaenraalnytsemde.taPpohlayspelofiidguarneds er recorded. f"oTreneiganticveicoondftrpeoollncyuplcltoueirdesmaentdacpuhlatsuerecselels,toeudt wof 0t0ihemheitgtahpehsatsdeocselle,v]waosftdheetetersmtisnuebdsqiuaanncteiutastidveliyn heanalysis for chromosomal sberaions. "TvhaleuneusmibnegrFoifsahbeerrrtaenstt m(Feitsahpehra1s9e73c)elsin achtreatment group was compared withthe solvent control
ny may
i: 000058
MIN 3150022356
SECOND TEST Cturletautrmeesntwwearse unsieidstinedthaenadbsmeanicnetoifne$d9 amsix.preIvniotuhselyprdeessecnrcieboedf. $9Inmitxh,isastehcroenedhoeusrt raeacuomneinntuwouass usPeerdf,lauorionoctthaenfeisrusltftoensyt.lTFlhueorhidea(rPOtSviFm)eewwesrsetaats2f0olhloouwrs:s forbothparsofthetes. Concentratioonfs
`Without S9 mix:
0.1,02,0.4,0.6,0.8,1,2and 5% v/v atmosphere.
With $9 mix:
06,08,1,2,5 and 7.5% viv atmosphere.
cDounpcleinctartaeticounltourfes 0.1wegr/em,usaenddfCorycelaocphhotsrpehaatmmeindte,antaditnhealsoclovnecnetntcronattriooln. ofM1i0togm/ymcli,nwCe.r, eatadadefdintaol duplicatecules
"Three hours ater dosing, the cultures containing S9 mix were centifuged. The cells were rinsed and riesnuscpeundfbeodraaintfufrertehsdehrm1e7dhiouurms.unCduelrtuarnesattrmeoatsepdheinrethoeafbsaierncienoufniSv9ermsialxwceonrteainienrcsu.batTedhefyor w2erheoutrhs.en
Aelnld couflttuhreesinwceurbeattiroenatpeedriwoidt.h CTohlecyemwiedre, tahtean fhianravlesctoendc,enftirxaetdioannodft0h.e1suligd/emsl,prtewpaorehdou2rss bpreefvoiroeustlhye described. The slides werethnexaminedmicroscopaiprceaviloulslyy described.
STABILITY, HOMOGAE NDN FOE RMUILAT TIY ON ANALYSIS
"dTehteersmtaibnieldityusapnadrthoofmotghiesnesiutdyy.ofAtnhaelytseissosfubascthaniceeveadncdonocfenttheratteisotnswuabsstannoctepeinrftohremseodlv2enptarvteorfe hniost study,
PETE
We
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MIN 3150022356
ASSESSMENT OF RESULTS
An assay is considered to be acceptable if the negative and posiive control values le within the current historical control range. "The test substance is considered to cause positive response ifthe following condions are met:
cSuhtriosmtocsalolmyessi(gneixfcilcuadnitnigngcarepass)erse(Po<b0s.e0r1v)eidnatthoenfereoqfumeoncryeoefstmectoancpehnatsreastiwoint.h aberrant "cTohnefiidnecrnecaeselsimeixtceed the negative control rangeofthi laboratory, taken at the 99% The increases are reproducible between replicate cultures "eTxhtereinmcerteoaxsiecsitayr.e not associated with large changes in osmolality ofthe treatment medium or Evidenceof a dose-relatonship is considered to support the conclusion. SAhonvegeactoinvceurrersepntoncsoentirsocllafriemqeudenicfineossatraetiosbtsicearlvleyds,iagniafnicyandtosienclreevaels.es in the number of aberrant cells A further evaluation maybe carried ouiftheabove crteia for a positive or a negative response are not met.
MAINTENANCE OF RECORDS All raw dats, samples and specimens (if appropriate) arising from the performance of this study will emin the propery ofth Sponsor Tanydpeasrcohfisvianmgplmeaayndbespdeicsipmoesnedwhoifcahfatree uthnesuiptearbiloed,sbsyatreeadsoinnofHunintsitnabgidloitny,LfiofrelSocnigenecrems Sreutnedntairodn Operating Procedures. Aalrlchoitvheerfosramppleersioadndofspfeicveimyeenasrsanrdoalm trhaewddaatteaownilwlhbiechrettahienSedtubdyyHDuinrteictnogrdosnigLnisfethSecifeinnaclesreipnoritt.s `After such time, the Sponsor will be contacted and his advice sought cn the tum, disposal or further retention of the materials. If requested, Huntingdon Life Sciences will continue to rea the
`materials subject toa reasonable fee being agreed with the Sponsor.
HofutnhteinfgindaolnrLeipforetSicnietnscaerscwhiilvlerientdaeifnintihteelQyu.ality Assurance records relevant o this study and @ copy
15:
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RESULTS
MIN 315022356
FIRST TEST
Toxicity data
aMritsothiocwinnidincTesaobflec2u.lured human lymphocyte tested with Peflorooctanesufony Fluoride (POSE)
Iinndtehxeta0b5s7e5n%ceoftofheS9somlivxe,ntPecornftlruoolrovoacltuaeneastudlfoosnyllevFelluoorfi5de5(vPvOSaFt)mocsapuhseerde.a Trhedeucdtoiosn Iivnetshesmliteoteicd. forth metaphase analysis were 1.25, 2.5 and 5% viv atmosphere. Iinntdhee(p0r.e5s2e%ncoeftohef$9somlviexn,tPceofnlturoorlovoacltuaenaetsudlofsoenyl!evFelluoofri5d%e (vPivOSaFtm)oscpahuesreed. aTrheedducotsieonlienvetlhs semlietcotteidc forthe metaphase analysis were 1.25, 2.5 and 5% viv imsphere. "fTihgeureqsuwanhtietnactivoemanpalaysrtiosethfdeorsoplovleynpltcoiodnytsoh.owed no increase in the number of polyploid metaphase
Metaphase analysis IyTmhpehoecffyetcetss rofePsehrofwunorinooTcaabnlees3uafnodryslummFlauroirsiededi(nPTOaSbPle) 1o.n th chromosomes of cultured human Isntibsoitchaltlhye asbigsneinfciecanatndintchrepsrsesiennhceeopfr$o9pomriio,noPfeclculosroowcittahnecshurlofmoonsyo|mFalluoarbiedrera(tPiOonSsF)atcaaunsyeddosneo level, when compared with he solvent contol. sBiogtnhifipcoasnitiivnecrceoasnetsro(lP<c0o.m0p0o1u)nidst,heMpitromoypcionorfCatbaeinrdroaCnnytcellolpsh.osTphhiasmdidee,mcaousnedstlhaetregfericsataacttiysoteifcatdlhley 9 mix and the sensitivityofthe test system.
SECOND TEST Toxicity data aMritstehinoidnicwTeasbnolfec4u.ltured human lymphocytes trated with Perluorooctancsufony) Fluoride (POSE)
Iinndtehxetoab6s1e5n%coeoftfhSe9somlivxe,ntPecornftlruoolrovoacltuaeneastudlofsoneyllevFelluoofri2d5e (vP1OSaFt)mocsapuhseerdea. Trehducdtoisoen lienvetlhsesemlietcotteidc forthemeaphase analysis were 0.8, 1 and 2% viv atmosphere.
116:
BRE
000059
MIN 315022356 mIinendtteahxpeh(pa0rse3es9eann%acolefoystfihsSwe9esromeli2vxe,,n5tPaecrnofndltur7oo.rl5oo%vcatvliaven.eastuldfoonsylleFvleuloroifde7.(5P%OSviFv). cTauhseeddoasereldeuvcetlisosnelinecttheed mfiotrottihce: `"WThheenqcuanotitmatipveoatanhaleryssoielsvfedonrtpocloyntprloolidy showednoincrease inthenumbeorfplyploid metaphase cells
Metaphase analysis
TIyhmephefofceycttss oarfePserhfoluwoinrnooTcaabnelse5ulnfodnys]umFmlauroirsideed i(nPTOaSbFl)e 1o.n the chromosomes of culured human Iunstbiostichaltlhye saibgsneinfciecanatndintchreeapsreesineatcheeopfroSp9ormtiixo,n oPfecrleulorloowcittahnecshurlofmoonys!omFalluoarbiedreat(iPoOnSsE)atcaaunsyeddonsoe level, when compared vith thesolvent control. Bsiogtnhifipcoasnittiivnecrceaosnetso(lPc<o0mp.o0u0inn)dtsh,epMriotpoomryteiionnoCfsabnedraCnytccllosp.hosphamide, caused lage, saiscally
CONCLUSION
1ofsclcaosntcolguendiecd tahcaitvtihye teisnt tshubsstianncierPoeflcoyrlooogcetnaeniecsutelsftonsyylseFlmu,oruinddee(rPOthSeF)exhpaesrismheonwtnalnocoenvdiidteinocnes described
BY
Banu
000080
REFERENCES
MIN 3151022356
EVANS, HJ. and ORIORDAN, M.L. (1975) Human peripheral blood lymphocytes for the analysis of chromosomeaberrations in mutagen tests. Mutation Research, 31, 135. FISHER, R.A. (1973) The Exact Treatment of 2.x 2 Table in: Statistical MethodsforResearch Workers. `Hafner Publishing Company, New York. GALLOWAY, SM, DEASY, D.A, BEAN, CL. KRAGNAK, AR, ARMSTRONG, MJ. and BRADLEY, M.0. (1987) Effects of high osmotic strength on chromosome aberations, sister chromatid `exchanges and DNA strand breaks, and the relation to toxicity. Mutation Research, 189, 15. 'HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formationof chromosomal aberrations. Mutation Research, 9, 117. ISCN (1985) An Intemational System for Human Cytogenetic Nomenclature, HARNDEN, D.G. and KLINGER, HP. (Eds). S. Karger AG, Basel. MARON, DM. and AMES, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173. NATARAJAN, AT, TATES, AD., van BUUL, PP.W., MEUERS, M. and de VOGEL, N. (1976) Cytogenetic effects of mutagenslcarcinogens afte activation in a microsomal system in vitro. Mutation Research, 31,83. NOWELL, PC. (1960) Phytohaemagglutinin: an initiator of mitosis in cultures of normal human leukocytes. Cancer Research 20, 462. SATYA-PRAKASH, KL. HSU, T.C. and PATHAK, S. (1981) Chromatid lesions and chromatid core `morphology. Cytogenetics and Cell Genetics, 30, 248. SCOTT, D., DEAN, B., DANFORD, N.D. and KIRKLAND, D.J. (1990) Metaphase chromosome aberration assays in vitro in KIRKLAND, D.J. (EA) Basic Mutagenicity Tests: UKEMS Recommended Procedures. Reportof the UKEMS sub-committee on guidelinesformutagenicity testing. Report, Part | revised p. 62. Cambridge University Press, Cambridge.
Ganfipn
18: 000061
in TABL1E
TT oe ETEEE ETr ETl (hours)
(%viv atmosphere) [Sad
Tra Mean Index
=| | a Ln |BE
a Tew
yz
75
fo 1
07 to 2
13
ES)
a 510ug/mi(Cyclophosphamide)| *18 *18_ 180% ['20 '20 200%"
3
50cellswereanalysed from thi culturedueto insufficientmetaphasespresentonslide
appt
000062
MIN 315022356.
TABLE2
Mitoticindex data- first test
`Without S9 mix, 3 hours treatment and 17 hours recovery
PerlCoornocoecnitarnaetsiiolnfoofnyl FluIovriademo(sPpOheSr) o ain
Moteindex # |Reliivnedmexine Incidence %Mean ) ssn000 90 100 21000
Poiypioy Incidence __%Mean 100 02 500
125
Ss00o0 sa
25
no 6s
n
55/1000
5
5s7m10o00 51
5
20550000 02
10
a
2
bb
"
obe
n
#3 CVoelrcyuoiwomnesthaapvhaesbeesepnrmeasdeneosnisnigdreounded values b Nocels,no meaprheseatoensisde
ANB
Cio:
000063
:
MIN 315/022356
TABLE2
Mitoticindex da -fit rstta est (continued)
`With$9mix, 3hourstreatmentand 17hours recovery
PerCfoomracoecrarnacosnloffon! F5oirsdaem(oPOSF) =0)
Vion index | Relaindmeox le
Incidence %Mean
10o01m000 99
10
Folio; Incidence %Mean u5s0o0 02
125
1w131i000 91
0
25
s5311u000 79
0
5
26e00100000 51
2
5a0s0o 00
0
.
2
5b
"
bb
0
#3b4 NCoV otlcelteonmsom hrcvelbsee leonmmepdarwt epesheaintel ogse nrrsoisudineadeodne vsioes Do elma
En
Lane
000064
Mv s02356
TABLES Metauaplshduaa Ssst fest Without $9mi,3hwrswstm17ehounssaedr
rCoegeom ononr |yNr o Tin | TNEoaber] _[Rpa--aon
oon
rwewe1|.onmreee| ogo on
>5 w-|
Lrols[2 | w
us wwl:|s |e
2 w| s
5
@w| |
CETs asl so] w : 2|e s] Pi asm 2 l:s w]e: sels
ormiene || o0w |[5 | 2|S
oPasle mwoan wo] -
3SaO Smam mm, one pFoio
oS3BweemCe SoemmT nte,ErioE nveR ts ii
Pan
.
000065
TABLES Metaphase analysisdua fs est. continued) With $9 mi, hours etmentand 1 hows ecovey
VN 5102356
roCmonaeneacoienossnf)|[Ncooemle]t Crome CrToomearnieoOnT | Go|sNboeorfacll_[Reliijoeeo]
(% viv atmosphere)
crypee.e| ovpreee | on on
%
=. w- | 2[3
Ps oE[2 w]e sw] w
us w-| lh 2 w-| |s s www| [1
2 |LaliPs som vo|ls aesfe wesw CtCaee l us| w
Coopopwmannity|| 00 [[71 2
a2 os{io mWol e ar] -
oSDoSm ESmTee.
ow Tr
EuC SmemmmeepE ,g ero? peels
Er
onoer prion
ERG
000066
TABLES
Mitte Indesdaa secon test
With $9mix, 20houscontinousream
PeuCmoamcearnmsoanieiton Nowiers |Remneres
`F(lRueorviadmeo(gPhOeSrF)) | bide Men
aon
wwoooo 17 10
MIN 3150022356
Food bide em w50o0 00
01
io3n0o0 109 5
0
inwao 109 5
0s
ssaonmo 94 "
[3
oaonom 6s
os
Sw0o10w)
'
naiwooe 77
2
Twooo 7a a 0a50o0 00
s
2w5o10n0 22
Cute bebe mdeigroundes
-
a:
000067
TABLE4
MIN 3157022356
Mitotic index da-st ecoand test (continued)
With$9mix, 3 hours restmentand17 housrecovery
PerCuoonrcoeomcrtanisosnlfoofny Fvluiorvide(PamOorSF)
ai0n
Hiro index | Relatiivnedemxote Incidence Mean 9e41n10w00 52 100
Folmiody Iodence Mean 5s0o0n 01
0s
m74o100e0 82
08
m79o100o0 16
"
1
sS1s0o0 70
5
2
ws51o10n00 60
"
s
s6w1i0c0o0 57
3
7s
35m10n00 52
oona 00
# Caletions hvebessmdewinroundedves
(heey
125:
000068
PerfoCrosmoecnurnaeisolnfoofny!
Fluwiondsiem(osFpOhSeFr)e) 2o)
MIN 3150022356
TABLES
Metanaalyspisdhata-asecsondetest
`Without $9mix, 20 hourscontinuous treatment
Chromae|Groomososn|
wre | we ab colon ee
w100 |
2
Ga |Noor[RMt iatotic
in%dex es es)
20 10|2 10] 00
0s
100 wo [2
'
wo [2
100
1
2
10
10
0 100 us| e
2
3
2 us| 20 es
1
1
0 o0fo oof a 0
01pgm) oiomyene)
w | 00
|e 31 [3 22
5 soo sof 7 ee [7 we
@S& GCooosmdteeks & Cromstew Obi"e poF0o01
aa CChwoemsoiodsooddmuse GGes GCnadlopeesarpmseeceohmnisnhr:ions, persed els
t8aty
2:
000069
Mi ss2356
TABLES
Mitotaneslits stony With59min, 3hors vent nd 17 hs covery
r [CCrornoermmaa arirontsly |frNoo R oe | l omeTe |[TNrieo ofsben rmcle [RioplYoeiwe]
lt) fed
w.n TM-
worleesz ww
2
-
s
wo
"0
2y0 t owls asm vyo ole es] 2 us| ow vf0 oro: us|
Cropopapmanias|| 30
saiso wmefln mol -
33 CT m omim e te n Le
onewt rrono
Qm ma STeeEEo mdD persionpitts
exits
7:
090070
rh
oe S-- g --eT ber
tr onr p----S --T a
=>
sgml |]
fi:
I
$5SILl | I M l o| l.
a
Satine
tana oS--d--a9d Dope eSY
heb A
a,
gaa l |
B8 20Liq | fi
. dl iA|en nn.
cor
+
090071
APPENDI2X
MIN 3157022356
Historical positive controWl idatna (oJ3aunmuitaxry 1999 - December 2001)
Excclhuudineggaapep,, loowweerr909%9%cocnofnifiddeennccee kit==180..50%%,,mmeaann == 1975.152%%
w
= zzg
Aifi
g2
EH
H2n0
i HTA H g
10:
l H| i
HHA
:
10 AC
0 5 0 15 2 2% mf 6 0 5 0 6
Freofaqberurantemetanphacsesy(4)
me
istoicl postive controlWdetia5J9amnniuxary 1583 December 2007) Bcinggas, ower56%coniance i 7.0%,mean = 14.55% ching cae,kor99% contd i =8.5%,moan = 8.40%
eozs 2
8s o 0s
|
iE
fm = f(H| t[oELLtEs L2WT ws e0m 6 ww
Froofqseruraemnetaephayses
: 000072
seo, S TATARs reonn e i
er fm niigng on
@
Rotating bottle holder
@ ve
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ote
"THE DEPOA FHER ALTHT OFTM HEGE OVEN RNMT ENT `OFTHE UNITED KINGDOM
SEEBoTemianE m a m R RE mEi eEvarie Eye
Econystems
a EEREaen, Mutagenicity
Ea E A faRm AS E 29January2001
Be fees
SEihR feet
Sut
000072
Ete 2
PCTL T-74"1 Huntini gdon
Life Sciences
PROTOCOL
/
PERFLUOROOCTANESULFONYL FLUORIDE (POSF)
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST
IN HUMAN LYMPHOCYTES
Sponsor 3f SSipMMCCoena mteart eToxicology fSoN Sh132.3220
Tost mbarof pages 12
Research Laboratory fH-- SuonamtiiinggidroonneLife Sciences Ltd CFoEmore
ENGLAND
000075
trmd
Enquiry monber, 23938
Huntingdon
Life Sciences
PROTOCOL APPROVAL
PERFLUOROOCTANESULFONYL FLUORIDE(POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST
IN HUMAN LYMPHOCYTES
ViEmg
n
(
e
Ye
7
la
Yor)
HomiagtonLs Sciences Ld
nr. Ri-- ley
wSeraa
mp51/7 Jor
Please sign both copiesofthis page, retain one for your records and return one to the Study Director at
Huntingdon Life Sciences.
SvyebeDirvcetorsprovaenfhpeeepdrhoeccogivpen oepohse swlatyeeoatsaegeofOtheepprroooeclooecscih do's
000076
.
priser. TO
Huntingdon
Study number:
Se `Test substance Identity: JRExpiry:
i
STUDYDETATS PAGE
MIN/315
Jin
i Perfluorooctanesulfonyl Fluoride (POSE) orrd Sponsor's responsibility; assumed stable for duration of
r tS--oilvent--:a--r--a
Sed `Sponsor's Monitoring Scientist:
Study Director:
Person acting in the temporary absence of the
Study Director:
Location of study:
plaa
`Completion:
Draft report:
ST------------------_ To be determined Tey John Butenhoff
`Ms Linda Allais
Mr Lincoln Pritchard
Dept. Genetic Toxicology, Huntingdon Life Sciences
Ltd., Eye, Suffolk, IP23 7PX, England
Pan
ca 15 March 2002
`Within four weeksofexperimental completion
STUDY DIRECTOR APPROVAL OF PROTOCOL
_
Sei
-
loayfanz --
Huntingdon Life Sciences Ltd
Ganda
000077
: Enquirynumber: 23973
`CONTENTS
LHiufenStciinegndcoens Page
yeeros... inp EA ? 8
GOODLABORATORY PRACTICE ....
QUALITY ASSURANCE... J ett tessa anes, 7
soni
000078
7
`Enquiry number:
23923E
Life SCi i ences
1. INTRODUCTION
The object cytogenetic
of this study test system
is to using
acsusletsusretdhehmuumtaangelnyimcphpoocteyntteisa.l
oTfhithseptreosctesduubrsetacnocmepliineasnwiinthvitthroe
following guidelines:
`OMEaCmmDalGiuaindeClhirnoemofosromteheAbTeersrtaitnigonoTfesCth,eGmuiicdaellsi.ne(417939.7) Genetic Toxicology: In Vitro
CUhSroEmPoAso(m1e99A8b)erHreaatlitohn TEefsfte.ctsEPTeAst71G2u-iCd-e9l8i-n2e2s3,. OPPTS 870.5375 Jn Vitro Mammalian
2. BACKGROUND Maboesrtratcihoenmsi.caAl wmeultlageestnasblpirsohdeudcteestrescyosgtneismabulseincghcruolmtuorseodmahlumdaanmalgyempehxopcryetsessedhaassfsrterquucetnutrlaly Kbiereknluansded119090o)b.serTvheethaebseerraabteirornastiaornes b(eEsvtanosbsaenrdveOd'Ratiotrhdeanme1t9a7p5h,aSsceotstt,agDeeaonf, cDelalnfdoirvidsiaonnd, twhheenmittohteicchirnohmiboistoorm,esCoalrceecmoindtr,acwtehdi.chDipvriesvieonntsofftohremacteillosncoafn tbheeamrirteosttiecd astpimnedtlae.phaTsheeusbienstg tesytpiemsaotef doafmtahgeeabweirlrlatbieonlosftrebqeufeonrceysiusbsaetqtuheentficsetllcedlilvisdiiovniss.ionThafetecrulttrueraetsmewnitl sbienchearcveersttaeidn after periodof time which is approximately 1.5 times the cell cycle after initiation of treatment (usually 18-21 hours). sMyasntyemssubthsattanacrees ndootnfootuenxderitnacumluttuargeednicecllesf.fecTthuenrteilfotrheeythheavceultbuereens maentdabtoelsitsesdubbsytaenncezyamree finrcoumbartatesd pirnebvoitohustlhyetarbesateendcewiatnhdaprseusbesntcaenocfe (aArsoucplpolrem1e2n5t4e)dklnivoewrnfrtaoctiinodnu(cSe9amhiixg)hplreevpealreodf enzymic activity (Maron and Ames 1983, Natarajan et a. 1976).
3. EXPERIMENTAL PROCEDURE
Culture of lymphocytes
B1l6o4o0dcowniltlaibneintgak1e0n%frfooemtahleaclatlhfysemraulme,dhoenpoarrsinanadnddialnutitbeidotwiicst)h. tiLssyumephcuolctyutreesm,ewdhiiucmh (dRoPnMoIt
`oncocurrmrailnyg
umnidteorggeon,cepllhydtiovhiaseimona,ggwliultlinbienst(iEmvualnastedan1d0
Od'oRisoordbayn
the addition of 1975, Nowell
the naturally 1960). The
ciunlstturielsewuinlivberesaplrecpoanrteadinaesrs5 amnidailnicquubotaste(d0.a4t m3l7bCl.ooTdh:e4.c5ulmtluremsewdiillo:be.o1cmclasiPoHnaAllsyolsuhtaikoenn)
to resuspend the cells.
5, AEE
000073
EI i
Life 0
postive cote nthesbsnce of Sin ----"
Supplier:
Picamn CoInnctehnetprraetsieonnc:e of9 in fpeeniitm:na
Concentration:
Propacution of89 fonction
MoiromeinC BDH Biochemical or other suitable supplier
0.1-1.6 pg/ml
[ SoEma-- dt othesale spp ae
5-30 pg/ml
Species:
Rat
:
Sex:
Male
yoSftrnaion:
EGoop Sprague-Dawley derived
e59 sa ttomn twillabevprpeepaairteldefnsotmeaacgrodounp oafbyudsAeuvmaaallsocnoaf 131300540a.nHiamgbdaolmd.iyrMmiecndiedgihnf.oannctiCsoopnprohoxepirdoaishnee
eihena romeomg on ose. sane, hs ts be et and ke rs
The following steps will be carried out at 0-4C under aseptic conditions. The livers will be
placed in 0.15 M KCI (3 ml KCI: 1 g liver) before being transferred to a homogeniser.
BeovonningeprAeNppaoeoenofto5rs3mocghhrotne Swhiebe.eceomdefvogreid yS0a0nd5hfeese10 mines To `supernatant fraction (S9 fraction) will be dispensed into aliquots and stored at -80C or below
reed veo $9 mix contains: S9 fraction (10% v/v), MgClz (8 mM), KCI (33 mM), sodium phosphate
`buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors will
ia
000080
-
`Enquiry number: 23923E
Huntingdon" Liife Sci: ences
Culture treatment
long stmiaton wih PHA. bere peste 10 st eb Te rs es "Two sets of cultures will be established, one set to be treated in the absence of S9 mix, the
other in its presence. All lymphocyte cultures will be incubated for approximately 48 hours, centrifuged and the cells will be resuspended in fresh culture medium (final volume 5 ml,
containing $9 mix (1 ml) where appropriate). Atmospheres of the test material at concentrations of 1.25, 2.5, 5, 10, 20, 40 and 70% v/v (70% v/v is the maximum practicable
cpnPooinndcgnenstrwasitiopn) rwmilleobreeietsitabwleiissnhgeeqddingssoeyeannleredmgAeloahsnsmbeottnliesep(e16a0eAmrl iantAbesmoaoelfRvsomleumppe)ooAwinth asAenptum iaEinedrrebqmueipliaborsaptieeon).ofitwheeiahtbmet5oseo7phenrec2seaeit 3om7cCpa,otphge tlnygmepr vhooochyntet ecuTlbhtouerl6esse(eoB5 dmlDs,ocmoeonmsntecaeiEmncinegoySe9nmix ile taienrond oO hone 4nd ptnteSsaoolr)sohfwm eil cioimnne At he Higher conenrtons ter is a ners risk of sfc cess in sberons atmosphere of air. Positive control cultures will be treated with MitomycinC (in the absence of
.. S9 mix) and cyclophosphamide (in the presence of S9 mix).
due to osmotic effects (Galloway er al. 1987). Change of pH in the medium, indicated by a
ceOolrEouireKchiannogie,,mmaaey nalcso nc1a2u0sgeToaorntsefa5cvtu9nal cdaRmfaogeee(Mmomritia,anWrataanaabet,eroTrankyeedcnaahatnmdddOekeounmenuesrya eoies
In th absence an presence of 59 ris, cles wil be cubed for des hous in th peorevseeencnse osfcatihneetnes.t csuoTbssotawnrcie. sbeAtrwithee henodoobfeothcis mtthraeedheouohrnpd3eriodH,otShpe Eceultnaurevsofewiall be.
(usually 18-21 hours).
Harvesting
Too hows before te cls ae Bavetd, mitotic activity wil be aed by he sain of
Colcemid at a final concentration of 0.1 pg/ml. The cells will then be harvested by
cl--enter--ifuagatsion ancdoltdremateed waithna htypotsoneicss3olu8tio0n (04.)07SaMnKCdI)rto 4 Cone cause swelling. sThedceallrs
Rr
000081
`Enquiry number: 23923E
Huntingdon
Life SCi ences
Side preparation A a townie Shes and homogenous cell suspension will be prepared and 0 ir. aliquots of Tthies crellesuespenhsiioen wwiilll bxe stained in ae10% eetGiemsa, orotate Shi left to air-dry and then mounted yi ik en in DPX. All of cm. the remaining cell Microscopic amination ne rp iceisl bevcapninodtbpy hst cisnors)caop.eThuereisne of oi ls er T ram hesri esie, eostshhserlnydcoontvto lvechfaoorinsnbhsqeuoenst SmooergResaHnkldrseewoislfabheeectgle3si0.%
Soi Dre. To memati ds ee i aly so some cvidnce Fn.eoskoeroenncepaoisoens loroaardenfto.TaSpdaosyoaEwPiAb.s The dose levels used in the repeat test may differ from those used in the initial assay. The Tai come compen secu ays ill be he oven concentration dosed unless a preliminary scan of metaphase figures indicates an insufficient Flowing maiaonnwdweookavsemsesasmdeonrtabvuttyhspeiropirsi10tccmoueeetrpaapophyaewsidealnbadybnthhseesmsSlC,dlNsr(iSNsCofNoIc1hr0rbo8nem.ocssooOdmrealodlyf
lb renmin.e wit oe lyst. oe er sigs of seman espe TT eh cmeeE aofmpilondoFmoeaeetdlslmvwelilslOFo1dhectltesrtmhinaeadsbfrsoegsis)ivdelcnlohbls caalnreyisss1ofnordr oo Dept idence stonSc inag soonmaeofgtmpsa eos5.b5eBileedcivsosfstecusorsnoefnrDaSNAhl(Heasddlh5e2aond Berodryicaostne
evr itn vgn of oval
Exngas
000032
.
Enquiry number: 23923E
Life Sci0cces
Second test
Ftorelaltomweinntgptehreiordesfourltthoefstetheofficruslttuerset,s atosbeecotnredatienddienpetnhdeeanbtsteensctewoifll$9bemicxonwdiulcltebde.deTpheendetenstt
on If
the results a negative
of the result
first test, i.e. is obiained a
3 hour treatment in different treatment
broetghimtehewiplrlesbeenceempalnodyeadb.senNcoeromfalSl9ymtihxi.s
wbielluseentdaiflorcocnutlitunrueosusintrtehaetpmrenetsoenfcceulotfurSe9smiinx.theTahbesenneceeod ftoSv9armyixt,repautlmseenttrceoantdmietnitownisllwialglaibne
evaluated harvested
on at
a case-by-case the same time
basis as in
at the discretion of the first test. The
the Study Director. procedures used are
All as
cultures will be those described
previously. The choice of test concentrations may vary from the first test e.g. a narrower range
maybeusoentdhe basisof the toxicity observed in the first test.
4. ASSESSMENT OF RESULTS
An assay is considered tobe acceptable if the negative andpositive control values lie within the current historical control range.
T`choempnaurmedbewristhotfheabseorlrvaenntt caonndtrpoollvyaplluoeidusmientgaaphoanes-etafiilgeudreFsishienr'esatchest(rFeiasthmeernt19g73r)o.upThwiilslibsea
uosbesfeurlvetedstevfeonrtsanfaallysiinntgodoantea owrheotnhecromopfatrwiongmuttwuoalilnydeepxecnludseinvtescalmapslseess..
It is used when the The test determines
cwlhaestsihfeircattiohnes.two groups differ in the proportions with which they fall into the two
"The test substance will be considered to be positive if the following conditions are met: Scthartiosmtoicsaollmyess(iegxncilfiucdainntg gaipncsr)eaarseesobsienrvetdheat ofnreeqouremncoyreotfest cmoentcaepnhtarsateison.with aberrant The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit. `The increases are reproducible between replicate cultures. The increases are not associated with large changes in osmolality of the treatment medium or extreme toxicity. Evidenceofadose-relationship willbe considered to support the conclusion.
Aabenrergaantticveellrseasbpoonvseecowinlclurbreenctlcaoinmterdoliffrneoqusetnactiisetsiciaslolybsseirgvneidf,icaatntaniyncdroesaeselsevienlthe number of
A further evaluation may be carried out if the above crieria for a positive or a negative. response are not met,
anny
000083
:
Enquiry number:
23923E
Li3 fe Sci+99egnces
-
5.
REPORTING
Te sopon ieteaiLtaualnyehissBroHfimeoetansinQldumyasndA,prtmhaansseseearnotna esdwelfsboped ehpxcaoh ctdroeswiolf
6.
MAINTENANCE OF RECORDS
All raw data, samples and sr specimens(if appropriate) arising from the performance of this study oTypens ofasnahmpilenansd stpecoimenahpicth aoefsnhseittbhlep,ebyoreesodn ofFionsinago,onfLrieoSnegireerems
`Standard Operating Procedures.
All thr samplesand specimens snd ll rw data wil be rend by Htinglon Li Sciences ee Eo She ee vi be coma an sda song oh seo,
disposal or further retention of the materials. If requested, Huntingdon Life Sciences will
continue to retain the materials subject to a reasonable fee being agreed with the Sponsor.
Ee Huntingdon Life pScaienncesewilrrettainnthe lQualyity Assurance records relevant to this study and a
7 GOOD LABORATORY PRACTICE The sod will be conducied in complisnce ith he prncpes of Good Laborary Practice
`Standards as set forth in:
BThCe DUK GonocdpLlaebosratoorfy PraGcotiocde RegLualbaotriaotnosry1999P(rSatcattiucteory (IsnsstrurmevniteNdo in3106). 1997)
'ENV/MC/CHEM(98)17.
EC Commission Directive 1999/11/EC of 8 March 1999 (Official Journal No L 77/8).
ana
090081
om
Lifasagcon
8. QUALITY ASSURANCE
"The following will be inspected or audited in relation to this study.
Protocol Audit
+ Study specific protocol.
Processbased inspections: Routine and repetitive procedures willbe inspected on representative studies, not necessarily on this study.
Report Audit
+ oTfhtehedradfrtafrterpeoprotratn(d0stthuedSypdoantsaorw.ill be audited before issue
QA findings will be reported to the Study Director and Company Management promptly on `acpopmrpolpertiiaotne CoofmepaacnhyacMtainona,geemxecenpttonfloyr. process based inspections which will be reporied to
9. HEALTH AND SAFETY
In order for Huntingdon Life Sciences to comply with the Health and Safety at Work etc. Act
1974, and the Control of Substances Hazardous to Health Regulations 1994, i is a condition of
uinndfeorrmtaatkiionng atvhaeilasbtluedy(0thitatretghaerdiSnpgonksnoorwnshaolrl poptreonvtiidale hHauznatridnsgadsosnociLaitfeed Swciitehnctehse hwaintdhlianlgl
and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor
shall also comply with all current legislation and regulations concerning shipmentof substances
by road, rail, sea or air.
Such information in the form of a completed Huntingdon Life Sciences test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance canbe handled in the laboratory. At the discretion of Safety Management SienrfvoircmeastioantmaHuynbteinagcdceopntabLlief.e Sciences, other documentation containing the equivalent
090085
Bulge, We
Life Sciences )
10. REB VEAFNESR,EN3C.ESande ORIORDAX, ML.t(a19g75e) M Homant peripherRalscorocdh Slym5ph5ocytes for the FH ISHER,R R.A. (1m97)aThte ECxotmpransyeotwoSfo2rx 2 Tal ns: Seical Methodsfor esearch BE GALLOWAY, SM., DEASYn,tBD.BAi.A, BaoEtrAWiNin,srCaLhc.emKeRanAsdGoNtuAomKee,loAno.Rns..o1A0mRoMocSsT.ROsNtMeGe,atiMo.nsJ.,
Research, 189, 15.
To ----_-- HEDDLE, J.A. and BODYCOTE, D.J. (1970) On the formation of chromosomal aberrations. SEONY(E198E 9 on emae tonal System for Huan Cognac Norcia, <i, Hamden, KmITeCHEoNG,s1.0t, MASsOaN,gC.ensd1J5O,N10E6S E. (597) pH and casgeiey - s elevant 10 i RA 7. 'MARON, DM. and AMES, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mr ORITAi , T. Wo ATANAn BE, Y,. TAKEDMA,iKo. nsnRd OeKUhMUR2A3,5.K5. (198) Effi of si on NATARa AJAN, AeTa, TrATE1S.,3a5A.D.mvanmBULoL, PsPhe,s sMeiEvUaEtRonS,nsM.eassd doe sVOGpEoLe,mXi. Ne OWELLs , P.C. (1C96a0)yPhRyettohaacmaSgog.lAteisn: an nso of miosis n clues of normal esos. SATYA-PRAKASH, KL., Cognit HSU, T.C. aanndd PClAlTHGeAnKet,icsS.. 3(01,928413) Chromatid lesions and Se SCOTT, D., aDEANo , B.e, nDAsrNFeOisRnDgto,oipNes.AnDR.oRfaL6in5Ad.NRCKDaIE,RmVKbDSLr7AdSNs(uD5U4,C)noDBJni.siecr(sWr19eso9sn0s)esCneaMcemlytbaepeihnoeaosseer
000085
:
Study Number:
Minas
Protocol AmendmentNombes: 1
H]unti3 n) gdon Life Sciences
T6063 PERFLUOROOCTANESULFONYL FLUORIDE (POSF)
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES
Study Director
Total number of pages: 4
Numberofagesfointernaldiebution: 4 Me. Linda Allis DEATox, DESS Pha, Vet, Face
heinrgeeonhts,y design ahfoedroe of i eboing mms wi Somer un `The signatureofthe Study Director authorises the implementation of this amendment to protocol. In
this amendment, deleted statements are struck through and new statements are underlined. Any
AMENDMENT APPROVAL
For Huntingdon ite SinceLia
Autrey:
(Study Director)
--
Approvedby: Pl
Klimt
SE cS
000687
Dae 21.1.pop,
Study Number:
MINB1S
Protoss Amst Number: 1
Hu: nti. n] gdon
Life Sciences
PERFLUOROOCTANESULFONYL FLUORIDE (POSF)
IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES
Reasons for amendments:
Toreythe allowing paragraphs, sdetntheProcol:
Original statement:
Studs Detailspage page) Sali of test substance formation:Nt ssi itissdy 5. Reporting GThoestreLa poirtmwioellncronaiainsndntfdliQtoufaethlebAoeskst ntsukbasgtasnchcee, onmettshsolwdyoilslosbgsyf,mtreeafsoudsceaacnhduntrpreailtliobnsohfoVthEe
5. QuallyAssurance The followinwill be nspcted radi fnlion is sad. Protocol Audit + Sulyspecific roto.
+ paca
090083
: Study Number:
MIN/31S
Protocol Amendment Number: 1
Hunti. ngdon Li.fe Sci. ences
PERFLUOROOCTANESULFONYL FLUORIDE (POSF) IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST
IN HUMAN LYMPHOCYTES
Revised statement:
----
Stabilityahnodmogeneity of test substance formulation: Not assessed in this study
5. Reporting
GG`oThtsetreLTpioorbtrbwsilrllaycPconrtraiiensldeatonafdilmsuoefythmeAtdesst nssudbtsstasncgee,phmtetashowdoilobgeyF,moreitsisssandeinterTpretation of the inthdbinne.hofoanoen comuicatons, HoneitnonALife SScneeness serriveess ttheesisehgthtotonallse Stor ole nal sep Sil the ba eg cotfcalons. omens of
appropriate) andwillbesubjecttoadditionalcost.
`inal report: _ AfterapprovalbytheSponsor
Repo ilbe upled on AG per dts lowing nmeofgswilspi Dru pont bound Goble sted
10thereport,
Lunbound(single sided)
090089
SttuocdoylNumbAemren:dmentarNumber: 2MIN/315
H!unti. n) gdon
Life Sciences
5. QuiltyAsus
The following will be inspected or audited in relation to this study.
Protocol Audit
: Studyspecificprotocolaanmdendments.
120 ON NIVINOD
0900990
