Document QXK4NDkNXGd5MyYv1JxjKqB4k
WW SAAaSSHHIaINNGeGST aOTsONS
MUTAGENICITY TEST ON ns
IN AN I VIVO RAT MICRONICLEUS ASSAY
Dama sEouIERUENT 5.5. SPA FIFRA Guideline 84-2
Fn spor
sumios Hemalatha Marl, Ph.D.
PERFORMING LABORATORY Hazl9e2t0o0n LWeacsshbiunrggtoFni,keInc. Vienna, Virginia 22182
:
LABORATORY PROJECT ID
iA Study Nous 13515-0-456
supe 10
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St. Pau3lM,0CMoircnpononercsaeotrtiaon 55144
stuov cowpreTion pate April 30, 1993
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STATEMENT OF DATA CONFIDENTIALITY CLATHS
oIfnfForImFaRtAio n 10cl(a4i)n(e1d)(Ac)o:n(fBi)d,ent0ial(C)onhatshe bebeansisreomfoveidts tofa3llicnognfwiidtehntiinaltheappsecno-pe dix, and is cited by crossreference number in the body Of the study.
Company: Company Agent:
Typed Name)
mms
me
The
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GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
TPRheoiprsueliascctaiuodnylsiwaanPsdaarztcdosn1d60ua.cstesdAetnyinfoscriotgmhnpilfiiinecnaTcniettlwedietv4hi0attoifhoentsEhePAfUr.FoSIm.FRtAChoeGdoepordooftoLcaFobelodrearatanoldr/yor GLP
oorertohamecenihcneitoednrepdarsetraaentgiuaolpnapteionfodnitxwhheitocrhesthuailfstfsecrteipenodrttht.ihse
Tqhuearleitwyeroer report.
niontdeegvriiattyioonfs thferomstuthdey
Study Director:
IHeemalaachbaoMturi,MePhd.D:. VDaenpnaarltimsenntCyoftogMeonleetciuclsar and Cellular Toxicology Study Monitor:
i
Applicant/Submitcer:
4)30)43 Tate
Thee
Tp T ate
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&@ WHAASZHLINEGTTOONN
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WASHINGTON QUALITY ASSURANCE STATEMENT
PROJECT TITLE: IN VIVO RAT MICRONUCLEUS ASSAY
PROJECT NOL: 20996
Assay wo.: 15315
Protocol Ko.: 4s
orton No: 5
QOBrueaeelnietiymeeAsesPurroacfneecdceuerenisncsepodfectptrihooejnesQcut.aofliwtetyrheeAcssostnuuddryuacntcaeenddlUonarictcroaervndiidnegwaccotfoordttihheengfSittonaanldtahrerdeport
so OeRmaenteoraonnCdvetmhteeonttishnes.opfescttutihdeoynsadpiparnredocptrofiriantaoeln
GroeopdortLabroervaiteowrywerPeracrteipcoerterdegutloations. the following dates:
FIBeneisstpeneicntRgieopnoo/frbtaTtoReoetviAerwt-i4c/l1e6-,31/92//11999533Findi43n//g20s32//11R99e98p33orted
x. AuNgeiwtloarnd 5 Millet
Final Report Review-4/3012993 4130/1993
c. Lee
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. YD[atZe[1R3etenied
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STUDY COMPLIANCE AND CERTIFICATION TAooao gegorrnocasibaust.Sibseparualy7v9ry% gaSnudton4nt0nelCFdR.4e16beS0U;MEcLhLeESOSrrpgSanhizesthieonJe ofrodr tPr eaohveecmaitcory EeTeoamenL,itevestehnere197w9e-re 1e98n0.o s(iegrsfiaFcitciavS net1S9e83r)i.itioTon.sthFerobmesttheofhthem HCE rEeattemheanctytoDneeedn. roevgiuolnantdiD onbsy otrhethBeiedsyipmDeidrecptroort,ocowlhotchoarrtiwfoiunlsd. athfafte.ctthetheun Cetiation Cicha in the context of the' study di esign and evaluationscrrivearpuitee:ts AtaLeLa,teTsTeetsoooraianmdesncct"oanttihroenoh,l rFFeeecscounrlidtss.naisnprotbtheoiescnolrrsee,pvoirontvdedacaeotyosputyhpepoorfStteutddhyebDyfiiraneanlcteoxrrpesepcoritnRIeLntaslev aptreobTEaetteiSoEe Tnats Drrl eedAypwill begharrpchived:fin uthe dstofagge epeciliptnies Eby A E n Reston rigiFhg ts olTor lhiga li, ig
[r---- GT Reseeae riAblay ocoiae, tet Besonars oNNaermaa)aitanotC,ysyopiMneacikcss Depertnenc. bf HoTecaiar and Cellular Toxicology 1s1sao0mase
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TABLE OF CONTENTS
Page No.
1. SPONSOR + cov nt eee.11
II. MAA.TERIACLlie(nTtE'SsTAIRdTeInCtLiEf)ica+ ti. on+. + oo ooo vo oon vnn 11 B. Dates Received CD.. GPehnyestiiccasl ADsessacyripNot.ion
IIT. TYPE OFASSAY oo wwii
eee ee
1
IV. PROTOCOL NO. + ov owovvvii nena
AD
v. AS.TUDY DIAnTiEtSiat. io. n Davtev toons eee
11
B. Experimental Start Date
C. Experimental Termination Date
VI.
SUPERVISORYPERSONNEL . . . . . . oo ooo ovvvvnn no. 11 A. Study Director
B. Laboratory Supervisor
VEL. OBJECTIVE . . . v vve oon ee eee eee 1D
VIII. MATERIA.LSoo oo ooon ee eee
12
IX. SOLUBILITY AND STABILITY . . . + + soo vvvvv vn. 12
x. DOSESELECTION STUDY + + + + + + o nov neeeo ooo 13
AB.. DDoosseingSelIencftoiromnation
C. Results and Interpretation
|
D. Conclusion
XI.
MICRONUCLEUS STUDY . . oe eee ee vvvvvvnnns. 15 A. Dose selection
B. Micronucleus Assay Dosing Information
XII. BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS . . . . . . . 16
XIII. EVALUATIONCRITERIA . . + + ooo ovvv nnn ooo 17 AB.. GDaetnaeraPlresentation and Interpretation
XIV. RESULTS AND INTERPRETATION . . . . . . o.oo... .. 18
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TABLE OF CONTENTS (Continued)
XV. CONGLUSION + + ww enon eee.18
XVI. REFERENCES . oo coo e see
19
XVII. DEVIATIONFROMTHE SIGNED PROTOCOL + + + + ow oo ooo. 19 XVIII. EXPERDDATMAETANBLTES . . . oo oe ooe oto. 20
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smear Mutagenicity Test on T-5711 in an in vive Rat Micronucleus Assay
hSyeiesobjoefcytSipvraeagsoufe-tDthaoivsiIenyiduncreavtismv.iocraosTnshueatyltevelasstinatortboiencvelaelmuaawtraesiowtshuepsoplaeybndcielhdietoyniantoif0c.5t%heeryttheesot-
Clehexymethyiceiiutose (high viscosity).
fboyobrsoerrtavhleeddgeoaisveacghese1 lamteaclt5ei0o0,nat s16t32u05d0,y0, m2g735/0mk,aglea3sn8d75a,n1d afne3dmafle5em00a0laetsng3/p8ke7gr5.
dosMeortlaelveiltywevraes ng/kg.
dosed
BSGaavsaeegde.urpneone12t5h0e.saen2d5ro0en0sl,uyltasna,dssir3ga0nt0se0dwmeatr/okega.dcohseTdednosfoear/nhiamtrahlevsesmti(cFritovineneucmlagelrueossup.aansdsaVyeFhivibeycleoraalnd
eTorieihituiintveaedtiscieondnttrhuoeplparsgosrxaoiyua.ptseleTyhueth2aa4,nnaitm4iabclseadnwdaepr7pe2rodhxooisumeradstwealifytther24tdheohosuitrnesgstaffaotrretriecxldteorsaicanntgdiovwneerroeef
The Bove macro.
hiTheeizsotmaesssctelseymsataenirdniah1los,necTom-na5s7ci1rd1oe,vreddpiodlnyecgnohattiiovnieantdiuiccnesthtaeytshiTaigtonciyfbtioecnseantmuanrdiernrocwretamhseiecrcoionnnudcilteiuosns of
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Mutagenicity Test on T-5711 in anin vivo Micronucleus Assay
I. seonsoR: aM Corporation
II MATERIAL (TEST ARTICLE): A Client's Tdentifications L-1276 (T-5711) 5. Dates Received: February 26, 1993; March 12, 1993 C. Physical Description: Cream-colored granular material D. Genetics Assay Nou: 1515
III TYPE OF ASSAY: In Vivo Rat Micromucleus Assay
Iv. PROTOCOL No.: 456, Edition 5
v. stony oats: A. Initiation Date: February 2, 1993 5. Experimental Start Date: March 2, 1993 GC. Experimental Termination Date: March 23, 1993
VI. SUPERVISORY PERSONNEL: A. study Director: Hemalatha Murli, Ph.D.
:
B.
Laboratory Supervisor: Jos Arriaga, B.S.
viz. omsscrive: CTTheresyttharaborjcteiyccitleiesv,eofTof5S7tp1hr1ia,sgutieon-Daviuniidvueocyeasrsmataisyt.rwoamsuTchlitesoi secvinuadlyubaowtnaeesmtahcerornaodwbuicltpieotdlyycuhsofrinogmtahteic okificarions of the procedures suggested by Heddle et al. (1983).
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visa. areas:
for the dose selection study and at 72#6F and 50201, respectively for
maintained. A commercial diet (Purina Certified Laboratory Chow*
:
by CO, inhalation, followed by penetration of the thorax. Any extra
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uSnbdielrictyheofpretphaerastpioonnsora.nd dosing conditions of the assay is the respon-
X. DOSE SELECTION STUDY A Dose Selection: SMSoioom:itnoixTsiectasie.tryed27i5nb0yf,ororm3aa8lt73i,ognavavanagdse3a0fv0o0railamagbd/lokesgeobnosdeyTl-e5wc7et1ii1gohntanswdteurddyeo.se levels of
5. Dosing Information:
TThueesdaanyi.malMsarcuhsed2, in199t3h.e the dose range finding
asdsoTashyee
waseesilgehc2tt7i6o.rn6anga-ess3ao1yf9.w3tehregeraandsiomseaadlnsdonu1s9e9d.8in-
2S4u4s.p9ensgiroanmss by Raking a
wf3eo0rr0emthpger/emmpaalrseetsdockajnudsftorfepmrtaihloeersh,itoghrdeosdsopiseencgti(va5en0ld0y0.wmegr/eDkogsp)ir.negparTehdis
tZessubprrienpgareidn 557306 mp/ni.
abyfiandadlingvol4u1.m4e This opaque,
cmolfreao6fm0-.0cCHoCmllorfeoadnd30sa.u0s0pf1ei2nnaslgioncofovnaTcs-e5n7mtiir1xa,etidonfor
bDioluuttio2nsminouftetshisinsatocTkissweureenizperresparteodenfsorurethehom3o8g75e,nei2t7y5.0, 1625,
and 500 mg/kg dose levels.
.
wDeorseingabwoauss a9chwieeevksedoludsinatg osing scheme is found in
athe10 tmiln/ekgof dodsoisinngg.voluAmne.outlALiLne anoifmatlhse the following table.
A total of 30 animals was used in this assay.
ReaTENT Dose Groupws oF
:
T-5711
500 mg/kg
3
3
1625 malig ss
2750 make ss
2075 mglke FE
5000 mg/kg ss
1A1nimdaolssesangdivweenreare- owneeaknsacouldteat(ontehe-titmiemeonolfy)dosbiansgi.s.
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C. Results and Interpretation: AdLuLratainoinmalosf wtehreesecxuadymin(etdhraefetedraysd)osifnogr atnodxicdaielfyfectthsrouagnhdo/uotr the rortalities. Nloatetloyxicandeffaebcotust v3erheournsoteadfteirn daonsyinogf. the animals observed immedASopF0p0r0tohmxeigm/eakyteges.ldyosaen2d1g.5raophupopeuarwrsaesd afeltxaepnrgeurididoe.snicnignA,LgL 1coomntavhlueelrsia(on6ni1sm6a0la)snd afrplopamecarritehmdeation normal and healthy at this time. a5p0p0r0moxgimkagteldyose47ghroouurpswaasftefrounddosidnega,d. thiAsLLmaolteher(#a61n6i0m)alsfroamppetahered normal and healthy at this time. An3op8rp75mraolmxigm/aakntgdeldhyeoasle6t9hgyrhoouuarptswtaahsfistefrotuindmdoesidanengad,d.r1emAafLieLnmeadloethseo(r6u1an8nt4ii)lmalftshreoampepntdehearoefd
FE TShuemmoabrsiezrevdatiinontheperfiooldl.owinTghetmabolret:ality data for this assay-- are SuTmnarRyateofDMoosretdalAictuiteeslyWiwtihtihnT357D1a1ys
.
observations
Treatment
vale
Female
500 mg/g
ors
or3
1625 malig
ors
ors
2750 mg/kg
or3
ors
3075 malig
ors
us
5000 mg ig
us
ors
e E e ------------
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D. Conclusion:
Based study
utpheonmatxheimmuomrttaollietryateadnd dotsoexic(MTsDi)gnswasinestthiemaatneidmaltso
in be
this about
S50u0f0fimcgi/ekngt. toThseelercetsuldtossesoffotrhisa rraatngemicfrionnduicnlgeuasssaayssawye.re
XI. MICRONUCLEUS STUDY:
A. Dose Selection: Tsheeledcotsieonlesvteuldsy aunsdedaniinmatlhsiswearsesaydowseerdeatbas12e5d0,on25t0h0e,doasned 5000 mg/kg body weight, administered by oral gavage.
B. Micronucleus Assay Dosing Information:
The animals used March 16, 1993.
Ciyncltohephomsipchraomniudceleu(sCP,assSaiygnaw,ereLotdos#e7d0H0o9n48T)u,esdtahye,
positive (Lot#13,
control, prepared
was solubilized at HWA) and was
in sterile deionized administered by oral
water gavage
at
60 mg/kg. The 054i. prepared
ovnehi1c/l2e0/1c9o9n3t;rolexpciornastiisotneddaotfeCM1C/20(/S1i9g9m4a),
Lotil2lFand was
a1d0miminfiksgt.eredThecowneciugrhrtentrlayngewitofh
the the
test article animals used
at in
a volume of the micro-
ntuheclemuaslesassaanyd wfaesmal2e3s6,.1re- sp2e8c7t.i3veglrya.ms aTnhde d17o5s.i5ng- s2u7s4p.e4nsigornasmsfofror
dtohseeas(s5a0y00wmegr/ekgp)r.eparTehdisbwyasmakpirnegparaed500bymga/dmdlingsto1c3k3.5fomrl tohfe
high GC to
50.0002g of T-5711, inal concentration
resulting in a of 500 mg/ml.
final volute This opaque,
of 180 ml and cream-colored
a
suspension vas mixed ensure homogeneity.
for about Dilutions
o5fmitnhuitsesstoinckaweTriesspureempiazreerd
to for
the
.
2(5d0e0siagnndate1d250Semcgo/nkdgarydosDeoseleGvrelosu.p)
A second was also
group of assigned
animals to the study
and was animals
dosed with the were only used
high dose of in the assay
atshe retpelstaceamretnitclse.for Thaenysewhich
died in the primary dose group.
T4h8eantdest72 ahrotuircsle afdtoesredaadnmiimnailsstrwaetrieoneuofthatnhaetitzeesdt
approximately article. The
24,
positive and vehicle control approximately 24 hours after
atnheimaaldsminwiesrteraetuitohnanaoftiztehed
control
afrotlilcolweisn.g tAabnleo:utline of the dosing scheme is found in the
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Dosing Scheme for Micronucleus Assay
A total of 120 animals vas used in this assay.
Nunber of Animals Assigned
Primary Dose Groups SecoGnrdoaurpys*Dose 208 48 HC 72 Hr
Treatment
M OF MF MF Male Femle
e eee ee.
T5711 1250 mg/kg, 2500 mg/kg,
ss 0s 5s 5s - ss sos ss - -
5000mg/kg
ss ss 50s 5 5
Vehicle 1Co0ntrmoll/,kg0.52 high vsiscsosi.ty carb-ox-ymethylo.cellulo-se -
Positive 6C0ontmrgo/lk,g Cyclophosp5hamside. o- - - = -
.
* oThnelyanuismeadlstoasrseipglnaecde atnoimtahles swehciocnhdardyieddosien gtrheouppsriwmearrey ddoosseedgarnodupvearte the
ehuitghhandaotsiezeldevealt. thAeLLcomepxltreatioannimOaflsthenottriuasle.d as replacements vere
The age of the animals at the time of dosing was =8 weeks.
Volunes dosed were 10 ml/kg and were based upon individual animal
.
weights.
XII. BONE MARROW HARVEST, SLIDE PREPARATION AND ANALYSIS: aAtndthteheapapdrheorpirnigatesofhtarvteisstsuetimaen,d tehpeiphaynsiemsalsofwebroetheuttihbiaaneatwiezreed wrietmhoveCdO. T0h.e1 mmalrrfoeutawlascaalsfpisreartuend tforofmortmhea bsounsepeannsdiomni.xedThien caelslysriwnegreewtihtehn Mpalya-cGedrunownalsdlidseoslutanidonaifro-ldlroiweedd, byfiGxieedmsian m(eStchhamniodl,, 1a9n7d5).staiTnheedaiinr-dried slides were coverslipped using Depex* mounting medium.
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T(hPeCE)codteodnosrlmidoecshrwoemraeticthe(nNCEs)corceedll forratimoi.cronSutcalnediardandfortmhse
polychromatic were used to
record quency
otfhesmeicrdoatnau.cleaOtneed tcheolulssandwasPOEesxprpeersseadnimasal pweerrceentscmoircedr.onucTlheeatferde-
Cneolrlmsalbafsreedquoenncytheof tmoitaclronCuEcsleipreisnentthisin ratthe stsrcaoirned isoptaibcoutfie0l.d0.-0.4TZh.e
oTfhePCfErsequanedncyNCEosf PoCbEsservveedrsuisn NtChEesowpatsicdeftieerldmsinewdhilbey ssccoorriinngg tthhee nfuimrsbter 1000 erythrocytes.
XIII. EVALUATION CRITERIA:
A. General:
TShcehmicdrit(e1r9i7a6).forMitchreoniudcelnetiifwiecraetidoanrkoflymisctraoinnuecdleaindwegreenertahlolsye of
round, occur.
aMlitchroounguhclaelimonhdadansdharrpingb-orsdhearpsedamnidcwroenrueclgeeineroaclclaysiobneatlwleyen
m1i/c2r0onaundcle1a/t5edthecelsli,ze nootf
the the
mPiCEc.ronuTchleeuusn;it thofus scthoerinogccvaassiontahle
cell with nucleated
more BCE,
ntohtantwonoe (moircrmoorneu)clemuiscrwoansuclceoiu.ntedTheas sotnaeinmiincgro-
p(rbolcueidsuhr-egrepyermanidtterded,theredsipfecfteirveenltyi)a.tion by color of PCEs and NCES
B. Data Presentation and Interpretation
Deta are points.
summarized Individual
by sex animal
and dose data are
garlosuopsprefsoernttehde. difTfheereanntalytsiimse
of the square
data root
wsarscsipneerfotrrmaendsfoursmiangtioann
wAhniaclhyswiass ofpeVrafroiramnecde onon thtehe
pazrcospionretiopnropoofrtcieolnl)s. witOhncemictrheonuAcnlaeliysipserofaniVmaarlian(csequahraed
root been
;
pfeorrfomrumletdi,pleTukceoym'psariSstoundsent(iSzoekdal raanndge Rothelsft,
(HSD) with 1981) was
adjustment used at each
hsairgvneisfticatnitmely todifdfeetreernmtine(pw<h0i.c0h5) dofsreom grtohuepsv,ehiicfleanyc,ontwreorle.
Acnoamlbyisneatsiowne,re anpderfaolrsomedat seeapcahrahtaerlvyesftor teiamech fhorarvtehset setxiemse
and sex combined.
Tshaeticsrtiitcearlilayfosrigdneitfiecramnitnindgosae-rpeolsaitteidve inrcersepaosnese ininmvioclrvoenducaleated Csisgn,ifiocrantthepdoestieticvteionresofponaserepfroroduactiblleeastandonestdaotsiestilcevaelll.y A test
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appeared languid. All animals in the 1250 mg/kg dose group and all
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wi. sermRmNGes: HNeedwdellel,, 3G.HA... Hunidte,SalM.a.monKei,rkhHaFre;, 3T.heLairnsdeunc,tioK.n. oMfacmGircergoomrs,cleJl.Ta.s a ensure of genotexiciny. Tutation Res.s A23:60.110, 1083. Schmid, W.: The micronucleus test. Mutation Res., 319-13, 1975. CShcehmmiidc,alW.H:ucagTohnes:micrPorniunccliepulsesteasntd Mfeotrhocdys.togfeornetTihceiranaDleyrsaicst.ion.In,Vol. & Ch oltaender, e400: Fionums pp. 31-33, 976. Sokal. RR. and RGM, J.J. Biometry, Freeman, 1981.
AIL. DEVIATION FROM THE SIGNED PROTOCOL: The following deviation was made from the protocol.
The temperature was maintained at 74.1 to 80.4F for the dose selection
GSstitffuefdcyltcuaintdthieensoatniatmmaalitsnhteaiiannneiadmnaylatwafya7.2c#i6liFt.y. ThTihsiswasslidguhet dteovitaetcihonnicadlid not
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ms 1 JICROMICLEUS DATA SOBARY TABLE
sT > st anes ng o:rh
moosr me wOWaErowsveal2P vacmA eraoATL sD eeS s5.5
i|1i
sTamnorEeEEaE mes ons
2o0d.
Poggme oui omies sms; mien |ouiea emsen
SKEoRmREAam Gey % 210i 2a2i0ae 230; |} omioon esren
geo
oEnlnc
HW omic; Lomi cates
oamsome 0:0
omsoo ONt0
||i
oeten 06x
owsom 0Miom
aa =
oW7n m oomsisooms
001003
omsom
0820.02 | 0630.05
omiom | eetem
0432036
omsom
:
smi 8 Gwioe omiom omeom | 010m Gaze
7 omiom mice omiem | owsom onion
% omsom omiom omieo | cmon omion
smi #8 cisom omiom omio@ |omsou omsow
2 omsom omios orion | amie omson
Sianiticancly different from the corcasponding vehicle control, 0.05.
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MICRONUCLEUS TEST - TNDIVIDUAL ANIMAL DATA
SpousoR: 3 Corporation
TEST ARTICLES T3733
Assay wo. 15313 MALES i FEMALES
mena
aWmEaR FGOR.oEMwN EmGmoE]'| MWmSaRL NoOE.oEM)N rmoome
E vBenoicwleecornvereosl y 6GGe39730.1 E eogsmeiyd eeewsw 0o0 onooss
GGae 11 oosmei wmww 4oomods
ptoshitoivne cmoenesoel) GSG72e5 m 128 omoomeyi eeemwsws1 ooosmw
GGem oaw oomsy esans 3ow oomx
Ta est aceicmtaemvest) Genw 11 oewmii eemm 13 oomw
1250 mg/kg G6G3oe6s3 00 0aT.5ma6ii| es636wa4 103 0oo.4ww6
omg GsGa anz:1 oewmmiii eeewmw z12 onoonm
'
G Gezo oOmei sewe 11 oomo
sms GGs an o1o Looomily eoeemwm 111 ooonmm
GGxe 31 ovmeyi eewm 1oonan
PRoerx=HnwircoBreloiynseuhcr=ioenMuaistcirconuecciyeeahteeodcyFeCEs
NCE = Normacheomatic eryehrocyie
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TABLE 3
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 34 Corporation
TEST ARTICLE: 17-5711
ASSAY NO.: 15515 TREATMENT
MALES
}
wom
T
ANNMDBIELR (C10E0s0) PRCAET:NICOE ||
FEMALES
MANDAL
To. W CEs
RATIO
NMBER (1000) PCE:NCE
T(e4s8thAorutrichlaervest)
6325 6027
2
0.59 1 0.79 1
6328 6374
2 o
0.43 0.66
1250 mg/g 6062083.
21
00..5680 || 66345266
1 2
0.39 0.6
6647
0
0.60 | 6430
1
0.55
2500 mg/kg
6319 6326
0 0
0.39} 056 1
6313 6338
1 1
0.38 0.45
6327 6380
2 1
110m 66336522
1
00..7285
S601
2
0.80 | 6387
5000 ng/kg
66336289
21
00..4943 1| 6633518
1
0.25 0.35
'
6377 613
2 2
00..8199}| 63su6112
12
01..2077
022
1
0.69 | 6614
o
0.66
PMNCE==MiPcorloyncuhcrloemuastic erythrocyte
No. MN PCEs = Micronucleated PCEs
|
NCE = Normochromatic erythrocyte
15515-0-454
23 004453
WASHINGTON
.
TABLE 4
MICRONUCLEUS TEST - INDIVIDUAL ANIMAL DATA
SPONSOR: 3 Corporation
TEST ARTICLE: T-5711
ASSAY NO.: 15515 TREATMENT
MALES
i
ANNUIMMBAELR
no. m
i
CEs (1000)
RATIO | PCE:NCE |
FEMALES
ANDAL NBMBER
wom CEs RATIO (1000) PCE:NCE
T(e7s2thAorutrichlaervest)
6354 6370
0 2
0.45 1 0.46 |
6345 6350
0 1
0.46 0.13
1250 mg/kg
6425 6429
0
0.63 | 0.43 |
6393 6433
1 2
0.36 0.15
661
1
0.32
6435
1
1.06
2500 mg/kg.
6320 6398
0 2
0.57 1 0.25 |
633 6366
2
00..3907
6404 6017
1 o
0.20 | 403
0.72
6405
1
0.33 0.36
6437
1
0.69
6445
1
0.71
5000 mg/kg
6342 6383
2 1
0.20 | 0.90 |
6315 6323
3 1
0.53 0.16
6391 6392
2
1.29 | 0.81 |
6385 sil
1 1
00..1363
664s
1
0.48 | 6436
0.75
MPNCE==MiPcorloyncuhcrloemuastic erythrocyte No. MN PCEs = Micronucleated PCEs NCE = Normochromatic erythrocyte
15515-0-454
2 004454
WASHINGTON
I EFRTormIoocNor No. 4
IN VIVO RAT MICRONUCLEUS ASSAY
HnadzleFtAonGVaaosdhiLnigseoovna,orIync.Pra(cHtUAi)cewi(lGlLE)coGnudiudcetlicnheiss. stTuhdiysipnroctoomcpolli,sncceriwtiiccaklEPA DL PyhasQeusY aliotfytBAhesesvuCororannkacuecIcneidphrboaygcrceoMsrisdaanaactned92wt0ih0tehefiSanOsaFhlsurargtepHoPraitkzel,veitloVlinenVbnaeas,hsiunVbgijereocgnti,ntcoaInea5s.u21d8l2eT.he
PART 1. SPONSOR INFORMATION AND APPROVALS.
1. SPONSORIDENTIFICATION
Gonpany Nase:
TI. TESTARTICLEIDENTIFICATION:
---
IIT. IESTARTICLEANALYSIS
CDheatgearcmtienraitsicoincsofasthedefcoisntedaritnictlhee GsLcPabirleigcuylatainodnstheoftFesOt a(r2t1icClFeR t5h8e.10r5e)s,ponEsPiAb-iTlSiCtAy (o4f0 CtFheR S7p9o2n.s1o0r5.), and EPA-FIFRA (40 CFR 160.105) is
IV. NOTIOF FRI EGUC LATA ORT YSUI BMIO SSIN ON
.
5If8no.r3eo0rl:dtenrEPaAgtoeTnSccCioAem,sp,l4y0cwoinGtsFhuRltU7.i9Sn3..g10lf;eadbeoErrPaaAlc-oFrIrFieRegAsu,lmau4ts0itonGbFeRcodn1e6os0t.i1f(0Fi)DeAd,anLdf21aclCelrFcRaotrn
BpTaarrrdttiecraScFescawhhesidtuculhdsyaogisfencstyco,udbieLcfssauunbynm.licthBtIeEdfhaeltloTeutcnheedievraegernetcghyue.larteosIruAyltsmraeivoinfetwat.ihnissPlsaetuadsye:
[3 vntecernines 3 roa CF eoazscn C3 eenroema
CJ were C3 sow CJ ozo CJ omimm
asa 1m
Lotto
004455
WASHINGTON
V. STUDY DATES
Proposed Experimental Start Date: Pumpured Bxpertuental Terstraston Detes
VI. ARPOFR STO UDYV PROA TOCL OL
seudy Director: TemaTatha Foril, PhD. Sponsors
"EPbRTOTTIOoCNOL5NO. 454
Dace: ovae
ise - 19
[ 04456
2et 10
g WASHINGTON
PERpOITTOICOONL5No. 4'54
PART 2 + STUDY PROTOCOL
IN VIVO RAT MICRONUGLEUS ASSAY
I. omEcTvE
:
Tchleasotbojgeecntiicveacotfivitthyisansdtuddyisrisupttioonevaofluatthee amittoetsitcarstpipcalreatufsorin Polychfonatic erythrocyte stem cells in rat bons marrow in vive.
I DEFINITIONS Mcihcrroonnouscolneeu(ss:) aand/somrallofchsrcoemnattriinc bcohdryo,moscoomnesisftrianggaenotf(se)n,tiwrheich cTahgrsomobseohnien(ds)atanmditoftriacgueannta(psh)asem.ay Anofttebreteilnocplhuadseed, Inthetshee daughter nCuyccloepil,isaa.nd may form single or multiple micronuclei in the
III. RATIONALE
Tahgeenmtsicraonnductleesuts atretsitclecsanwhsiecrhve ianstearferraepidwitshcrenoernmaflor mcitloatsitcogecneilcl
daFnioavrpimhseaidsoenfrao(nmSdchcmchiardno,mboes19o7ms5ec;sorHoeredddcdlhuerroiemntogsoainln.et,erfpr1h9a8ag3sa)ee.nbtesMcailucesrfetonbutechhleeiyinpdeardrseuirsitng
i(nSchtmhied,bon1e975m)a.rrowInartehisscaosrseady,foproltyhechrpormeasteincceeroyfthmircorcoyntuecsle(iF.CEs)
Dexutrriundgedm,atwuhrialteionmicfrroonmucalreyic,hroibflaprsetsetnot,eryrtehmraoicnytien tthhee mcyutcolpeluassmi.s
DFeatceilcittiaotnedo,f manidcrotnimuecleiinvoinlvneodn-innucsleeaartcehdincgelflosr misetatphuhsase spreads
:
aifnfetcrteiantgedscpeilnldlpeo-pfuilbaetriofnusnctisioenliomrinfaotremda.tioTnesast awretlilcalses
C(lSacshmciodg,eni1c975a)g.ents can be detected through micromicleus induction
IV. MATERIALS
A bnimals YSoturanign,adu8l-t10mawleeeksandoldfemastlecherattsineofofthedosSipnrga.guwei-lDlavbleey purchased fron Charles River Laboratories, Inc.. of
asi = 19
00445,
3010
@ HAZLETON
WASHINGTON
B. ControlArticles
prick PROTOCOLNO. 454
:
ones sistonsiores torohe
Vv. [EXPERIMENTALDESIGN
A. AnimalHusbandry
temperature, 72 6'F; humidity, 50% % 20%; light
0044553
WASS H IN GTON
ETON PROTOCOL sNO. 454
santcaey Ecoaghes.oaenda.brCeeadardskitinengegdwwivlislthwbieeenrutshseeudl.tsonbtlo_eaFlersomel
tive potent ans coutpacnt.
B.
Dose Selection
TIhe ahighSdoesne igeonerrasl)ly
wi"Tlhle
EE oaeion of eoxicity
(hboiergbhs,eldeodescaettehds,hoasduolpd60rp8arsoeodfiuoctnehe
E EetEoyHToma ufsoeeodmnaotenmsotchehea.rsonemeesrrtmiaosfdeiytaihthiesiocraiyngidha.sLdoowsedose
TELre ihTianrmenatsoectietvherartey'c.somve"enGdrdeedco.lfaastioggohn wdioslel biencrdeeatseecsted,
1H6oontoonappTroouphryiaetaem baengpeerffionrdmiendg data ave availible, a range
DIT OhSeEdoRsAeN-GE cEaFnIgNeDfIiNnGdStiThnaUgDiYsveeudyyewoiuplsl bweillcocndouncstiesd ousfin3gmaflieveantdrea3tment
fone cae
Group Designation and Treatment Regiasns
coon votoSuber of"heRmaatyse mows -- Coase
'
ss 3
2
ss w 3
>
ss 0 3
.
ss w 3
s
ss w 5
IThe reoute ioefcLhaednhmeeinihcseotvrriaoacyteeiosorrntowti"Tilhclesseb"eprrooorucattleusdgaovofargaseld.asianvIiansgiecrshaeci1o7envehnave
ise 1/9
sof 10
004459
@ HAZLETON
WasRINGT OW
Biles [PROTOCOL NO.454
be used to estimate the Maximum Tolerated Dose (MTD). Doses will
MICRONUCLEUS STUDY
C. DosingScheduleandRouteofAdministration
004469
?
NGTON
WASHI
o
sti ! PROTOCOLNO. 454
NUMBER OF ANIMALS USED FOR MICRONUCLEUS ASSAY
Marvest Tines Afcer Treatment
(Males and Females)
.
Treatment 2H4ours 4H8ours 72Hours Total
NPeogfaittiivvee Ccoonnetrraoll 534455 oo B=. s5e0s3
TWoeodtubonDseose
SS1I5s ssiss ssiiss Bmiinn
High Dose
313 sis 3s sels
ToTaL
seas 1541s 1541s 5545s
TThhee ervoeuntte otfhatadsTeisntiscarrtaitciloen vcihlalrabceteroirsatlicgsavapgree.cludIen oToruatesgavoafgea,daiInPisiintjaetcitoinonhvaivlelbebeenemspelloeycetde.d beTchaesuese TThhiesy atreesttpherocmeodsutrec.ommoOntheTroutreosuteosf aodfmiandimsitnriasttiroantiofnor nay be used as indicated by selentific evidence:
D. Extractionof BoneMarrowandPreparationofSlides
ATnheima`tlhsorwaxi,llabned hkiinldled1ivnibtbhonoeys, wfiollllobweedrebnyovpeedneftorratmiaornroowf tTrraancstfieroene,d tTohecemnatrrriofwugweiltlubbese Fclounsthaeidninfgron3 mtlhefbaotnael acnadlf SToerupmell(eotnethteubetisfsoure,sacshomeaniomfalt)h.e suFpoelrlnoawtianngtcweinltlribfeugdartaivonn GTEiEi,destheancdelalisr-drreiseuds:pended, and the suspension spread on
Tahceridsilniedesorawniglel, tahnedn baenaFliyxseedd uinndemerchfalnuoolr,esscceanttned in
'
mFiocxroasncaolpyya.is. For control of bias, all slides are coded
E.
ScotrheiSln idg es
Aannimaaclc.ampeThweilflrebqeuemnacdye oftomisccroroenuocnlee-accheoduscaenldls FwCiElsl pbeer
pTorteaslsPeEdsapserpceerncte.nt MThiecronnourcmailcatbeadckgcreiolusndbafsreeqdueonncythe
oF mlcronucle in the fac strain 1s around 0.00.41,
asa 1m
7010
004461
2 WASHINGTON
'
EPRbOITTOICOONL5Ko. 456
heCeEksaEarneiqduiNelCnEcsbyeoofbdseetFCreEvrseaidvneeirdnsbutyshesmacotopurtriiencgfeltrehyelcdhsnruoswcbyhetirelseof
Scoring the firet 1000 erythrocytes on the slide.
.
vi. parma STSoheaebraetcshr,it(`e1ir5ti76na).sfhoarMriptchreboonriuddceelrnestlifainadrceatabirroeinggheotnferymeaillcllroyown,buectluewslueaelnalrye1/tr2ho0ousanedndof1/5 eotoritTeiE'easlaof cthehelelmwlPicCEtr,honwumhcoirlceehattWehidalnlceolnslet,aminnioctbrrointguhhcetlemuiocrsarnoignseu.ccloeuuTnsht;eedutnahsiuscontoefhe Sicronucicated PCE, mot two (of more) micronuclei. Tohteyeshtraoimnairnigcpraoncdedmuerremocpehrraoincasticthesrydtihfrfoecryecnetsiat(ibornigbhyt ocorlaonrgeofand Those like, dark green respectively).
DaPsraesensation TIThooenneb.edracaanodfrmetpihoeerrcorenadutciwloielaoltfedpionlcPylCEucsdh,eiomttahhteeicnpuesrtbcoeernmtoaorgfmeoBcCohEfrsommiascctroirocendu,cletaheted Crychrocyces for each experimental animal.
Evaluacion Criteria
To Mhieonica triictaenrteieadeofosrefe-prarepolodasuticetidibvlieencarreneasdspeosntsaientismistiiccaraolsnltuyactliessaittgiencdiafliPlcCEyasn,t porosittheive
TeCteiatethniessrceicaafolsrleyaatcissicTgeinacisaftlilcoyannetsidgaonnsidefircelaepvnretold.udocsieAblerteessitpnocanrrseecasiecnloeratathoante
induces dose
:
e12eblaseids ucpoomnidsecvieedntinfeigactijvued.gemeInnt.either case, che final decision
VII. IIENSTERPRETATION ieThteihnanmeailcycssonin'sacclohefel tahrpicesesindaeanciaatarlwa.inlslfoIbrfematptheieornfasonralmoyefsditsuhesionfpgrvoaparoniratanincaoelnysoiifss ceoflls nioynithairceEansti.gn1i(6fp1ie)c0.a0wn5it)ll,lyadbeiffuestreeesdtnttwoiftdrheotmmeurtlmhtieinpenleegvahtciiocvmhepadrocisosenotnrgsorlou(pSso,kalif pcoembeinastioanc.e perforned separately for each harvest time and sex
asa + 1/93
004462
80 10
WASHINGTON s
PEDRIOTTIOOCNOL5NO. 454
VIII. REFERENCES
1. HMeadcdGlree,gorJ,A4,.T.H,iteN,eweHl.,l, KiG.cUk.haraen,d BS.a,lamLoanres,en,H.EK..!, The induction of micronuclel as 4 measure of genotoxicity,
.
Mutation Res., 123:61-118, 1983.
2. Schmid, W.: The micronucleus esc. Mutation Res., 31:9-15, 1975.
3. Sacnhamliyds,is.W.: In,TheChemmiiccraolnucMluetuasgentse:st fPorrincciyptloegseneatnidc Heedt)h.odsPlfeonrumT,heiprp. De3t1e5c3t,ion1,576V.ol. 4 (A. Hollaender,
4. Sokal, RR. and Rohlf, J.0.: Biometry, Freeman, 1981.
IX. REPORTFORMAT
Hi employs a standard report forma for each assay design. aRirentpfioocrrltmeas.tiwoinlE:lacbhefiinsasluerdepionrdtivwiidlulallpyrobvyidetypteheoffoltelsotwiangnd by test
+1 QSupaolniscoyr Aisdseunrtainfciecatsitoant.ement.
1 SSitgantaetmuernetsofofGLsPtudCyompdliiraenccteo.r and sentor
+ Tleasbttaercthincilcieanl:dencification and HiA Study
Ntuessbtera.rticAlephyasnidcdalaedeosfcrriepcteiiopntowfiltlhebe
:}
+ Tiynpceludofedasisnaythiasndsepcrtoitoonc.ol number. Dates of scudy initiation and completion.
{Do StuNedthyodsd.irector and senior fechnician:
II 1
EIvnacleuraptrieotnaticerntteorfiar.esulcs Conclusions.
10 RTeefsetrernecseusl.ts presented in cabular form.
4s = 19
5 of 10 604463
WASHINGTON
PROTOCOL No. 454 EDITION 5
Xx. CHAON RREG VIE SIOS NS
AsniygnecdhanbgyestheorStruedvyisiDiornesctoofr,thidsateadp,proavneddmparionttoacionledwivlilthbethdioscupmreonttoecdo,l.
The Sponsor will be notified of any change or revision.
fi
XI. REC TOBO EMR AIND TAIS NED
AgLeLnerraatweddataas, adorceusmuelnttaotfiotnh,isresctourddys,wiplrlotboecoalrsc,hivaendd fininatlheresptoorrtasge ffiancaillitrieepsortofcHoazthleetsopnonfsoorr.at Alfetaesrt otnhee yoenaeryefaorllopweriinogd,subtmheissspioonnsoorf mtahey eflaeccitlitcioehsavoef Htahzeleatfoonremfeonrtiaonneadddmiattieorniaallsperrieotdainoefdtiimnethoer ssteonrtagteo a storage facility designated by the sponsor.
us = 1/93
6044,a
10 of 10
3M Internal Correspondence
ce:
To:
From:
Subject:
Date:
R. G. Perkins - 220-2E-10
B. C. Copley - 53-35-02
R. D. Howells - 53-35-02
Key to FC Alcohol Tox Samples
September 14, 1995
T5877
`Wide Range EFOSE
Analytical Request 41220
FM 3924 Lot 547
Retain from 2 year feeding study ~~ L-13203
T5710
`Narrow Range EtFOSE Lot 884
Typical Raw Material
for FC-807
Analytical Request 41220
L-10059
T5711
Wide Range MeFOSE.
Lot 555
Typical Raw Material
for FX-845
Analytical Request 41220 L-1276
TS794
Narrow Range MeFOSE
Analytical Request 41343
Notebook 97900-1072
Lab Prepared Sample
L-13097
T5795
Wide Range EtFOSE
Notebook 97900-112-2
Lab Prepared Sample
Analytical Request 41343 L-13098
TS878
`Wide Range MeFOSE Lab Prepared from
`Washed POSF
Analytical Request 42607
PDs
Attachments
004465
REQ 41220
R. Howells
-
GC/MS analysesofthese samples were accomplished using a 25 m X 0.32 mm HP-1 GC column to
introduce the samples into the Finnigan SSQ-70 mass spectrometer. The sample components were ionized using chemical ionization with methane as the reagent gas. The GC column was operated from 40 to 300 C ata rate of10degrees per minute. The resultsofthese analyses combined with their corresponding GC/FID area percents show the following:
main [ive [pars | NR. N-EtFOSE
`W.R. N-EtFOSE
[-- r-- E I Component LD.
Lot
Lot 547
ncosamie orsomeyy
lows lone |
lossomleoua w n Jonoe n|
lomsoneuoonne c[muno s n|
loosonelm ousenimos s|
lonJoe wx [ir es | lorsosecowme o|mawo e s|
"Jorsoseeamms elmowo s s| leesonaoc opsm[ er| on =
00446
Component LD.
L-1276
-
N-MeFOSE
A.
Lot
C2FsSO2N(Me)CH2CH20H
J es cso A
C6F13302N(Me)CH2CH20H
J---- CsF17802N( )CH2CH2C1
N-MeFOSE_ CsF17SO2N(
CH20H
CoF19S02N(Me)CH2CH20H
cotrsopurs (C8F17SO2N(Me)(CH2CH20)2H
OtherHigh Boilers
[se| lowe| Los|
loss
|
Los| 0.13%
83.88%
087%
034%
155%
Further work has been done by GC on these samples which involves derivatization of the alcohols with
wifluoroacetic anhydride (TFAA) and again with BSA (to give the trimethylsilyl ethers). This work was designed to investigate the potential problems that could be overlooked by any one method of analysis.
Preliminary results show that analyzing the EtFOSE underivatized could hide asignificant amountof N-EtFOS
`Amide under the C-3 alcohol peak in wide range material. However, analyzing the same material that has been
derivatized with TFAA shows that any E(FOSE-chloride that is present in the sample is now completely masked
by the derivatized C-8 alcohol. The BSA derivative has not been evaluated yet, but similar problems are
expected becauseofthe number of different components in the sample. The same sort of problems will most
likely exist with MeFOSE and will be even more complicated in the analysis of MeFOSEA.
712/93
004467
REQ 41343
J. Grant
=
GC/MS analyses of these samples were accomplished using a 25 m X 0.32 mm HP-1 GC column to iunstirnogdcuhceemtihceaslaimopnliezsatiinotnowtihtehFmientnihgaanneSaSsQt-he70remaagesnstsgpaesc.trTohmeetGerC. cTohleusmanmwpalse ocpoemrpaotneednftrsowme4r0eti0on3i0z0edC ata rateof10 degrees per minute. The results of these analyses combined with their corresponding GC/FID area percents show the following:
Component ID.
cssone N-Ethyl-carboxamides (R-C(OIN(EY) crson@anazon cruson
Ep ---- cimasoane:
crsonEmaon
CaF9SOIN(EN)CH2CH20H
CcsrF1uIsSOoNn(EE)CcHc2CrHo20nH . w|eCIsF1wsSONE_NcCHv2CsH0oH nccon
WR NEFOSE |WR N-EFOSE
Precut
`main cut
97900-112-1
97900-1122
68.34%
o12s7%
I
I
PF
ws Jue|
[nx [one
062%
042%
[ml aise s os | 21w 6% n|
[Ji35a2%num25a 5% ]
Component LD.
NR N-MeFOSE BP. 132 97900-1072
004468
}
lomsoneecleonaom|
lomsomo Jewen|] loco Joms s |]
Amide
[----P-- Y97900-111
lomsonee
Jew |]
e Ee e I=| that include hydrides, chlorine in the backbone, and
unidentified high boilers
004469
To:
From:
Subject: Date:
1. Mugg
53-65-02
R. M. Payfer
236-2B-11 (612)733-4212
SA&C Analytical Request No. 42607
Dec. 21, 1993
GinCt/roMdSucaentahleyssiasmopfltehiisntsoatmhpeleFiwnanisgaanccSoSmQpl-i7s0hemdasussisnpgeact2r5ommetXer0..3T2hme msaHmPp-l1e cGoCmpcoonleunmtnstwoere. fironoimz4ed0utsoi3n0g0chCemaitcaalraitoenoizfa1ti0ondewgirtheemsetpehranmeinaustet.heGrCeaagneanltysgiass.witThheflGamCe cioonliuzmatniownadsetoepcetriaotnewdas amlassosdosnpee,cawnordkt.heTahreearepseulrtcsenotf tvhaelsueesanfarloymstehsis(wwohirckhwaerreenaoptpnleiceedstsoartihleypqeuaanktiitdaetnitivtei)esshforowmtthhee
following:
Mol. Weight |Component LD.
L-13202 N-MeFOSE
N-Methyl-carboxamides CrF1s-C(OIN(Me)H | 0.06 %
|ser lowrasomumer
[sn orosomnman
ozs|
[sr loessonmmorcon
C5F11SO02N(Me)CH2CH2OH
[ose |
C6F13S02N(Me)CHzCH2OH
338%
:
CrF15502N(Me)CHzCH20H
216%
N-MeFOSE _CoF17802N(Me)CHeCHzOH
CoF15S02N(Me)CH2CHzOH
CoF19802N(Me)CH2CHzOH
[osx |
[ess Jo`mC8uFs16rCIeSOs2oN(nMem)oCHo2CoHr2OoHn
604479
[JCoerrusomuaicrcion [ lovertumoues
004471