Document gDDmEnk2JBB6kkon9qja1QJJ9
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ARR26 - 1104 sret (2234
Study No. T-6295.30; ST69
Oxygen Saturation and pO2 Levels in Rat Pups Borne to PFOS Exposed Dams
Study Location: 3M Strategic Toxicology Laboratory
Corporate Toxicology 3M Medical Department 3M Center, Building 270-SB-314
St. Paul, MN 55144
Study Director: 3DeManMneadJi.caLluDeebpkte.r,/ MC.oSr.p,orAadtveaTnocxeidcoRleosgeyarch Toxicologist
3M Center, Building 220-2E-02
oe
Saint Paul, MN 55144
Ph: 651-737-1374 FAX: 651-733-1773
Study Toxicologist: Andrew M. Seacat, Ph.D., DABT, Toxicology Specialist 3M Medical Dept. / Corporate Toxicology
3M Center, Building 220-2E-02 Saint Paul, MN 55144
Ph.: 651-575-3161 FAX: 651-733-1773
x 32 :
`Sponsored by33MMCSepnetceiraBltlydgCh2e3m6icals Division
ES
St. Paul,MN 55144
=S3o3x
z 55
KR
ER
CONTAIN NO CBI
000168
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T629F5i.n3a0l;ReSpTo6r9t
StudyObjective T`hmieschsatnuidsymwoafsptehrienfaitrastlomforatasleriiteysionfraetxsplaonrdatmoircyepbilootresttuodtieismdeesdi-gpnerdetgo nidnavnemssttidgaotseedthweith PexRcOhSandgueriinngthgeesptuatpisonw.asThinevehsytpiogtahteesdi.s ItnhaatddPiRtiOoSn,iantmenrifoetriesc fwliutihdnwoarsmaalnarleyszpierdatfioornPaFnOd/Sorlegvaesls and specimens were gathered for possible future analysis (i.c., to investigate the ability of PFOS tointerfere with the glucose metabolism).
MethodSummary
`(T1h)isansdtucdlyaswsaifsiepderasfoar"mCeldaisnstBheSt3uMdyS"traasteegxipclaTionxeidcoilnoTgOy XLaSboOrPat0o9r5y0,unSdterrataedgeifciTnoexdicporlootogcyol LAnaibmGaLlPRePvrioegwraCmomPmrioctetdeuere(3(2M).LAAlRl Cin)-laifnedparroeceddetuarielsedweinreanaipmparlovuesdagbeyatphpeli3caMtiLoanbo2r0a0t1o-r0y182
A).
Ucunmduelrattiheveindiotisael osftu1d0y0prmogt/okcgo.l (T1h),ediantmesntwwearse dtoosaeldloowntdhaeysesa1n4i-m1a7lsoftgoelsittatetri,oanntdotroeatchhenof
Svaecrryifhiicgehboratthesdoafmsstialnldbopmuppsupwistahnind 2ea4rlhyoudrelsoivfebriirest,h.greTahtilsydcoosmipnlgicraetgiinmge,thheowsetvuedyr,anrdesulted in
p(rpe0v2en)tfinrgomthbeeeinndgpeoxianmtisonfedi.ntIenrersetsp(oonxsyeg,etnhesasttuuradtyipornoatnodcoplarwtaisalaomxeyngdeendpraensdsuarseeicnobnldosotdudy as
pPeFrOfoSrmmeady. hAasvedibseceunssdeedliivnertehde ptorotthoecoplupasmaenndd/moernttha(t4)d,ositiinsgtpheoruigohdtmtahaythaanveexbceeessnivte0odolastee oifn
gPeFstOaStipoenr.kUgnbdoedrythweeiagmhetnpdeerddsatyudfyorpr3otcoocnosle,cuatniivmealdsaywser(edatyrsea1t2e-d1v4ioaforgaelstgaatviaong)etwoirteha2c5h amg
jcuusmtuplraitoirvteoddoeslievoerfy7.5 Pmugp/skgw.erAellreamniomvaelds,viiancclauedsianrgeadna-msse,ctwieorne(ce-ustehcatinoinz)e,dtvoisatdaencdaapridtiazteiotnhe
steitmteoomfadianttaacionllaetcteimopne,raatnudrmeaonfu~al9l5ytsoti9m8udleatgerdeetso bF.reaBtlhopordioarndtoadmenciaoptiitcatfilounidun(pdoeorlaedhefartolmamp
`fmruolmtieplaechamdiaomtifcorsPacRsOuSsianngalaysniesed(lseeeaAnpdpseynrdinigxe1prfioorrdteotarielsmoovniPngFOcSacahnpaulypt)icwaelrmeectohloldec)t.ed.
L`piovsesribwlaesfaultusroecaonlalleyscitse.d fPruopmsewaecrhecd-esceacptiitoanteedddwaimthiannd5fmlianshutfersozbeenining rlieqmuoidvenidtfrorgoemntfhoer
placental sac. Immediately heparinized weigh boat and
following decapitation pipetted into an -Stat
of the pups, cartridge for
blood was drained ontao determinationofpO2 and
other
b`mleoaosdugraesmeesntbsyutshienIg-tSthaetpmuelstehoodxi(mdeetsecrriwbeerdeinobtthaeinsetdu.dyOpnroetoacdodli,ti1o)n.alNdoaomxywgaesnrescaemirvaetdiofnrom
tlihtetevrewnedroer.thIetnwmaosnditoosreeddwitothasPsFesOsSviaasbidleistcyarfitbeerdbiarbtohveusainndgatlhlisowsetdudtyodleisteirg.n.`This dam and her
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Results
T629F5i.n3a0l;Re5pTo6r9t
Initial Study
IPnF-lOiSfe torbesaetrevdaatnioinmsalfsrwoemrteheinicnlituadledstiundtyhicsasntbudey.fouOnndeicnoTntarbolle a1n.dFoonuertcroenattreodlaanniimmaallswearnednsoitx
pregnant. Pregnant control animals gained an averageof 33.0 + 7.8 grams from day 14-17 of
gaensitmaatlisonliwthtierleed torneadtaedyp2r1egofnagnetstaantiimoanlasngdaailnlerdeoantleydaanniamvaelrsagleittoefre8d.6on+e1i2t.h2ergdraamys.20Aolrl2c1onotfrol
sgehsotrattliyoanf.teCrobnitrrtohl(wliitttherisnaapppperaorxeidmantoerlmyal10whmiilneutterse)a.tLedivleitrerasndwesreeraeiwteherrebgoatrheerdeedafdorormdiseodme
`doaxmysgeannsdatpuurpastiaonndusfilansghafrpouzlesne ofxorimpeotsesri,blaenfduptuOr2e alneavleylssisw.itAhnanatIt-Setmapttbwlaosodmgaadseantaolmyezaesruorne
ptuhlossee pouxpimsebteornmeeatlhivoed. wNaos unsoetfusluidtaatblaewfaosr ugsaethoenresducfohrsemiatlhlerppuaprsamaentdert.hat1tfuwtausredbelcoidoeddgtahsat the
`analysis would be limited to the I-Stat method.
:
Amended Study
I4nP-lFifOeSobtsreeravtaetdiaonnismfarlosmwethreeaimncelnuddeedd isntutdhyiscsatnudbye.fAolulndaniinmTaalbslwee2r.eTphrreegenacnotn.troClonatnriomlalasniamnadls gained an average of 99.5 0.7 grams from day 12-21 ofgestation while treated animals gained onDleyWaaynnaevWearlakgeoref(734.M8L%ab11A.n4igmraalmsV.etDeraimnasriwaenr)eapnadlppiutpastewdeornegdeestteatrimoinneddayto(bGeD)to1o9imbymaDtru.re (ton=s1u2r)viwveerief duenlaibvleeretdo.brOenaethcaonntdrosluravniivme.alCw-assecdteicoanpiintgaotfedthaendrecm-asienctiinogndedamosn GwaDs2t0huasnd pups `pTohsetrpoenmeadionnint2hgecroenctroomlmaennidmaatlisonwoefreDrde.caWpailtkaetredunatnidl eca-rsleyctmioornnedinogn(GD251--,:6a:n33d0la0imtt)erosnofGD102a1n.d `1a1ndpcu-pssecwteiroeneddelainvderleidt,taerlsloafpp1e0a-1r3inpgunposrwmaelr.e dTehlrieveeroedf.thBelotroedatweadsdaevmisdewnetrienatlhseoudteecraipniethatoemds omfienauctehsofafttherebPirFtOh.SIttrweaatseddedtaemrsm.inTerdeatthaetdtphuepsse htraedatdeidffpiucpulstywebrreeantohtiangsamnadtsuormaesedtiheedcwointthrionl pups. The remaining PFOS damwasallowed to litter. She delivered 13 pups (10 on GD22 am, 3 on GD23 am), all bore deadordying within minutes of birth.
1Stat data from the pups can be found in Table 3. The only parameter that significantly different between control and PFOS treated groups (p<0.05) was pH, with controls averaging 7.14 0.08 ((Nm=m2H)ga)ndorPOF2OSsattureraatteidonav(e%r)agwiansgf7o.u0n7d+b0e.t0w3ee(nN=t8r)e.ateNdoansdigcnoifnitcraonltpduipsf.feCroennticrneolppOO22lleevveellss averaged 30 + 4 mmHg while levels in PFOS treated pups averaged 35 6 mmHg. Oxygen rsaetpurreasteinotnsatvheerOa2gleedv3e9lsat1 t%heienxcaocnttrtoilmseatnhde 4bl4o+od1i3s%colilnePctFeOdSantdreiastreedapduiplsy.alSteyrsetdebmyicapf0e2w dbraemasthasr(e5~).60LimtemraHtugrewvhaelnuetsafkoern pwOit2hifnro3m0 csoenctornoldspuofpsbidretlhiavenrdeadffterroamt cl-esasetcotnioenberdedaetchap(i5t).ated This is equivalent to vaginally born pup PO2 levels (5). The pO2 levels found in the current study de'substantially lower than those cited in the literature. `This may be a result of the
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T629F5i.n3a0l;ReSpTo6r9t
increased time the pups were allowed to breath on their own prior to decapitation (5 minutes as. opposed to 30 seconds).
`flDuaimd PseFrOaSanldevaemlnsiaovteircafgleudid0P.0F1O7S+l0e.v0e0ls3 cuagn/mbleafnodun0d.0i1n1Tab0l.e40.03 Cuogn/tmrlorlessepreactainvedlya.mnTiohteicratio
of sera to amniotic fluid PROS levels amniotic fluid PFOS levels averaged
for controls averaged 1.7:1. PFOS treated sera and 68.3 15.0 ug/ml and 16.4 1.2 ug/ml respectively.
The,
ratio of sera to amniotic fluid PFOS levels for the PFOS treated dams averaged 4.2:1.
Conclusions
f`rTohemlboewinngudmrbaewrsn.ofTahneimreaslusltussoefdtihnetIhiSsteatxpalnoarlaytsoirsyspuiglgoetsts,tuhdoywperveevre,ntthsadtePfiFnOitSivdeoceosncnloutsions
ienxtpeorsfeerdedwaimths.noRremsaulltrsesopfirtahteidonaamndse/roaragnads eamxncihoatnigcefilnuindePonFaOtaSlarnaatlpyusipsssbuoggreesttothPaRtOPSROS
tdirsetartiebduatneismmaolsr)e.rePaFdiOlSy iwnatso,thheowseevreart,hadnetiencttoabtlhee ianmntihoetaimcnfiloutiidc(falpupirdooxfiamlaltaenliym4a:l1sitnePsteOd.S
Tthheisamsnhioowtsicthfaltuinde.onTahtealdroastepuuspesdbionrtnheetaomPenFdOedtrsetautdeyd,
dams are exposed 25 mg/kg/day on
to PROS in utero days 12-14 of
via
ogefsbtiarttiho.nA(cduomsuelaptriovdeudcoisneg odfea7t5hmwgi/tkhgi/ndtahye)fpirros6dtu-ce2d4dheoautrhsoinfliivvee bboirrtnheipsudpessiwrietdhitno amlilnouwtetsime
effofredcattaascmoullltecitpiloendaanildyodbosseersv.atTihoun.s,I2t idsoaslesoretshpooungshet stthautdyawsiilnlglbeedpoesrefomramyedprtoodduecteertmhienseaimfethe
dosing regime can be limited to a single administration and to find a dose level that will extend
life to 6-24 hours following birth.
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ara
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List of Tables Table 1: Initial Study In-Life Data `Table 2: Amended Study In-Life Data `Table 3: Amended Study I-StatData (Pups) `Table 4: Amended Study Maternal Sera and Amniotic Fluid PFOS Levels
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Signatures:
Prepared By:
Se T-6295.30; ST69 :
Deanna Luebker, MS `Advanced Research Toxicologist Study Director
Lox chs1) Sad
`Andrew Seacat, Ph.D.
Toxicology Specialist Study Toxicologist
-
Reviewed By:
Far Botte
John Butenhoff, Ph.D.
Corporate Toxicology Management
rtHwe- e7 dQos.
Dan Hakes
`Sponsor Representative
Date
i.fafer
Date
20s Serre
Date
fy fon
pal
000177
retorts
.
T629F5i.n3a0l;ReSpTo6r9t
References
1. 3M Medical Department, Corporate Toxicology Protocol for Study No. T-6295.30 DT-69 Oxygen Saturation and pO2 Levels in Rat Pups Boe to PFOS Exposed Dams, 2001.
2. 3M Medical Department, Corporate Toxicology Strategic Toxicology Laboratory Standard Operating Procedure (TOX SOP) No. 0950 ~ GLP Program Procedure, 1999.
3. 3M Lab Animal Review Committee Animal Usage Application No. 2001-0182, MECHANISTIC RESEARCHIN TIMED PREGNANT RATS ANDMICE -- AMENDMETNOT AUA # 2001-0182, 2001.
4. 3M Medical Department, Corporate Toxicology Amendmen#t1 for Study No. T-6295.30 DT-69 Oxygen Saturation and pO2 Levels in Rat Pups Borne to PFOS Exposed Dams, 2001.
S. Vaillancourt C., Berger N., and Boksa P. (1999) Effects of vaginal birth versus Caesarean section birth with general anesthesia on blood gasses and brain energy metabolism in neonatal ats. Experimental Neurology 160:142-150.
Ren
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Page 110f 15
Appendix 1 3MAnaClyotricpaloLraabtoreaTtoorxy iBlcdogl2o36gy
T629F5i.n3a0l;Re5pTo6r9t
|
To: From: Compound:
Date
DAenadnrneaw LSueeacbakter
David J. Ehresman BOoxryngeentoSaPtFuOraStiEoxnpoasneddpD0a2mLsevels in Rat Pups 33MM MSterdaitceaglicDTeopxairctomleongyt LSatubdFyoNlduemrbNeurmTb-e6r29D5T.3-069 December 10, 2001
aPtrteegmnpatnttofdeemtaleermriantes wraetrpeuepxopoxsyegdentolePveOlsSattobiervtah.luPaatretthoef pthuips sfettuadlyliunnvgomlavteudrdiettyeirnmainning tcohrerceolnacteenwtirtahtitohneoOfxPygFeOnSSianttuhreatfieomnalleeverlastosfetrhuemeaxnpdosinedthaenadmcnoinottrioclfpluupisd.post exposure to `deSlaimvpelrestoofttheheStrraattesgeircuTmoaxnidcoalmongiyotLiacbofrlauitdofrryofmobroatnahlyesxipsobsyedLaCn-dMSco.ntTrohleaanminmiaoltsicwefrleuid samples were collected at the time of Caesarean section delivery for both the control and dosed animals. CCoonnttrrooll aanminmiaoltsicwfelrueidparnedgnsanetrufmemwaelreescroaltlsewcittehd tfhreomsatmheemapfporroaxniamlyasties.gestational status.
AnalyticalMethod:
ePxFtrOactlieovnelisn wetehryeldaecetteartmei.neTdhefrpoHm booftthhethteesrtaetdsseorluutmiaonnd(1a0m0niuoLt;icseflruuimd oursianmgniaostiincglfel:uid) bwuafsfeardjsuosltuetdiotno(apnHa=pp3r.o0x).imIantteerpnaHlosft3a.n0dawridt(hPtFhHeSa)ddwiatisoanoddfe4d0p0urLioofotahre0.e2xNtrafcotrimonicanacdid tahte:3m0i0x0truprme'wsafsors5hamkiennuotensaamnedcthhaeniecthayll sahcaekteartefotrra1nshfoeurrr.edT1h0ea tculbeeasn wpeorleyptrhoepnylceenntertifbueg.ed Tdrhieer.ethTylheacreetsautletawnatsredsiiedduetwoadsryrnee-sdsisussoilnvgedaignen2t5l0eusLtorefa2m 5of%niactertoogneintrgialse oanndth7e5N%-E1v0ampm ammonium acetate buffer. This mixture was then filtered through a 0.2 micron syringe filter
"and placed in a polypropylene injection vial. 25 uL was injected on to the LC-MS system.
"os
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T-6295.30; 5T69
Final Report
Appendix 1
`uTsheedlwiqausida cKheryosmtaotnoegFrlaupohruospehdasweas(1t5h0e xFi2n.n1imgamn)MsApeTciaSlpietcytcroalsuymsntefmeaPt4u0ri0n0g. a Tsheleecctoilveu,mn
`flmuUo/rmiinna.teAd,gbrraadniecnhtedf-lcohwaoifnaaclektyolniptarcikliengw.asTuhseedfltoow rate was maintainedat a constant 0.4
celountceentthreactoiomnpoofun7ds%0ofacienttoenrietsrtilsetaorvtienrgawistixh a1n5d%onaceethoanliftrmiilneuatnedgernaddiienngtwpirtohgraam. There
pwhaassea bcoenisntgan1t0mmemthaanmomlocnonicuemntarcaettiaotneobfuf1fe0r%. mTehtehainniotlialwibtuhfftehrecboanlcaenncteroaftitohnes wmoebriele
erqeutiulniberdat1i0osntpaertriinogd6w.5asmitnhuetne2s.5inatdodtihteiornuanl amnindurteeascbheefdoarte8t.h0emnienxutteisnj.ecCtoiolnufmonr a cycle time
of 10.5 minutes per the gradient run.
injection.
All
compounds
of interest eluted
within
the first
5
minutes
of
i`nThtehFeinnengiagtainveTiSoQnizsaytsitoenmmwoadseowpietrhataedcoinnsttahnetMsSou/rMcSe pmootednetiuaslionfg3t.h0ekEVlecatprploiSepd.raTyhseource
reactantgasin the Q2 region of transition of the following mass
the triple ions:
quad
system
was
argon.
The
method
involved
the
Compound
Base Peak (Q1 mass) MS/MS lon
Int. Su. (PFHS) ~~ 3990
80
02 Offset Voltage 60 volts
PFOS
4990
80
60 volts
The capillary heater was held at a constant 300 degrees C. All quantitative calculations were
based on Standard
the MS/MS (PFHS).
fon
ratios
between
the
compound
of
interest
(PFOS)
and
the
Internal
Study Results:
Serum PFOS Level Amniotic Fluid Level
[6030
TComwol TiSmgmL
[GmgmL
|
EL __-- 1
"|
71
[6| 031DoseAnd imal |56.0 ug/mL
16.0 ug/ml
[6032
| Dosed Animal _|64.0ug/ml
17.7 ug/ml
[6| 033DoseAnd imal |85.0 ug/mL
[155 ug/ml
Duetothe high concentration of PFOS in the serum a 1:500 dilltion was required to bring
ftohre tchoencdeonsterdatainoinmadlosw.n`tTohebdeowsietdhiannitmhealquaamnntiiottatiicofnluliidmiwtsaosfdtihleuteesdta1b:l1i0s0hebdefsotraendtahred curve.
concentration was within the acceptable curve limitations. The routine standard curves were
+ go established using 6points between 10 ng/mL and 500 ng/mL.
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Page 13015
Appendi1x
T62Fi9a5t:Re5p7o6r9t
The Rsquared onthecurveused forthecalculatedresults was 0.9926forthestraightserum andamnioticfluidresultsrunonthe controlanimalsandthe 1:100 dilutionofthe dosed
animal amniotic fluid extracts. The 1:500 dilutionof the dosed rat serum curve had an R squared valueof0.9997.
El oe
Ey
o fEI=ee mEe EfmWEoEEEn 2momT r ooEmEmEoe ooOCT mEoRmCaZEIORFlm= nR em 1lmmwFmEE E E :io f f28|
[apatnaam mieam tmme xeonms ttmmsa Lea
Rea
rr
|i psp
ee
1
BE.
|i.
OTSATII
TINY RECN WEAK
Fee EEE
Straight serum, diluted serum, and control dams amniotic fluid standard curve graph from
original Finnigan Xcalibur program.
:
000181
Page140115
,
T-62F9i5n.a3l0;RSeTp6o9r.t
Appendi1x
s
Sh. nee ae
EE k E
haaces
[Semesnvimedne
saimeooessm e dA l isE i .m ||
i1
S; ee SxSLe IER h ne E a a
RD ver ABARS AOR es Sernl]
=
i|
i
Tchuirsvegrwaapshbarseedponr5etphoseinsettsansntdtaarrtsdincgaurtv2e5usnegd/mwLitth thhe1r:55o0000duinlggu/tmehLd.spTechieme1n0ss.taNnodtaer:d wTahiss
:
droppedonthis rundue tolowdetector response.
`Therawdata and printed reportsarecontainedinlabeled 3 ringbindersinthe Bldg. 236 Strategic Toxicology LC-MS Laboratory. All original methods and raw dataarestored as
finleeesdtthoabtearree-blaocakdeeddutopatocZoimppadtiisbclsefFrionmnitgheanFiXncnailgiabnurXscaylsitbeumrtsoyfsutnecmt.ioTnhpersoepefrillyes. would
ean
000127
Page15of 15
}
PCTL {p55
3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-6295.30 DT-69
OxvgenSaturationand p02LevelsinRatPupsBornetoPFOSExposedDams
`STthiusdsytOubdjyeicsttihvee:firstof aseriesdesignedtoinvestigatethemechanismofperinatal g`emsotrattailiotny. Tinhreathsyapontdhemsiicsetbhoamtweitllobte tiesmteedidnt-hipssdtraumdesy gidsotnsheaadtwnPiFttOhSPiFnOteSrfdeurreisnwgith `normalrespirationand/orgasexchangeinthepups. Inaddition,specimenswillbe gatheredtoinvestigatetheabilityofPFOStointerfere withtheglucose metabolism.
Research Client: .
3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220
Sponsor:
3M Specialty Chemicals Division 3M Center, Building 236 `Saint Paul MN 55133-3220
Study Location:
3M Strategic Toxicology Laboratory 3M Center, Building 270-SB314 Saint Paul, MN 55144-1000
Study Director: -
Deanna J. Luebker, MS. `Advanced Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55144-1000 Ph. 651-737-1374, FAX: 651-733-1773
Study Toxicologist:
:
Andrew M. Seacat Ph.D. 3`TMoxMiecodliocgaylSDpeecpita.li/sCtorporate Toxicology 3M Center Building 220-2E-02 Saint Paul, MN 55144-1000 Ph: 651-575-3161, FAX: 651-733-1773
ProposedStudy Timeline: In-Life Start Date: September 24, 2001 In-Life End Date: October 2, 2001
Regulatory Compliance: Thisstudy willbeperformedinthe3M
Strategic ToxicologyLaboratoryinthe
"spiritof
GLP". Adefinedprotocolwillbeinplaceandthestudywillbeclassifiedas"ClassB"as
explained in TOX SOP 0905, Strategic Toxicology Lab GLP Program Procedure. All n-
000183
Page ofS
T629530;S69
life procedures havebeen approved by the 3M Laboratory Animal Review Committee (3M. LARC)andaredetailedinanimalusage application 2001-0182. Test Material: `Perfluorooctane Sulfonic Acid, Potassium Salt (FC-95) Lot 217 CAS#2795-39-3 CiF0SOK* Animals:
Species: Rat Strain: SpragueDawley Source: CharlesRiverLaboratoriIensc,. Age atinitiationoftreatment: Day16ofGestation Numberandsex: 3,female Identification: unitaqilmuarek Husbandry: HousinAgl:l ratswillbe individuallyhousedinstandardsolidbottomcagesand
provided nestingmaterial. Diet/Water:
Allratswillbeprovidedtapwaterandratchowad libitumthroughoutthe: study. Environment: `Environmentalcontrolsfortheanimalroomwillbesettomaintain a temperaotfu7r2e 3F, humidityof 30-70%, aminimumof 10 exchanges . ofroomairperhourand a 12hourlightdarkcycle. Doseand DosingProcedures: `Studies conductedby the EPANHEERLhavedemonstratedthatthe PFOS-induced increasesinperinatalmortality observedinpreviousrat reproductionstudies,inwhich dosingoccurredfrom42daysprior tomatingthroughendoflactation,canoccurinrats andmiceby dosing during gestationalone. Thiseffect appearstorelate directlytobody burdenandcanbereproducedbydosingduringthelastseveraldaysofgestation. A cumuldosaeotfriouvghely 100 mg/kghasbeen showntoreducepupviability by approxim0a%twiethliynthefirst 5daysoflactation.Theseobservationsprovidean effective exposure model for mechanistic research. Fouroftheanimalswillarriveat 3Mongestationday9,andfouranimalsongestationday 10. Animalswereorderedinthismannersolitering wouldoccuronedayapart. Dosing willoccurfollowing aone-weekacclimationperiodasoutlinedin Table 1. Fourrats will betreatedviaoralgavagewith25mgPFOSperkgbodyweightperday,for four consecutive days (days 16of-ges1tati9on)toreach acumudloseaoft10i0mvg/keg. A `Smg/mL stocksolutionof PFOSin 2%Tween 80willbepreparedimmediatelypriorto
000184
Pagedofs
1
T-6295.30; ST69
do'sPFiOnSgtarenadtm5emnltdgorsoiunpg.sCoolnuttiroonlr/aktgsbwoildlyrewceeiigvhetSwmilll/bkeg 2ad%mTiwniesetnere8d0toonedaacyhsra16ti-n1t9hoef
gestation.
5
`TABLE1:Dosing Regim
ree JTe] aAmControel. EE EEE HEE (25 mg/kg/day PFO
[TmoS om 0 mATiN= one Mondays TOOT|
B2:925-9801
B2:N= onTu2 esday 10/2/01
ObservofaAtniimaolsn:
Clinica`lEOabcshearnviamtailownisl:lbeobserveddaily (excludingweekends)formortalityand
`morbidity and notable findingswillberecorded.
Body Weights: `Damswillbe weighedimmediately priortoeach dosing.
Terminalbody
weights willberecorded on damsandlitters.
Sample Collection: Num& Fbreqeuenrcy v`AinaiCmaOl2sawisllpbhesyacxriifaaicnteddipwuioptnhsinvi2a4dheocaupristoatfidoenl.ivA ery.n Damhi asvweibm lelebnea oerudtehl raendiszs eudch thMaotndfoauyrnwiiglhltl,itOtcertoSbuenrda1y.niTghhte,aSneipmatlesmbtehart3l0i%t.teTr/hareerbeommaeinoinngSuf ndao wyilnlilu gihtttr weirl"l besacrificed on Monday and theanimalsthatliter/arebomeMondaynightwillbe s`apcurpisfwiicleldboenbTourenspdearyl,itwtietrh.iInt2is4phroeusruomfetdhtehiarbtitrhtehd.aImtsisweilslctanniibtmahlaiatz1etp0utepos1dt4hat a`rpeersltiitlelrbwoirlnolbrtehuasteddiiendtuhreisntgutdhye.nTihgrhte.eEoifgthhteocfitghhetrpupes fmromapeuaipcshl(niiftaeivrawiinllalbblgee): biedemnotniifiteodr(edatmsevaearsarltriemckeo-rcdoeeudrtsdeimpeu)ppso.in`tTshethOrxoyuggehnoustthaedtayuiusnritnhgaetshetpeouixpisomweitnlelr ``Tmheetohod(tdepshucrpiseboendtrbheeloswt)udtyowdieltlebremimnoencihtaornegdesfiornooxxyyggeennssaattuurraattiioonnbyovtehretime. `oximeter method,then immediatelysacrificedtoobtainbloodforpartial oxygen Nperaesrstuhreeeinndbolofotdhe(sptOu2d)y,dtehteetirmme-icnoaubrtysietophunepsI-wSitaltlmbeetshacordi(fdiecsecdrtioboebdtabienlobwl)o.od forpO2 determbyithne Ia-Sttatmietohond. Any remaipupnsmianybge sacrificedtoobtain serumandtissuesforpossible futureanalysis.
Bu ern
Method of Collection/Measurement Oxygensaturation willbemeasuredwith
a
pulseoximeter
(Ohmeda),
andpO2
will
bemeaswiuthrane-stdat blgaosanoalydzer (AbbotLabs). Oxygen saturation
andpulseratewillbemeasuredoneach pup usingan Ohmeda Biox 3740Pulse
000185
Page3of5
T-6295.30;5T69
Oximeter,accordingtothemanufacturers" directions.Thepupwillthenbe
euthanizedby decapitationand blood willbedrainedontoa slide.Theblood will
betransferredfromthesidetoan I-Statcartridgeusing aheparin-washedsyringe.
`ThecartridgewillimmediatelybeinsertedintotheI-Statanalyzerandthe pO2
leveldetermined. Systemicbloodfromcachdam (= 5mi)willbecollectedviathe
abdominalaorta andtransferredtobloodcollectiontubes withoutanticoagulant.
All bloodsampleswillbeallowedtoclot for aperiodof 15to 30minutesatroom
temperatureandthe clotwillbespundown in acentrifugeat 1100 x gfor 5
:
`minutes. `Theserumwillbetransferredtolabeled1.5mlmicrofugetubesand
centrifugedagainat2000 x gtoremove anyremainingredbloodcells. Theserum
willthenbetransferredtolabeledpolypropylenemicrofuge tubesand flash-frozen
inliquidnitrogen.Liversfromallpupsanddams wilberemoved,weighed,
`placedindividuallyintolabeledtubesandflashfrozeninliquidnitrogen.
SampleHandling: + Liverandserasampleswillbestoredat 70CintheStrategicToxicologyLab,
building270.
Analysis: `The oxygensaturationandthepO2techniqueswillbecomparedtodetermine which methodwillbeusedin futurestudies.Theresultsfromeachanalyticalmethodwillbe `comparedbetween treatedandcontrolanimalsandanalyzedtodeterminestatistical differencesusingthe students T-test. These results wibelusledtodetermine the validity ofthe hypothesisthat PFOScaninterferewithnormal respirationand/orgasexchangein thepups.Liverandserawillbe retainedfor analysisofgluconeogenic enzyme activity, glycogenstores,andserum glucoselevels.Resultswillbeprovidedforinclusioninthe _ finalreport.
000186
Pagedof5
.
Signatures:
Tessas
Mics
Deanna J. Lu
r, M.S.
-
``SAtduvdaynDcierdecRteosrearch Toxicologist
oicial
Date
Lrdhes WM. Gon i
TAonxdirceowloMg.y SSpeeacciaatl,isPth.D. `Study Toxicologist
Y/ia/o1
Date
A. Ja Kn
`DSapnonHsaorkeRsepresentative
9=zo/or
ite
~
"o
000187
PageSof5
.--
PCTL 13334
1029101
T-6295.30; DT-69 Amendment #1
3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Amendment #1forStudy No. T-6295.30 DT-69
OxvgenSaturationandpO2Levelsin Rat Pups Borne to PFOSExposedDams
Sponsor: Study Location:
Study Director:
3M Specialty Chemicals Division 3M Strategic Alternative Toxicology Laboratory
DeannJa. Luebker, M.S.
.
"BTahecikngitrioaulnsdt:udyperformed underthisprotocolresultedinveryhighratesofstillborn
`pupsandearlydeliveries. This greatlycomplicatedthestudy,preventing the endpoints
ofint froe mber inge exas mint ed.Itisbelieve thatthesecompliwceraeatreisuoltnofsa
ddeossierebdeidngedelciveirrnedpetuopvtaihaebspiulipetsy.tAhastwwaasssitnaetxecdeisnsthoefwohriagtiwnaalssnteucdeyspsraortyotcoola,c"hsiteuvdeitehse conductedbytheEPANHEERLhavedemonstratedthatacumulativedoseof roughly 100mg/kgwilreducepupviabilitybyapproximately 50%withinthefirst 5days of lactation". Inthe EPAstudies,however, Tween 20wasusedfor avehicleratherthan ``Twaeemno8o0fu,PanFsOhtSasisbpereensuusmeedidnttohbeemabasojrboeodrwfihPeFtnOySuosirnagldTowseienngs8t0udaisecs.omApgarreeadtetor
Tween 20.Thus,webelieve acumulative dose of 100mg/kgwith Tween 80isin excess
ionfwgehsattatiisonndoesic ng e oiccns utrhses,stea hsetgur rdeiay etse.rItnheadrdaitteioofn,ptuhpemEorPtAalhiatsy.dTehempoensrottfihadrtoostaihdntegleatder `usedtheundertheoriginalstudyprotocol, days 14-17ofgestation,mayhavebeentoo latetoadministersuch a highdose ofPFOS.
=
O`bTjheecptuirvpeo:seof thisamendmeisnttoad.justthedosingregimeusedundertheoriginal
Protocol to prevent in werodeathandearlydeliveries.Inaddition, amnioticfluidwil be
collectedandanalyzed for PFOS and caesarean-sections (c-sections) will be performed
ratherthanallowingthedamstodeliver.Thepurposeofthec-sectiiosntso standardize
thetimeofdatacollection.A follow-upstudywillthusbeperformedundertheoriginal
studyprotocol with the following modifications:
1. IPnaagdedlit.iSotnutodtyhOebojriegcitniavlely:statedobjective,amniotic fluidwillbecollectedfrom
c-sectioned rats to measure PFOS levels.
2. Pagel. Proposed Study Timeline: "Thein-lfestartdateforthefollow-upstudywill be November7, 2001andthein1ifeenddatewillbe Novem14b,2e00r1.
3. SiPaxgaen2i.maAlnsiwmilallbNeuumsebdeirn:thefollow-upstudy.
4. Page2-3. Dose and Dosing Procedures:
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.
T-6295.30; DT-69 Amend1m0e2n9t0#11
AVlila osriaxlagnaivmaalgse wwiiltlha2r5rimveg/atkg3MPFoOnSdapeyr1k1gobfogdesytwateiiognh.tTpherredearyatfsorwi3ll be treated cmoyn/skegc.utPivFeOdSaywsill(dbaeyspr1e2p-a1r4eodfagsesdteastciroinb)edtoinretahcehoraicgiunmaullaprtoitvoecodlo.seTohfe75 redmaayionfi2n%gtThrweeeenrat8s0woilnldsaeyrsve1a2s-1c4onotfroglesstaantidonr.eceAinvye Samdidi/tkigonbaoldryatwseriegchetivpeedr flirttoemr.thPeupvsenadnodrdwialmlsbweidllosbeedmwointihtoPrFeOdStharsoudgeshcdraibyedoafbolvacetaatnidonatloloawsseedstsothe dosing regime or sacrificed to collect tissues for additional analyses.
5. PAlalgean3i.maSlsa,mpinlceluCdoilnlgecdtaimosn,s,wiNlulmbbeeerutahanndizFerdeqviuaendeccyapitation on gestation day
19. Pups will In addition to
tbheatrdeemsocvreibdevdiainct-hseecotriiogniinnagl.
protocol,
amniotic
fluid
will
be
collected from Liver and sera
each may
bdeacmolfloelcltoewdifnrgodmecsaopmitea,tnioont.all,ofthe
dams
and
pups.
6.
Page4. Sample Handling Amniotic fluid will temporally
be
stored
at
~70C
in
the
Strategic
Toxicology
Licaeb,tobuDialvdeinEgh2e7r0s.maFno,rbPuFilOdSinagn2al3y6s.isL,itvheeraamnndiosteirca wfillulidbweilhlanbdelsehdipaspeddesocnridberdy.
inthe original protocol
7. IPnagaedddi.tioAnnatloytshiesanalysis outlined in the original protocol, PFOS level will be compared between PFOS treated and control dose groups and analyzed for statistical differences.
Page20f3
100189
. Signatures:
10/29/01 T-6295.30; DT-69 Amendment #1
ADdveanaceJnd.nLRueebaksere, archM.ToSx.icologist StudyDirector
02/0
Date
{go A Lege
AndrewM.Seacat,PhD.
Date
ToxicologySpecialist
StudyToxicologist
10oafo!
3
0
DanHakes `SponsorRepresentative
dod . Date n/e/=f
Page3of3
000190