Document ey9mEnym0mzxLGrpep3keNKM
:
DEseanch
SS
ARI 056\
Argus Research Laboratories, Inc.
PROTOCOL 418-008
`SPONSOR'S STUDY NUMBER: 6295.9
STUDY TITLE:
Combined Oral (Gavage) Fertility, Developmental and
Perinatal/Postnatal Reproduction Toxicity Study of PFOS in
Rats.
PURPOSE:
The purposeof this study is to test for toxic effects/
disturbances resulting from PFOS treatment of Cr:CDBR 'VAF/Plus male and female rats before cohabitation
through mating, gestation and lactation. This study
evaluates ICH Harmonised Tripartite Guideline stages A
through F of the reproductive process and should detect
effects on the estrous cycle, tubal transport, implantation, gestation, parturition, lactation and maternal behavior in
female rats, on the development of the offspring of the
treated male and female rats, and permit detection of
functional effects (.g., effects on libido or epididymal sperm
maturation) that may not be detected by histological examinations of male rat reproductive organs. Because manifestations of effects induced during this period may be
delayed in the offspring, observations will be continued
through production of F2 generation litters.
ESTING FACILITY:
Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 Telephone: (215) 443-8710
Telefax:
(215) 443-8587
STUDY DIRECTOR:
Raymond G. York, Ph.D., DABT
Associate Director of Research
SPONSOR:
3M Toxicology Services 3M Center, Building 220-2E-02
St. Paul, Minnesota 55144-1000
006061
Protocol
418-008
Page 2
STUDYMONITOR:
Marvin T. Case, D.V.M., Ph.D.
Telephone: (612) 733-5180
Telefax:
(612) 733-1773
ALTERNATE
STUDY MONITOR:
Andrew M. Seacat, Ph.D.
Telephone: (612) 575-3161
Telefax:
(612) 733-1773
REGULATORY CITATIONS:
SInttuedrynaDteiosniaglnCaosnfMeordeifnicceatoinonHaofr:moUn.iSs.atFiooond; aGuniddeDlriunge Aodnmidneitsetcrtaitoinonof(t1o9x9i4c)i.ty to reproduction for medicinal products. Federal Register, September 22, 1994, Vol. 59, No. 183.
U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule.
21 CFR Part 58.
Japanese for Safety
Ministry Studies
of on
Health Drugs,
aMnHdWWelOfradriena(n1c9e97N).umGboeord21L,abMoarractohr2y6P,ra1c9t9i7c.e
Standard
EacucreoppteaannceEcboyntohmeiEcuCroompmeuanniEtcyo(n1o9m8i9)c.CoComumnucniiltdyecoifsaionnOoEnC2D8 Jduelciys1io9n8/9reocnortmh-e
mendation on the European
Ccoommmpulniiatniceesw:ithLepgriisnlcaitpiloens.of32go(Nood.
laboratory L 315; 28
practice. October):
Official 1-17.
Journal
of
REGULATORY COMPLIANCE:
This study will be conducted in compliance with the Good Laboratory Practice (GLP) regulations cited above.
All changes Director and
or revisions of this protocol shall be the Sponsor, dated and maintained
documented, signed with the protocol.
by
the
Study
Tahnde wQiulalliintsypAesctsucrriatinccalepUhnaitse(sQAoUf)thweilsltauuddyitinthaeccproortdoacnolc,etwhiethrtahwedSattaanadnadrdthOeperreapotritn,g
Procedures of Argus Research Laboratories, Inc.
Tachceurfaintaellyrerpeofrltecwtislltihneclruadwe daatsataotbetmaeinntedsidgunreidngbtyhtehpeeSrtfuodrymaDnicreecotfotrhtehatsttuhdeyraenpodrtthat
all applicable GLP regulations were followed in the conductofthe study. Should
significant deviations from GLP together with how the deviation
regulations occur, each will be described might affect the quality or integrityofthe
in detail, study.
000662
.
Protocol 41P8a-g0e038
STUDY SCHEDULE: See ATTACHMENT 1 to the protocol. TEST ARTICLE AND VEHICLE:
Identification:
TestArticle:
NaPhmyesi:cal
Description:
~~
PFOS. Light-Colored
powder.
SLpoetcBiafitcchGrNauvimtbye:r:
217. ~06.
Pury:
98.9%.
Expiration Date:
May, 2000.
Information on the identity, composition, strength and purity of the test article is on file with the Sponsor.
Vehicle:
0De.i5o%niTzweedeWnat8e0r)i.n RSeuvpeplriseerdaOnsdmloostiisdeMnteifmibcartaionneofPrTowceeesnse8d0DteoiboneidzoedcuWmaetnetre(dR.i0n.the raw data.
tNoeibteheprretsheenStpionntshoervneohrictlheetShattudwyouDlidreicnttoerrfiesraewwairteh tohfearneysuplottsenotfiathliscosnttuadym.inaThnetrsefliokreel,y no analyses other than those mentioned in this protocol will be conducted.
SafetyPrecautions:
fGolromvuelsa,timoanskpr,epaaprpartoiporniaatnedeydeospargoteecatdimoinniasntrdataiounn.ifTohremiMaabtecroiaatl aSraefetotybeDawtoa mShdeuerting (MSDS) is attached to the protocol (ATTACHMENT 2).
Storage:
VBeuhlikcTleesCtoAmrtpiocnlee:nts: RRoooomm tteemmppeerraattuurree..
Prepared Vehicle:
Room temperature.
Prepared Formulations: ~ Frozen (-20C).
AJlullitaenstGauritbiicnlseksi,hiMpamnenatgsertootfhFeoTremsutliantgioFnasc,ilaittytshheopurledviboeusaldydcrietsedseadddtroetshse
attention and
of
telephone number.
00CC63
Protocol 41P8a.g0e084
Shipments should cartons should be
include labeled
aipnpfroorpmraitaitoenlyc.onTcehrenirencgipsiteonrtagsehocuolnddibteionnostiafinedd
shipping in advance
of
shipment.
FORMULATION:
Frequency of Preparation: Formulations (suspensions) wil be prepared daily at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3).
Adjustment for Purity:
The test article will be considered 100% pure for the purpose of dosage calculations.
Te
cility Re
amples:
The Sponsor the course of
will reserve this study.
a sample (1 The Testing
g) ofeach Facility will
lot of the bulk test reserve a sample
article (5 mL)
used during of each lot of
the vehicle components used during the course of this study. Samples will be stored
under the previously cited conditions.
ANALYSES: Samples additional to those described below may be taken if deemed necessary during the course of the study.
Bulk Test Article Sampling:
No analyses of the Information on the
bulk test article will be stabilityofthe bulk test
conducted during the course of this article is on file with the Sponsor.
study.
nalyses of Pr
lation:
Stability:
Stability data for prepared formulations bracketing the range of concentrations and tcohnedciotinodnusctooffthtihsisstsutduydya.reSounspfeilneswiiotnhsthwiellSbpeonpsroerpaarneddwdilalilnyoattbtehedeTteesrtimnigneFdacdiulrtiyn.g
CCeesa
Protocol 41P8a-g0e0s8
HomogeneityAnalyses:
Homogeneity course of this
of the study.
test articie A syringe
in prepared will be used
suspensions to withdraw
will be verified samples (5 mL
during each)
the from
the
tEoapc,hmisdadmlpelaen(d5 bmoLt)towmilobfethdeivhiidgehdesitntcootnwceonatlriaqtuiootns,oonnteheoffi2rsmt dLaaynodf oprneepaorfat3imoLn..
OatntehealTieqsutoitn(g2 FmaLc)ilwtiyllabseasbhaicpkpeudp fsoarmapnlael.ysiBsa;ctkhueposthaemrplaleisquwoitll(b3emsLt)orwieldl ubnedreertatihneed
previously cited conditions and discarded at the Testing Facility upon the requestof the
Sponsor.
ConcentrationAnalyses:
oCfotnhciesntsrtaudtyi.onAofsytrhienpgreewpiallrebde tuessetdarttoicwlietshudsrpaewnssiaomnpslewsil(5bemvLereiafciehd)dfurroimngetahcehcourse
c(5onmceLnteraacthi)onwildlurbiengditvhiedefidrsitnatondtwsoixatlhiqwueotesk,
of dosage administration. one of 2 mL and one of3
Each sample mL. One aliquot
(T2esmtLi)ngwiFlalcibleitsyhaisppaebdafcokruapnaslyasmipsl;e.theBaotchkeurpalsiaqmupotle(s3 mwiLl)bweilstboererdetuanidneedr atthethe
previously cited conditions and discarded at the Testing Facility upon the request of the
Sponsor.
ShippingInstructions:
Samples to be analyzed will be shipped (frozen on dry ice) to: Kris J. Hansen, Ph.D. 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-3-09 St. Paul, Minnesota_55133-3331 Telephone: (612) 778-6018 Telefax: (612) 778-6176
Both the recipient and the Study Monitor will be notified in advanceof sample shipment.
DISPOSITION:
Prepared article will
formulations be returned
will be discarded at the to the Study Monitor at
Testing Faciity. All the previously cited
remaining address.
bulk
test
020065
Protocol 418-008 Pages
TEST SYSTEM:
cies! a
n for Selection:
The Cr:CDBR VAF/Plus (Sprague-Dawley) rat was selected as the Test System baencdaduesvee:lo1p)mtehnitsasltrtaoixnionfs raantdhahsasbebeenendewmiodnelsytruasteedd tthorboeugsheonustitiinvdeusttorryefporroductive rexeipsrtodautctthieveTeasntdindgeFvaecliolpmieynt;aalntdox3i)cittyheevtaelstuaatritoinclse; i2s)phhiasrtmoraiccoallodgaitcaalalnydacetxipveeriinetnhcee species and strain.
Number:
Initial population acclimated: 195 virgin male and 205 virgin female rats. Population selected for study: 175 male rats (35 per dosage group) and 175 female
rats (35per dosage group). Ten mated female rats will be assigned to Caesarean-sectioning on day 10 of presumed gestation; the remaining female rats will be permitted to deliver litters. 250 F1 generation pups (25 per sex per dosage group) will be selected at weaning on day 21 postpartum for continued postnatal observation.
BW odyeiganhdAt ge:
Male rats will be ordered to weigh from 300 g to 325 g each at receipt, at which time. they will be expected to be at least 60 days of age. Female rats will be ordered to weigh from 200 g to 225 g each at receipt, at which time they will be expected to be at least 60 days of age. Actual body weights will be recorded the day after receipt and will be documented in the raw data. The weight ranges will be included in the final report.
Sex:
Both Fo and F1 generation male and female rats will be evaluated. Only Fo generation male and female rats wil be given the test article.
Source:
Charles River Laboratories, Inc., Raleigh, North Carolina. The rats will be shipped in filtered cartons by air freight and/or truck from Charles River Laboratories, Inc. to the Testing Facility.
CCCose
Protocol 41P8a-g0e0?8
Identification:
EGoeneration:
Rats are permanently identified using Monel self-piercing ear tags (Gey Band and Tag
Croe.c,eiIpntc.a,nNdog.iMveSnPuTni2q0u1e01p)e.rmMaanleentaniddenfteimfaiclaetiroantsnuarmebearsssiwghneedn taesmspiogrnaerdytnoutmhbeesrtsudayt
before administration of the first dosage of the test article.
F1/F2 Generations:
Pups will not be individually identified during lactation; all parameters will be evaluated iindetnetirfmiseodfwtihthealiMttoern. eAltwseeafnfi-pnige,rceiangchearrattasge.lected for continued observation will be
ANIMAL HUSBANDRY:
All cage sizes and housing conditions are in compliance with the Guide for the Care `and Use of Laboratory Animals*.
Housing:
Fo Generation Rats/F1 Generation Litters:
Feofxocrgeaetpsntewdriulalrtiibnoegnhtrohaeutsscewoidhlalibnbiettahitenidominaviladenuadrlaltpy'osshctoapugasere.tdumBienpgseitrnainioindlnseg.ssnDosutrleiaetnlegrwtichorahena-bbidotatatytoi2omn0e,doefcaacghespair pInrdeivsiudmuaeldlygehsotuastieodn,inFnoesgteinnegrabtoixoens.femEaalcehrdatasmasasnidgndeelditvoerneadtulritatlerdweillilvbeeryhwoilulsbeed in a
`common nesting box during the postpartum period.
Ahfotuesrewdeainnipnagi,rst(hoenFe1mgaelneerraattipoenrrfaetmsawlilel rbate)idnudriivindguaclolhyabhiotuatsieodn,beafnodreincdoihvaibdiutalaltyion,
hFoougseenderaafttieroncorhaatbsi.taBteigoni.nniTnhgensoalmateertytpheanofdacayg2in0gofwipllrbeesuumseedd
as described gestation.
for
the
F1 generation female rats will be individually housed in nesting boxes. Each dam and
deliveredlitterwill be housed in a common nesting box during the postpartum period.
oCeee?
'
Protocol 418-008 Pages
NestingMaterial: Bedding material (bed-o'cobs) will be supplied to female rats assigned to natural delivery.
ABneadldyisnegswiflolrbpeoscshibalnegecodntaasmoifntaetnioans anreececsosnadruyctteodkeaennputahlelyanainmdaldsocdurymeanntdecdleinant.he raw data.
RT ooe mAimr,peraat ndu Humr idie ty:
The animal room fresh air that has
is independently supplied with been passed through 99.97%
at least ten HEPA filters
changes per hour of (Airo Clean room).
100%
cRoonsotmanttelym.perRaotoumrehwuimllidbietymawiilnltaalisnoedbeatm6on4itFor(e1d8cCo)nsttoa7nt9lyFa(n2d6mCa)inatnadinmeodniatto3r0ed% to
70%.
Light:
Amaninatuationmeatdi.caElalychcodnatrroklpleedri1o2d-whiollurbelgiignhta:t121-8h0ou0rhdoaurrksfEluSoTr.escent light cycle will be
Diet:
Rats will be given Certified Rodent Diet #5002 (PMI Nutrition Intemational) available ad libitum from individual feeders.
Water:
Waantaeurtowimlaltbiec wavaatielraibnlge aacdcelisbsitsuymsftreomm. iAnldlivwiadtuaelr wbioltltlbees fartotmacaheldoctaol tshoeurccaegeasndorpfarsosmed
through a reverse osmosis membrane before use. Chlorine will be added to the
processed water as a than 1.2 ppm chlorine
bacteriostat; processed at the time of analysis.
water Water
is is
expected analyzed
to contain no more monthly for possible
bacterial contamination and twice annually for possible chemical contamination.
Contaminants:
tNoeibteheprretsheenStpionntshoercnerotriftiheed
Study Director is diet, the drinking
aware water
of or
tahneynpeostteinntgiamlatceornitaalmiatnalnetveslslitkhealyt
`would interfere with routinely performed
bthyetrheesufletesdosfutphpilsisetruodry.thTohseeremfeonrtei,onnoedaninalthyissepsrootthoecroltwhialnl
those be
conducted.
CCess
Protocol 4P18a-g0e0s8
RANDOMIZATION AND COHABITATION:
EGoeneration:
Upon arrival, rats will be generated random units.
aAsfstiegrnaecdcltiomiantdiiovni,dumaallheouasnidngfeomnalteherabtasswiisllofbecosmepluetcteerd-
for
study on the basis of physical appearance and body weights recorded during
acclimation. The rats will be assigned to dosage groups based on computer-generated
(weight-ordered) randomization procedures.
Within each dosage group, cohabitation, one male rat
consecutive order will be used to assign rats to per female rat. The cohabitation period will consist
of
a
mvaagxiniamlucmonotfe1nt4sdaanyds./orFeamcaolpeulraattosrwyipthlusgpoebrsmeartvoezdoainsoibtsuewrivledbein
a smear of considered
the to be
at
dmaayte0dowfitphrienstuhmeefdirsgtes7tdataiyosnofancdohaasbsiitgatnieodntwoiilndbieviadsusalighnoeudsianlgt.emFaetmeamlaelerartastnsotthat
have mated (same dosage group) and will remain in cohabitation for a maximum of
seven additional days.
Tashseigfinrsetdtteon Cfaeemsaalreearnat-ssepcetridoonsinaggeongrdoauyp1w0itohfaprcoensfuimremdedgedsattaetioofn.matTihneg rweilmlaibneing female rats will be permitted to naturally deliver litters.
Astaambpllee ocfolrlaecntdioonmautnsitcshewidlul lbeedussaecrdiftioceasasfitegrncfoimveplreattsiopnerofgtrhoeupcothoaabipthatairomnacpoekriinodetic (male rats siring litters with dams allowed to naturally deliver a litter) or on day 21 postpartum (female rats allowed to naturally deliver fitters).
FAIF2 Generation Pups:
Day 1 of lactation (postpartum) is defined as the dayofbirth and is also the first day on
awfhtiecrhalalllppuupspsininaaliltittetreraraeredeilnidviveirdeudalalnydwegirgohoemded(pbuyp
body weights the dam).
will
be
recorded
On day 4 and litters
postpartum, a table of random units will be reduced to eight pups each.
will be used to select Whenever possible,
pups to be culled, the same number
of
male and female pups per litter will be continued on study.
AwitllwbeeanuisnegdotfotsheeleFc1t g2e5nemraalteioanndpu2p5sfoenmadlaeyp2u1pspopsetrpagrrtouump,, aretsaubllteinogfirnaantdootmal uonfit2s50 F1 generation rats (125 per sex) chosen for continued evaluation. At least one male pup and one female pup per litter, when possible, will be selected.
0CC069
Protocol P4a18g.e00180
I ON:
RR outee andasfoo rChon ice:
Tdiheetaorryalro(ugtea,vatghee)erxoaucttedwoassagseelceacntedbefoarccuusreatbeelcyauadsmei:ni1s)teirnecdo;mapnadri2s)onitiwsitohntehoef the possible routes of human exposure. Method and Frequency: Dosages will be adjusted for the most recently recorded body weight and given at approximately the same time each day. Eo Generation Male Rats M(amlaexiramtusmwi1ll4bdeaygsi)veanndthecotnetsitnauritnigcltehornocueghdtaihleydbaeygibnenfionrge 2s8acrdiafyicse.befMoarlee croahtasbwiitllatbieon sacrificed after completion of the cohabitation period.
Fo Generation Female Rats:
Female rats will be given the test article once daily beginning 28 days before cohabitation (maximum of 14 days) and continuing through day 9 of presumed
gestation (rats assigned to Caesarean-sectioning), day 24 of presumed gestation (rats
assigned to natural delivery that do not deliver a litter) or day 20 postpartum (rats that deliver a litter).
EG1eneration:
F1 generation pups will not be directly given the test article, but may be possibly
exposed to the test article during maternal gestation (in utero exposure) or via matemal
.
`milk during the lactation period.
Dosages will with the test
be selected article.
by
the
Sponsor
on
the
basis
of
previous
studies
conducted
5
id
060070
'
Protocol P41a8g-e00181
DC osaogen Levcele s, ntratai ndVooln umess:
I
gay|concantratmopnm) |V(omliugm)e
_|
Tos Jovan] |&[ocoommommomnen]
Eo[os [ooToms[onemoonmnren]
Co[os |oo[ow |&[ouscoscommemm]
[ov[os [ wo | ow | 5 eswasoommomnren]
Tos TseToe Ts[ouwomconmennven]
"Th test ricevil beconidred 100% pur forthe purposeofdosagecalciaors.
RY
TS - Fo GE
:
Viability -
ale Rats:
All Periods:
Atleast twice daily.
Clinical Observations and/or General Appearance - Male and Female Rats:
Acclimation Period:
Atleast once.
Dosage Period:
Twice daily. Prior to dosage administration and once approximately one hour postdosage.
Maternal Behavior:
Days 1,4, 7, 14 and 21 postpartum. Any observed abnormal behavior will be recorded daily.
Clinical observations may be recorded more frequently than cited above,if deemed appropriate by the Study Director and/or Study Monitor.
Body Weights - Male Rats:
Acclimation Period:
Atleast once.
Dosage Period:
Weekly.
Sacrifice:
Terminal weight.
00CCTL
Protocol4P1a8g-e00182
Body Weights - Femal Acclimation Period: Dosage Period:
Sacrifice: d Cor ion Values
Dosage Period:
Atleast once. Weekly to cohabitation. Daily during presumed gestation and on Days 1, 4, 7 and 14 postpartum (rats assigned to natural delivery). Terminal weight. -Mal (recorded and tabulated): Weekly.
FC eedonsum Valp uest -Fei malo eRan ts (recorded and tabulated):
Dosage Period:
Weekly to cohabitation. Daily during presumed gestation. Days 1, 4, 7 and 14 postpartum (rats. assigned to natural delivery).
Feed consumption not tabulated after day 14 postpartum, when it is expected that pups will begin to consume maternal feed.
Feed Consumption Values - Male and Female Rats:
Feed consumption values may be recorded more frequently than cited above if it is necessary to replenish the feed. During cohabitation, when two rats occupy the same cage with one feed jar, replenishment of the feed jars will be documented. Individual values will not be recorded or tabulated.
Estrous Cycling and Mating:
A table of random units will be used to select 15 female rats per group for evaluation of estrous cycling by examination of vaginal cytology for 14 days before the start of the cohabitation period.
During cohabitation, all female rats will be evaluated daily until spermatozoa are observed in a smearof the vaginal contents and/or a copulatory plug is observed in situ.
Dura oft Gesitao tionn:
The durationofgestation is calculated from day 0 of presumed gestation to the day the first pup i$ observed.
0CO07R
Protocol 4P1a8g-e01038
FertilityParameters:
Fertility Index (percentage of matings that result in pregnancies).
Gestation Index (percentage of pregnancies that result in birth of live liters).
Number of offspring per litter (live and dead pups).
Number of implantation sites.
General conditionof dam andliter during the postpartum period. Viability Indices (percentage of pups born that survive 4 and 7 days). Lactation Index (percentageof pups born that survive 21 days).
Caesarean-SectioningObservations:
RaaptpseawrillabbneoCrameasla(rseiazne,-sceoclotrioonresdhaopned)awyill10beofnoptreedsuinmetdhegresatwatdiaotna..
Placenta that The rats will be
examined for number and distribution of:
Corpora Lutea.
Implantation Sites.
V(iAavbilaeblaendemNbornyvoiaibsloevaElmborrycorse.scent amniotic sac filled with clear fluid. A
shaped, pink, fin nonviable embryo
aisndameonrcplhoosuesd,insmaanll,
pale
pink to tanordeep red to black, soft and enclosed in an amniotic sac filled with
clear, cloudy, or opaque fluid.)
Natural Delivery:
Female rats will be evaluated for:
Clinical Observations During Parturition.
Dobusreartvieodn)o.f Gestation (day 0 of presumed gestation to the time the first pup is
fLiersntgptuhpofdiPvairdteudribtiyonN-(1tipmueposf
delivery in each
of last liter)
pup
minus
the
time
of
delivery
of
the
Litter Size (defined as all pups delivered).
'
0CCe73
Protocol 4P1a8g.e00184
Pup Viability at Birth.
+
D Of \CRIFICE -
NERATION:
Rats will be sacrificed by carbon dioxide asphyxiation. Embryos will be discardedafter `examination.
NECROPSY - Fo GENERATION:
Gross lesions will be retained in neutral buffered 10% formalin for possible future
esveaxlufartoimonwh(iacthaballletoifssruaensdeoxmamuniintesdwiallt bneecursoepdsytowisllelbeectroetnaeinceodn,trionlogrrdeoruptoraptroovfiedaech control tissues for any possible histopathological evaluations of gross lesions). Unless
specifically cited below, all other tissues will bediscarded.
Male and Female Rats Assigned to Pharmacokinetic Sample Collection:
Awittshcdhaedmusleadllsoawcerdiftiocenaafttuerralcloymdpelleitvieorna
of the liter)
aconhdabointadtaiyon21perpioosdtp(amratluemra(tfsemsairliengraltitsters.
allowed to naturally delivera litter), five rats per group will be assigned to a
pbehlaorwm,acbolkoiondetsiacmpslaemspl(eapcporlolexcitmiaotne.lyIn4amddLitpieonr troatt)hweilalpbperocporlilaetceteedvfalruoamtithoensindfeesricorribed
vena cava into (approximately
serum 2 mL)
separator tubes and centrifuged. will be immediately frozen on dry
The resulting serum ice and maintained frozen
a(n7d0aC)saumntpilleshsiecptmieonnt(tloattehraelSlpooben)swoirllfobreafnraolzyesinsa.nTdhreetlaiivenredwilaltb-e70exCciusnetdi,l swheiipgmheendt, to
the Sponsor for analysis.
Abfetesrhicopmppeldet(firoonzeonf osnamdprlyeicceo)llteoctKiroins, Js. eHraunmseann,d Plhiv.eDr.,seacttitohne (plraetveriaoluslloybec)itseadmapdldersewsisll for analysis. Both the recipient and the Study Monitor will be notified in advance of
sample shipment.
Scheduled Sacrifice of Male Rats:
Anfetcerrocposmyploefttihoentohfortahceicc,ohaabbdiotmaitnioanl paenrdiopde,lvmiaclveisractesrawiwlilllbebesapcerriffoircemdeda.ndTahegrfooslslowing organs will be excised and weighed and retained for possible histologic evaluation:
testes, epididymides, prostate and seminal vesicles (weighed with and without fluid). The testes will be fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10% formalin for possible histopathological evaluation. The remaining organs will be retained in neutral buffered 10% formalin.
C0074
Protocol 4P1a8g-e00185
heduled Sacrifice - Female Rats Assi
Caesarean-Sectionin
Oannddaaygr1o0ssofnpecrreospusmyedofgtehsetatthioorna,cifce,maabldeormaitnsawlilal nbde psealcvriifcivceids,ceCraaewsiallrebaen-pseercfotrimoende.d, Uctoenrfiiromf atphpeaarbesntelnycenoonfpirmepglnaannttatriaotnsswiiltlebse. sUttaeirniedofwniothnp1r0eg%naanmtmorantisuamndsuallfliodveatroies will be retained in neutral buffered 10% formalin for possible future evaluation.
Scheduled Sacrifice - Female Rats Assigned to Natural Delivery:
eRxatasmitnhaetddfoorngortodseslilveesrioansl.itteUrtweirlil wbiel sbaecrsitfaiicendedonwitdhay1205%oafmpmroensuimuemdsuglefsitdaetitooncaonnfdirm the absence of implantation sites.
A`gfrtoesrscnoemcprloeptisoynofoftthheeth2o1r-adciacy,paobsdtopmairntaulm
period, female rats will be sacrificed, and pelvic viscera wil be performed.
and a The
number and distribution of implantation sites will be recorded
Dams with No Surviving Pups:
Dams with no surviving pups will be or presumed cannibalized. A gross
sacrificed necropsy
after the last pup is found of the thoracic, abdominal
dead, missing and pelvic
viscera will be performed. Postpartum data for these dams will be excluded from
summary tables.
Rats Found Deador Moribund:
Rats that die examined for
otrhearceasuasceriofficdeedatbhecoarumsoeroifbumnodricboundnidticoonndointitohneodraaybotrhteioonbsweilrlvabteion
is
made. The rats seminal vesicles
will of
be examined male rats will
bfeoregxrcoisssedleasniodnsi.ndiTveisdtueasl,orepgiadniwdyemiigdhetss,wiplrlobsteate
and
rBeocuionr'dsedso(lsuetmioinnaflorve4s8ictloe9s6wehiougrhsedanwditthhaenndrewtiatihnoeudt filnunide)u.trTalhebutfefsetreesdwi1ll0b%efofrimxaeldini.n
Tsthaeturseamnaidniutnegrionregacnonstweinlltsbeofrfeetamianleed riantsnewuiltlrablebruefcfoerrdeedd.10A%bofrortmeadlifn.etuPsresegannadn/cory
delivered pups will be neutral buffered 10%
examined to the formalin. Uteri of
extent possible. Ovaries apparently nonpregnant
will rats
be retained in will be stained
with
10% ammonium sulfide to confirm the absence of implantation sites.
CCOC7S
Protocol 4P1a8g-e00186
TM ESTSE ,ANAA LYSS ESAU ND REME -F1N GENET RATS ION:
Viability:
Preweaning Period:
Litters will be observed for dead pups at least twice ddaailyy.. The pups in each litter will be counted once
Postweaning Period:
Twice daily.
ClO inicbal serv and/aorGt enei ralAo ppen arans ce:
Preweaning Period:
Once daily.
Postweaning Period:
Once weekly.
Matemal Behavior:
Days 1,4, 7, 14 and 21 postpartum. Any observed
abnormal behavior will be recorded daily.
aClpipnriocpalrioabtseebrvyatthieonSstumdayyDbireecrteocroarndde/domrotrhee fSrteuqduyenMtolnyittohra.n cited above,if deemed
Body Weights:
Preweaning Period:
Days 1 (birth), 4, 7, 14 and 21 postpartum.
Postweaning Period:
Weekly.
Presumed Gestation Period: Days 0, 7, 10, 14, 17 and 20 (female rats only).
Lactation Period:
Days 1, 4,7, 10 and 14 (female rats only).
Sacrifice:
Terminal weight.
Feed Consumption Values (recorded and tabulated):
Preweaning Period:
Postweaning Period: Presumed Gestation Period: Lactation Period:
Not recorded.
Weekly except during cohabitation. Days 0, 7, 10, 14, 17 and 20 (female rats only) Days 1, 4,7, 10 and 14 (female rats only).
0CC76
Protocol 4P1a8g-e00187
Freepeldencioshnstuhmepfteieodn.
values During
mcaohyabbietarteicoon,rdwehdemnortewofrreaqtuseonctclyuipfyittihse
necessary to same cage with
one
feed jar, values will be documented when feed jars are filled. These intervals will not be
tabulated.
PreweaningDevelopmentalObservations:
Tcohnetinnuumebseurntoilftphuepsdamyeethteincgritthereiocnriitseraitotnaiisnerdecboyradleldpuopnseaincthhedalyittoerf.testing. Testing
Surface Righting Reflex (ability to right in 5 seconds): From day 1 postpartum.
Pinna Unfolding: From day 2 postpartum.
Eye Opening: From day 12 postpartum.
Acoustic Startle Response: From day 13 postpartum.
Air Righting Reflex: From day 14 postpartum.
Pupil constriction is evaluated once, on day 21 postpartum.
Postweaning Developmental Observations:
`Sexual Maturation: Fpoesmtaplaertruamt.s wiMlallbeereavtsalwuilaltebde feovratlhueataegdefoorf tvhaegiangale poaftpernecpyu,tibaelgsienpnairnagtioonn,dbaeyg2in8ning on day 39 postpartum.
Passive AvoidanceTesting:
Beginning at 24 where possible,
+1 will
day postpartum, one male be evaluated in a passive
rat and one female rat from each liter, avoidance test for leaming, short-term
retention and long-term retention.
PTlheexipgalsassiveliadsv.oiOdnaencceoampppaarrtamteunstciosnsfiitsttesdowfitah tawbor-icghotmpliagrhttmaenndtPclheaximgblearswiftlhoorh.inTgheed ocutrhreerncto(m1pmaArt)mceannt bsefdiettleidvweirtehd.a Tgrhied tfwlooorctoomwphaircthmaenbtrsieafr(e1sseepca)raptulesdeboyfmaisliddienlegctric disooorp.enOend eaancdhttheestlitgrhiatl,istthuerrnaetdisonp.laTchede irnattoitshaell"borwiegdhtt"oceoxmpplaorrtemtehnet,aptphaerasltiudsinugntdioloirt etnutmeerds tofhfea"nddartkh"ecobrmipeafrptumlesneto,f cTuhreresnltidiisndgeldiovoerreisdtthoetnheimgmreiddifalotoerl.yTchloeserdat, itshtehleinght is removed from the apparatus and placed into a holding cage for 30 seconds before the startof the next tral. Trials are repeated until the rat remains in the "bright"
Cee??
Protocol 4P1a8g-e00188
compartment 15 trials have
for 60 been
seconds on completed.
two The
consecutive trials (the criterion for learning) or latency to enter the dark compartment or the
until
- maximum 60-second interval is recorded for each trial
Each rat is tested twice. The test sessions are separated by a one-week interval, and the criterion is the same for both days of testing.
Dosage groups are compared for the following dependent measures:
The number of trials to the criterion in the first session~this measure wil be used 10 compare groups for overall leaning performance.
The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the first test session--this measure wil be used to `compare groups for activity levels and exploratory tendencies in a novel environment.
The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 2 in the first test session--this measure will be used to compare groups for short-term retention.
`The number of trials to the criterion in the second test session~--this measure will be used to compare groups for long-term retention.
`The latency (in seconds) to enter the "dark" compartment from the "bright" compartment on trial 1 in the second session---this value is another indication of long-term retention.
WatermazeTesting:
Beginning at approximately 70 days postpartum, one male rat and one female rat from each litter will be evaluated in a water-filled M-maze for overt coordination, swimming ability, leaming and memory. Each rat is tested in a watertight 16-gauge stainless steel modified M-maze. The maze is filed with water to a depth of approximately nine inches, and the water is monitored for temperature (range of 21C 1C). On each test tial, the rat will be placed into the starting position (base of the M-maze stem farthest from the two ams) and required to swim to oneofthe two goalsofthe M-maze, in order to be removed from the water. On the first tral, the rat is required to enter both arms of the maze before being removed from the water. The initial arm chosen on trial 1 is designated the incorrect goal during the remaining trials. Rats that fail to makea correct goal choice within 60 seconds in any given trial are guided to the correct goal and are then removed from the water. A 15-second intertrial interval will separate each trial. Each rat is required to reach a criterion of five consecutive erroriess trials to terminate the test session. The maximum number of trials in any test
Cee?s
Protocol 4P1a8g-e00189
session is 15. Latency (measured in seconds) to choose the correct goal or the maximum 60-second interval is recorded for each trial, as is the number of errors. (incorrect turns in the maze) during eachtrial
Each rat is tested twice. The test sessions are separated by a one-week interval, and the correct goal and the criterion are the same for both test sessions.
Dosage groups are compared for the following dependent measures:
"The number of trials to criterion on the first day of testing--this measure will be
used to compare groups for overall leaming performance.
The average number of errors (incorrect turns in the maze) for each trial on the
first day of testing--this measure will also be used to compare groups for overall
learning performance.
Ttehsetilnagt--etnhciys m(ienasseucroendwsil)l tboeruesacehd ttoheccoomrpraecrteggoraoluopns tfroirals2hoorft-ttheerfmirrstetdeantyioonf.
The number of trials be used to compare
to criterion groups for
on the second day long-term retention.
of
testing~this
measure
will
Tmehaesauvreerawigle anlusmobbeeruosfeedrrtoorscofomrpeaarcehgtrrioaulposnftohrelosnegc-otnedrmdraeyteonftitoens.ting--this The latency (in seconds) to reach the correct goal on trial 1of day 2 of
testing--this is another indicator of long-term retention.
Reproductive Capacity:
Abteaapspsriogxniemdatteolcyoh8a0bidtaaytsioonf, aognee, mtaheleF1ratgepneerrafteimoanlerartast,wbitahsiendeaocnhcdomopsuatgeer-ggreonueprwaitlled rcoahnadboitmatuinoitnspoefriroadnwdiolmcounnististtabolfesa, mwaitxhitmhuemexocfl1us4idoanyosf. siFbelimnaglmeatriatnsgsw.ithThe sobpseerrmvaetdozionasiotbuswielrlvbeed cionnasisdmeeraerd otfo tbhee avtagdianyal0 coofntpernetssuamnedd/ogresatactoipounlaatnodryaspsluiggned
to individual housing. Female rats that do not mate within the first 7 days of
cohabitation will be assigned alternate male rats from the same dosage group that have mated. Female rats will be allowed to naturally deliver and maintain litters through a
21-day postpartum period.
MatingPerformance: As cited above for Fo generation rats.
Cee?
Duration of Gestation: As cited above for Fo generation rats. Fertility Parameters: As cited above for Fo generation rats.
Protocol 4P1a8g-e00280
Viability, clinical observations and body weights for F2 generation pups will be recorded as cited above for F1 generation litters. METHOD OF SACRIFICE - F1 GENERATION RATS/F2 GENERATION PUPS:
As previously cited for Fo generation rats.
NECROPSY- F1 GENERATION RATS:
Gross lesions will be evaluation (a table of
retained random
in neutral buffered 10% formalin for possible units will be used to select one control group
future rat of
each
scoenxtrforlotmiwsshuiecshfaolrl atinsysupeossseixbalemihniestdopatatnheoclorgoipcsayl weivllalbueatrieotnasinoefd,grionsosrdleesriotnosp).rovUindleess
specifically cited below, all other tissues will be discarded.
Scheduled Sacrifice - F1 Generation Male Rats:
Rats will be necropsyof
stahceritfhiocreadciacf,tearbcdoommpilneatlioanndofptehlevi1c4v-idsaceyraclohwaibllitbaetipoenrfpeorrimoedd..
A gross Testes
and
Tephiediedpyimdiiddeysmiodfemsawlilel rbaetsrweitlalibneedeixncinseuetdraalndbuifnfdeirveiddu1al0%orfgoarnmawleiing.htTshweiltlebsteesrewciollrdbeed.
fixed in Bouin's solution for 48 to 96 hours and then retained in neutral buffered 10%
formalin.
Scheduled Sacrifice - F1 Generation Female Rats:
Female number
rats and
will be sacrificed after completion of distribution of implantation sites will
the 21-day postpartum be recorded. Rats that
period. do not
The deliver
a
liter 10%
will be sacrificed on day 25 of ammonium sulfide to confirm
presumed gestation and uteri will be stained the absence of implantation sites". A gross
with
necropsy without a
of the thoracic, abdominal and confirmed mating date that do
pelvic viscera wil not deliver a litter
be will
performed. Female be sacrificed on an
rats
estimated day 25 of presumed gestation.
06COS0
:
Protocol P41a8g-e00218
al
tion Rats Found Dead or Moribund:
Rats that die examined for
otrhearceasuasceriofficdeedabthecoarumsoeriofbumnodricbounndidticoonndointitohneodraaybotrhteioonbsweilrlvabteion
is
made. The rats will seminal vesicles of
be examined male rats will
bfeoregxrcoisssedleasinodnsi.ndiTveisdtueasl,oerpgiadnidwyemiigdhetss,wiplrlobsteate
and
Broeucionr'dsedso(lsuetmioinnaflorve4s8ictloe9s6wehiougrhsedanwidtthhaenndrewtiatihnoeudt ifnluinde)u.trTahlebutfefsetreesdwi1ll0%befofrimxaeldiinn.
Tsthaeturseamanidniuntegroinregaconnstweinltsbeofrfeetamianleed riantsnewuiltlrablebruefcfoerrdeedd.10A%bofrortmeadlifn.etuPsresegannadn/cory
dneeluitrvaelrebdufpfueprsedwi1ll0b%efeoxrmaamliinn.edUtteortihoefeaxptpeanrtepnotslsyibnloen.prOevganrainets
will rats
be retained in will be stained
with
10% ammonium sulfide to confirm the absence of implantation sites.
F1 Generation Dams with No Surviving Pups:
Dams with no surviving pups will be sacrificed after the last pup is found dead, missing or presumed cannibalized. A gross necropsy of the thoracic, abdominal and pelvic: viscera will be performed. Postpartum data for these dams will be excluded from summary tables.
FA/F2 Generation Pups Found Dead on Day 1 Postpartum:
Pups that die before examinationofthe litter for pup viability will be evaluated for vital status at birth. The lungs will be removed and immersed in water. Pups with lungs that sink wil be identified as stillborn; pups with lungs that float will be identified as liveborn, and to have died shortly after birth. Pups with gross lesions will be preserved in Bouin's Solution for possible future evaluation. Should postmortem autolysis preclude these evaluations, it will be noted in the necropsy data.
FA/F2 Generation Pups Found Dead or Moribund on Days 2 to 21 Postpartum:
Pups found dead or sacrificed due to moribund condition will be examined for gross. lesions and for the cause of the moribund condition or death. Pups with gross lesions found on days 2 to 4 postpartum will be preserved in Bouin's solution for possible future evaluation; gross lesions of pups found on days 5 to 21 postpartum will be preserved in neutral buffered 10% formalin. Should postmortem autolysis preclude these evaluations it will be noted in the necropsy data.
[LIT
Protocol 4P1a8g-e00282
i
n
lg
r Cont
rvation:
F1 for
and F2 generation pups gross lesions; pups with
culled gross
on day lesions
4 postpartum will will be preserved
be sacrificed and examined in Bouin's solution
fNreocntraolp-spayriweitllalinscultuudreeaansdineglxeamcirnoasts-isoencotfiotnheofctrhoessh-esaecdtaitontehdeblreavieln ofofrtahpeparent
hydrocephaly.
All F1 gross
lgeesnieornas;tigornospsuplsesciuolnlsedwilolnbdeapyr2e1seprovsetdpianrnteuumtrwialll
be sacrificed buffered 10%
and examined formalin.
for
fNreocntraolp-spayriweiltlalinscultuudree aansdineglxeamcirnoasts-isoencotfitohnoefctrhoessh-esaecdtaitontehdeblreavienl foofrtahpeparent
hydrocephaly.
ScheduledSacrifice-F2GeneratPiuposn:
ONencdroapys2y1wiplolsitnpcalrutduema, spiunpgslewiclrlobses-ssaeccrtiifoincoefd atnhed heexaadmiantetdhefolrevgerloosfstlheesions. fhryodnrtoalc-eppahrailetya.l suture and examinationof the cross-sectioned brain for apparent
GCO0S2
Protocol 4P1a8g-e02038
PROPOSEDSTATISTICALMETHODS "2: Aapvperroapgrieastea.ndAdpdeirtcieonnatlagpersocwieldlubreescaalncdu/loarteadn.alLyitsteesr vmaalyuebsewiplelrbfeorumseedd, wifhaeprperopriate.
TyofpTee st"
|. Parametric"
A. Bartlett's Test!
Il. Nonparametric
A. Kruskal-Wallis Test (575% ties)
Significant at ps0.05 Not Significant
Significant at p<0.05 Not Significant
an n of Variance aTest
Significant at p<0.05
Not Significant ~~ B. Fisher's Exact Test (75%ties)
Dunnett's Test
Hil. TPesrtfoorportiDoatna Variance Test for Homogeneity of the Binomial Distribution
a. Statistically significant probabilities are reported as either ps0.05 or ps0.01. b. Used only to analyze data with homogeneity of variance. c. Proportion data are not included in this category. d. Test for homogeneityofvariance.
C0053
Protocol 4P1a8g-e02048
DATAACQUISITION,VERIFICATIONAND STORAGE:
Data will be hand- and/or computer-recorded. Records will be reviewed by the Study Director and/or appropriate management personnel within 21 days after generation. All
original records will be stored in the archives of the Testing Facility. All original data will
breequbeosutn.dParnedseirnvdeexdedt.issAuescowiplyl bofeaslltorraewd daatttahewilTlesbteinsgupFpalciieldtytoatthneoScphoanrsgoerfourpoonne year after mailing of the draft final report, after which time the Sponsor will be contacted
to determine the dispositionof these materials.
RECORDS TO BE MAINTAINED:
Protocol and Amendments.
TAensitmaAlrtAicclqeu,isViethiiocnl.e and/or Reagent Receipt, Preparation and Use. Randomization Schedules. Mating History. TGreenaetramlenCto(mifmpernetssc.ribed by Staff Veterinarian). Clinical Observations and/or General Appearance. BBloodoydWSeaimghptlse. Collection, Processing and Shipment. FCaeeesdarCeoanns-uSmepcttiioonniVnagluOebss.ervations. Natural Delivery Observations. LRietftlerexObasnedrvPahtyisoincsa.l Development and Behavioral Observations - F1 Generation Pups.
Gross Necropsy Observations.
OPhrogtaongWreaipghhst(sif (rief qrueiqrueidr)e.d). Study Maintenance (room and environmental records). Feed, Water and Bedding Analyses. Packing and/or Shipment Lists.
KPEEYRSONNEL:
Executive Director of Director of Research:
RAelsaenarMc.h:HoMbiedrrmeadn,S.PhCh.rDi.s,tiDaAn,BPTh.D.,
ATS
Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT
Director of Laboratory Operations: John F. Barnett, B.S.
Manager of Study Coordination: Valerie A. Sharper, M.S.
Manager of Animal Operations and Member, Committee: Dena C. Lebo, V.M.D.
Institutional
Animal
Care
and
Use
MCoannsaugltearnto,f VReetgeurliantaorryyPCaotmhpollioagyn:ce:W.KRatahyleBernowAn.,MDo.rVa.nM,.,M.PSh..D., ACVP
000084
Protocol 418.008 Page 25
EINALREPORT:
Abecfoimnpalriezheednfsoillvoewidnrgafctofnisnualltraetpioornt
wwiitllh
be prepared on completionof the study and the Sponsor. The report will include the
will
following:
Summary and Conclusion. Experimental Design and Method.
Evaluation of Test Results.
ADpatpae,ndPircoetso:colFiagnudreAss,sSocuimamtaedryAmaennddImnedinvtidsuaalnTdaDbelveisatSiuomnsm,arSitzuidnygDtihreecAtobro'sve GLP Compliance Statement, Reports of Supporting Data (if appropriate) and QAU Statement.
1
I
CARE AND USE C!
i
`The procedures described in this protacal have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All procedures described in this protocol that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or pain to the animals.
The Sponsor's signature below documents the fact that information concerning the necessity for conducting this study and the fact that this is not an unnecessarily duplicative study may be obtained from the Sponsor. No altemative (in vitro) procedures were available for meeting the stated purposes of the study.
GOOOSS
Protocol 4P1a8g-e02086
REFERENCES:
1. CThersitss.tianE,nvMi.rSo.nmaenndtaVloyPtreokt,ecPt.iEo.n (A1g9e8n2c)y.,IWnaVsihviongRteopnr,odDu.cCt.ivNeatainodnaMluTteacghenniiccailty Information Service, U.S. Department of Commerce, Springfield, VA 22161.
2. Cnharlitsrteixaonn,eM(.PSr.o(c1e9e8d4i)n.gsReofprNoadlutcrteixvoenetoSxiycmitpyoasnidumt,erNateowloYgoyrekvaAlcuaatdieomnsy ooff Sciences, November 7. 1983), J. Clin. Psychiat. 45(9):7-10.
3. Lang, P.L. (1988). Embryo and Fetal Developmental Toxicity (Teratology) CInocn.t,rWoillDmaintgationn,thMe ACha0r1l8e6s7-R0i6v3er0.C:(DCaDtaBbaRseRaptr.ovCihdaerdlebsy RAirvgeursLRabeosreaatrorcihes, Laboratories, Inc.)
4.
Institute of Laboratory
Laboratory Animal Animals. National
RAecsaoduermcyesPr(e1s9s9,6)W.asGhuiindgetfoonr,thD.eCC.are
and
Use
of
5. ISmaplleawnstkait,ioEn.ss(t1e9l6l4e)n. aFmarUbteemreusthdoedreRzatutme.maArkcrho.skPoatphioslc.hEexnp.NaPchhawremiaskovlo.n 247.367.
8. Snedecor, G.W. and Cochran, W.G. (1967). Variance test for homogeneity of
tPhreesbsi,nAommieasl,dipspt.ri2bu4t0i-o2n4.1.Statistical Methods, 6th Edition, lowa State University 7. Sokal, RR. and Rohl, F.J. (1969). Bartlett test of homogeneity of variances.
Biometry, W.H. Freeman and Co., San Francisco, pp. 370-371.
8. MSneetdheocdosr,,6tGhWE.diatnond,CloocwharaSnt,atWe.UGn.iv(e1r9s6i7t)y.PrAensasl,ysAimseosf,Vappr.ia2n5c8e-.27S5t.atistical
9. tDruenantemtte,ntCs.wWi.th(1a95co5n)t.roAl. muJl.tAimpeler.coSmtpata.rAisssooncp.r5o0c:e1d0u9r6e-1f1or29c.omparing several
10. WSoHka.l,FrR.eRe.maanndanRodhlCfo,.F.SJ.an(1F9r6a9n)c.isKcrou,skpapl.-W3a8l8l-i3s89T.est. Biometry,
11.
Dunn, OJ. (1964). 6(3):241-252.
Muttiple comparisons using
rank sums.
Technometrics
12. SMiceGgrealw,-HSi.l(l1,95N6e).w YNoornkp,arppa.me9t6r-i1c04S.tatisticsfortheBehavioral Sciences,
CcC0056
:
PROTOCOL APPROVAL:
FOR THE TESTING FACILITY
OcS - rey AGsesoorcgieatEe. DDieraercltoovreo,f PRhe.sDe.a,rDcAhBT
Zt
A
Raymend G. York, Ph/5., PABT
Associate Director of
arch
Study Director
ChaaribraprearsJ.onP,atInsthount,ioBnaAl.Animal Care and Use Commitee
Protocol 4P1a8g-e02078
2/opmmg ofS
Date
2/9 9
Date
= Me, - 98
Date
FOR THE SPONSOR
M4 iT7~G
Marvin T. Case, D.V.M., Ph.D.
Study Monitor
2
POR
Date
crocsT
.
v ICA r,PRIMED!
a Argus Research Laboratories, Inc.
A
PROTOCOL 418-008
COMBINED ORAL PERINATALPOSTNATAL
(GAVAGE) FERTILITY, DEVELOPMENTAL AND REPRODUCTION TOXICITY STUDY OF PFOS IN
RATS
SPONSOR'S STUDY NUMBER: 6295.9
eet Amendment 8 - 18 May 1999
1 Concentration Analyses (page 5ofthe protocol):
The concentration samples were sent to the Sponsor. Sample analyses will be
conducted at the Testing Facility.
discretion
of
the
Sponsor
and
no
report
will
be
sent
to
the
Reason for Change:
This change was made at the request of the Sponsor to clarify the protocol.
2 oMfatlheeapnrdotFoceoml)a:le Rats Assigned to Pharmacokinet tic Sample Collection (page 14
Tanhaelylizveedr aant dthseedriusmcrsetaimopnloefstwheerSeposnesnotrt.o the Sponsor. Samples will be Reason for Change:
This change was made at the request of the Sponsor to clarify the protocol. 3. AE1m/eFn2dGmeennetra5tioofnthPeupprsotNooctol)S:elected for Continued Observation (page 22 and
TSthoemsatcohmacocnhtecnotnstewnitlls boef tahneasleyzpeudpsatwtehreedissecnrtettiootnhoefStphoeSnpsoonrsfoorr.analysis.
0600S8
ProtAomcoelnd4m18e-n0t088 Page2
Reason for Change:
This change was made at the request of the Sponsor fo clarify the protocol.
$<) rd 0H orge(E.
Deariove,
Ph.D.,
(81-77 fem cX
DABT Date Rafiond G. Yor
AEN
(Pro. DABT
Associate Director of Research
ASstsuodcyiaDitreecDtiorerctorof Research
(gry 99
Date
Olen Windd
rel Klan [oa
V7
CDheaniarpCe.rsLoenb,o,InVst.iMt.uDti.onal Animal CaDraete MSatruvdiynMTo.niCtaosre, D.V.M., PhD. Date
and Use Committee
C0089
3MENVIRONMENTAL LABORATORY
PROTOCOL - ANALYTICAL STUDY Potassium Perfluorooctanesulfonate in
Two Generation Rat Reproduction
In-vivo study reference number: Argus 418-008
Study number: FACT 052798.1 Test substance: Potassium perfluorooctanesulfonate (PFOS)
Name and address of Sponsor:
Marvin Case 3M Toxicology Services
3M Center
`Building 220-2E-02
St. Paul, MN 55144
Name and address of testing facility:
3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106
`Experimental start date:
Expected termination date: December 31,1998
Spectrometry Method numbers and revisions:
FACT-M-1.0,
Extraction of Potassium Perfluorooctanesulfonate or Other Anionic
`Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass
FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-
Electrospray/Mass Spectrometry
'FACT-M-3.0,
Extraction of Potassium
Surfactants from Serum
PfoerrfAlnuaolryosoicstaUnseisnuglfHoPnLatCe-Eolre`cOttrheorspArnaiyo/nMiacss
Spectrometry
FACT-M-4.0,
Analysis of Fluorochemicals in Serum Extracts UsingHPLC-
Electrospray/Mass Spectrometry
Author: Lisa Clemen
Yul
Kris Hansen
Study Director
745/7
Date
Pa Tlai
jin Case
`Sponsor Representative.
[pF 129
Date
000030 1
10 Purpose
`The analytical portion of this dosing study is designed to evaluate levels of potassium
perfluorooctanesulfonate (PFOS), or a metabolite of PFOS designated by the study director, in
the liver of the parent and subsequent generationsofthe test system, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories.
2.0 REGULATORY COMPLIANCE
"This study is conducted in compliance with the Food and Drug Administration Good Laboratory
Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report.
30 0Tes0 rMat0 emats0000000000
31 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum
32 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability
information will be included in the final report
3.2.2 Analytical reference substance matrix: Argus Research Laboratories;
traceability information will be included in the final report
3.2.3 Analytical control matrix: 3.23.1 Ratliver -- Argus Research Laboratories; traceability information will be
included in the final report 3232 Rat serum - Sigma Chemical Company; traceability information will be
included in the final report 33 Number of test and control samples. Liver samples for testing were received from 40
test animals and 10 control animals. Serum samples will be tested at the discretion of the
Study Director. 34 Identification of test and control samples: The samples are identified using the Argus
Research Laboratories identifiers, which consist of a letter followed by the Argus project
number, the animal number, the group designation, and the draw date.
35 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor.
000091 2
36 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor.
37 Storage conditions for test materials: Test materials are stored at room temperature. `Samples are stored at 20% 10C.
38 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation.
39 Safety precautions: Refer o the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis.
4.0 EXPERIMENTAL - Overview
_
Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-008 are
received for analysis of fluorine compounds. At the discretion of the Study Director, aseries of
analytical tests wil be performed on select tissues.
Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry
(ES/MS). On the basis of findings from these analyses, additional sample matrices may be
evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol
amendment will be written.
5.0 EXPERIMENTAL - Analytical Methods 51 FACT-M-L0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic
Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 52 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-
Electrospray/Mass Spectrometry 53 `SFuArCfaTc-tMan-t3s.f0r,omExSterarcutmiofnorofAnPaoltyassissiUusmiPnegrfHlPuLoCr-oEolcteacntersouslpfronaayt/eMaosrsOtShpeerctArnoimoentircy 54 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC-
Electrospray/Mass Spectrometry
6.0 DATA ANALYSIS
--
61 Data transformations and analysis: Data will be reported as the concentration
(weightweight) of PFOS per tissue or sample, or of PFOS per unit of tissue of fluid.
62
Statistical analysis: Statistics used may include regression concentrations over time,and standard deviations calculated
analysis of the serum for the concentrations
within
cach dose group.If necessary, simple statistical tests, such as Student's t test, may be
applied to evaluate statistical difference.
0eeoR 3
7.0 MAINTENANCE OF RAW DATA AND RECORDS 7.1 The following raw data and records will be retained in the study folder in the archives
according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.12 Study comespondence,
7.13 Shipping records
714 Raw data
7.5 Electronic copies of data
72
Supporting recordstobe according to AMDT-S-8
retained separately from the study will include at least the following:
folder
in
the
archives.
7.21 Training records
722 Calibration records
7.23 Instrument maintenance logs 7.24 Standard Operating Procedures, Equipment Procedures, and Methods
7.2.5 Appropriate specimens.
80RewpmeNcES 81 3M Environmental Laboratory Quality System Chapters 1, 5 and 6 82 Other applicable 3M Environmental Laboratory Quality System Standard Operating
Procedures
90Atracevents 91 `SFuArfCaTct-aMn-tsL0fr,omExLtirvaecrtifoonr oAfnaPloytsaisssUisuimnPgerHfPlLuCo-rEolocetcatnreossuplrfaonya/tMeasosr OStpheecrtrAonmieotnriyc 92 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-
Electrospray/Mass Spectrometry 93 SFuArCfTac-tMa-n3ts.0f,romExSterarcutmiofnorofAnPaoltyassissiUusmiPnegrfHlPuLoCr-oEoclteacntersouslpfroanayt/eMaosrsOtShpeerctArnoimoentircy 94 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC
Electrospray/Mass Spectrometry
06Ces3 4
PCoAtSasNsuimumbePre2rt7s5r5oo3c5ia3nesufonste
ATTACHMENT F:
PREPARATORY AND ANALYTICAL METHODS
EnRvepeoraLabToorxat0o1r2y LaboratoryRecuest Number(RIH2006
Proprietary and Consent
006094
BR 3M EO NVIRONMEENNTTAALLLLAABBOORRAATTOORRYY
ee
METHOD EXTRAFCLTUIOORNOOCFHPEOMTIACSASLISUUMRPFEARCFTLAUNOTRSOFORCOTMANLEISVUELRFFOONRATAENAOLRYSOITSHUESRIANNGIONIC
HPLC-ELECTROSPRAY/MASS SPECTROMETRY
Method Number: FACT-M-1.0
Author: Lisa Clemen
Adoption Date: 5/20/57
Revision Date: 3/4
-- Approved By: J ] | NO
Laboratory Manager
Yrakee te
Group Leader
Os A Doma,
`Technical Reviewer
) s/e c/7 Date
Shu /4
Date
S[23/9
Date
1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction ofPotassium Perfluorooctanesulfonate (PFOS) or
other fluorochemical surfactants from liver.
1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds.
1.3 Mvaaltirdiacteiso:n reRpaobrbti.t, rat, bovine, and monkey livers or other livers as designated in the
Microsoft 7.0.1/95
FACT-M-1.0 Extractionof PFOS from Liver
Page 1 of8
000C95
2.0 SUMMARY OF METHOD
2.1 Tfhliuosrmoechtehmoidcadlesscurrifbaecstahntoswftrooemxtlriavecrt upsoitnagssiiounmpapierrifnlguorreoaogcetnatneasnudlf5o.n0amteLs(oPfFeOtSh)yolr other
acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLsofextract is removed to a centrifuge tube and put onto a nitrogen evaporator
until dry. Each extract is reconstituted in 1.0 mL methanol then filtered througah 3 cc. plastic syringe attached to a 0.2 um filter into glass autovials.
3.0 DEFINITIONS 3.1 None.
4.0 WARNINGS AND CAUTIONS
4.1 Health and Safety Warnings:
4.1.1 Upastehougneinvser.sal precautions when handling animal livers, they may contain
5.0 INTERFERENCES 5.1 There are no known interferences at this time.
6.0 EQUIPMENT
--
6.1 Tachceepftoalblloew.ing equipment is used while carrying out this method. Equivalent equipment is
6.1.1 6.1.2 6.13
Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or [EC
6.1.4 Shaker, Eberbach or VWR.
6.15 Nitrogen Evaporator, Organomation
6.1.6 Balance
7S 0 uepLiESANDMaTERIALS
7.1 Gloves
7.2 Dissecting scalpels 7.3 Eppendororf disposable pipettes
7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks
7.6 40 mL glass [-CHEM vials
7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL
7.9 Labels
ExtractioFnoAfCTP-FMO-S1.f0rom Liver
Page 2 of 8 CCe96
7.10 Syringes, capable of measuring 10 pL to SO uL
7.11 Glass, type A, volumetric pipettes
7.12 Graduated pipettes
7.13 Electronic pipettor, Eppendoorrf equivalent
7.14 Timer
7.15 Disposable plastic 3 cc syringes
7.16 Filters, nylon syringe filters, 0.2 um, 25 mm
7.17 Crimp cap autovials
Note:
PQriMor
wtaotuesri.ngRignlsaessswyarriengaensdabomtitlneis,murimnsoe f39
times times
with with
mmeetthhaannooll,an3dri3nsteismefsrowmit3h
Milliseparate
vials.
8.0 REAGENTS AND STANDARDS
8.1 Reagents
:
8.0.1 2S0o0dgiruammHsydNraoOxHid.eP(oIuTr iBnatkoear 1or00e0qumivLalbeenta)k,er(NcoanOtHai)ni1n0gN:50w0eiligthersap(pLr)oMxiilmlait-eQly
`water, mix until ll solids are dissolved. Store ina 1 L nalgene bottle.
8.12 MSeoadsiuurmeHy10drmoLxiodfeth(Je.T1B0aNkNeraOorHeqsuoilvuatlieonnt)i,nt(oNaa1O0H0)mILN. voDliulmuetteri1c0Nfla1:s1k0,and
dilute to volume using Milli-QTM water. Store ina 125 mL nalgene bottle.
8.1.3 Taepptrroaxbiumtaytlealmym1o6n9iugmrahmysdroofgTeBnAsulifnattoea(|KoLdvaoklourmeetqruiicvacloenntt)a,in(iTngBA5)000.L5MMi:lWlie-iQgh
Vwaotleurm.eAdwjiutshtMtiolpliH-Q10TM uwsatienrg.apApdrdoxNimaaOteHlysl6o4wlmyLwh1i0lNe aNdadOinHg atnhed ldaisltut1emtLo of
NaOH because the pH changes abruptly. Store ina | L nalgene bottle.
8.13.1 nTeBeAderdequusiirnegs IaNchNeacOkHprisoorluttoioena.ch usteo ensure pH = 10. Adjustas
8.1.4 {SNoad,iCuOm,c/aNrabHonCaOt,e)/S0o.d2i5uMm: BWieciargbhonaaptperoBxuifmfaetrel(Jy.T2.6.B5akgeorfosroedqiuuivmalceanrtb)o,nate
(N2,CO,) and dillte
atnodv2o1l.u0megowfistohdMiiulmlib-iQcTMarwbaotneart.e
(NaHCO; Store ina 1
into a 1 L volumetric L nalgene bottle.
flask
8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538.
8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade.
8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade.
81.8 Liver and control liver, received frozen from testing laboratory.
8.1.9 bMeilplrio-vQidTMewdatbeyr,a aMlillwlait-eQrTuOseCdPilnutshissysmteetmh.od should be Milli-QTM water and may
82 Standards 8.2.1 Prepare PFOS standards for the standard curve.
FACT-M-10 Extraction of PFOS from Liver
Pa3gofe
00097
82.2
Weigh approximately the actual weight.
100 mgof PFOS
intoa
100 mL volumetric flask and record
8.23 Bring to volume with methanol foar stock standard of approximately 1000 ppm
(ng/mL).
8.24
Dilute the stock solution approximately 50 ppm.
with
methanol
foar
working
standard
1
solution
of
8.2.5
Dilute the stock solution approx. 5.0 ppm.
with
methanol
for
a
working
standard
2
solution
of
8.2.6
Dilute approx.
the stock solution 0.50 ppm.
with
methanol
for
aworking
standard
3
solution
of
9.0 SAMPLE HANDLING
.
9.1 All livers are received frozen and must be kept frozen until the extraction is performed.
10.0 QuALITY ConTROL 10.1 Matrix Spikes
10.1.1 tPhreepaacrceuraancdyoafntalhyezeexmtartarcitxiosnp.ike and matrix spike duplicate samples to determine 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver.
10.1.3 Expected concentrations will fallin the mid-rangeofthe initial calibration curve. 102 Continuing Calibration Checks
10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearityofthe initial calibration curve.
10.2.2 One check is prepared per groupoften samples. For example,if a sample set = 34, four checks are prepared and extracted.
10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve.
10.2.4 The expected concentration will fall within the mid-rangeofthe initial calibration curve.
11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare Liver Homogenate to Use for Standards
11.1.1 Weigh approximately 40 gofliver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution.
11.12 140 gis not available, use appropriate amounts of liver and water in keeping with 21:5 ratio.
11.13 See section 13.0 to calculate the actual densityof liver.
FACT-M-1.0 Extraction of PFOS from Liver
Page 4 of 0C098
11.1.4 Add | mL of homogeneous solution toa 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen | mL aliquots of homogeneous solution in 15 mL centrifuge tubes.
11.15 Two 1 mL aliquots serve as matrix blanks, Use the standard concentrations and spiking amounts listed in table 1 10 spike, in duplicate, two standard curves for a total of fourteen samples. Table 1 Approximate Spiking Amounts for Calibration Standards
`Working Standard
WL "Approx. final cone. of
(Approx. Conc)
PFOS in liver
[Bak
0.010 ppm
0.50 ppm_-
0.050 ppm
0.100 ppm
5.0 ppm. [1 | 0 | 0250pm
5.0 ppm.
0.500 ppm.
[50pm[50 | 0750pem
0 ppm [4| T000ppm
11.1.1 See section 13.0 to calculate actual concentrationsof PFOiSn calibration standards.
11.2 Extract spiked liver homogenates following 12.14-12.24ofthis method. Use these Standards to establish each initial curve on the mass spectrometer.
12.0 PROCEDURES 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with
other tissues 122 Cut approximately 1 g of liver using a dissecting scalpel.
123 Weigh the sample directly into ataredplastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vialwiththe study number, weight, liver ID,dateand analyst initials 126 Add 2.5 mLsof water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or
until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10Cap the sample and vortex for 15 seconds.
FACT-M-10 Extraction of PFOS from Liver
Page S of
000099
12.11 tPuibpeettweit|hmthLe hidoemnotgiceanlaitnefoirnmtoataio1n5amsLthpeoslaymppruolpyelveinale. c(eSneteriWfuogrekstuhbee.etLfaobreldotchuemceennttriinfguge the remaining steps.)
12.12
Spike liver homogenates section 11.1 or Table 1.
with
the
appropriate
amount
of
PFOS
standard
as
described
in
12.13 Piinpsettrtuemetnwtob1lamnkLs.aliquots ofMilli-QTMwaterto centrifuge tubes. These will serve as
12.14
Add I buffer.
mL
0.5
M
TBA
and
2
mLof
the
0.25
M
sodium
carbonate/sodium
bicarbonate
12.15 Using a volumetric pipette, add mLs ethyl acetate.
12.16Capeach sample and put on the shaker for 20 minutes.
12.17
Centrifuge for 20 to approximately
to 25 3500
rmpirniu.tes,
until
layers
are
well
separated.
Set
power
on
the
centrifuge
12.18
Remove 4 centrifuge
mLsoforganic tube. Label this
lfaryeesrh,
tuusbiengwiath5
tmhLe
sgarmaeduiantfeodrgmlaatsisonpiapsetitne,12t.o5.a
clean
15
mL
12.19
Put each hours.
sample
on
the
analytical
nitrogen
evaporator
until
dry,
approximately
2
to
3
12.20 Add 1.0 mLofmethanol to each centrifuge tube using a graduated pipette.
12.21Vortex mix for 30 seconds.
12.22 Attach a 0.2 um nylon mesh filter to a3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial.
12.23 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction.
12.24 Cap and hold for electrospray mass spectrometry analysis.
12.25 Complete the worksheet and tape to pageofstudy notebook.
13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations:
13.1.1 Calculate the densityofliver (mg) in 1.0 mL homogenate using the following equation: gof Liver x Average weight of ten | mL aliquots (mg) (gofLive+r gofWater)
FACT-M-1.0 Extraction ofPFOS from Liver
Page 6 of
000200
13.1.2 Cfaolloluolwaitnegaecqtuuaatliocno:ncentrations of PFOS in calibration standards using the
WLof
Standard x mg Liver
Concentration (ug /mL) / | mL homogenate
=
Final Concentration of PFOS in Liver
(g/g
or
mg/kg)
m*ALvehroamgoegewneaitgehstooffalpipvreorxiinmsaotluetliyon40asmdeetleirvmeirniend2i0n01m3.L1.o1,f
by weighing ten Milli-Q water.
|
14.0 METHOD PERFORMANCE 14.1 The method detection limit is equal tohalfthe lowest standard in the calibration curve.
15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 hSiagmhplBeTwUasctoentiasindeirssp,oasendd iunsebdioghlaazsasrpdipceotntteaiwnaesrst,efisladmimsapobsleedsionlvbentrwagso lteasisskcdoinstepaoisnneedrsin
located in the laboratory.
16.0 Recorns
_
esti
16.1 Complete the extraction worksheet and tape into the study notebook.
17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 The validation report associated withthismethod is FACT-M-1.0 & 2.0-V-1.
18.0 REFERENCES
_
18.1 AMDT-EP-22, "Routine MaintenanceofUltra-Turrax T-25"
19.0 AFFECTED DOCUMENTS 19.1 FMaAsCsT-SMp-ec2t,ro"mAentarlyy"sisofLiver Extracts for Fluorochemicals using HPLC-Electrospray
20.0 Revisions Revision Number.
Reason For Revision
ReDvaitseion
FACT-M-10 ExtractionofPFOS from Liver
Page 7of
60101
.
Extraction Worksheet for FACT-M-1
Study #
NSuammpbleer
setd
|
apprPoxF.O0.S5
ppm|
apprPoxF.O5Sppm
|
approxP.FOS0
ppm|
Date and
Initials
actual ppm| actual ppm| actual ppm|forStd.
HW.
HW
HW.
-- C--C--TwomGker emw1 - r T7 -T r T rr
r 1 r 1-- r rT T rT r r r r r T r r 1 T ] r rr rr r -- --r rr r r
rr rT 1
r r r T T r ] r rT r r r rr T T r r -- rr r r --rrr
ER --
A
EE
MSMSDIP--om.CoMntS.MChSecDksu:seSdpiskaemdple ul. ofa
|
----
~ pCoonmt.stCdhecks wed same hToomrogTeinnaatcaeosncfeonrttrdatciuornveo.f
FACT-M-1.0 Extraction of PFOS from Liver
Page 8 of8
00e102
3M ENVIRONMENTAL LABORATORY
METHOD
ANALYSIS OF FLUOROCHEMICALS IN LIVER EXTRACTS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY
Method Number: FACT-M-2.0
Author: Lisa Clemen Approved By:
D1 Jpa
Laboratory Manager
Yorker Hr
Group Leader
ae ACh
Technical Reviewer
Adoption Date: 5/2/48
Revision Date: Ja
Sac fy
Date
5/2/98
Date
s/23/g
Date
1.0 SCOPE AND APPLICATION
reece
1.1 sSucrofpacet:anTthsiussimnegtHhPoLdC-isefloerctthreosapnralayys/imsaofssexstpreacctrtosmeotfrlyi.ver or other tissues for fluorochemical
1.2 SAuprpflaictcaanbtlse, oCroomtphoeruniodnsi:zabPloetcaosmspiouumnpdesr.fluorooctanesulfonate, anionic fluorochemical
1.3 Matrices: Rabbit, rat bovine, and monkey livers or other livers as designated in the
validation report.
Word 7.0.1/95
FACT-M-2.0 AnalysisofLiver Extract Using ESMS
Page 1 of8
000103
2.0_ SUMMARY OF METHOD 2.1 This method describes the analysis offluorochemical surfactants extracted from liver using
HPLC-electrospray/mass spectrometry. The analysis is performed by monitoringa single
ion characteristic ofa particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to
verify compound identification.
3.0 DEFINITIONS 3.1 None.
4.0 WARNINGS AND CAUTIONS
4.1 Health and Safety Warnings:
41.1 Use caution with the voltage cable for the probe, When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is riskofelectrical shock.
42 Cautions:
4.2.1 Do not run solvent pumps above capacityof 400 bar (S800 psi). If pressure goes over 400 bar, the HP1100 will initiate automatic shutdown.
422 Do not run solvent pumps to dryness.
5.0 INTERFERENCES
5.1
Teflon should not be used for sample storage or any partof instrumentation that comes in contact with the sample or extract.
60Equewenr
6.1 Equipment listed below may be changed inorderto optimize the system.
6.1.1 Micromass Electrospray Mass Spectrometer 6.12 HP1100 low pulse solvent pumping system and autosampler.
7S 0 uepLiEsaNDMatemaLS 7.1 Supplies
7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.12 HPLC column, specifics to be determined by the analyst. 7.13 Capped autovials or capped 15 mL centrifuge tubes.
8R .0 EAGENTSANDSTANDARDS
8.1 Reagents
811 Methanol, HPLC grade or equivalent.
`Word 7.0.1/95
Analysis of LiFvAeCrTE-xMtr-a2c.t0Using ES/MS
Page0200f180
8.1.2 Milli-QTM water, all water used in this method should be Milli-QTMwaterand may be provided by a Milli-Q TOC Plus system.
8.13 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards
82.1 Typically one HO blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1.
9.0 SAMPLE HANDLING
9.1 Fstroersehdliinvecrasptpaenddaarudtsovairaelsproerpacraepdpewdit1h5emacLhcaennatlryisfius.ge Etxutbreascutnetdilstaannaldyasridss. and samples are
92 Ifanalysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed.
10.0 QUALITY CONTROL 10.1 Matrix Blanks and Method Blanks
10.1.1 Analyze a method blank and matrix blank priotor each calibration curve.
10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis.
a. 10.2.2 Expected concentrations will fall in the mid-rangeofthe initial calibration curve. Additional spike concentrations may fall in the low-rangeofthe initial calibration
10.2.3 See section 13 to calculate percent recovery.
10.3 Continuing Calibration Checks
103.1
Analyze a mid-range calibration standard after every tenth sample. If a significant change (+ 30%) in peak area occurs, relative to the initial standard curve, stop the run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed.
10.3.2 See section 13 to calculate percent difference.
10.4 System Suitability
10.4.1 System suitability (e.g. peak area, retention time and peak shape, etc.) will be. assessed for each run.
11.0 CALIBRATION AND STANDARDIZATION. 11.1 Analyze the extracted liver standards prior to and following each setofextracts. The. mean
oftwo standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software.
FACT-M2.0
y AnalysisofLiver Extract Using ESMS
Page 3 of 8
060105
11.2 The 7 valueforthe data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst.
11.3Isfttahnedacrudrcvuerdvoee(sifnnoetcmeseseatrrye)qaunidrermeeanntasl,yzpee.rform routine maintenance or reextract the
12.0 PROCEDURES
12.1 Acquisition Set up
12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign
a(MfSil)efnoarmeacuqsuiinriglnge,ttaenrd-tMyOp-eDiAnY-slaamsptledidgeistocrfipyteiaorn-s.sample number, assign a method
12.1.2 STIoR.creSaette IaonmieztahtioodncMliocdkeonasscaappnrbouptrtiaotneian ntdhemAacsqsuitsoi4t9io9nocroonttrhoelr paapnperolparinadteselect masses. A scan is usually collected along with the SIRs. Save method.
12.1.3 Typically the sample list begins with the first setof liver standards and ends with
the second setofstandards.
12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such.
12.2 Using the Autosampler 122.1 Set up sample tray according to the sample list prepared in section 12.1.1.
12.2.2 `Saenta-luyspttchoensHiPd1e1r0s0a/papurtoopsraiamtpeleforraotptthiemaflolrleoswpionngsec.onRdeictoiorndsaocrtuaatlccoonnddiittiioonnsstihne the
instrument logbook:
12.2.2.1 Sample siz=e 10 pL injection with a sample wash
12.2.2.2 Inject/sample = 1
12.2.2.3 Cycle time = 15 minutes
12.2.2.4 Solvent ramp =
Time
20mM Ammonium acetate
[[Somm [00ow%e[ | 1100 % %|| [smn| 6% | 5% |]
Note: In this instrument configuration, the run must be set up on the electrospray psroefstsweadreownitthheaH"PWaWiotriknsgtaftoiroinn.let start" message before the "Start" button is.
12.2.2.5 Press the "Start" button.
FACT-M:2.0 Analysis of Liver Extract Using ESMS
Page 4 of 8
00106
123 Instrument Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 1232 Check the solvent level in reservoirs and refill ifnecessary. 12.33 Cthheectikp.thTehsetatiinplseshsousltdeeblecafpliatllwairtyhatnothjeaegngedodfetdhgees.prIofbteh.eUtsipeiasnfoeuynedpitoecbeeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 SOebtseHrPvLeCdrpopulmetpstcoo"mOinn"g.oSuettotfhtehfeltoiwptooft1h0e-p5r0o0beu.L/Amlilnowortoaseqaupiplriobprraitaetef.or approximately 10 minutes. 12.3.5 Taurorunnodnthtehetinpitorfogtehne.prAobfei.ne mist should be expelled with no nitrogen leaking 123.6 Tchhaenignestinruomrednetrutsoeosptthiemsiezepatrhaemreetseprosnsate:the following settings. These settings may 123.6.1 Drying gas 250-400 liters/hour 123.62 ESI nebulizing gas 10-15 liters/hour 123.63 LC constant flow mode flow rate 10 - 500 uL/min 123.64 Pirnesstsruurmeen<t4i0s0opbearra(tTihnigscporarreacmtelyt.e)r is not set, it is a guide to ensure the 12.3.7 fCaarrtehferu.llCyognuniedcettthheepvroolbteagientcoatbhleesopteontihneg.proIbnes.ert probe until it will not go any 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysisofbiological matrices. 123.10toCpliocfksoanmpslteartlsbtu.ttEonnsiunrethsetaArctqaunidsietnidonsCaomnptlreolnuPmanbeelr.Pirneclsusdtehsealsltasratmbpultetsontoatbe: analyzed.
13.0 DATA ANALYSIS AND CALCULATIONS
13.1 Calculations:
13.1.1 Calculate matrix spike percent recoveries using the following equation:
%Recovery=
Observed Result - Background Result x 100 `Expected Result
13.1.2 Calculate percent difference using the following equation: 9% Difference = Exepeceted Conc.c-eCadlCculoatend Ceon,e. x 100
FACT-M-2.0 AnalysisofLiver Extract Using ESMS
Page 5 of8 |
Ceo?
13.1.3 Calculate actual concentrationof PFOS anion in total liver (mg):
(= PFOS anion cal. from stpd e)
solvedforanlyss J ootos oflive(9 1000ug/1 mg
14.0 METHOD PERFORMANCE
14.1 The method detection limit is equal to at least tree times the baseline noise in the matrix
blank.
14.2 The practical quaniitation limit is equal to the lowest standard in the calibration curve.
15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
15.1 hSiagmhplBeTwUasctoentiasindeirssp,osaenddignlbaisoshpaizpaetrtdecwoansttaeiniesrsd,isfploasmemdaibnlbersooklevnengtlawsasstcoentisaidniesrsp.oseAdllin containers are locatedinthe laboratory.
16.0 RECORDS
16.1 iStnofroermcahtrioomnaitnocglruademdsienitthheer isnttuhdey hfoeladdeerr. oErahcahndchwrroimtaetnogornatmheshcohurlodmahtaovgertahme:fosltluodwying number, sample name, extraction date, and dilution factor (fapplicable).
162 Plot calibration curve by linear regression and store in the study folder.
16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog.
16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook.
166 Summarize data using suitable software and storeinthe study folder. 16.7 Bloaccaktiuopnoeflbecatcrkonuipc edlaetcatrtoonaipcpdraotpar.iate media. Record in study notebook the file name and
17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V-1.
18.0 REFERENCES 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer"
FACT-M-2.0
AnalysisofLiver Extract Using ES/MS
Page 6 of8 090108
19.0 AFFECTED DOCUMENTS 19.1 UFsAiCnTg-MH-P1L.C-0E,l"eEcxttrroascptiroanyo/fMaPsostaSspseicutmroPmeertfrlyu"orooctanesulfonate from Liver for Analysis
20.0 Revisions Revision Number.
Reason For Revision
ReDvaistieon
FACT-M-2.0 Analysis of Liver Extract Using ESMS
Page 7of 8 60109
Laboratory Study #
Study: Test Material: MatixFinal Solvent: MAneatlhyotidcRaelvEiqsuoinp:ment System Number: Instrument Software/Version: Filename: ReSquared Value: Slope: Intercept: DDaatteeooffEAxntarlaycstiiso/nA/nAanlaylsyts:t:
Group Dose
ugml.
TartnarLVol. Factor
Finuagl/Cmolne.
Sioper Taken rom linear egresion equation.
Group/Dose: Taken fom the study folder.
Sample#: Taken frothm study folder.
Concentration (up/mL): Taken fom the MassLynx integration summary.
Initial Volume (mL): Taken fromthestdy folder.
Dilution Factor: Taken from the study folder. Final Cone. (g/mL): Calculated by dividingth
initial volume from the concentration
FACT-M-2.0
AnalysisofLiver Extract Using ESMS
Page8 of8
60140
ME!
TALL,
Y
METHOD
EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER ANIONIC FLUOROCHEMICAL SURFACTANTS FROM SERUM FOR ANALYSIS USING
HPLC-ELECTROSPRAY/MASS SPECTROMETRY
Method Number: FACT-M-3.0
Author: Lisa Clemen Approved By:
J
Laboratory Manager
futo 1--
Group Leader
Adoption Date: 472]a3
Revision Date: Ng
Soe 55
Date
tray
Date
Technical Reviewer
Date
0 SCOPE AND APPLICATION 1.1 Sotchoepre:fluTohrioschmeemtihcoadl sisurffoarctthanetesxftrraocmtisoenorufmp.otassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, and bovine serum or other sera as designated in the validation report.
Microsoft 7.01/95
FACT-M3.0 Extraction of PFOS from Serum
Page 10f8
00111
2.0 SUMMARY OF METHOD 2.1 aTnhiiosnmicetfhloudordoecshcermiibceaslhsouwrtfaocteaxnttsrafcrtopmotsaesrsuimumuspienrgflaunoriooonctpaanireisnuglfroenaagteent(PaFnOdS5).o0rmoLthoefr
petahrytlitaicoenteadtei.ntoAnethiyolnapcaeitraitneg. rFeoaguernmt Lisofadedxetdratcottahreesraemmpoleveadndantdhepuatnaolnyttoe ainoinptariorgeins tevharpoourgahtoar3 ucnctiplldarsyt.ic Esayrcihngeextartatctacihserdetcoonast0i.t2utjeudm innyl1.o0nmfiLlteorfimnettohgalnaosls,autthoevniaflisl.tered
3.0 DEFINITIONS 31 None.
4.0 WARNINGS AND CAUTIONS
RE
41 Health and Safety Warnings:
4.1.1 Uhasnedluininvgearsnailmaplrescearuutmio,nist,measpyeccioanltlayinlapbaotrhaotgoernysc.oats, goggles, and gloves when
5.0 INTERFERENCES 5.1 There are no known interferences at this time.
6.0 EQUIPMENT 6.1 The following equipmentisused while carrying outthis method. Equivalent equipment is
acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.12 Centrifuge, Mistral 1000 or [EC 6.13 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance, (0.100 gm)
7.0 SUPPLIES AND MATERIALS 71 Gloves 7.2 Eppendorofr disposable pipettes 7.3 Nalgene botles, capableof holding 250 mL and 1 L 7.4 Glass, type A, volumetric flasks 7.5 40 mL glass I-CHEM vials 7.6 Polypropylene centrifuge tubes, 15 mL 7.7 Labels 7.8 Syringes, capableof measuring 10 L to 50 uL 79 Glass, type A, volumetric pipettes 7.10 Graduated pipettes
----
FACT-M-3.0 ExtractionofPFOS from Serum
Page 20f8
00112
7.11 Electronic pipettor, Eppendoorrf equivalent 7.12 Timer 7.13 Disposable plastic 3 cc syringes 7.14 Filters, nylon syringe filters, 0.2 um, 25 mm 7N.o1t5e: QCPrPriiwomarptteocrau.psiaRnugtiongvslieaaslsssywrairngeeasnadmbiotnliems,umrinosef93ttiimmeesswwiitthhmmeetthhaanonlo,l3anrdin3setsifmersomwi3thseMpairlaltie-:
vials. 8.0 REAGENTS AND STANDARDS. 8.1 Reagents
8.1.1 2S0o0digurmamhsydNraoOxiHd.eP(oJ.uTrBiantkoer2o1r00eq0uimvLalebneta)k,e(rNcaoOntHa)in1i0nNg:5w0e0ilgihterasp(pLr)oMxiilmlia-tQe'ly `water, mix until al solids are dissolved. Store ina 1 L Nalgene bottle.
8.12 SMoedasiuurmeh1y0drmoxLidoef (1J0.NT BNaakOerHosroelquutiivoanleinntto),2(1N0a0OmHL) vINo.lumDeitlruitce f1l0aNsk1a:n1d0.dilute to volume using Milli-QTM water. Store in a 125mL Nalgene bottle.
8.1.3 aTpeptrroaxbiumtaytlealmym1o6n9iugmrahmysdroofgTeBnAsulifnattoe (K1 oLdavkolourmeetqruiicvacloenntt)a,in(iTnBgA5)000.L5MM:ilWlie-iQgh Water. Adjust to pH 10 using approximately 64 mLof LON NaOH and dilute to NvoalOuHmebweictahusMeitlhlei-pQHTMcwhaatnerg.esAadbrdupNtalyO.HStsolroewliynawh|ilLeNaadldgienngethboettlalset. mL of 8.13.1 TneBeAderdequusiirnegs IaNchNeacOkHprisoorluttoioena.chuseto ensure pH = 10. Adjustas
8.1.4 (SNoedi,uCmOycNarabHoCnOat,e)/s0o.d2i5uMm:bWiceairgbhonaaptperbouxfifmeart(e1l.yT.26B.a5kegroofrsoeqduiiuvamlecnatr)b,onate a(Nnad.bCrOi;n)g taondvo2l1u.0mgeowfitshodMiilulmib-iQcTMarwbaotenra.teS(tNoraeHiCnOa;1iLntnoaalg1eLnevbooltutmlee.tric flask
8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Other fluorochemicals, as appropriate. 8.1.7 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.8 Methanol, Omnisolv, glass distilled or HPLC grade. 8.19 Serum, frozen liquid from Sigma. 8.1.10 Control serum received with each sample set. 8.1.11 bMeilplrio-vQidTMewdatbeyr,aMalilllwiat-eQrTuOseCdPilnutshissysmteetmh.od should be Milli-QTM water and may
FACT-M3.0 Extraction of PFOS from Serum
Page 3 of 8
60213
82 Standards
82.1 Prepare PFOS standards for thestandard curve.
822 Prepare other fluorochemical standards, as appropriate.
82.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record
the actual weight.
8.2.4 Bring to volume with methanol for astock standard of approximately 1000 ppm
(g/mL).
825 aDiplpurtoexitmheatsetloyck50soplpumt.ion with methanol for a working standard 1 solution of
82.6
Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm.
82.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm.
9.0 SAMPLE HANDLING 9.1 All sera are received frozen and must be kept frozen until the extraction is performed.
0.0 QUALITY CONTROL 10.1 Matrix Blanks and Method Blanks
10.1.1 Taswmoat1r.i0xmbLlanaklsi.quoSteseofstehcteiosner11u.m1.a2r.e extracted following this procedureand used 10.1.2 Two 1.0 mL aliquotsofMilli-QTM water are extracted following this procedure and
used as method blanks.
102 Matrix Spikes 10.2.1Prepareand analyze matrix spike and matrix spike duplicate samples to determine the accuracyofthe extraction. 10.2.2 Prepare each spike using serum chosen by the analyst, usually control serum received with each sample set. 10.2.3 EAdxdpietcitoendalcosnpcieknetsramtaiyonbsewiilnlclfuadlelidnatnhde mmaidy-rfaalnligneotfhtehleowin-irtaianlgceoaflitbhraetiionnitciuarlve. calibration curve.
103 Continuing Calibration Checks 103.1 Pcroenptairneueadnldinaenaarliytzyoefctohnteiinnuiitniaglccaalliibbrraattiioonncchurevcek.samples to determine the 10.3.2 One check is prepared per groupoften samples. For example, iaf sampleset = 34, four checks are prepared and extracted.
FACT-M-3.0 ExtractionofPFOS from Serum
Page 4 of 8
000114
103.3 Prepare each continuing calibration check from the same serum used to prep the initial curve.
10.3.4 The expected concentration will fall within the mid-rangeofthe initial calibration curve.
11.0 CALIBRATIONAND STANDARDIZATION
111 Prepare Serum Standards 11.11 Transfer 1 mLofserum to a 15 mLcentrifuge tube. 11.1.2 Iufstihneg msaejrourmitvyoolfumseesriunmtshaempsltaenvdaorldusmeeqsuaarletoletshsethsaenru1m.0vmoLl,umeexstrianctsasmtpalnedsa.rdsDo notextract below 0.50 mL of serum. Record the serum volume on the extraction sheet. 11.1.3 M in 15oirmsLhx ackeentrbieftuwgeeetnubaelsi.quots while preparing a totalofsixteen aliquotsofserum
11.1.4 Tstwaonda|rmd Lconorceanptprraotpiroinasteanadlisqpuioktsinsgearvmeouanstmsatlriisxtedbliannktsab.leT1yptiocaslpliykeu,sientdhueplicate,
two standard curves for a totaloffourteen samples.
11.15
Referto the the working
validation ranges for
report FACT-M-3.0-V-1 calibration curves.
and
FACT-M-4.0-V-1
which
lists
Table 1 Approximate Spiking Amounts for Standards and Spikes
Using 1.0 mL of Serum
Working Standard (Approx. Conc) -- ~~
0.500 ppm 5.00 ppm 5.00 ppm. 50.0 ppm. 30.0 ppm 30.0 ppm.
WL "Approx. final conc. of PFOS in serum
TT Bak | 0.010 ppm 0.025 ppm
[10 0050ppm 0.100 ppm 0.250ppm
[10 0500pm [15 0750ppm
11.1.4 See section 13.0 to calculate actual concentrations of PFOS in calibration standards.
11.2
Extract spiked establish cach
isneitriualmcsutravnedaorndsthfeolmlaoswsinsgpe1c2t.r6o-m1e2t.e1r6.of
this
method.
Use
these
standards
to
FACT-M:3.0 ExtractionofPFOS from Serum
Page 5 of 8 000145
12.0 PROCEDURES
pr----------
12.1 Obiain frozen serum samples andallowto thaw.
12.2 Vortex mix for 15 seconds then remove 1.0 mL or appropriate volume toa 15 mL.
polypropylene centrifuge tube.
12.3 Return serum samples to freezer after extraction amount hes been removed.
12.4 Record the serum volume on the extraction worksheet. The final methanol volume will
equal the initial serum volume.
125 wLaobreklshteheettfuobredwoictuhmtehnetsitnugdythneurmebmeari,nisnegrustmepIsD., date and analyst initials. See atiached
12.6 STapbilkee Isefrorumthweictahlitbhreataipopnrocpurrivaetestaamnodaurndtso.fAPlFsOoSspiskteanmdaatrrdixassdpeiskcersiabnedd cinonsteicntuiionng11.1 or
calibration standards.
12.7 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds.
12.8 Add 1 mL 0.5 M TBA and 2 mLofthe 0.25 M sodium carbonate/sodium bicarbonate buffer.
12.9 Using a volumetric pipette, add 5 mL ethyl acetate.
12.10Cap each sample andputon the shaker for 20 minutes. 12.11Centrifuge for 20 to 25 minutes, until layers are well separated. Set poweron the centrifuge
to approximately 3500 pm.
12.12
Transfer 4 centrifuge
mL of organic layer, tube. Label this fresh
using a 5 mL tube with the
graduated glass pipette, same information as in
to aclean 12.5.
15
mL
12.13 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3
hours
12.14
Add 1.0 pipette.
m(TLhiosrvaoplpurmoperieaqtuealvsotlhuemienoiftimaletvhoalnumoelotfoscearcuhmceunsterdiffougrethteubeexutrsaicntgioan.g)raduated
12.15 Vortex mix for 30 seconds.
12.16AFtilttaecrhinato0.a2 1p.5mmnLyloglnamssesauhtofviiltaelr.to a3 cc syringe and transfer the sample to this syringe.
12.17 Lmaabterilx,thfeinaaultosvoilvaelnwti,tehxttrhaecsttioundydantue,mbaenrd,aannaliymsatl(sn)uwmhboerpaenrdfogremneddert,hesaemxtprlaecttiiomne.point, 12.18Cap and hold for HPLC-electrospray/mass spectrometry analysis. Extracts may be stored
at4 C until analysis.
12.19 Complete the extraction worksheet, attached to this document, and tape to page of study notebook.
FACT-M3.0 ExtractionofPFOS from Serum
Page 6 of8 000146
Extraction Worksheet for FACT-M-3
Study #
Sample
PFOS
PFOS
PFOS
Date and
`Number aapcpturaolx. 0.5 ppppmm|| aapcpturaolx. 5 ppppmm|| aapcpturaolx. 50 ppppmm|| IniSttidal.sorfor
sett
#W
#W
HW,
Comments
----1 --woessxemma [o 1 o
r rr rr r
-- r r r r r
r rr r
-- ---- r rT r
r rr rr r
-- r r T r
r rr T r
-- r r r rr r
r -- r r rr r
-- r r r r r
r --r r rr r
r rr T rT
r rr r r
rrr 1
Ansar
[FicTnLoro NapCOy0TMNaiCO woe Sif
[Foemcsolotemimeeme
moa
Memon
a ewe wwe
MSMSDIPo-m.CoMnSt/CMhSecDksu:seSdpskaemdple WL of3 pCopnt.sCdhesswed same seorrums1Tsfrolrscodncceunrtvrea.tion oF
FACT-M3.0
Page 8 of8
Extraction of PFOS from Serum
6001177
3
NME LAB!
METHOD ANALYSIS OF FLUOROCHEMICALS IN SERUM EXTRACTS USING
HPLC-ELECTROSPRAY/MASS SPECTROMETRY
Method Number: FACT-M4.0
Author: Lisa Clemen
Approved By:
LF (ee n) N
Laboratory Manager
Adoption Date: 473/43
Revision Date: |
q[>2/9% Date
Group Leader
ise Cluman
`Technical Reviewer
Date
41s
Date
1.0_ SCOPE AND APPLICATION
1.1 Ssucrofpaec:tanTthsiussmiengthHoPdLCi-sefloerctthreoasnparlayys/imsaofsesxtspreaccttrsoomfets ry.eorrtu issmue for fluorochemical 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical
surfactants, or other ionizable compounds.
1.3 Matrices: Rabbit, rat, and bovine serum or other seraas designated in the validation report.
`Word 7.0.1/95
`Analysis of SeFrAuCmT-ExMt-r4a.ct0Using ESIMS
Page 1 of8
000118
2S .2.01 TuU hsiisngHmeM PtLhoCd-edM leescctrriobA sepsrtahye/R amnaaslsyssipY seocftfrolmuO eotrroyc.hF TemhiecaanlM alsyusrifsacE itsapntesrfeT oxrtrmaecdtH ebdyfmroonO mitsoerriunD mg a spienrgflleuofroonocchtaarnaecstuelrfiosntiacteo(afPFpOaSrt)icaunliaornf,lMuo/rZo=ch4e9m9i.calS,amspuclhesasmtahye palostoasbseisucmreened to verify compound identification.
3.0 DEFINITIONS 31 None.
cr eee eer
4.0 WARNINGS AND CAUTIONS 41 Health and Safety Warnings:
41.1 Uinsteo cthaeutpiroonbweiDthOthNeOvToltTaOgeUcCabHleTfHoEr tPheROprBoEbe,.tWhehreenistrhieskvoolftaelgeectcraibclale isshopclku.gged
42 Cautions: 42.1 Dovoerno4t00rubnars,oltvheenHtPp1u1m0p0s waiblolvienictaiaptaeciatuytoomfa4t0ic0 sbhaurt(dSo8w0n0. psi). Ifpressure goes 422 Do not run solvent pumps to dryness.
5.0 INTERFERENCES
_--
5.1 Teflon should not be used for sample storage or any partofinstrumentation that comes in
contact with the sample or extract.
6.0 EQUIPMENT
_
6.1 Equipment listed below may be changed in order to optimize the system.
6.1.1 Micromass Electrospray Mass Spectrometer 6.12 HP1100 low pulse solvent pumping system and autosampler.
7.0 SUPPLIES AND MATERIALS 7.1 Supplies
7.11 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.13 Capped autovials or capped 15 mL centrifuge tubes.
8.0 REAGENTS AND STANDARDS 81 Reagents
8.1.1 Methanol, HPLC grade or equivalent.
FACT-M-4.0 Analysis of Serum Extract Using ES/MS
Page 2of 8
Ge119
8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may `be provided by a Milli-Q TOC Plus system.
8.13 Ammonium acetate, HPLC gradeor equivalent.
82 Standards
82.1 Typically one H,0 blank, one serum blank,and seven serum standards are prepared during the extraction procedure. See FACT-M-3.
9.0 SAMPLE HANDLING
9.1 Fresh serum standards are prepared with each analysis. Extracted standards and samples
are storedincapped autovials or capped 15 mL centrifuge tubes until analysis.
9.2 Ifanalysis will be delayed, extracted standards and samples may be refrigerated at 4 C until analysis can be performed.
10.0 QUALITY CONTROL 10.1 Matrix Blanks and Method Blanks
-- ---- ----------
10.1.1 Analyze a method blank and a matrix blank priotoreachcalibrationcurve.
10.2 Matrix Spikes
10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis.
curve. 10.2.2 Expected concentrations will fall in the mid-rangeofthe initial calibration curve. Additional spike concentrations may fall in the low-rangeofthe initial calibration
10.2.3 See section 13 to calculate percent recovery.
10.3 Continuing Calibration Checks
10.3.1 Analyze a mid-range calibration standardafterevery tenth sample. Ifa significant change (+ 30%) in peak area occurs, relative to the initial standard curve, stop the
run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed.
10.3.2 See section 13 to calculate percent difference.
10.4 System Suitability
10.4.1 System suitability (e.g., peak area, retention time, peak shape, etc.) will be assessed for each run.
110 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted serum standards prior to and following each setofextracts. The `meanoftwo standard values,at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software.
FACT-M-4.0
" Analysis of Serum Extract ne Using ES/MS
Page3 of 8
00012-0
11.2 Ther value for the data should be 0.98 or greater. Lower values maybeacceptableatthe discretionofthe analyst.
113 Ifthe curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze.
2.0 PROCEDURES
SE
--
12.1 Acquisition Set up
12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign
(aMfSi)lefnoarmeacuqsuiinrginlge,tatnedr-tMyOpe-DiAnYs-laamsptledidgeistcorfiypetaiorn-ss.ample number, assign a method
12.1.2 To create a method click on scan button intheAcquisition control panel and select
oStIhRer(SaipnpgrloeprIioanteRemcaosrsdeisn.g)A. sSceatnIoisniuzsautailolny Mcooldleectaesdaaplpornopgrwiiatteh athnedSmIaRss.s tSoa4v9e9 or
method.
12.1.3 Typically the sample list begins with the first setof serum standards and ends with the second set of standards.
12.1.4
Samples
sample.
Saroelvaennatlybzleadnkwsitshhoaulcdonbteinaunianlgyzcaeldipberraitioodniccahlelycktoinmjoencittedorafptoesrseivbleeryantaelnytthe
carryover and are not considered samples but may be included as such.
12.2 Using the Autosampler
12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1.
12.2.2
Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook:
12.2.2.1 Sample size = 10 uL injection with a sample wash
1222.2 Inject/sample = 1
12.2.2.3 Cycletime = 15 minutes
12.2.2.4 Solvent ramp =
Time
2.0mM
[Comm| a% |Amm sw onium | acetate
[E omra |I-- so | -- 1% |
Note: In this instrument configuration, the run must be set up on the electrospray software with a "Waiting for inlet start" message before the "Start" button is pressed onthe HP Workstation.
12.2.2.5 Press the "Start" button.
FACT-M-4.0
Analysis of Serum Extract Using ES/MS
Page 4 of 8
000121
123 Instrument Set-up
123.1Refer toAMDT-EP-31 for more details.
12.32 Check the solvent level in reservoirs and refill if necessary.
12.33 cChheecckktthheetsipt.ainTlhesestsitpeeslhocualpidllbaeryfalatttwhiethenndoojfatghgeedpreodbgee.s.UIsfethaenteiypeispifeocuendtoto be unsatisfactory, disassemble the probe and replace the stainless steel capillary.
12.3.4 Set HPLC pumpto "On". Set the flow to 10 - 500 uL./min or as appropriate. Observe droplets coming outofthe tipofthe probe. Allow to equilibrate for approximately 10 minutes.
12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tipofthe probe.
123.6 The instrument uses these parameters at the following settings. These settings may
:
change in order to optimize the response:
12:3.6.1 Drying gas 250-400 liters/our 123.62 ESI nebulizing gas 10-15 liters/hour 123.63 HPLC constant flow mode flow rate 10 ~ 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is aguide to ensure the
HPLC is operating correctly.)
12.3.7 Cfaarrtehfeurl.lCyognuniedcettthheepvroolbteagientcoatbhleesotpoentihnegp.roIbnes.ert probe until it will not go any
123.8 Record tune parameters in the instrument log.
1233.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysisofbiological matrices.
12.3.10Click on start button intheAcquisition Control Panel. Press the start button at top `ofsample list. Ensure start and end sample number includes all samples to be. analyzed.
13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations:
13.1.4 Calculate matrix spike percent recoveries using the following equation: %Recovery= Observed Result - Background Result x 100 Expected Result
13.15 Calculate percent difference using the following equation: % Difference = Expected Conc. - Calculated Conc, x 100 `Expected Conc.
FACT-M-4.0 Analysis of Serum Extract Using ES/MS
Page Sof8
00122
13.1.6
C(aslgciumlLayt:e actual concentration of PFOS, or other fluorochemical, anion in serum
g of PFO calc. from std. Curve x Dilution Factor x Final Volume (mL) Initial Volumeof serum (mL)
14.0 METHOD PERFORMANCE 14.1 The method detection limit is equal tohalfthe lowest standard in the calibration curve.
142 The practical quantitation limit is equal to the lowest standard in the calibration curve.
5.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
15.1 Spiapmepttleeweaxsttreacitswdaisstpeosaenddifnlbarmomkaebnleglsaoslsvceonnttaisindeirsspolsoecadtiend hiingthheBlTaUborcaotnotrayi.ners, and glass
Ie SORECORDS ooo ee
16.1 Store chromatograms in the study folder. Each chromatogram must have the following
information included either in the header or hand written on the chromatogram: study `number, sample name, extraction date, and dilution factor(if applicable).
16.2 Plot calibration curve by linear regression and store in the study folder.
16.3 Print sample list from MassLynx and tape into the instrument runlog.
16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and
tape into appropriate study notebook.
16.6 Summarize data using suitable software and storeinthe study folder.
16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and locationofbackup electronic data.
17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA 17.1 Attachment A: FACT-M-4 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.0 & 4.0-V-1.
18.0 REFERENCES
A--
18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer"
19.0 AFFECTED DOCUMENTS 19.1 FACT-M-3.0, "Extractionof Fluorochemical Anions from Serum for Analysis Using
HPLC-Electrospray/Mass Spectrometry"
FACT-M-4.0
A Analysis of Serum Extract 3 Using ES/MS
Page 6 of8
Ce123
2e 00Revistons ee
Revision
Revision
Number.
ReasonFor Revision
Date
FACT-M4.0 AnalysisofSerumExtract Using ESIMS
Page 7 of 8 000124
Laboratory Study #
TSeusdtyM:aterial MMeatchioxdFRienvaisSioolnv:ent: IAnnasltyrtuimceanltESqoutiwparmeelnVteSryssiotne:m Number: RFielSeqnuaamreed Value: SloIpnee:rcepe: DDaatteeooffAEnxarlaycstiosnA/nAanlaylsyts:i:
(Doosre [ snl [amL a | oFacetor |Tsmgl |
`GSroopuerpDToaskee:n rTaokmenlifnreaormetghreesstiuodynfsoqldueari.n. Sample Taken from the study older. `DICniioltnuictaielonVntorFalatucimtooenr:((umgTL/)am:kLe)nT:afkrTeoanmkfetrnhoefmsrttouhmdeytshtfeuolMddyaerso.slLdeyrnx integration summary. Final Cone. (sg/mL): Calculated by dividing th intial volume from the concentration
FACT-M4.0
Analy sis ofSerumExtractUUssiis ngES'MS
Page 8 of
000125
EPostaNssoimmaPrear1t5uc5r3o5c5nesstorae
Labora ResEtmNu`mrRbeeprea(tFhoNLa)bTo0or2xat00o10r28y
FE
ATTACHMENT G:
RESULTS OF CONFIRMATORY DOSE ANALYSES
Proprietary and Confidential
0C0126
EFmer EEL.
&ess EEre --[=
5z
Jay
EE
Ss
PC E gE e EE e e
E=E LL E]
EFEre
ea e EE
g8
Fi
JERE
g
ETTEE
E|Ereet]e:E]
er