Document zQB3VGj0QOnpeznZxpjbD6O7R
Pathology Associates International
A Company of ScienceApplicationsInternationaClorporation
AnarO&AOOWUd CW4POnY
Sponsor: 3M
St.Paul,Minnesota
PROTOCOL
Study Title: CellProliferatiSotnudy withN-EthylPerfluorooctanesulfonamEitdhoanol(N-EtFOSE; 3M T-6316.11), PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295.16)a,ndN-EthylPerfluoroctanesulfonamide
(PFOSA 3M T-7091.1)inRats
Date: January12,1999
Performing Laboratory R.O.W. Sciences 15 FirstfieRlodad
GaithersburgM,aryland 20878
Laboratory Study Identifi*cation:
Study Number: (1132-100)
PAI ProjectNumber: (Histolognyumber tobe assignedby PAI by protocolamendment)
is Wormants Mill Court, Suite I Frederick, Maryland 21701 * (301) 663-1644
(301) 663-8994 FAX
Study Number 1132-100 Page 2
Study CellProliferatiSotnudy withN-EthylPerfluorooctanesulfonamiEdtohanol(N-EtFOSE; 3M T6316.11),PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS; 3M T-6295-16),and N-Ethyl Perfluoroctanesulfonami(dPeFOSA 3M T-709 1.1)inRats
Purpose To assessceU proliferatiaond peroxisomeproliferatiionnratsadministeredtestmaterialinthe diet.
Sponsor 3M CorporateToxicology Building220-2E-02,3M Center St.Paul,MN 55144-1000
Study Representative Marvin T. Case,D.V.M, Ph.D. 3M CorporateToxicology Phone No.: 651733-5180 Fax No.:651733-1773 Email:mtcase@nu=.com
Alternative Study Representative Andrew M. Seacat,Ph.D. 3M CorporateToxicology Phone No.: 651575-3161 Fax No.:651733-1773 Email:amseacat@mmm.com
Study Location R.O.W. Sciences 15 FirstfielRdoad GaithersburgM,aryland 20878
Study Number 1132-100 Page 3
Study Monitor Sandra R. Eldridge,Ph.D. PathologyAssociatesInternational Phone No. 301624-2036 Fax No. 301663-8994 Email:srepaisaic@aol.com
Study Director Gary W. Wolfe,Ph.D.,D.A.B.T. R.O.W. Sciences Phone No.:301330-3723 Fax.No.: 301330-3738 Email:gwolfe@lab.row.com
Principal Investigator Sandra R. Eldridge,Ph.D. PathologyAssociatesInternational Phone No. 301624-2036 Fax No. 301663-8994 Email:SREPAISAIC@aol-com
Study Pathologist CarolynMoyer, D.V.M., Diplomate,A.C.V.P. PathologyAssociatesInternational Phone No. 301624-2928 Fax No. 301663-8994
Proposed Study Timetable in-lifSetartDate:To be added by protocolamendment; Day 0 In lifeEnd Date:To be added by protocolamendment AuditedDraftReportDate:To be added by protocolamendment
Regulatory Compliance This studywillbe conducted inthespiriotfGood LaboratoryPractice(GLP) regulations.
Study Number 1132-100 Page 4
Animal Care and Use Statement All proceduresin thisprotocolareincompliance with theAnimal Welfare Act Regulations,9 CFR 1-4. Inthe opinion of the Sponsor and studydirectort,he studydoes not unnecessarilyduplicate any previouswork.
Quality Assurance Not applicable.
Test Materials
Test Material:
N-EtFO-S-E (completedby 3W
Identification: N-ETFOSE
Lot Number:
FM 3929
Purity: Stability:
99.2% > 5 years
Storage
room temp.
Conditions:
Characteristics:waxy solid
PFOS (completedby 3M
PFOS 217
99% > 5 years room temp.
white powder
PFOSA (tobe added by protocol iaiendinent)
PFOSA
Wy-14,643 (tobe added by protocol amendment)
WY
> 5 years room temp. amber waxy solid
Reserve (Archive) Samples A reservesample (approximately5 g)ofeach lotwillbe takenand stored-artoom temperature. These samples willbe transferretdotheSponsor aftercompletionofthe in-lifpehase tobe retained in accordance with 40 CFR 792.195.
Dispositionof Test Material Afterauthorizatiofnrom the Sponsor,any remainingtestmaterialwillbe returnedto:
Marvin Case, D.V.M., Ph.D. 3M CorporateToxicology Building220-2E-02,3M Center St.Paul,Minnesota 55144-1000 Phone No.: 651-733-5180 Fax No.: 651-733-1773
StudyNumber 1132-100 Page 5
Animals
Species: Strain:
Rat Crl:CD*(SD)IGS BR
Source:
C7harleRsiver LaboratoriesI,nc.,Raleigh,NC
Age atinitiatiofnTreatment: Preferabl6e weeks of age,but notmore than 8 weeks of age
Weight atInitiatiofnTreatment: 150 to300 9
Number and Gender:
males
Identification:
liniquiedentificatiboynindividuaclartagsand cagecards
StudyNumber 1132-100 Pa-e 6
Husbandry Housing: Diet:
Singlehoused inhano,ing stainlesssteelwirecages
Teklad 7012 CertifieRdodent Diet.Freshfood willbe providedweekly.
Feed isanalyzedby themanufacturerforconcentrationosf specifiedheavy metals,aflatoxinc,hlorinatheydrocarbons,organophosphates,and specified nutrientsS.pecifiednutrientasnalysesareon fileatR.O.W. Sciences.
water
Tap water,providedad libittuvniaan automaticwateringsystemor water
bottles.The waterisanalyzedatleasttwo timesperyearforcontaminantsand
specifimcicrobes.The resultosf theseanalysesareon fileatR.O.W.
Sciences.
I
Contaminants:
The studydirectoarnd/ortheSponsor have consideredpossibleinterfering substancespotentiallpyresentinanimalfeedand water,includingthetest materialitselofr possiblestructuralrleylatemdaterialsas wellas the items listeidn (2)and (3)above.None ofthesecontaminantsarereasonably expectedtobe presentinanimalfeedor wateratlevelsufficienttointerfere with thisstudy.
Environment:
The targetedtemperaturesarebetween 64 and 79F witha relativheumidity between 30% and 70%. Temperatureand humidityaremonitored continuouslyA. 12-hourlight/12-houdrarkcyclewillbe maintained.Ten or
greaterairchanges/hourwillbe maintained.
Acclimation:
Animals willbe acclimatedtothefacilitfyora minimum of 7 dayspriortothe startofdosing.Animals willbe observedforgeneralhealthand suitabiliftoyr testingduringthisperiod.Animals thatarediseasedorunsuitablfeortesting willbe removed from thestudy.
Randomization:
Usin- computer-0-eneraterdandom numbers witassignmenttogroups,At the time0of randomizationt,heweightvariatioonftheanimalsof each sexused shouldnotexceed 2 S.D. of themean weight,and themean body weights
foreach group ofeach sexwillnotbe statisticadlilfyferent.
Justification: Ratswillbe usedbecauseoftheextensivehistoricdaaltabase,and theFDA requirementsfora rodentspecies.
Study Number 1132-100 Page 7
Group
Designations, Dietary Levels and Scheduled Sacrifice Time Num@ber of-&Fal Rats
Points
Group Number Control
N-ETFOSE
PFOS
(Time Point) (0ppm) 300 100 30 ppm 20 ppm
PFOSA Wy-14,643 TotalNO. 100ppm 1000ppm ofAnimals
1
10
10 10 10
10
10
5
65
(48 hrs)
2
10
55
5
5
5
5
40
(7 days)
3
10
55
5
5
5
5
40
(14 days)
4
10
55
5
5
5
5
40
(1wk recovery
5
10
55 5
5
5
5
40
(4 wk recovery,'
TotalNo. of
Animals
50
30 30 30
30
30
25
225
Dosing Procedures
Method of Administration Dietary.AnimalsinGroups I through3 willreceivetestdietfor48 hours,7 days,and 14 days, respectively.
Animals in Groups 4 and 5 willreceivetestdietfor 14 days followedby a I or 4 week recoveryperiod,respectively.
Reason for Dosing Route The potentialhuman exposure isby theoralroute.
Dose Preparation Before initiatioonf treatmentd,osepreparationofeach testmaterialwillbe mixed. All dose preparationswW be storedatroom temperature.Dose preparationwillbe documented and reported.See Attachment I fortestdietpreparationprocedures.
Study Number 1132-100 Page 8
Retention Sample Samples (approximately100 g)willbe takenfrom thedose preparatioannd storedatroom temperature.Unless used foranalyses,thesesampleswillbe discardedatleast1 month aftercompletionofthein-lifpehase.
Observation of Animals
Clinical Observations Each animalwillbe observedtwicedaily(am. and p.m.)formortalitaynd moribundity; findingswillbe recordedas theyareobserved.
Body Weights Priortotreatment(atrandomization)w,eekly forWeek I through4 weeks of recovery.
Food Consumption Weekly forWeek 1through4 weeks of recovery.
ClinicalChemistry Animals willbe fastedovernightbeforeanimal'sschedulednecropsy;blood willbe collectefdrom ajugularveinintoan EDTA-coated tube.Serum enzyme levelsof alanineaminotransferas(eALT), alkalinephosphatase,aspartataeminotransfemse(AST), cholesteroalnd triglyceridweisllbe determined.
Termination Unscheduled Sacrifices and Deaths Necropsieswillbe done. Animals tobe sacrificewdillbe anesthetizewdith CO,, weighed,and exsanguinated.
Study Number 1132-100 Page 9
Scheduled Sacrifices
Interim Sacrifices At 48 hrs,7 days,and 14 days,animalswillbe fastedovernight,bled forserum samples, anesthetizedwith C021 weighed, and exsanguinated.
NOTE: Two serum sampleswillbe needecl(1)a 0.5ml sampleforclinicaclhemistryand (2)a 1.5n-dsample forcompound levelanalysis.
The abdominalcavityofeach aniirwwillbe opened,theliverwiU be removed and weighed,and liversampleswillbe collectedA.nemql wfflbe discardedafterliver collection.
Terminal Sacrifices AfterI and 4 weeks of recovery,animalswillbe fastedovernightb,ledforserum samples, anesthetizewdith C02, weighed,exsanguinated,and necropsied.
NOTE: Two serum sampleswillbe needed,(1)a0.5 ml sampleforclinicaclhemistryand (2)a 1.5ml sampleforcompound levelanalysis.
Postmortem Procedures
Necropsy The necropsywiU includean examinationof theexternalfeaturesof t@ecarcass;allexternal body orificest;heabdominal,thoracica,nd cranialcavitieso;rgans;and tissues.
Cell ProliferatioTnissue Collectionand Immunohistochemical Evaluation. Representativseamplesof theleftlaterallobeof theliverand any macroscopiclesionsof the liverwillbe collecteadnd preservedinzincfor-malin.
Afterfixatione,ach sampleof livewrillbe deliveretdo: Sandra R. Eldridge,Ph.D. PathologyAssociatesInternational 15 Worman's MRI Court,SuiteI FrederickM,aryland 21701
Study Number 1132-100 Page 10
Proliferaticoenllnuclearantigen(PCNA) evaluatiownillbedone on thesamples. In addition, liversectionpsreparedfrom thesame tissueblock willbe stainedwith hematoxylinand eosin and examined microscopically.
Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately500 mg) oftherightlaterallobeoftheliverwillalsobe collectefdrom selectanimalsand flash-frozeinnliquidnitrogen.See AttachmentH forprocedure. The liver tissuewillbe storedina freezersettomaintain-60 to-80*C untilanalyzedby Covance for pal@toyl-CoA Oxidaseactivity.The liversamplesto be analyzedwillincludeallstudy animals,EXCEPT for the Wy-14,643 animalsand allanimalsfrom the 4-week recovery groups. In additionto thisstudy,samplesfrom a previous3M studywillbe analyzedfor palmitoyl-CoAOxidase activittyh;esesamplesconsistof liversamples from 35 ratsand 35 guinea pigs.
Tissue Collection for Electron Microscopic Evaluation Sectionsof liverfrom allanimalswillbe collectedm,inced to approximatelyone millimeter cubesand placedina fixativaeppropriatfeorelectromnicroscopy. The containersand fixative willbe providedby PAI. Electronmicroscopywillbe performedon one animalperftrat=nt group exhibitintghe highestcellproliferatirveesponseas well as one controlanimalatthe discretioonf theSponsor,from one timepointas wellas the4-week recovery. Thus, EM will be performedon one animalfrom thecontrolN,-ETFOSE (one dose only to be determined), PFOS, PFOSA, and Wy groupsatone of thetimepoints,aswellasthe4 week recovery,fora totalof 10 animals.
Remaining Liver Tissue The remaininglivertissuewillbe frozen and storedat -60 to -80* C for possiblefuture analysis.
Organ Weights At thescheduledsacrificetsh,eliverwillbe weighed.
Histopathology Liverfrom each animalthatisexamined forcellproliferatiwoinllbe stainedwith hematoxylin and eosin,and examined microscopicallfyorhistopathologicchanges.
Study Number 1132-100 Page II
Reports One copy of thedraftreportwillbe senttothe Sponsor. The reportwillincludethefollowing information:
Experimental Design and Methods
Results dose analyses mortality clinicaolbservations body weights body weight changes foodconsumption testmaterialconsumption clinicaplathologyresults palmitoyl-CoAoxidaseactivities macroscopic observations microscopicobservations ultrastructuroablservations cellproliferatiaosnsessments
Record Retention AU raw data,documentation,records,protocol,specimens,and finalreportgeneratedas a resultof thisstudy willbe archivedin thestoragefacilitioefsPAI fora periodof 1 year followingsubmissionof thefinalreportto theSponsor. One year@ftersubmissionof thefinal repom allof theaforementionedmaterialwsillbe senttothe Sponsor and a returnfee willbe charged.Allraw datastoredon magneticmedia willbe retainby PAI.
Study Number 1132-100 Page 12
PROTOCOL APPROVAL
MStaurdvyin'Cas-eD,.V.M., Ph.D. Representative
3M CorporateToxicology
Gary W V,6,fe,Ph.D.,D.AR.T StudyD'irehtdr R.O.W. S nces
/A L2..::m Date
liz /9?
Date
Sandra !Z.Eldridge,Ph.D. PrinciplIenvestigator PathologyAssociatesInternational
Z:/0 @2-2 Date
Study Number 1132-100 Page 13
Attachment: I Test Diet Preparation Procedures
1) Determine the amount of testdiet(feed)thatis to be prepared and weigh out that amount of feed.
2) Calculatethe amount of testarticltehatis needed to preparethe testdiet at the desired concentration.
3) Accuratelyweigh out the necessaryamount of testarticle. 4) Transfertheweighed testarticlteoa containerand add a small volume of acetone to container.
Manually mix to dissolvethe testmaterialadding acetoneas necessary(typicalratioof test materialto acetone is 1 g- 15-20 ml acetone). Visuallyinspecttestmaterial/acetonfeor
I solubilitoyf testmaterial. 5) Preparea pre-mix by ftwsferringthe dissolvedtestmaterialinto4 kg of feed in a Hobart niwng bowl. Mix for 10 minutes. Transferthe premix to a largermixer, add remaining amount of weighed diet,mix for.30 minutes.
Study Number 1132-100 Page 14
Attachment: 11 Collection of Tissue Samples for Biochemical and Molecular Analysis
Because of the extreme instabilitoyf certainenzymes and biomolecules,itis essentialthattissues be harvestedas soon afterdeath as possibleand flashfrozen inunediateliyn liquidnitrogen. Failureto follow theseproceduresmay leadto lossof theentiresample and allthe energiesand resourcesthatwere investedintogeneratingthe samples. Therefore,make every effortto comply with the following:
1) Harvest the tissuesamples as soon as possibleafterdeath. Delays may allow for biodegradationand/orinactivatioonfthedesimd endpoinl
2) Immediatelysubmerse the tissuesample directliynto liquidnitrogen. Dry ice or other alternativewsillnot suffice.Itis importantthatthe tissuebe immersed directlyin liquid nitrogenTransferringitto a dry vessel(orsample containers)uspended in liquidnitrogenwill not suffice.The tissuemay fteezeto thevesselwalland willthenbe impossibleto remove withoutcompletelydestroyingthevessel(orsample container).
3) Be absolutelysureto maintainthetissuefrozen. Itshould be storedin a sealedcontainerat70'OC and shipped or transferreodn dry ice. Ifneeded, the frozensainplecan be fractured (brokenintoportionsfordifferenatpplicationsb)y placingin a cruciblwehich containsliquid nitrogentokeep the sample frozenwhile grinding/fracturing.