Document 3eya1YRGqbLQ39dYO4mg3jq2x

10/29/98 T-6295.16;DT-31 Low LzvelPFOS 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Protocol for Study No. T-6295.16 DT-31 lkw Level PFOS Dose versus Rat Serum and Liver PFOS Projected Start Date: October 29'h,1998 Study Objecdve: The objective of thisstudy isto obtain data from which to construct standard curves of singleoraldose versus serum and liverPFOS concentrationin male and female rats. These curveswillbe used to interpreftutureoralabsorption,distributiomne,tabolism and excretion(ADMIE) studieson POSF-based polymericfluorochemicals(FCs) which may containlow-levelcontaminationwith PFOS. These curveswillbe used to compare serum PFOS concentrationaftera singleoraldose of PIOSF-based polymeric FCs containing known quantitiesof PFOS to expected serum leveland liverlevelincreasespredictedby the standardcurve based on an equimolar dose of the residual.These curves willhelp evaluatewhether an increasein serum and Ever concentrationof PFOS isa resultof metabolicdegradationof the polymer or absorptionof residuals. Research C7ient. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul MN 55133-3220 Sponsor. 3M SpecialtyChemicals Division 3M Center,Building236 SaintPaul MN 55133-3220 Study Locati@on: 3M StrategicAlternativeToxicology Laboratory 3M Center,Building270-SB-181 SaintPaul,MN 55133-3220 Study Director. Andrew M. SeacatPh.D. Sr. Research Toxicologist 3M Medical Dept. / Corporate Toxicology 3M Center Building220-2E-02 SaintPaul,MN 55133-3220 Ph.: 651-575-3161, FAX: 651-733-1773 Study Toxicologist: Deanna Nabbefeld,MS Advanced Research Toxicologist 3M Medical Dept. /Corporate Toxicology 3M Center Building220-2E-02 SaintPaul,MN 55133-3220 Ph.:651-737-1374, FAX: 651-733-1773 Proposed Study 7-imeline. In-LifeStart Date: October 29th, 1998 In-Life End Date: November 19th,1998 Regulatory Compliance. This isa rangefinder study and thus non-GLP. 10/29/98 T-6295.16;DT-31 Low LevclPFOS Test Mate7ial.The sponsor willprovide a sample of PFOS. Analytical documentation of the starting materialwillbe the responsibilitoyf the sponsor. A chemical composition specification sheet willbe kept on file. Identirication: Name: PerfluorooctaneSulfonicAcid, Potassium Salt;CAS # 2795-39-3 Molecular Formula: C&@170S02-K' Lot Number: The lotnumbers willbe maintainedin theraw data. Purity: Documentation willbe kept in on file. Stability: Documentation willbe kept on file. Storage Conditions: Upon receipt,testmaterialwillbe storedtightlysealedatroom temperature. Characteristics: Information on synthesismethods, composition or othercharacteristictshat definethe testmaterialwillbe kept on file. Animals: Species: Strain: Source: Rat Sprague Dawley Harlan Laboratories,Inc. Age at initiationof treatment: 6-8 weeks old Weight at initiationof treatment: appro)dmately 150-200g Number and sex: 75 males,75 females GROUP GROUP GROUP 1:control 2: 3 gg/kg 3: 30pg/kg - 15 male, 15 feinale - 15 male, 15 female - 15 male, 15 female GROUP 4: 1OOgg/kg - 15 male, 15 female Identirication: GROUP 5: 30OAg/kg - 15 male, 15 female ear tag Husban&y.Housing: AR ratswillbe group housed in standard cages. Diet/Water: All ratswillbe provided tap watet and Harlan Teklad LM-485 Mouse/Rat SterilizablDeiet (Harlan Teklad, Madison, WI) ad libitumthroughout the study. 2 10/29/98 T-6295.16;DT-31 Low LcvclPFOS Environment: Environmentalcontrolfsortheanimalroom willbe setto maintaina temperatureof 72 3'F,humidityof30-70%, a minimum of 10 exchanges of room airperhour and a 12 hour fight/darckycle. Dose and Dosing P@ocedures.A Img/mL stocksolutioonf PFOS in2% Tween 80 wiflbe preparedimmediatelypriorto dosing.Serialdflutionosfthestocksolutiownillbe performedto reachtheappropriate concentratiofnoreach dose level.A singledose ofcompound, usinga dose volume of 5 mi dosingsolutio/nkg body weight,willbe administerevdiaoralgavage to ratsingroups 2-5 on day zeroofthestudy.The controlratswiH receiveno treatment. Dose SelectionJustirication: The doses selectefdorthislow dosePFOS studyaredesignedto yieldserum PFOS leveltshatareator nearthelimitof quantificati(o1n5 ppb). Based on previousdata,I mg of ingestedPFOS yieldsapproximately5 ppm PFOS inthe blood. Therefore,usinglineaerxtrapolatio3n,gglkg wflltheoreticauyyieldabout 15 ppb PFOS intheblood. Three jiglkgwas thusselectedas thelow dose. Mixing procedure: A I mg/mi stocksuspensionofPFOS willbe preparedby homogenizing 10 mg PFOS in 10 ml 2% Tween 80 usinga 15 n@dglasstissuegrinder.A seriadlilution wfllbe performedbasedon thefoffowingcalculations: Assume a dosingvolumeof (5ml/kg)( 0.3kg)= 1.5mi/rat 0.3mgl Kg dose group: (0.3mg/kg dose)(0.3 kg rat)/1.5ml/rat= 0.06mg/ml PFOS solutioinn 2% Tween 80 neededDflute6 ml ofthe I mg/n@ solutiotno 100 tnlin2% Tween 80. 0.1mg/kg dose group: (0.1 mg/kg) (0.3kg rat)/1.5ml/rat= 0.02mg/mi PFOS solutionin20/o Tween 80 needed. Dflute33.333 ml of 0.06 mg/ml solutionup to 100 n-Ain20/aTween 80. 0.03 mg/kg dose group (0.03mg/kg) (0.3kg)/ 1.5ml/rat= 0.006 mg/mi PFOS solutionin2% Tween 80 needed. Dilute30 mi ofthe0.02mg/ml solutiounp to 100 mi in2% Tween 80. 0.003 mglkg dose group (0.003)(0.3kg)/1.5H / rat= 0.0006 mg/ml PFOS solutionin2 % Tween 80 needed. Dilute10 ml of the0.006mg/inisolutiounp to 100 ml in2% Tween 80. Dose Verirication: Samples oftheconcentratedI mg/ mi PFOS stock,the0.06mghnl stock(forthe 0.3mg/kg dose group)and the0.02mg/n@ stock(forthe0.1 mg/kg dose group) wHI be analyzedby LCMS fordoseverificatiboynKrisHansen,3M Environmental Lab. Itwillnotbe possiblteo sufficientdliylutethe0.006 and 0.0006 mg/n-Adose solutionasnd stilqluantifythePFOS. Extrapolatiotno thelower dose levelswill 3 10/29/98 T-6295.16;DT-31 Low Lmel PFOS therefore,be performed based on the quantificafioonf the more concentrateddose solutions.All doses willalsobe confirmed by totalorganicfluorineanalysis performed by Dr. Ventakaswarlu Pothapmgada (Dr.V.),3M SCD Lab.Observation ofanimals.ClinicalObservations: Each animal willbe observed dailyformortalityand morbidityand notable findingswillbe recorded. Weekly, each animalwillbe removed from its cage and a record of abnormal findingsor normalitywin be made. Additionalfindingswillbe recorded as they are observed. Body Weights: Each animalwillbe weighed immediatelypriorto dosing,weekly thereafter and immediatelypriorto euthanasia. Sample Collection: Frequency and Number of Animals: Scheduled sacrifices: Five males and fivefemales from each group willbe euthanized on days 1, 4 and 14 post dose. See Table I - schedule. 4 10/29/98 T-6295.16;DT-31 Low LevelPFOS TABLE I -SCHEDULE Nov 1 Nov 2 day 4 post dose NECROPSY 5K 5F each,grps 2 & 3 (20) Nov 8 Nov 15 Nov 9 day 4 post dose NECROPSY 5K5F each, grps 1,4 & 5 (30) Nov 16 1 1 Nov 3 Nov 10 Nov 17 1 Nov 4 Nov 11 Oct 29 Oct 30 day 0 day I post DOSE dose grps2 & 3 NFCROPSY (60) 5 M, 5 F each,grps 2 & 3 (20) Nov 5 Nov 6 day 0 day 1 post DOSE dose grps1 4 NECROPSY i 5 5K5F (90) each,grps 1,4 & 5 (30) Nov 12 Nov 13 day 14 post dose NECROPSY 5 M, 5 F each, grps 2 & 3 (20) Nov 18 Nov 19 day 14 post dose rqECROPSY 5 K 5F c-ack grps 1,4 & 5 1 (30)1 Nov 12 1 Oct 31 Nov 7 Nov 14 Nov 21 1 Method of Simple Collection: At all designated times, animals will be euthanized by C02 and gross necropsyperformed.Duringnecropsy,systemicblood(--5 ml) willbe collectevdiatheabdominalaortaand transferretdo blood collectiotnubes without anticoagulant.Allblood samples willbe allowed to clotfora periodof 15 to 30 minutesatroom temperatureand theclotwillbe spun down ina centrifugaet I100 x g for5 minutes.The serum willbe transferretdo labeled1.5ml rnicrofugteubesand centrifugedagainat2000 x g to remove any remainingredblood cefls.The serum willthenbe transferretdo labeledpolypropylenemicrofugetubesand flash-frozeinn liquinditrogen.Liverswillbe removed,weighed,placedindividualilnyto labeledtubesand flashfrozeninliquidnitrogen. Sample Handling: Samples willtemporarilybe storedina freezersetto maintain-60 to 800C. For analysist,hesesampleswillbe packed indryiceand shippedto: 5 10/29/98 T-6295.16;DT-31 Low LevelPFOS KrisHansen, Ph.D. 3M Environmental Technology and SafetyServices 935 Bush Avenue St.Paul,NIN 55133-3331 Telephone No.: 651-778-608 1,FacsimileNo.: 651-778-6176. The number, type and date of.samples to be generated for analysisare as follows: TABLE Sample Serum Liver 2 - S"PLES Oct 30 20 20 Nov 2 20 20 Nov 6 30 30 Nov 9 30 30 Nov 12 20 20 Nov 19 30 30 Total 150 150 Analysis.Samples willbe analyzed by 3M Environmental for PFOS provided forinclusioninthe finalreport. concentration. Results Willbe Report. Best-fitcurveswillbe obtainedby linearregressionbased on mean serum and liverPFOS concentrationsfor each group. Responsibilides.- Andrew M. Seacat:Study Director,Deanna Nabbefeld: Study Toxicologist Deanna Nabbefeld and Andrew Seacat are responsiblefor dosing the animals, performing the necropsy and collectingand sending tissuesamples for analysis. Kris Hansen, 3M Environmental,willbe responsiblefor the analyticaalnalysis. Andrew Seacatwilldrafta reportand ensurethatitreceivesappropriate3M review before a finaldraftisissued. Signatures.- Dr. Andrew Seacat SeniorResearch Toxicologist Study Director Date 10(lb@ Deann, Nabb@'@:pe:@lIdd.,MS Advanced Toxicologist Study Toxicologist Date 6 11/5/98 T-6295.16;DT-31 Low LevelPFOS Protocol Amendment # 1 Study No. T-6295.16, DT-31 Low Uvel PFOS Dose versus Rat Serum and Liver PFOS Sponsor.Study Locadon: Study Director. 3M Specialty Chemicals Division 3M StrategicAlternative Toxicology Andrew M. Seacat Ph.D. Laboratory This amendment modifiesthefollowingportionsof the protocol: EffectiveNovember 5, 1998 I. Page 2. Animals, Number and Sex: To strengthenthelow-dose portionof the dose response curve,Dose Group 5 was replacedwith a 10 ug/kg dose group. The dose groups will now be as follows: GROUP 1:control - 15 male, 15 female GROUP 2: 3 jig/kg - 15 male, 15 female GROUP 3: 30gg/kg - 15 male, 15 female GROUP 4: 1OOgg/kg - 15 male, 15 female GROUP 5: 10 gg/kg - 15 male, 15 female 2. Page 3. Mixing Procedure: The 0.0 10 mg/kg dosing solutionwill be made by maldng a 10 fold ciuufion of the 0.1 mg/kg dosingsolutiorlTherefore,10 ml of the0.02 mg/mi PFOS solutionin 2% Tween 80 willbe dilutedto 100 ml in2% Tween 80 to yieldthe 0.002 mg/ ml PFOS solutionin 2 % Tween 80 needed. 3. Page 4. Dose Verification: The 0.002 mg/ n@ PFOS solutionin2 % Tween 80 willbe confirmed by totalorganicfluorineanalysisperformed by Dr. Ventakaswarlu Pothapragada (Dr.V.),3M SCD Lab. 4. Page 6. Analysis: Serum and liversamples willbe analyzed by BattelleLaboratoriesfor PFOS concentration.Resultswillbe provided forinclusionin the final report.Shipping Address: BattelleLaboratories Attention:Angela Capers 505 King Avenue Columbus, Ohio 43201 Phone: (614)424-5588, Fax: (614)424 -3268 7 Amendment Avt)rova Signatures: Dr.Andrew Seacat SeniorResearchToxicologist Study Director Deanna Nabbde/l@ MS Advanced Toxicologist Study Toxicologist 11/5/98 T-6295.16; DT-31 Low Level PFOS Date Date 8 04/20/99 T-6295.16;DT-31 Low LevelPFOS Amendment #2 ProtocolAmendment 2 Study No. T-6295.16,DT-31 Low Level PFOS Dose versusRat Serum and LiverPFOS Sponsor. Study Locadon: Study Director.- 3M SpecialtCyhemicalsDivision 3M StrategiAclternativTeoxicologyLaboratory Andrew M. SeacatPh.D. EffectiveApril 26'b,1999 The purposeof thisamendment isto reflectthedecisionof theSponsorto add a dose group of 5mg/kg body weightand a necropsydateof 28 dayspostdoseto theoverall studydesign. Allmodificationasreadditionstotheoriginaplrotocoland Amendment 1. The modificationasreasfollows: 1. Page I Protocol,Proposed Study Timeline: AdditionalProposed Timelinespecifitco Amendment #2: In-LifeStartDate: 3/2/99 In-LifeEnd Date: 5/24/99 2. Page 2 Protocol/Page 1 Amendment Nl, Animals, Number and Sex: The following2 dosegroups willbe added totheprotocol: GROUP 6:vehiclecontrol - 12 nWe, 12 fenwe GROUP 7: 5mg/kg - 20 nWe, 20 female 3. Page 3 Protocol/ Page 1 Amendment #1,Mixing Procedure: A single5mglkg dose of compound willbe administerevdiaoralgavage to ratsin groups7 on day zeroof thestudy.The compound willbe preparedas a 1% (1 mg/mL) uniformsuspensionin2% Tween 80 usinga 15 n-dtissuegrinderA. volume of 5 n@ suspension/ kg body weightwillbe administeredR.e-suspension of solidswillbe performedwith5 strokesofthetissuegrinderpestlebeforeeach sampleisdrawn-up inthesyringefordosing.The 10 controlrats(group6) win be dosedwith 5 mykg 2% Tween 80 (vehiclceontrol). 4. Page 4 Protocol/Page 1 Amendment #1,Dose Verirication: Thevehiclceontroslample(2% Tween80)andtheImg/n@PFOS solutiion2 % Tween 80 willbe confirmedby totalorganicfluorinaenalysipserformedby Dr. VentakaswarluPothapragada(Dr.V.),3M SCD Lab. 5. Page 4 Protocol.Sample CollectionF,requency and Number ofAnimals: Frequency and Number ofAnimals: Scheduledsacrifices: Five males and fivefemalesfrom group 7 and 3 males and 3 femalesfrom group 6 willbe euthanizedon days 1,4, 14 and 28 postdose.See Table I schedule. Page Iof3 TABLE I - SCHEDULE April26 day 0 DOSE grps6 & 7(64) April27 day Ipost dose NECROPSY (16) April28 May 2 May 3 May 4 May 5 04/20/99 T-6295.16;DT-31 Low LevelPFOS Amendment #2 April29 April30 day 4 post dose NECROPSY (16) May 1 May 6 May 7 May 8 May 9 May 16 May 23 May 10 day 14 postdose NECROPSY (16) - May 17 May 24 Day 28 postdose NECROPSY (16)1 May 11 May 18 May 12 May 19 May 13 May 20 May 14 May 21 May 15 May 22 Page 2 of 3 Amendment Aipiprova Signatures: Andrew M. Seacat,Ph.D. Senior Research Toxicologist Study Director Deanna Nabbefell,MS Advanced Toxicologist Study Toxicologist 04120/99 T-6295.16;DT-31 Low LevelPFOS Amendment #2 Date /.;1@3/C/Cf Date Page 3 of 3