Document dVV7dXdG0ZJMynoEw53BrKpe

April27,2000 One GenerationReRroduction Study ofPFOS Mevalonic Acid/CholesteroClhallengeand NOEL InvestiL-atiionnRats Draft Study Outline Study ObjectivesT:hisstudywillserveasa follow-upstudytoCombined Oral(gavage FertilitDye,velol2mentaland Perinatal/PostnaRteaplroductionToxicityStudy of PFOS in Rats(ArgusProtocol418-008,3M studynumber T-6295.9).The objectiveosf thestudy areasfollows: 1) todeterminewhetherornotco-administratiofnmevalonicacidor cholesteroclan preventor antagonizetheadversematemal and fetaleffects(reducedmatemal body weightgain,increasedpercentstillbordne,creasedbirthweightand decreasedpup survivalo)bservedinthepreviousstudy;and 2) torefmetheno observedeffectlevel(NOEL). GLP Status:A GLP studywithappropriatQeA auditsa;signedQUA statementobe includedinthe fmal report. Animals: Female HarlanCD rats. Dose Groups: Thirteen:VehicleControl;Mevalonic Acid Control;CholesteroClontrol; 0.4mg/kg PFOS; 0.8mg/kg PFOS; I mg/kg PFOS; 1.2mg/kg PFOS; 1.6mg/kg PFOS; 1.6mg/kg PFOS + mevalonicacid;1.6mg/kg PFOS + cholesterol2;mg/kg PFOS; 2 mg/kg PFOS + MevalonicAcid;2 mg/kg PFOS + cholesterol Number Per Group: 22-28ingroups1-3& 8-13;16-20ingroups4-7.TOTAL: 246312 Test Article:Compound wiH be furnishedby the Sponsor who isresponsiblefor compound identitaynd purity.Reservesample to be retained. Compound AdniinistrationF:emales only,dailyoralintubationd,ose volume 5ml/kg. The vehicle(0.5% Tween 80 indeionizedwater)win be preparedweekly. PFOS willbe prepareddailyas a suspensionindeionizedwaterwith0.5% Tween 80. Mevalonicacid willbe preparedfreshtwicedailyindeionizedwater.Cholesterowlillbe preparedfresh dailyinoliveoil.Compound administratiownillstart6 weeks priortomatingand continuethroughday 4 postnatal(seetable1). Page Iof5 D.J.Luebker April27,2000 Table I - Dose Groups & Dosing Schedule Dose Groups lotdosing Dosing schedule 2nd dosing Name N 1 VehicleControl 30-40 2 MevalonicAcid 22-28 Control CholesteroClontrol 22-28 Deionized H20 500 mg/kg Mevalonic Acid *500 mg/kg 1/2 hr after1 0.5 % Tween 80 0.5 % Tween 80 0.5% Tween 80 Cholesterol 4 0.4mg/kg PFOS 5 0.8mg/kg PFOS 6 1 mg/kg PFOS 7 1.2mg/kg PFOS 8 1.6mglkg PFOS 9 1.6mg/kg PFOS + Mevalonic Acid 10 1.6mg/kg PFOS + Cholesterol 16-20 16-20 16-20 16-20 22-28 22-28 22-28 NA NA NA NA NA 500 mg/kg MevalonicAcid *500 mg/kg Cholesterol 11 2 mg/kg PFOS 12 2 mg/kg PFOS + Mevalonic Acid 13 2 mg/kg PFOS + , Cholesterol Cholesteroldosingmay 22-28 NA 22-28 500 mg/kg MevalonicAcid 22-28 *500 mg/kg Cholesterol occurbeforeor afterPFOS orTween 0.4mg/kg PFOS 0.8mglkg PFOS 1 mg/kg PFOS 1.2mg/kg PFOS 1.6mg/kg PFOS 1.6mg/kg PFOS 1.6mg/kg PFOS 2 mg/kg PFOS 2 mg/kg PFOS 2 mg/kg PFOS 80 dose. 3rddosing 5 hr afterTd DeionizedH20 500 mg/kg MevalonicAcid NA NA NA NA NA NA 500 mglkg MevalonicAcid NA NA 500 mg/kg Mevalonic Acid NA Samples of dosing preparationswillbe collectedonce during the sixweeks priorto mating, once during gestationand once during thepostnatalperiod. A portionof each of these samples willbe frozenand shipped to theSponsor forpossibleanalysis.A backup sample of each willbe retainedby thetestingfacilityR.ecords willbe maintained on how dosing preparationswere made. Chnical Observations: Twice daily- approximately one hour afterdosing and again 4-6 hours later. Body Weights: Weekly except when females are cohabitingwith males during mating. Food Consumption: Weekly except when females are cohabitingwith males during mating. Mating: After 6 weeks of compound administration, one male (Argus breeder colony) willbe co-inhabitedwith each female. A female willbe determined as mated upon the presence of sperm positivevaginalsmear. The day of mating shallbe considered day 0 of gestation. Pre-Birth Sacrificeand Examination: Just priorto delivery(morning of day 21 of gestation)6-8 pregnant females in groups 1-3 and 8-13 willbe sacrificedby C02 asphyxiationand cesarean-sectionwillbe performed. A terminalbody weight foreach dam and a pooled terminalbody weight foreach littewrillbe obtainedand recorded. Animals willbe necropsied and any gross lesionswillbe describedand recorded. Page 2 of5 D.J.Luebker Apria27,2000 Serum/plasma and Ever samples willbe taken from each dam and her pups (pup samples pooled by littera)nd milk willbe collectedfrom each dam. Liver and serum willbe analyzed forLDL, HDL, totalcholesteroland triglyceridePsl.asma mevalonic acid levelswillbe measured. Milk willbe analyzed fortotalcholesterolAdditionalliverand serasamples willbe frozen(-20'C) and shipped on dry iceto Kris J.Hansen, Ph.D.,3M Environmental Technology and Safety Services,935 Bush Avenue, Building 2-3E-09, St. PauL MN 55133-3331 forPFOS analysis.A portionof each liversample willbe flash frozen in liquidnitrogenand shipped on dry iceto the Sponsor forpossiblefuture analysis(i.e.l,iveradeninenucleotides& IMG-COA reductaseinhibition)A.dditional livertissuewillbe collectedand fixedin gluteraldehydeforpossiblefutureanalysisby electronmicroscopy. Female Parturition:Remaining ratswillbe allowed to delivertheirlitters. Pup Examinations: Pups willbe counted, sexed,weighed and observed for moribundity/mortahty on postnataldays 0, 1,2, 3 and 4. Rats Found Dead or Moribund: Rats thatdie or are sacrificedbecause of moribund conditionor abortionwillbe examined forcause of death or moribund conditionon the day the observationis made. Aborted fetusesand/or in-uteropups willbe examined to the extentpossible.When possible,the followingspecimens willbe collectedand analysis performed: Rats willbe necropsied and any gross lesionswillbe describedand recorded. Serurn/plasmaand liversamples willbe taken (individuallfyoreach dam and her pups pup samples pooled by litterL)i.ver and serum willbe analyzed forLDL, HDL, total cholesteroland triglyceridePsl.asma mevalonic acid levelswillbe measured. Milk will be collectedfrom each dam and analyzed fortotalcholesterol Additionalliverand sera samples willbe frozen(-20'C) and shipped on dry iceto KrisJ.Hansen, Ph.D., 3M Environmental Technology and SafetyServices,935 Bush Avenue, Building 2-3E-09, St. PauL MN 55133-3331 forPFOS analysis.A portionof each liversample willbe flash frozenin liquidnitrogenand shipped on dry iceto the Sponsor forpossiblefuture analysis(i.e.l,iveradenine nucleotides& HMG-COA reductaseinhibition)A.dditional livertissuewillbe collectedand fixed ingluteraldehydeforpossiblefutureanalysisby electronmicroscopy. Myocardial tissuewillbe collectedforpossiblehistologyand electronmicroscopy. Day 4 Postnatal Sacrificeand Examination: Sixteento 20 dams in each dose group and allof theirpups wiU be sacrificedby C02 asphyxiationon day four postnatal A terminal body weight foreach dwn and a pooled terminalbody weight foreach littewrillbe obtainedand recorded. Animals willbe necropsiedand any gross lesionswillbe described and recorded. Serum/plasma and liversamples willbe taken Erom each dam and her pups (pup samples pooled by sex foreach littera)nd milk samples willbe collectedfrom each dam. Liver and serum willbe analyzed forLDL, HDL, total cholesteroland triglyceridesP.lasma mevalonic acid levelswillbe measured. Milk will be collectedfrom each dam and analyzed fortotalcholesterol Additionalliverand serum samples willbe frozen (-20"C) and shipped on dry iceto Kris J.Hansen, Ph.D., Page 3 of 5 D.J.Luebker Apr.U27,2000 3M EnvironmentalTechnology and SafetyServices9,35 Bush Avenue, Building2-3E09,St.PauL MN 55133-3331 forPFOS analysisA. portionofeach liversample willbe flashfrozeninliquidnitrogenand shippedon dry icetotheSponsorforpossiblefuture analysis(Le.,liveradeninenucleotide&s HMG-COA reductaseinhibitionA)d.ditional livertissuewillbe collecteadnd flxedingluteraldehydfeorpossiblefutureanalysisby electronmicroscopy.Myocardialtissuewillbe collectefdrom each dam and fourof her pups (2 males and 2 females)forpossiblehistologyand electronmicroscopy. Table 2 - Scheduled Sacrifice(sN damstgroup/Sacrifice) Grp 1 2 3 4 5 6 7 8 9 10 11 12 13 Group - Nanie 9 > Pre"Birffi6-8 (dy Dy 4 16-20 Postnatal TOTAL 22-28 N / grp -b=0o >4> 2 6-8 16-20 22-29 2 0= 6-8 16-20 22-29 R0. 16-20 16-20 C6 00 16-20 16-20 0 0 16-20 16-20 16-20 16-20 rA 6-8 16-20 22-28 00C+4 R 6-8 16-20 22-28 + 00 Go -tc Ot R- I C4 6-8 r,8 o .a2 a 0 "a tg o + C4 + 6-9 6-8 16-20 16-20 16-20 16-20 22-28 22-28--rF-28- 22-228081 Page 4 of5 D.J.Luebker Study Director: Deanna J.Luebker,M.S. 3M CorporateToxicology 3M MedicalDepartment 3M CenterBldg.220-2E-02 SaintPauL MN 55144-1000 Telephone 651n37.1374 FAX 65in33.1773 AlternativeStudy Director: John Butenhoff,Ph.D.,DABT, CIH 3M CorporateToxicology 3M MedicalDepartment 3M CenterBldg.220-2E-02 SaintPauL MN 55144-1000 Telephone 651n33.1962 FAX 651n33.1773 In-LifePhase Investigator: Argus AnalyticalPhase Investigator: 1)Argus 2)KrisJ.Hansen,Ph.D., 3M EnvironmentalTechnology and SafetyServices, Bldg.02-3E-09 935 Bush Avenue, Building2-3E-09, St.Paul,MN 55133-3331 Telephone 651/778.6018 FAX 651n78.6176 April27,2000 Page 5 of5 D.J.Luebker