Document q3EZxYjqdKpEZLdy7zr6rpeQj

FINAL REPORT PROTOCOL 418-013 ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.12 FINAL REPORT DATE: 24 JUNE 1999 00780 I-- PROTOCOL 418-013 ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.12 TABLE OF CONTENTS SUBJECT PAGE I. SUMMARY AND CONCLUSION - A. Methods - B. Results 12 C. Conclusion "4 Il. DESCRIPTION OF TEST PROCEDURES 1-1 A. Conduct of Study 11 A. Sponsor 11 A2. Testing Facility 1-1 A3. Study Number 11 A4. Sponsor's Study Number 1-1 AS. Purpose of the Study 11 AS. Study Design 1-1 i 00781 SUBJECT AT. Regulatory Compliance A8. Ownership of the Study A. Study Monitor A10. Alternate Study Monitor A.11. Study Director A12. Technical Performance A.13. Report Preparation A.14. Report Review A.15. Date Protocol Signed A.16. Dates of Technical Performance AA7. Records Maintained B. Test Aticle Information B.1. Description B2. LovBatch Number B.3. Date Received and Storage Conditions 8.4. Special Handling Instructions B.S. Analysis of Purity C. Vehicle Information Cu. Description C2. Lot Numbers C3. Date Received and Storage Conditions C4. Special Handling Instructions i PAGE 11 2 1-2 Ih2 2 12 2 12 1-2 3 3 1-3 1-3 3 3 3 4 4 4 4 14 4 000782 SUBJECT C5. Analysis of Purity D. Test Atticle Preparation and Storage Conditions D1. Sample Information D2. Analytical Results E. TestSystem E1. Species E2. Strain E3. Supplier (Source) E4. Sex ES. Rationale for Test System E6. Test System Data E7. Breeder Male Rat Data E:8. Method of Randomization ES. System of Identification F. Husbandry F.1. Research Facility Registration F.2. Study Rooms F3. Housing F4. Lighting F5. Sanitization F6. Feed F.7. Feed Analysis Ly PAGE 14 14 15 15 115 Is 1-5 15 15 16 1-6 16 16 7 7 7 7 7 7 1-8 1-8 18 000783 SUBJECT F.8. Water PAGE 1-8 F.9. Water Analysis 1-8 G. Methods 1-9 G.1. Dosage Administration 1-9 G.2. Rationale for Dosage Selection 1-9 G.3. Route of Administration 1-9 G.4. Rationale for Route of Administration 1-8 G.5. Frequency of Administration 1-9 G.6. Length of Study 1-9 G.7. Method of Study Performance 1-10 G.8. Gross Necropsy I-11 G.9. Statistical Analyses 1-13 I. RESULTS n-1 A Mortality, Premature Delivery, Clinical and Necropsy Observations 1li-1 A.1. Mortality and Premature Delivery n-1 A.2. Clinical Observations n-1 A.3. Necropsy Observations n-1 B. Body Weights, Uterine Weights and Body Weight Changes n-1 B.1. Precohabitation n-1 B.2. Gestation n-2 C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values n-2 C.1. Precohabitation v m-2 00784 SUBJECT C2. Gestation D. ObCsaeesravraetaino-nSsectioning, Litter and Fetal Gross External D.1. Day 15of Gestation D.2. Day 21 of Gestation REFERENCES APPENDIX A - REPORT FIGURES Figure 1. Body Weights - Precohabitation Figure 2. Matemal Body Weights -- Rats Caesarean-Sectioned oon Day 15 of Gestation Figure 3. oMantDeranyal21BoofdyGeWsetiagthiotns -- Rats Caesarean-Sectioned APPENDIX B - REPORT TABLES Table 1. Clinical and Necropsy Observations `Summary Table 2. Body Weights - Precohabitation - Summary Table 3. Body Weight Changes - Precohabitation - Summary Table 4. Maternal Body Weights and Gravid Uterine Weights Gestation - Summary Table 5. Maternal Body Weight Changes - Gestation - Summary Table 6. Table 7. Absolute Feed Consumption Values (g/day) Precohabitation - Summary Relative Feed Consumption Values (g/kglday) - Precohabitation - Summary Table 8. GMeastteartniaolnA-bsSoulmumtearFyeed Consumption Values (g/day) - PAGE 2 103 n-3 m3 in-4 A1 A2 A3 B-1 B4 B-5 B-6 B-10 B-12 B-13 B14 v C60785 SUBJECT PAGE Table 9. Matemal Relative Feed Consumption Values (g/kg/day) - Gestation - Summary B-16 Table 10. Caesarean-Sectioning Observations - Summary B-18 Table 11. Litter Observations (Caesarean-Delivered Embryos or Fetuses) - Summary B21 Table 12. Clinical Observations - Individual Data B22 Table 13. Necropsy Observations - Individual Data B-29 Table 14. Body Weights - Precohabitation - Individual Data B34 Table 15. Matemal Body Weights - Presumed Gestation - Individual Data B-44 Table 16. Feed Consumption Values - Precohabitation - Individual Data B49 Table 17. Maternal Feed Consumption Values - Presumed Gestation - Individual Data B-54 Table 18. Caesarean-Sectioning Observations - Individual Data B59 Table 19. Litter Observations (Caesarean-Delivered Embryos or Fetuses) - Individual Data B63 Table 20. Embryonal Vital Status or Fetal Sex, Vital Status and Body Weight - Individual Data B67 Table 21. Placental Weights - Individual Data 8-73 APPENDIX C- PROTOCOL AND AMENDMENT C1t0C-34 APPENDIX D - DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY D1 APPENDIXE - TEMPERATURE AND RELATIVE HUMIDITY REPORTS E-110E-3 APPENDIXF - HISTORICAL CONTROL DATA F-110 F-13 vi 00786 SUBJECT APPENDIX G - STATEMENT OF THE STUDY DIRECTOR APPENDIX H - QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT PAGE G1 H-1to H5 vii 00787 418-013:PAGE I-1 TITLE: ORAL RATS (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN APRRGOUTSOCROELSNEUAMRBCEHR:LAB4O18R-A0T1O3RIES, INC. SPONSOR'S STUDY NUMBER: T-6295.12 I. SUMMARY AND CONCLUSION A. Methods aEsigshitgynevdirgtionffiveemadloesaCgre:gCrDouBpsR(VGArFo/uPpIsu|sthr(oSupgrhagVu),e-1D6awrlatesy)perratdsowseargee group. The rats were administered the test article, PFOS (FC-95), or the vehicle, 0.5% (R.0. DTewieoneinzed80WaitnerR)e,veorraslelyO(svmiaosgaivsagMee)m,bornacneedaPirloycbeesgsiendniDnegio4n2izdeadysWaprtieorr to cohabitation through either day 14 or 20of presumed gestation (DG 14 or DG 20). Dosages were 0 (Vehicle), 0.1, 0.4, 1.6 and 3.2 mg/kg/day. dosage volume was 5 mL/kg. The Tclhineicraaltssiwgenrseofobefsfeercvtesdoffotrheviatbeisltitayrtaitclleeabsetfotrweicaenedacaphpdraoxyiomfattehleysotundeyhaonudr faofrter dosage dosage and on the period and day sacrificed. Body weights were recorded at sacrifice. Feed consumption values were daily during the recorded weekly to cohabitation and daily during presumed gestation. Urine and fecal samples were collected one day prior to initiation of cohabitation ftorotzheenfaonlldowsihnigppmeodrntiontgheanSdpoDnGssor6ftoor 7a,na1l4ystios.15Blaonodd2s0atmopl2e1.s wSearmeplcoelslewcetreed coennttrhiefudgaeyd caonhdabtihteatsioenruwmaswaisnitfiraotzedenanadndonshDipGpsed7,to15thaenSdp2o1n. soBrlofoordawnaalsysis. Rats were sacrificed on DG 15 or 21, as described below, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. A liver section and the milk-secreting glands from the axillary, thoracic, abdominal and inguinal a. Detailed descriptions of all procedures used in the conduct of this study (arPeRpOrTovOiCdeOdLiAn NthDe AapMpEroNpDrMiaEtNeTs)e.ctions of this report and in APPENDIX C 030788 418-013:PAGE 1-2 regions of analysis. each dam were collected, frozen and shipped to the Spansor for Eight randomly selected female rats, with a confirmed date of mating, per dosage group were Caesarean-sectioned on DG 15. The gravid uterus was excised corpora and weighed. Rats were examined lutea, implantation sites, viable and for number and distribution of nonviable embryos. A sample of the `amniotic fluid of each viable viable embryo was removed embryo was collected and pooled from the uterus with the attached by liter. placenta Each and pooled by litter. These samples were frozen shipped to the Sponsor for analysis All remaining female rats in DG 21 or estimated DG 21. each The dosage group were Caesarean-sectioned gravid uterus was excised and weighed. on dIinsdtirviibduutailonploafcceontraplorwaeilguthetas, wiemrpleanrteactoridoend.siteRs,atlsivwe earned edxeaamdinfeetdusfeors naunmdbeearrlaynd and late resorptions. Each fetus was removed from the uterus, weighed and weexraemicnoelldecfotredsewxheanndpogsrsoisbslee,xtaenrdnaploaollteerdatbiyonlsi.tter.SaPmlpalceesntoafetwheeraemnciooltliecctfelduid and litter pooled by litter. and centrifuged. Blood samples were collected All samples were frozen and from each shipped to fetus, pooled the Sponsor by for analysis. Lung and five litters liver samples were per dosage group. collected Sections from from three fetuses per litter from two to the half of the lungs and one lobe of the liver were shipped to Pathology Associates Intemational, Durham, tNhoertohthCearrollaitneraa,lfloorbpeososfitbhleelfiuvteurrweeervealfuraotzioenn.anTdhsehoitphpeerdhtaolftohfe tShpeonlusnogrsfaonrd analysis. The livers of the remaining fetuses, plus any remaining liver from the three csaerleccatsesd(faentdushees,adw)eorfeeCaocllhecfteetdu,s pwoaoslefdrobzyenliattnerdasnhdipfproezden.to tThheeSrpeomnasionrinfgor analysis. B. Results No deaths or premature deliveries rats (one each in the 0.1, 0.4 and were attributed to PFOS treatment. 1.6 mg/kg/day dosage groups) were Three sacrificed because the eye was injured during orbital sinus bleeding. One rat in the vehicle control group did not have the study. Two rats in the a confirmed mating date and 3.2 mg/kg/day dosage group delivered delivered on on day DG 66 21. of All other rats survived to scheduled sacrifice. C00789 418-013:PAGE |-3 wAlelraedvceornssiedcelrienidcaulnorbesleartevdattioonthsedtuersitngarttihcelepraencdohnaobigtraotsisonleasnidongsewstearteiornevpeearlieodds by necropsy. Body weight gains for the entire precohabitation period (DSs 1 to 42) were 88.8%, 80.8%, 66.3% and 17.4% of the control group value in the 0.1, 0.4, 1.6 a96n.d33%.,28m3g./6k%g/adnady 8d5o.s3a%geofgrtohuepcso,ntrreoslpegcrtoivueplyv.alBueodinytwheeisgehtfsouwrerreesp9e8c.t0i%ve, dosage groups on DS 42. As the result of random selection for assignment to Caesarean-sectioning on either DG 15 or DG 21, groups assigned to Caesarean-sectioning on DG 15 weighed less Reflecting the on DG 0 random than groups assigned to Caesarean-sectioning on selection and differences in average body weights DG 21. of the two subsets, as well as the differed in the two subsets. relatively small group sizes, body weight In both subsets, body weight gains were gains reduced on DGs 010 7 in groups administered 0.4 mg/kg/day and higher dosages of PFOS, after which body weight changes did not demonstrate a clear dosage-dependent pattern. Absolute (g/day) and relative (g/kg/day) feed consumption values in the 0.4, 1.6 and 3.2 mg/kg/day dosage groups in each week of were the. reduced prerleactoihvaebfieteadticoonnpseurmiopdt.ionRevflaelcuteisngwetrheesreeedfufceectdsfoofrtthhee teensttiraertpicrleec,oahbabsiotlauttieoannd period (DSs 110 42) in the 0.4, 1.6 and 3.2 mg/kg/day dosage groups. rGerdouucpesdaadbmsionliustteeraendd0r.e4lamtgiv/ekgf/edeadycaonnsduhmipgthieorndvoaslaugeessdoufritnhge ttheestfiarrsttiwcleeehkadof trheedugceestdaftoironthpeerrieodm.ainAdbesroloufttehefedeodscaognesupemrpitoidoninvtahleue0.s4coanntdin1u.6edmgto/kbge/day dosage groups assigned to DG 15 Caesarean-sectioning and in the 3.2 mg/kg/day dosage group for rats assigned to either DG 15 or DG 21 Caesarean-sectioning. No Caesarean-sectioning or litter parameters were affected by dosages of the test article as high as 3.2 mg/kg/day administered to rats Caesarean-sectioned on DG 15. tThhee3l.i2ttemrga/vkegr/adgaeysdfoosraigmpelagnrtoautpioCnas,eslaitrteerasni-zseesctainodnelidveofnetDuGses21w.erVealrueedsuwceedrein below the ranges observed historically at the Testing Facility. Fetal body weights wseecrteioanlisnog roerdluicteerdpianrtahmeet3e.r2smwg/ekrge/dafafyecdtoesdabgyedgorsoaupg.esNoof tohtehetresCtaaerstaicrleeaans.- CCO790 418-013:PAGE |-4 high fetal as 3.2 mg/kg/day administered gross extemal alterations were to rats Caesarean-sectioned observed. on DG 21. No C. Conclusion TphhaermpaucropkoisneetoifctshiosfsPtuFdOy,Saisn sFtoatgeedneinratthieonprdotaomcosl,anwdasF1togeevnaelruaattieonthfeetuses following PFOS treatment to female rats during premating and gestation. This reports includes study data from the in-life portion which was conducted at Argus Research Laboratories, Inc. The PK samples that were collected during the inlife portion of the study were sent to the Sponsor for analysis and will be reported sceopmabriatneelyt.he Iitni-slitfehererseuslptosnswiibtihlitthyeoafntahlyetiScpaolnsreosru,lt3sMinCtoorapfoirnaaltephTaorxmiacoclookgiyn,ettioc report. The results for this study mimic those reported in the Combined Oral S(tGuadvyagoef)PFFerOtiSlitiyn, RDaetvsel(oAprgmuesntRaelsaenadrcPherLianbaotraalt/oProisetsn,atIancl.,RePprrotoodcuoclti4o1n8-T0o0x8i)c,ity reported 10 June 1999. Mats J Yel ler yw Mildred S. Christian, Ph.D, Fellow, ATS Date Executive Director of Research / r - ede, Tuff Alan M. Hoberman, Ph.D., DABT Date Director of Research nd G. York, PRT, DABT ASstsuodcyiaDitreecDtiorrector of Rgsearch and 25Date 000791 Il. DESCRIPTION OF TEST PROCEDURES 418-013:PAGE II-1 A. Conduct of Study: AA. Sponsor: 3M Corporate Toxicology, 3M Center, Building 220-2E-02, St. Paul, Minnesota 55144-1000 A.2. TestingFacility: Argus Research Laboratories, Pennsylvania 19044-1297 Inc., 905 Sheehy Drive, Building A, Horsham, A3. Study Number: 418-013 A4. Sponsor's Study Number: T-6205.12 AS. Purpose of the Study: TFohegepnuerrpaotsieonofdtahmisssatnuddyFw1agsentoereavtailounatfeettuhseesphfaorllmoawcionkginPeFtOicSstorfeaPtFmOenSt ionf Cr:CDBR VAF/Plus female rats during premating and gestation. AS. Study Design: `The requirements of the U.S. as the basis of study design. Food and Drug Administration (FDA) were used A7. Regulatory Compliance: The study was conducted in compliance with Good Laboratory Practice (GLP) rMiengiusltartyioonfsoHfeatlhteh aU.nSd.WFeolofdaraen(dMDHrWu)gAdamnidnitshteraEtuironop(eFaDnAE)c?otnhoemiJcapCaonmemsuenity q(uEalEiCt)y.or Tihnteergreitwyeorfetnhoe sdteuvdiya.tioQnusalfirtoymAsthseurGaLnPcereUgnuiltaftiinodnisngtshadtearfifveecdtefdrotmhethe inspections provided to during the conduct of this study are documented and have the Study Director and the Testing Facility Management. been 00792 418-013:PAGE II-2 AB. OwneorftshehStiudpy: The Sponsor owns the study. All raw data, analyses, reports and preserved tissues are the property of the Sponsor. AS. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A10. Alternate Study Monitor: AndrewM. Seacat, Ph.D. A.11. StudyDirector: Raymond G. York, Ph.D., DABT (Associate Director of Research) A.12. Technical Performance: John F. Bamett, B.S. (Director of Laboratory Operations) Margaret M. Martin (Research Associate) Corrie L. Pabst (Quality Control Associate) A.13. ReportPreparation: Raymond G. York, Ph.D., DABT Jo Ann Frazee, M.S. (Study Coordinator) Susan K. Bradshaw, B.S. (Data Management Specialist) Karen G. Parker, AA. (Report Administrator) A.14. Report Review: Mildred S. Christian, Ph.D., Fellow, ATS (Executive Director of Research) Allan M. Hoberman, Ph.D, DABT (Director of Research) A.15. Date Protocol Signed: 9 November 1998 60793 418-013:PAGE Il-3 A.16. Dates of Technical Performance: Rat Arrival Date Dosage Period (42 days prior to cohabitation until DG* 14 or 20) Cohabitation Period Caesarean-Sectioning Period (DG 15) Caesarean-Sectioning Period (DG 21) 10 NOV 98 16 NOV 98-21 JAN 99 27 DEC 98 PM - 01 JAN 99 AM 12 JAN 99 - 13 JAN 99 18 JAN 99 - 22 JAN 99 A.17. Records Maintained: `The original report, raw vehicle components are data and retained reserve samples in the archives of of the Argus bulk test article and Research Laboratories, Inc. one Any year preserved tissues are after the mailing of the retained in drat final the archives of the Testing report, after which time the Facility for Sponsor will decide their final disposition. discarded at the Testing Facility. All unused prepared formulations were Unused bulk test article wil be returned to the `Study Monitor upon completion of all work with the test article. B. TestArticle Information: B.1. Description: PFOS (FC-95) - an off-white powder B.2. Lot/Batch Number: 217 (Expiration date: May 2000) B.3. Date Received and Storage Conditions: The test article was received on 21 October 1998, and stored at room temperature. B.4. Special Handling Instructions: Standard safety precautions (use of protective clothing, gloves, dust-mist respirator, safety handling the bulk goggles or test article safety glasses and a face-shield) and prepared suspensions. were taken when a. DGis used as an abbreviation for day of (presumed) gestation. 00791 418-013:PAGE 114 BS. Analysis of Purity: Information regarding the identity, composition, strength and purity of the test article is on file with the Sponsor. C. VehicleInformation: C.1. Description: 0.5% Tween 80 in Reverse Osmosis Membrane Processed Deionized Water (R.O. Deionized Water) C.2. LotNumbers: Tween 80 - M29477 and MO3H0S C3. Date Received and Storage Conditions: The Tween 80 was received from J.T. Baker, Phillipsburg, New Jersey, on 17 September 1998 (Lot Number M29477) and 3 December 1998 (Lot Number MO3HOS), and available from stored at room temperature. The R.O. deionized a continuous source at the Testing Facility and is water is maintained at room temperature. C.4. Special Handling Instructions: Standard safety precautions respirator, safety goggles or (use of protective clothing, gloves, dust-mist safety glasses and a face-shield) were taken when handing the vehicle components and prepared vehicle. C5. Analysis of Purity: Neither the Sponsor nor the Study Director was aware of any potential contaminants likely to be present in the vehicie that would interfere with the results of this study. D. TestArticle Preparation and Storage Conditions: Suspensions of PFOS were prepared daily at concentrations of 0, 0.02, 0.08, 0.32 and 0.64 mg/mL. Prepared formulations were stored at room temperature. 00795 DA. Sample Information: 418-013.PAGE Il-5 [`sComncmenotrsateion Toa ue [Rives [SStoreagenrShiTMpmng [sSupoensrorte T[aSeis TE NOSVE [BlrulkoevTeeelsst)TMAi=de To i2s0n0oNu8s8n | commas[mTesttingrFaTMciy 2008N% |asuovas| RVeehsiecrlveeComponents Tween 80 RO. deionized Sm water|Smi. | | 1102DNEOCV9968" | 12NOVS8 | Room Room temperature temperature |ArTcehstiivnegsFaciity | Testing Faciity | 2275 JNaOnV9998 | 34 Nova6 a `Daucphicsaettswsaasmpslehs wiafroperaapnkaelenysifdsr.omTTheerfermstaiannidnagstsarmepglaersawteorneornettahieAnreGcdahyaitvpetrsneepaTresetdingOnFsacSiatyriaes a hbaeckSuppos.nsoBrackup samples are sored frozen (70C or below) and wil be discarded at te regent of b.. LLoott NNuummbbeerr M2O0S4H7O7S.. D.2. Analytical Results: Data verifying the stability of the conditions of administration and test article in the vehicle for 48 hours the stability of the bulk test article are under on file the with the Sponsor. Homogeneity of prepared formulations is on file with the Sponsor. Results of the of this report. concentration analysis were not available at the time of the writing E. TestSystem: EA. Species: Rat E2. Strain: Cri:CDBR VAF/Plus (Sprague-Dawley) EJ. Supplier (Source): Charles River Laboratories, Inc., Raleigh, North Carolina Ed. Sex: Female (Note: not considered Male rats were part of the Test used only System.) for the purposes of breeding and are 000796 418-013:PAGE I-6 ES. Rationale for Test System: The Crl:CDBR VAF/Plus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat was used in the reproductive and TdeesvteilnogpmFeacnitlailtyto"x;icaintyds3t)udtihees;te2s)t hairstticolreiciasl pdhaatramaacnodloegxipcearlileyncacetiexviestinatthtehe species and strain. E6. TestSystem Data: Number of Rats Approximate Date of Birth Approximate Age at Arrival Weight (g) on the Day After Arrival Weight (g) at Study Assignment 122 07 SEP 98 65 days 193-223 208-224 E.7. BreMae led Rate Datr a: Number of Rats Approximate Date of Birth Approximate Age at Arrival Weight () on the Day After Arrival Weight (g) at Cohabitation 112 13 JAN 98 78 days 300- 356 558-871 E8. Methodof Randomization: Ugepnoenraatrreidvarl,anradtosmweunrietsa.ssAiftgenredactcoliimnadtiivoind,uavlirhgoiunsfienmgaolne the rats basis were of computerselected for study on the basis of physical appearance and body weight recorded during Va)c,cli1m6atriaotns.perFedmoaslaegeragtrsowuepr,eusaisnsgiganecdomtpouftievre-dgoesnaegraetgerdo(uwpesig(hGtr-oourpdser|etd)hrough randomization procedure. Within each dosage group, consecutive order was used to assign female rats to cohabitation with breeder male rats, one male rat cpoenrffiermmaeldemraatt.inAg tdaabtleesopferradnodsoamgeungitrsouwpasforusCeadestaoresealne-csteecitgihotnfeexmaamlienarattisownisth on DG 15. The remaining female rats in each dosage group were Caesarean- sectioned on DG 21%. a. See APPENDIX D (DEVIATIONS FROM THE PROTOCOL AND THE STANDARD item 1 OPERATING PROCEDURES OF THE TESTING FACILITY), 00797 418-013:PAGE II-7 ES. Syos fIdet ntife icatm ion: tMoaltheerTaetsstwienrgeFagciivleitny'usnibrqeueedpeerrmmaalneenratt ipdoepnutliaftiicoant.ionFneummableersrautspowneraessaisgsnimgneendt temporary numbers at receipt and given unique numbers when assigned to the study. Each rat permanent identification was individually identified with a Monelself-piercing ear tag (Gey inscribed with the rat's designated Band and Tag Co, Inc., No. unique permanent number. MSPT 20101) F. Husbandry: FA. Research Facility Registration: USDA Registration ot seq. No. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 F.2. Study Rooms: aThhealsltwuadyyarnodomisndweepernedmeanitnltyasiunpepdliuenddewirtchoandmiitinoinmsuomf opofstietnivcehaainrfgleosw rpeelrathioveurtoof t1e0m0p%erfarteusrheaairntdhahtumhiadditbyeweenrpeamsosneidtotrherdoucgohns9t9a.nt9l7y%thHrEoPugAhofiulttetrsh.e Rstoudoym. hRuomoidmittyemwpaesrattaurrgeetweadsatta3r0g%etetod 7a0t%8.4FSetoe 7A9PPFE(N1D8ICXtEo 2(6TCE)M;PrEelRaAtiTveURE AND RELATIVE HUMIDITY REPORTS.) F.3. Housing: AClalrceaagendsiUzsees aofndLahbooursaitnogrycoAnndiimtiaolnss.weFreemianlceomrpatlsiwanecree wiintdhivtihdeualGluyihdoeufsoerdthien stainless steel, wire-bottomed cages except during collection intervals for urine and fecal during the samples. cohabitation period and During cohabitation, each pair of rats was urine and fecal housed samples in the male the female rat's rats cage. were During collection individually housed intervals for in metabolism cages. No nesting materials were supplied sacrificed before parturition was expected because the female rats were Fd. Lighting: Alinght:au1t2o-mhaotuicraslldayr-kc,onwtirtohlleeadcfhludoarrekscpeenrtioldighbtecgyicnlneinwgaast m1a9i0n0tahionuerds aEtS1T2.-hours 00798 418-013:PAGE 11-8 F5. Sanitization: Cage were pan liners were changed approximately three changed approximately every other week. times each week. Cages F6. Feed: Rats were given ad libitum access to Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in individual feeders. F.7. Feed Analysis: Analyses levels exc eweedrienrgoutthienemlayxpiemrufomrmceodncbeyn ttrhaetifoenedlismiutpsplfioerrc.erNtioficedonfteaemdionrants a t deviations from expected nutritional requirements were detected by these analyses. Copies of the results of the feed analyses are available in the raw data. Neither the Study Director nor the Sponsor was aware of any agent present in the feed that was known to interfere with the results of this study. FS. Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad. libitum from individual water bottles and/or from an automatic watering access system. Chlorine was added to the processed water as a bacteriostat. F.9. Water Analysis: The processed water is analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, Pennsylvania) and monthly fPoernnpsoyslsviabnlieab)a.c raw data. teCroipailecsonotfatmhienraetsiuoints(AonfatlhyteiwcaalteLrabaonraaltyorsieess ,arIen ca.,vCahiallabfloentin, th e Neither the Study Director nor the Sponsor was aware of any agent present in the water that was known to interfere with the results of this study. C0799 418-013:PAGE II-9 G. Methods: G.1. Dosage Administration: [T rToEoEE omrrT[[ ee eew 0r e e A v Loww w Ow = [mwc weeereec eeee] Group| (mg/kg/day) (mg/mL) (mUkg) Female Rats Numbers G.2. Rationale for Dosage Selection: Dosages were selected on the basisof a previous study conducted with the test article (Argus Research Laboratories, Inc., Protocol 418-008). G.3. Route of Administration: Oral (gavage) G.4. Rationale for Route of Administration: The oral (gavage) route was selected for use because: 1) this was the route of administration in the developmental and reproductive toxicology studies; and 2) it is one of the possible routes of human exposure. G.5. Frequency of Administration: Appropriate dosages of the test article or vehicle were administered orally (via gavage) once daily to female rats beginning 42 days prior to cohabitation through either DG 14 or DG 20. The dosage volume was 5 milkg, adjusted daily on the basis of the individual body weights recorded before intubation. The rats were intubated once daily at approximately the same time each day. G.6. Lengthof Study: Approximately 11 weeks 0C800 G.7. Method of Study Performance: 418-013:PAGE Il-10 The and female rats for general awpepreearoabnscerevaetdlfeoarstvioabnicleityduartilnegasatcctlwiimcaetieoanc.h day The of the study rats were also perxeammaitnuerdefdoerlcilvienriiceasl aobnsderdveaattihosnsbeoffoerfefeacntds aopfptrhoextiemsattaerltyicloen,eabhoorutrioanfst,er dosage and once prior to sacrifice. Body weights were dosage period and recorded at at sacrifice. least Feed ocnocnesudmurpitnigonacvcalilmuaetsiowne,redairleycodrurdiendg the at least once during the acclimation presumed gestation. period, weekly to cohabitation and daily during pAfltaecre4d2indtaoycsohoafbtietsattiaortnicwliethad8m0inbisrtereadteironm,a8l0e healthy virgin female rats were rats (one male rat per female rat in the and male rat's confirmed cage)". by the Mating was observation evaluated daily of spermatozoa during the in a smear cohabitation period of the vaginal contents and/or a copulatory plug in situ. were returned to individual housing. Female rats considered to be at DG 0 Urine and intervals: ofencealdasaympprlioerstwo eirnieticaotilolnecotfecdofhraobimtfateimoanletoratthse at the following following morning and Fforlolmoweiancgheoafcthhe24m-ahtoeudr fceolmlaelcetiroantsinotenrvDalG,ssa6mtpol7e,s1w4etroe 1c5olalnecdte2d0 itnoto21c.entrifuge tubes, placed (on dry ice) to on dry ice and maintained the Sponsor for analysis. frozen (-70C or below) until shipment fBrloomodmestaambpolleissmwecraegicnogll(epcrtieodr tforotmesteaacrthicolfe tohrevfeheimcalleeardamtisnifsotlrlaotwiionng)roenmotvhaelday cohabitation was initiated female rats on DGs 7, 15 (prior to and 21. cOonhaabliltdataiyosn)ofacnodllfercotimoneaecxhceopfttDheGsma1t5edand 21 mL (dams each) at their terminal collection interval), blood were collected from the orbital sinus. On samples (approximately DG 21, blood samples 1 b(laopopdrocxoilmlaetcetiloyn4onmLDGeac1h5)wwaesrea fcionlallecbtleededvifaorthteheinrfeesripoerctvievneadcaavma,(i.e.Whtehne dam was was Caesarean-sectioned that day), the collected via the inferior vena cava. sample was approximately Blood was transferred into 4 mL and serum separator tubes transferred into and spun in a polypropylene refrigerated centrifuge. tubes labeled with the The study serum was number, rat identification, date of collection, were immediately frozen on dry study day and ice and stored collection (-70C or timepoint, All samples below) until shipment (on dry ice) to the Sponsor for analysis. a. See APPENDIX D, item 1 00501 G8. Gross Necropsy": 418-013:PAGE I-11 Rats were sacrificed by carbon dioxide asphyxiation on DG 15 or 21, as wdeasscrpiebrefodrbmeeldo.w.AAligverrossescntieocnro(prisgyhtolfattehrealthloorbaec)ica,nadbdthoemimniallk-saencdrepteilnvgicglvainscdesra from the axillary, thoracic, abdominal and inguinal regions (left side only) of each dam (except rats without confirmed dates of mating) were collected, frozen and stored (-70C or below) until shipment (on dry ice) to the Sponsor for analysis. To confirm the pregnancy status, uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide". Tissues with gross lesions were optrheesrermvaetderinnanleuttirsasluebsufwfeerreeddi1s0ca%rdfeodr.malRienprfoersepnotsasitbilvee fpuhtoutreogervaalpuhastioofn;maatilemal lesions are available in the raw data. G.8.a. DG 15 Caesarean-Sectioning Eight randomly selected female rats, with a confirmed date of mating, per dosage group were Caesarean-sectioned on DG 15. The gravid uterus was excised and weighed. Rats were examined for number and distribution of corpora lutea, implantation sites, viable and nonviable embryos. A viable embryo was defined as oval or crescent shaped, pink, firm and enclosed in an amniotic sac filled with clear fluid. A nonviable embryo is amorphous, small, pale. pink to tan or deep red to black, soft and enclosed in an amniotic sac filled with clear, cloudy or opaque fluid. A sample per litter, of the frozen amniotic fluid of each viable and stored (70C or below) embryo was collected until shipment, on dry and ice, pooled to the Sponsor attached for analysis. Each viable embryo placenta, pooled per litter frozen was removed from the uterus with and stored (70C or below) until the. shipment, on dry ice, to the Sponsor for analysis. G.8.b. DG 21 Caesarean-Sectioning All remaining female rats in each dosage group were Caesarean-sectioned on DG 21 (rats with confirmed dates confirmed of mating). dates The of mating) or gravid uterus estimated DG 21 was excised and (rats without weighed. Individual placental weights were recorded. Rats were examined for number and distribution of corpora lutea, implantation sites, live and dead fetuses and early and late resorption. An early resorption was defined as one in which organogenesis was not grossly evident. A late resorption was defined as one in a. Atable of random units was used to select one control group rat from which all tissues examined at necropsy were retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions. 000502 418-013:PAGE II-12 awdrehefiiccnhoendtshaiesdoeacrcetuderrtrmoebnfceeteudosefatohdartg(tarhneeosrgpeeownneedrseeidsntwooasdsteimagurdlio.sfestlNuysoeensvr)i.edsenpto.ndAinlgivteefremtufsewtauss.es erexstorrepmteioanustoalryesidsififnedriecntaitaetdedthbaty the the degreeofautolysis preDseeandt;fmetaurskeesdatnod fetus was a late resorption. late Each fetus was identified with a removed from tag noting the sthteuduytenruumsb,eprl,acleitderinnaunmbienrd,ivaidnudaltceornitnaginer and gdirsotsrisbuetxitone.malEaaclhterfaettiuosnsw.asLisvuebfseetquuseenstwleyrweeisagchreidfiacenddveiaxadmecianpeidtatfoirons.ex and wuSneatrimlepslcheoilsplemoceftntetdh,etoainmtdnhiievoiStdpiuacolnfllsyuoipdroof(olwrehdaennpalepyrossiisst.iebrl,Bef)lroaoozndednstaahmnepdlplesatcoernetda(-of70eaCchorfbeetulsow) gtuabcehs.feTtuhsevsiaamdpeclaepsitwaetrioen,sppuonoliendapreerfrliitgteerraatnedd cternatnrsiffeurgers.edwTeihrnteeo scsoeelrrluuemcmtwesadeypf.arroamtor wtidreearnnetsiffieimcrmarteeiddoinia.nttdeoaltpyoefloryfopzrceoolnplyeolcnetindoerny,tuisbcteeusdaynldadbaseytloeardnewddit(cho-l7tl0heecCtsiotornudbtyeimlneoupwom)ibnetr.,Alllittsear mples (on dry ice) to the Sponsor for analysis until shipment Lung and five litters liver were liver samples were per dosage group. cOonlleechtaeldfoffrtohmethlruenegsfeatnudseasnpeerlaltietrtaerl individually retained in neutral buffered 10% formalin from lobe two to of the vials. thick) Prior to fixation, of this half of the sleuvnegrsalan(dmitnhiismluombeofotfwtoh)e sections in scintilation (approximately 1 mm liver were removed using a tsschcieanltlpiulenllagtaianonnddvrilaelitsva)e.irnsTeadhmeipnllMeucnsDgoinawnendelullt-irvTaerlrubsmuepfc'ftseiroFenidsxai1tn0iMv%ecDf(ooswrtmoearllelid-nTurnudmepr'rsefFriixgaetriavteioannidn cold packs). microscopy, rfoersppeocstsiivbellye,ftuotuPraetehvoallougaytiAosnsobcyiealteecstrIonntemrinactrioowsnacelor,peyDsuohrrihpiapgmhe,td (on North TfClaharesohsliefnrafo.lzaesThnhfienroloziteqhuneirdluhnnaigltfraoongfdenthliaevnelrdusnsagtsmopraelndedsintwhheeeraoett-hmseaerianlltaatabeilrneaelpdloofubrceohzoeefnsthoen ldirvyeriwceere below) until shipment (on dry ice) to the Sponsor for analysis. (70C of The livers of the selected fetuses, remaining fetuses, plus were collected, pooled any remaining liver byliter and frozen. from The the three remaining carcass (and frozen (70C head) of each or below) until fetus was shipment frozen. (on dry These ice) to tshaemSppleosnswoerrfeormaanianltyasiins.ed G.8.c. Moribund Sacrifice or Premature Delivery wReatrse tehxaatmwienreedsfaocrritfhiececdaubseecaounsethoefdmaoyritbheunodbsceornvdaittiioonn worasprmeamdateu.reThdeelirvaetrsy 000803 418-013:PAGE 11-13 wseecrreeteixnagmgilnaenddsfofrrgormotshse lesions. axillary, A liver section (right thoracic, abdominal lateral lobe) and inguinal and the regions milkof eshaicphmdenatm((olneftdrsyidieceo)nltyo)thweerSepocnolsloerctfeodr, afnraolzyesinsa. ndPrsetgonraednc(y-7s0taCtuosr abnedlouwt)eruinnteil contents using the were recorded. same methods Delivered described pfourptserwmerfeeteusxeasm.inUetedritoofthaeppeaxrteennttlpyossible, nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites. GS. Statistical Analyses: Averages and appropriate. percentages were calculated. Litter values were used where 00804 Wl. RESULTS 418-013:PAGE Ill-1 A. (MoSrutamlmitayr,yP-rTeambalteur1e; IDnedliivveirdyu,alCDliantiaca-lTaanbldeNse1c2roapnsdy13O}bservations AA. Mortality and Premature Deliv rNaotsd(eoantehseaocrhprinemtahteu0r.e1,de0l.i4vearnides1w.e6 rmeg/atktgr/idbautyeddotso aPgFeOgSroturpesa)tmwenetr.e Three sacrificed bcoenctaroulsegrtohuepedyied wnoatshianvjuereadcdounrfiinrgmeordbimtaaltisnignudsatbeleaednidngd.eliOvnereedraotnindtahye 6v6ehoifcle the study. other rats suTrwvoivreadtstoinstchheed3u.l2emdgs/akcgri/fdiacye.dosage group delivered on DG 21. All A2. Clinical Observations Awlelraedvceornssiedcelrienidcaulnorbesleartevdattiootnhsedtuersitngarttihceleprbeeccoahuasbei:tat1i)otnhaenidncgiedsetnacteisonwpeerreiods nraotts. doTshaegsee-doebpseenrdveantti,onasnidnlcolru2d)edthmeisosbisnegrvoartisownololcecnurfroerdepian wondilgyito, nsecaobrstwoon funodreerpsaiwd,e,swcohlrloemnofrohrienpoarwr,helao,caclhirzeodmoadlaocprecyioarrohneat,heswloimlblse,n head, ears, back and/or dental problems h(meimsosrirnhg,agber,okceonmeaandl/oopramciitsya,liagxinleldariyncmiassorss,),exsowpolhltehnalsmnoosut,,ecnoorpnhetahlalmos, lenticular white film opacity, on eye, lacrimation, ungroomed missing right eye, large coat, traumatized eye, dark red eye, cornea, and lids cloudy, unable to close. A3. Necropsy Observations No gross lesions were revealed by necropsy. B. Body Weights, Uterine through 3; Summaries Weights - Tables and Body 2 through Weight Changes 5; Individual Data (Figures 1 -- Tables 14.and 15 B.A. Precohabitation Body weight gains for 88.8%, 80.8%, 66.3% tahneden1t7i.r4e%proefcothheabciotnattrioolngpreoruipodva(luDeSin1 to 42) were the 0.1, 0.4, 1.6 9a6n.d3%3.,29m3g./6k%g/adnady d85o.s3ag%eogfrtohuepsc,onrtersoplecgtrioveulpy.valBuoediyn wtehiegshetfsowurerrees9p8ec.t0i%v,e dosage groups on DS 42. 000805 B.2. Gestation 418-013:PAGE lll2 AeisthtehreDrGesu1lt5 oofrrDaGnd2o1,m gsreloeucptsioansfsoirgansesditgonCmaeenstatroeaCna-esseacrteiaonn-isnegctoinonDiGng1o5n weighed less on DG 0 Reflecting the random stehlaenctgiroonuapsndasdsiiffgenreedncteosCianesaavreeraang-esebcotdiyonwienigghotns DG 21 of the. two subsets, as well as the differed in the two subsets. relatively small group sizes, body weight In both subsets, body weight gains were gains reduced on DGs after 00 7 which ibnogdryowuepisgahdtmcihnaisntgeerseddi0d.4nomtgd/ekmgo/ndsatyraantde higher a clear dosages of PFOS, dosage-dependent pattern 0G.e1stmagt/ikogn/bdoadyydwoesiagghetsofatnhdebtoesdtyawrteiiclgeh.t gGarianvsidweutreeriunneafwfeeicgthetdsbwyerteheunaffected by dosages of the test article as high as 3.2 mg/kg/day. C. A(bSsuomlmuatreie(sg/d-aTy)abalensd6Retlhartoiuvegh(8a;lkIa/nddaiyv)idFuaeledDaCtoan--suTmapblteison16Vaalnudes17) CA. Precohabitation Ainbstohleu0t.e4,(g1/.d6aay)ndan3d.2remlga/tikvge/(dga/ykgd/odsaay)gefegerdoucposnsiunmepatcihonwevaelkueosf were the reduced cporencsouhmapbtiitaotniovnalpueersiowde.reRe9f4le.c8t%i,ng9t2h.e2s%e aenffdec8t3s.o8f%thoef ttehsetcaorntticrloel, garbosuolpuvtaelufee,ed aconndtrroellagtrioveupfeveadluceonfosrumthpetieontnirvealpureescowheabrieta9t5i.o1n%,pe9ri4o.d3(%DaSnsd19t0o.472%)oifn tthhe.e 0.4, 1.6 and 3.2 mg/kg/day dosage groups. Absolute and relative feed were unaffected by the 0.1 consumption values during the precohabitation mg/kg/day dosage of the test article. period C2. Gestation rGerdouucpesd aadbmsionliustteeraendd0r.e4lamtgi/vekgf/ededaycaonnsduhmipgthieorndvoaslaugeessduorfitnhge ttheestfiarrsttiwcleeehkadof the 1.6 mgegs/tkagt/idoanypedroisoadg(enogrvoaulpueassswiegrneedavtaoilCaabelseafroeraenv-asleucattiioonnifnogr the on DG 15). Athbesodloustaegfeeepdercioondsiunmtphteio0.n4vaanldue1s.c6omngt/ikngue/ddatyo dboesraegdeucgerdoufposr tahsesirgenmeadintdoer of aDsGsi1g5neCdaetsoaerietahner-sDeGcti1o5noirngDaGnd21inCatehsea3r.e2anm-gs/ekcgt/idoanyindgo.saRgeelagtirvoeupfefeodr rats consumption values tended to 1.6 and 3.2 mg/kg/day dosage be increased groups after over the DG 10. control group values for the 005806 418-013:PAGE III-3 Absolute and relative by the 0.1 mg/kg/day feed consumption dosage of the test varatliculees. during gestation were unaffected D. Caesarean-Sectioning, Litter and Fetal Gross External Observations 21)(Summaries ~Tables 10 and 11; Individual Data - Tables 18 through D.1. Day 15 of Gestation Pregnancy 8(100.0%) occurred in 6 of the rats in e(a7c5.h0d%)o,sa7g(e87g.ro5u%p)., 8 (100.0%), 6 (85.7%) and tNeostCaaretiscalreeaasn-hsiegchtaiosn3i.n2g mogr/lkitgt/erdpaayraadmmeitneirsstewreerdetoafrfaetcsteCdaebsyardeoasna-gseesctoifotnheed onnonDviGab1l5e. emTbhreyloisttear nadvepreargceesntfonrocnovripaobrlae elumtbera,yoimsplpaenrtalittitoernsw,ervieabcloempaanrdable `among the five dosage groups. embryos. No dam had a litter consisting of only nonviable D.2. Day 21 of Gestation 6Pr(e1g0n0a.n0c%y) orcatcsurwriethd ainc7on(f1i0r0m.e0d%)m,at7i(n1g0d0a.t0e%)i,n 5ea(c8h3.d3o%s),ag2e(g5r0o.u0p%.) aOnnde rat in each of the 0.1 and 0.4 mg/kg/day dosage groups was moribund sacrificed on DG 0 and the pregnancy status could not be determined and two rats in the 3.2 mg/kg/day dosage group delivered before Caesarean-sectioning on DG 21, as previously described. As aresult, Caesarean-sectioning observations were based on 7, 7, 5, 2 and 4 pregnant rats with one or more live fetuses in Groups | through V, respectively. The litter averages for implantations, litter sizes and live fetuses were reduced in the 3.2 mg/kg/day dosage group. Values were below the ranges observed h3i.s2tomrgi/caklgl/ydaatythdeosTeasgteinggroFuapc.ilitNy'o. otFehtearlCbaoesdayrweeaing-hstesctwieorneinaglsoor rlietdteurced in the parameters were affected by dosages of the test article as high as 3.2 mg/kg/day `caodrmpionriastleurteeda,toearraltys aCnadeslaatreearne-ssorepcttiioonn,edploanceDntGal21w.eigThhtes,lipteterrcaevnetrlaigveesmaflore fetuses and percent resorbed conceptuses were comparable among the five dosage groups. No dam had a litter consistingof only resorbed conceptuses, and there were no dead fetuses. No fetal alterations were identified at gross `external examination. a. See APPENDIX F ( (HISTORICAL CONTROL ) DATA). 00507 EFERENCES 418-013:PAGE lll4 1. U.S. Food and Harmonisation; DGruuigdeAldimnieniosntrdaettieocnti(o1n99o4f)t.oxiIcnitteyrntoatrieopnarlodCuocntfieonrefnorce on medicinal products. No. 183. Federal Register, September 22, 1994, Viol. 59, 2. RUeSg.ulaFtoioodnsa;ndFinDarluRguAldem.in2i1strCaFtiRonP.artGo58o.d Laboratory Practice 3. PJraapcatniceeseStMainnidsatrrdy foofrHSeaafletthyaSntdudWieelsfoanreDr(u19g9s7,).MHGWooOdrdLaibnoarnacteory Number 21, March 26, 1997. 4. E26urJoupleya1n9E89coonnomthiecaCcocmemputannicteyb(y19t8h9)e.EuCrooupnecainl dEeccoinsioomniconCommunity of gaonoOdElCabDordaetcoriysiproancrteicceo.mmOeffnidcaiatliJoonuronnalcoomfptlhieanEcuerowpitehanprCinocmimpulneistoifes: Legislation. 32 (No. L 315; 28 October): 1-17. 5. MChurtiasgteinain,ciMt.yST.esatns.d VEonyvtiekr,onPm.eEn.ta(l198P2r)o.tecItnioVnivAogeRnecpyr,odWuacsthiivnegatnond, D.C. SNpartiinognfailelTde,cVhnAic2a2l1I6n1formation Service, U.S. Department of Commerce, 6. nCahrlitsrteixaonn,eM(.PSr.o(c1e9e8d4i).ngsReofprNoadlutcrteixvoenetoSxiycmiptyosaindumt,erNateowloYgoyrekvaAlcuaatdieomnsy of of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 7. LCoanntgr.olP.DL.at(a19i8n8)t.he EChmabrrlyeos RainvderFeCtrall:DCeDvelBoRpRmaetn.taClhaTrolxiecsitRyiv(eTreratology) ALrabgoursatRoersieesa,rcIhnc.L,aWboirmaitnogrtieosn,, MA Inc.) 01887-0630. (Data base provided by 8. UInssetiotfuLteaboofrLaatboorraytAonriymAalnsi.malNaRteisonoaulrcAecsad(1e9m9y6).PreGsusi,dWeafsohritnhgetoCna,reD.aCn.d . ISmaplleawnstkait,ioEn.ss(t1e9l6l4e)n. aFmrUbteemreusthdoedreRaztutem.mAarkcrho.skPaotphiols.chEexnp.NaPchhawremiaksovlo.n 247:367. 0803 APPENDIX A REPORT FIGURES C00309 BODY WEIGHTS PRECOHABITATION Figur1e ul . 5 oe ln | GT es -| x Eo mn Za a wl i 2 3Eh ae be =5S 838 : MATERNAL BODY WEIGHTS RATS CAESAREAN-SECTIONED ON DAY 15 OF GESTATION Figur2e || el~]- | eT De | [re B& o[| alyool - lSl oao0 o . oan s -a x] [me i wl x | wx ox ox ox % x = = = | 3 { g g8g bobcat z | . MATERNAL BODY WEIGHTS RATS CAESAREAN-SECTIONED ON DAY 21 OF GESTATION Figur3e "i | "| . - P |] :| brn vo | 0eT Geea s a EEa T Le rdel ssu=ensonr wlexxxnsxnn ert i 5i B 21 m g ddr seh 8 2 z; APPENDIX B REPORT TABLES C0813 on re PRECOUSITATION(9AY1 OFSTVOYToTHEoAYorcommrTATION) we ee ew"e or"e or: e ra eo come - ti Fibre i 3 gi 2 ween ul a LE VN EN "ewe we mn an iowioogr a ao oyu: : tek 8 2 prevodi %. won mie wn wn en Yiowuourowr owe = owl oED yr yi err we uno oanom a pe pe wwveeo eauwewouwWnleooauanlomm x we ular oun -- EN a pom L oe UoeLUwa "os oouen 2 gQE hui rossinia nciomcy+ (oA 22 xWATE pmaen 5F MAF coin ane LA Et &3 2 i samy pg ener SOY neket a4 veoweoa.oulou + SE TE MOS SI 2 32g g : z g 22 3 2B Ww wen melas ali aeime aii le - g3 2 g 8 5 - mse maine weins mews woiws mies gy ji on sn men vine ma 3 3 Lon Mmann on z wi 1 $2 RLIT III: ca ssn, scrpr ns Cres ection hercios wre ms rete B 5 BRLLISLS, ano er pe recor. wisn) 23 @ g 8 ! S oh -- en wn ou gn ag Uk memgws nme wesw wsgun 3 BR E EE Te s ct msurT at writser e2t eyetteainyg oot esenrnemasrieonnn eps ss Z B2 Wh Ts me nl IE ef oQo F SERSSLAE TL LI I MI E i ot ent ston nd rite ey stn 2 i & 8 BE aetss Retna Sly Ton"hay 350 ence Lonbay went Rim heaha eevin weight & - g2 3 S a ? n& 3 ww men alu. wail aban iu mee FE mE eeE meen esT is IwilSe cUrNDtTi TD rmn eee og 3 3 Bechudes values' for rate shat 414 nox have contirmedmucing date. | | - or 3 8 erate alert kyle Tayo enn fo erpwd S87 SE hl ten ows as sents ne Rr i . oe 1. chiar atomsShas were Incorrectly recorded, su vell a8 those assclated wich spiiinge. . g gg2&g z8&2 won wee we bT albeit I DE tnttt ct, eh 1 es sce i ito & $2 3 2i R8g 8 SE I LE SS ne ee & 3 22g 2?3 85 wg BL o ERE EE IRE ELE JR 2 @ mirromsasioomen, Ms rs etn ey 3 2 a "ones oars orcesTRriM - - te 5 Ly A Gg gi 8 IS... g i: Ws mse rime wanna ein eR gb EEE TTL Beso to ene sions mms se 3 PR i vars Ce g Qo Sg moles BALE ihe fon ta shsens,Awici h uevdeb2ie eentis nreicmhaetisngoenteo 11 of aety du 3 of comman 3 Xclide 3 ' 8 n values that vere not Tecorded. as well 8 thous aesocibted wih - spiiiage niirasion az3 BRET ee sine wae umn umm QE ii aes io a es ion $d sei eo sestove n1 eer, 8 RB Tee g s oC EEdEEERERn LT 3 8 3 2 5 3g8 7 sn, os on omen son omen min PE En ese on aricn new wns ier f3 3 TURE 2g i z g 33 38 2g % - BPeEE p Eae s g 9 8 BSaLnEi E EERm E oo 82 EN 8 ; 2g :& g % Rs 3 823 _-- . _-- & 8 2 Bw mma ERE ge ne 8 2 o ) g ' 2 . : BRAT . g 3 2 g2 z% & 3g i = 3 g2 2g 23 8 & 2 2 2 8 2 & EE a LE RE oo - 3 & $ 33 38 a & - n RR m EE Fen rnry &2 5 3 & % LEELA ELEEDYE 3 1? E AERAEBBEDERRARENERSEREERRRERRYE 8 HES z Ee iy . gg 38 2 & #BOPEpEEE@EEEsEoEaEbELEREOBOR LDROLGE g HB OBERBERERRERESIREER & SZ HEE Z 5 : g g g;zS 8 l EmEhh L BEBE RREREERRIEEREBO3Z 3 g: 8 i gE UE i EE fd EC o HE OEmEEEESSD ELL RD RE 3 2 ALL WEIWEGRERHECTORDSED IN GRAS (G). T $ g3 23 g2 a | BOBEESEREGEEEEEERE OZ 8 HEE en wo z 2 ie MOON BY ONO BONN EW 9 8 ie EE RE TR I FE A 5 g a a @ : ! BEES rman 9 Em eee id RR 2 BgEOEOEm Eom OEon omomomomonom 3a2 $& D835 TB19530r oi ma. . o5s.mo. a Mne. -- ue on oe - priHii : za2 FgoOHEpE BE end E TOU U0 HEBEEL MB Mw a ow ow wo S Bodg HE i gI 33: BEBFnr EEBEEO2E moma omomomon g mTEy om % 3 ii i ~ i2 wina 3 8 | | ars. oe. ts ons sx re rt on on g I H Ebel ww eww iid g$ ] 2 3 8 5 CER g bE REE a ren eee ess se 3 & g8 3 i i 2 $ a & Bh ed ecs husesthe cobeuiboneotf ents vase. ia Z2 :: EEgoof rtrd 8 i BB wow ww errant Besson ;: oreteraas a gow : rm dm a i 5 i Fi fm a sc ss or g BEER OL ovououn i . E BEBE... E g 3Bm Hm IEA BRRN SRST ED rear ere : 3 - 8a EH. Sgr 3 LA @ Lgl geen DOLD NT _ PAAE LDRS CUBED Thin rape AVERAGE a 00 a :3 or 2> Eoopig o HE PPE 2 ttl, tis 3 bbE bid DPE iE 2 imine pi nad TR :3 aashrtasoness 2 - ny A oS - et : i LE td Ed EE -- g 8g gilLlLi LELE PLEA EEE g g JHRILLTT ceed QTc serra sowscaves naresoneenise. 8 & 3 32 8z3 & gg HE i :  25 % men i * :Si : - - o : oe - i. - of : SE BE re Hone Ff ines mn ssch sr ern HE Grew Lm LL 3 2g8 wtFi Hm [CE I or 88 g 2 @3 RR aki A Buttle BBE SH WEL MMA A A A A A A A ESA A A A A A a 2 82EniBSh i I &Qo vJmIE xk rh a tA sAeA a Aa asSRa - - 2 WO MAE A A A ALA AE A A A A a 3 22BEecm wt vere to ct8 3 & 2vinniemanos wowiAneDano +/+ omnis woeiotFcioovinx EE 28 a sa iis an sre Wao ie g 828 _-- iza Ld sane Sw UR WRT HT son ror C8 GR C8G000ih CC shche Th oe ST & gS2q mmmmar haEWhmRhW F en mR InR monn A2 BB &G uI ron"reakiuoumexr "KFeikss so Br rwEtowT s iL weak concko1 Nason same tor. Pt ii 8i # rosoricomnix 8 PC NEYJ Ph a main momo mm mn EE EE nr NORIBUNO SACRIFICED ON DAY 43 OF STUDY (BAY 3 OF CORAMTATION g gg rweae nEsRfEAfiRanARERATE anaa nawnn an,n & 2 REI LR ER RRL = 2 CE I LE RR 3 322& or & 3 & 3g hh RR EE REN TE So ne RR RRBRE ratmm, & 2 3 re hE A ER en, 8 G 3 H 8 Wier roi AL LE Ave Emaawersou SS i er ois i 5d ci - ssssssnin 3oSTab2ecm HaoS homom Oohh m ToT mL mTTmh HmOhBR SoHmim E moTmT fk ne mSmR mom mm 5 "3 QE wT eir lheeh terieLn iERiEor i eens reroroinot 2 g 3 382 &: APPENDIX C PROTOCOL AND AMENDMENT 0e8S9 2 PRIMEDICA 418-013:PAGE C-1 pea ar bBasemean rtns Tonpee I ERAeS PROTOCOL 418-013 SPONSOR'S STUDY NUMBER: T-6295.12 STUDY TITLE: PURPOSE: TESTING FACILITY: Oral (Gavage) Pharmacokinetic Study of PFOS in Rats The purpose of this study is to evaluate the pharmacokinetics of PFOS in Fo generation dams and F1 generation fetuses following PFOS treatment of GCersit:aCtiDonBR VAF/Plus female rats during premating and 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 Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, Minnesota 55144-1000 STUDY MONITOR: Marvin T. Case, D.V.M., Ph.D. Telephone: (651) 733-5180 Telefax: (651) 733-1773 ALTERNATE STUDY MONITOR: Andrew M. Seacat, Ph.D. Telephone: (651) 575-3161 Telefax: (651) 733-1773 60390 418-013:PAGE C-2 Protocol 41P8ag0e132 REGULATORY CITATIONS: U.S. Food and Drug Administration (1994). Intemational Conference on Harmonisation; Guideline on detection of toxicity to reproduction for medicinal products. Federal Register, September 22, 1994, Vol. 58, No. 183. U.S. Food and Drug Administration. Good Laboratory Practice Regulations: Final Rule. 21 CFR Part 58. Japanese MinistryofHealth and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Communityof an OECD decision/recommendation on compliance with principles ofgood laboratorypractice. Official Joumal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. REGULATORY COMPLIANCE: "This study will be conducted in compliance with the Good Laboratory Practice (GLP) regulations cited above. All changes or revisionsofthis protocol shall be documented, signed by the Study Director and the Sponsor, dated and maintained with the protocol. `The Quality Assurance Unit (QAU) will audit the protocol, the raw data and the report, and will inspect critical phases of the study in accordance with the Standard Operating Procedures of Argus Research Laboratories, Inc. `The final report wil include a statement signed by the Study Director that the report accurately reflects the raw data obtained during the performance of the study and that all applicable GLP regulations were followed in the conduct of the study. Should significant deviations from GLP regulations occur, each will be described in detail, together with how the deviation might affect the qualityorintegrity of the study. SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE: `See ATTACHMENT 1 to the protocol. 00391 418-013:PAGE C-3 Protocol 4P1a8g0e133 TEST ARTICLE AND VEHICLE: Identification: Test Ace: Name: PFOS (Synonym: FC-95). Physical Description: ~~ Light-colored powder. LotBatch Number: 217, Specific Gravity: ~06. Purity: 98.9%. Expiration Date: May 2000. Information on the with the Sponsor. identity, composition, strength and purity of the test article is on file Vehicle: 0De.i5o%niTzweedeWnater8)0.inSuRpepvleiresreaOndsmlootsiidsenMtiefmicbartiaonneofPTrowceeesnsed8D0etioonbiezdedocWuamteenrt(eRd.Oi.n the raw data, . Netio tbheerprtehseenStpionntshoervneohirctlheetShattudwyouDlidreicnttoerrfiesraewwairthe tohfearneyspuotsenotfiathliscosntutdaym.inTahnetrsefliokreel,y no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: fGolromvuelsa,timoanskpr,epaaprpartoiporniaatnedeaydemipnriosttercattiioonna. nTdahe uMnaitfeorrima/llaSbafceotayt DaarteatoShbeeewt o(mMSdDuSri)nigs attached to the protocol (ATTACHMENT 2). Storage: Bulk Test Article: Vehicle Components: ~~ Room Room temperature. temperature. Prepared Vehicle: Prepared Formulations: Room temperature. Room temperature. JAullitaenstGauritbiicnlseksi,hiMpamneantgsertootfhFeorTmeusltaitnigoFnasc,ilattythsehopurledviboeusaldydcrietsedseadddtroetshse aanttdention of telephone number. CO0E92 418-013:PAGE C4 Protocol 41P8a.g0e1s3 `Shipments shoud include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment. FORMULATION: Ereqouf Preepnaractioyn: Formulations (suspensions) will be prepared daily at the Testing Facility. Data verifying the stability of the test article in the vehicle for 48 hours under the conditions of `administration are on file with the Sponsor. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). Adjustment for Purity: The test article will be considered 100% pure for the purposeofdosage calculations. Testing Facility Reserve Samples: The Sponsor will reserve a sample (1 g) of each lotofthe bulk test article used during the course of this study. The Testing Facility will reserve a sample (5 mL) 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 the course of the study. described below may be takenif deemed necessary during Bulk Test Article Sampling: No analyses of the bulk test article will be conducted during the course of this study. Information on the stabilty of the bulk test article is on fle with the Sponsor. Analyses of Prepared Formulations: "Homogeneity and stability of prepared formulations is on file with the Sponsor. However, records will be maintained to document how the test article formulations were. prepared. 0e393 418-013:PAGE C6 Protocol 41P8a.g0e1s3 Body Weight and Age: Female rats will be ordered to have body weights of 200 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 cata. The weight range will be inciuded in the final report. Sex: Female rats will be given the test article. Male ratsofthe same source and strain will be used only as breeders and are not considered part of the Test System. Source: Charles River Laboratories, Inc. The rats will be shipped in fitered cartons by air freight andor truck from Charles River Laboratories, Inc., to the Testing Facilty. Identification Rats are permanently identified using Monei seff-piercing ear tags (Gey Band and Tag Co, Inc., No. MSPT 20101). Male rats are given unique permanent identification numbers upon assignment to the Testing Facilit's breeder male rat population. Female rats are assigned temporary numbers at receipt and given unique permanent identification numbers when assigned to the study. ANIMAL HUSBANDRY: All cage sizes and housing conditions are in compliance with the Guide for the Care and Useof Laboratory Animals. Housing: Female rats will be individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period and during collection intervals for urine and fecal samples. During cohabitation, each pair of rats will be housed in the male rat's cage. During collection intervals for urine and fecal samples, the female rats will be housed individually in metabolism cages. No nesting materials will be supplied because the female rats will be sacrificed before parturition is expected. 060394 418-013.PAGE C-5 Protocal 41P8a0g1e3s Conce ofn Test tArrtica leFt ormi ulao tion ns: Concentrationof the prepared formulations will be verified during the course of this study. Duplicate samples (2 mL. each) wilbetaken from the first and last preparation on the day prepared. One sample of each set will be shipped for analysis; the sraemmapilneisngwisllambpelsetsorweidlfbreozreenta(i-n7e0dCatotrhebeTleoswt)inagndFadciilsictayradsedbaactktuhpe sTaemsptliensg.FacBiaictykup upon request of the Sponsor. `Shipping Instructions: 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 2-36-09 St. Paul, Minnesota 55133-3331 Telephone: (612) 778-6018 Telefax (612) 778-6176 The recipient will be notified in advance of sample shipment DISPOSITION: ParrteicplaerweidlfboermrueltatuimoendstwoiltlhbeeSdtiusdcyarMdoenditaotrtahtetTheestpirnegviFoaucsilliytyc.iteAdllardedmraeisnsinugpobunlk test completion of all work with the test article. TEST SYSTEM: Soecies/Strain and Reason for Selection: `The Cr:CDBR VAF/PIus (Sprague-Dawley) rat was selected as the Test System because: 1) this strain of rat was used in the reproductive and developmental toxicity studies; 2) historical data and experience exist at the Testing Facility"; and 3) the test article is pharmacologically active in the species and strain. Number: Initial popuiation acciimated: Population selected for study: 120 virgin female rats. 80 mated female rats (16 per dosage group). 00395 | 418-013PAGE C7 | Protocol 41P8a.g0e1?3 m Air, Te and Humidity: Tfrheeshanaiirmtahlatrohoams biseienndeppaesnsdeednttlhyrosuugphpl9i9ed.9wi7t%hHaEtPleAafsittteernsc(hAiarnogCelsepaenrhroouorm)o.f 100% cRoonsotmanttleym.perRaotourmehwuimlidbietymawiilnltaalisnoedbeatm6on4itFor(1e8dcCo)ntsota7nt9lyFa(n2d6mCa)inatnadinmeodniatto3r0ed% to 70%. Light An automatically controlled 12-hour light:12-hour dark fluorescent light cycie will be maintained. Each dark period will begin at 1900 hours EST. Diet: Rats will be given Certified Rodent Diet #5002 (PMI Nutrition Intemational) available ad libitum from individual feeders. Water: Waanteaurtwoimlaltbiec wavaatielraibnlge aacdcelisbsitsuymsftreomm. iAnldlivwiadtuearl bwioltltlbees fartotmacaheldoctaol tshoeurccaegeasndorpfarsosmed tphrroocuegshseadrweavteerrseasosamobsacitsermioesmtbatr;apnreocbeefsosreed uwsaet.erCihsloerxipneecwtield bteo caodndteaidntnoothmeore tbahcatner1i.a2lpcopnmtacmhilnoraitnieonatatnhdettwiimceeoafnannuaallylsyisf.orWpaostseirblies acnhaelmyizceadl mcoonnttahmliynaftoiropno.ssible Contaminants: NtoeibteheprrethseenStpionntshoernceorrtitfhieed SditeutdoyrDtihreecdtroirnkiisnagwwaarteerofatanlyevpeoltsetnhtaitalwocounltdaimnitnearfnetrse lwiiktehly tbhyetrheesufletesdosfutphpilsisetruodry.thTohseeremfeonrtei,onnoedaninalthyissepsrootthoceorltwhialnl those routinely be conducted. performed RANDOMIZATION AND COHABITATION: cUopmopnutaemriv-agle,nmeraalteeadnrdafnedmoamleunirtast.s wAilfltebreaacsclsiimganteidont,oviinrdgiivnidfueamlahloeursaitnsgwiolnbteheseblaescitseodf afocrclsitmuadtyioonn. tThheebafseimsaolfeprhaytssiwcialll baeppaesasriagnnceed taonddobsoadgyewgerioguhptss breacsoerddeodn dcuormipnugtergenerated (weight-ordered) randomization procedures. 00396 418-013:PAGE C-8 Protocol 418013 Pages Within each dosage group, consecutive order will be used to assign female rats to cohabitation with breeder male rats, one male rat per female rat. The cohabitation period will consist of a maximum of five days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ will be considered to be at day 0 of presumed gestation and assigned to individual housing. A table of random unitsor a computer-generated randomization procedure will be used to select eight female rats per dosage group for Caesarean-section examinations on day 15 of presumed gestation. The remaining female rats in each dosage group will be examined on day 21 of presumed gestation. ADMINISTRATION: Route and Reason for Choice: The oral (gavage) route was selected for use because: 1) this was the route of administration in the developmental and reproductive toxicology studies: and 2) tis one ofthe possible routes of human exposure. Method and Frequency: Female rats will be given the test article or vehicle once daily beginning 42 daysprior to cohabitation through either day 14 of presumed gestation or day 20 of presumed gestation. Dosages will be adjusted daily for body weight changes and given at `approximately the same time each day. Rationale for Dosage Selection: Dosages were selected on the basis ofa previous study conducted with the test article (Argus Research Laboratories, Inc. Protocol 418-008). DoCsaogenLevcele s, ntratai ndVooln umess: lo ee | er[82]pn | oy rpm | mgmt |ming | o oe | [[6Jovan| oT 5 [|sonommonsomnre|n CeTeloo I om TsTsunonsonmmve|n [0[wToo[ow To[scwomcommmnmm|n [wlwlwT en [5[eeonoomsmmnme|n Col we TsTow [=|suonconmmene|n The testarici witbconsidered 100%surefor hopurseofdosagecaciaons. 6e3597 418-013:PAGE C-9 Protocal 418.013 Page Ss IALYSES AND ME) NTS: Viability: All Periods: Atleast twice daly. Clinical Observations andlor General Appearance: Acciimation Period: Atleast once. Dosage Period: Twice daily. Priorto administration and once `approximately one hour postdosage. Postdosage Period: Once priotor sacrifice. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director andor Study Monitor. Body Weights: Acciimation Period: Atleast once. Dosage Period: Daily. Sacrifice: Terminal weight. Feed Consumption Values (recorded and tabulated): Acclimation Period: Atleast once. Dosage Period: Weekly to cohabitation and dally during presumed gestation. Feed consumption values may be recorded more frequently if tis necessary to replenish the feed. These intervals will not be tabulated. MatingPerformance: Mating will be evaluated daily during the cohabitation period and confirmed by abservation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. 00598 418-013PAGE C-10 Protocol 4Pa1g8e01130 Pharmacokinetic Sample Collection: rine and Fecal Samples: Female rats will be housed individually in metabolism cages for collection of urine and fecal samples for the following intervals: one daypriorto inftiation of cohabitation to the following morning and days 6 to 7, 14 to 15 and 20 to 21 of presumed gestation. Following each 24-hour collection interval, samples will be collected into centrifuge tubes, placed on dry ice and stored frozen (70C or below) until shipment for analysis. In the event that a dam begins to deliver before completion of urine and fecal sample collection (day 20 to day 21 of presumed gestation), the dam will be removed from the metabolism cage and placed in a nesting box with sufficient bedding unt sacrifice. BloodSamples: Blood samples will be collected from each of the female rats following removal from metabolism caging (priorto administration) on eachofthe following days: on the day cohabitation is initiated (prior to cohabitation) and on days 7 and 15 of presumed gestation, as well as day 21ofpresumed gestation. The time of blood collection will be recorded in the raw data. On all daysofcollection except days 15 and 21of presumed gestation (dams at their terminal collection interval), blood samples (approximately 1 mL each) will be collected from the orbital sinus. If necessary, whole blood may be collected from an altemate site; if so, the altemate site wil be documented in the raw ata. On day 21 of presumed gestation, blood samples (approximatel4y mL each) will be collected via the inferior vena cava. If blood collection on day 15 of presumed gestation will be a final bleed for the respective dam (i... the dam will be Caesarean-sectioned that day), the sample will be approximately 4 mL and will be collected via the inferior vena cava, Blood will be collected and transferred into serum separator tubes. The samples will be spun in a refrigerated centrifuge. The serum will be transferred into polypropylene tubes labeled with the study number, animal identification, date of collection, study day. and collection timepoint. All samples will be immediately frozen on dry ice and maintained frozen (70C or below) until shipment to the Sponsor for analysis. `Shipping Instructions: All samples will be maintained frozen (70C or below) until shipment for analysis. `Samples wil be shipped frozen on dry ice via ovemight mail. A packing list will be included with the samples and sent to Kris J. Hansen, Ph.D., at the previously ited address. Both the recipient and the Study Monitor wil be notified in advance of sample shipment. 0e399 418-013:PAGE C-11 Protocol 4P1ag8e01131 Caesarean-Sectioning Observations - Day 15 Presumed Gestation: Eight randomly selected female rats per dosage group will be Caesarean-sectioned on day 15 of presumed gestation. The gravid uterus will be excised and weighed. Placenta that appear abnormal (size. color or shape) will be noted in the raw data. `The female rats (pregnant dams) wil be examined for number and distribution of: Corpora Lutea. Implantation Sites. Viable and Nonviable Embryos. (A viable embryo is oval or crescent shaped, pink, firm and enclosed in an amniotic sac filled withclearfluid. A nonviable embryo is amorphous, small, pale cplienakr,tocltoaundoyrodreoepparqeudetoflubilda.)ck, soft and enclosed in an amniotic sac filed with `Sample Collection: Caps and labeled tubes wil be weighed (combined weight, to the nearest .001 gram) before and after retention of pooled embryonic samples for subsequent use in pharmacokinetic analyses. These weights will be documented in the raw data, and copies of these weights will be included with the packing listpriorto shipment. Samplesof the amniotic fluid of each viable embryo will be collected, pooled (per litter), frozen and stored (-70C or below) until shipment to the Sponsor for analysis. Each viable embryo will be removed from the uterus with the attached placenta, pooled (per liter), frozen and stored (-70C or below) until shipment to the Sponsor. `Shipping Instructions: All samples will be maintained frozen (-70C or below) until shipment for analysis. Samples will be shipped frozen on dry ice via overnight mail. A packing lst will be included with the samples and sent to Kris J. Hansen, Ph.D., at the previously cited address. Both the recipient and the Study Monitor will be notified in advance of sample shipment. C0300 418-013:PAGE C-12 Protocol 4P1ag8e01132 Caesarean-Sectioning Observations - Day 21 Presumed Gestation: All remaining female rats in each dosage group will be Caesarean-sectioned on day 21 of presumed gestation. The gravid uterus will be excised and weighed. Individual placental weighed awnedigphltascewdilinbeinrdeivciodrudaeld.conTthaienefrest.useTshweilflebmealreermaotvse(dprfergonmantthedautmesr)usw,ill be examined for number and distribution of: Corpora Lutea. Implantation Sites. [Placentae that appear abnormal (size,color or shape) will be noted in the raw data]. Live and Dead Fetuses. (Alive fetus is defined as one that responds to stimuli a dead fetus is defined as daetaedrmfefteutsuessthdaetmdonosetsrantoitnrgesmpaornkdedtotosteimxutlrieamnedautthoatlyissinsoatremacroknesdildyeraeudtotloyzbeed; late resorption.) Early and Late Resorptions. o(Argcaonnocgeepnteussisis defined as a has occurred; late resorption if ifthis is not the itis grossly evident that case, the conceptus is defined as an early resorption.) EetalObservations: Caps and labeled tubes will be weighed before and after retention of pooled fetal (scaommpbliensedfowresiughbts,eqtuoetnhte nearest use in .001 gram) pharmacokinetic analyses. These weights will be documented in the raw data, and copies of these weights will be included with the packing list prior to shipment. Placental Samplesand Amniotic Fluid: cSoalmlepclteesd,oifntdihveidaumanliloytpiocollueidd ((pwehrelnitepro)s,sfirbolzee)naannddthsetoprleadc(en7t0aoCf oeracbhelfoewt)usunwtiilll be shipment to the Sponsor for analysis. C0001 418-013:PAGE C-13 Protocol 1Pa8g.e01133 Gross External Alterations, Sex, Body Weights and Identification: Fetuses will be examined for sex andforgross extemal alterations. Late resorptions and dead fetuses will be examined for gross extemal alterations to the extent possible. The individual body weight of each fetus will be recorded. Only body weights of live fetuses will be used to determine litter fetal body weight averages. Representative photographs of fetal gross external alterations will be taken. Blood Samples: Blood samples will be collected from each pup via decapitation, pooled (per litter) and transferred into serum separator tubes. The samples will be spun ina refrigerated centrifuge. The serum will be transferred into polypropylene tubes labeled with the study number, animal identification, date of collection, study day and collection timepoint. All samples will be immediately frozen on dry ice and maintained frozen (-70C or below) until shipment to the Sponsor for analysis. LiverSamples "The liver of each fetus will be collected, pooled (per litter), frozen and stored (-70C or below) until shipment to the Sponsor for analysis. Fetal Carcasses: The remaining carcass (and head) of each fetus will be collected, frozen and stored (70C or below) until shipment to the Sponsorfor analysis. `Shipping Instructions: All samples will be maintained frozen (70C or below) until shipment for analysis. `Samples will be shipped frozen on dry ice via overnight mail. A packing list will be included with the samples andsentto Kris J. Hansen, Ph.D., at the previously cited asdhdirpemsesn.t. Both the recipient and the Study Monitor will be notified in advance of sample METHOD OF SACRIFICE: Rats will be sacrificed by carbon dioxide asphyxiation. Live fetuses will be sacrificed via decapitation. 000302 418-013:PAGE C-14 Protocol 4P1ag8e01134 NECROPSY: Gross lesions will be retained in neutral buffered 10% formalin for possible future evaluation (a table of random units will be used to select one control group at from which all tissues examined at necropsy will be retained, in order to provide control tissues for any possible histopathological evaluations of gross lesions). Unless specifically cited below, all other tissues will be discarded. Scheduled Sacrifice: On either day 15 of presumed gestation or day 21 of presumed gestation, following the final collection interval for urine and fecal samples and blood sample collection, female rats will be sacrificed, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. The number and distributionof implantation sites will be recorded. A liver section (right lateral lobe) and the milk-secreting glands from the axillary, thoracic, abdominal and inguinal regions of each dam (left side only) will be collected, frozen and stored (-70C or below) until shipment to the Sponsor for analysis. Uteri of apparently nonpregnant rats will be stained with 10% ammonium sulfide to confirm the absence of implantation sites. Rats Found Dead or Moribund: dRealtisvetrhyatwidlliebeoreaxraemsianceridfifcoerdthbeeccaauusseeooffmdoeraitbhuonrdmcoornidbituinodn,coanbdoirttiioonn oonr ptrheemdaatyurtehe ~ observation is made. The rats will be examined for gross lesions. A liver section (right lateral lobe) and the milk-secreting glands from the axillary, thoracic, abdominal and inguinal regionsof each dam (left side only) will be collected, frozen and stored (-70C or below) until shipment to the Sponsor for analysis. Pregnancy status and uterine contents of female ats will be recorded. Aborted fetuses and/ordelivered pups will be examined 10 the extent possible, sing the same methods described for term fetuses. Uteri of apparently nonpregnant rats wil be stained with 10% ammonium sulfide to confirm the absence of implantation sites". All samples will be maintained frozen (-70C or below) until shipment for analysis. `Samples will be shipped frozen on dry ice via ovemight mail. A packing list wil be included with the samples and sent to Kris J. Hansen, Ph.D., at the previously cited address. Both the recipient and the Study Monitor will be notified in advance of sample shipment. 00303 418-013:PAGE C-15 Protocol 4Pa1g8e01135 STATISTICAL EVALUATION: Averages and percentages will be calculated. Litter values will be used where appropriate. Additional procedures andlor analyses may be performed,if deemed appropriate. DATA ACQUISITION, VERIFICATION AND STORAGE: DDiarteactwoilrlabned/hoarnadp-praonpdr/ioartceommapuntaegre-rmeecnotrdpeedr.soRneneclorwdisthiwinll21bedareyvsiaefwteedr gbeynetrhaetSiotnu.dyAll original records will be stored in the archives of the Testing Facilty. Al original data wil be bound and indexed. A copyofall raw data will be supplied to the Sponsor upon yreeqauresaftt.erPmraeisleirngveodf ttihsesdureasftwiflilnablersetpoorrte,daafttetrhewhTiecshtitnigmeFatchieltSypaotnsnoorcwhiallrgbeefcoornotnaected to determine the disposition of these materials. RECORDS TO BE MAINTAINED Protocol and Amendments. Test Article, Vehicle and/or Reagent Receipt, Preparation and Use. Animal Acquisition. Randomization Schedules. Mating History. Treatment (i prescribed by Staff Veterinarian). General Comments. Clinical Observations andor General Appearance. Tissue and Sample Collection, Processing and Shipment. Cap and Labeled Tube Weights. Body Weights. Feed Consumption Values. Caesarean-Sectioning and Fetal Observations. Gross Necropsy Observations. Organ Weights. Photographs (if required). SFeteudd,y MWaaitnetreannadncBeed(doionmg Aannadlyesnevsi.ronmental records). Packing and/or Shipment Lists. Ces04 418-013:PAGE C-16 Protocol 4P1a8ge01136 KEY PERSONNEL: DEixreeccuttoirvoefDRiersecetaorrcohf: RAelsaenarMc.h:HoMbielrdmreadn,S.PhCh.rDi.s,tiDaAn,BPTh.D., Fellow, ATS Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT DDiirreeccttoorr ooff SLatbuodryaMtoarnyagOpeemreanttio:nsV:alJeroihenAF..SBhaamrpeetr,, BM..SS.. MaUnsaegeCormomfiAtntiemea:l Operations and Member, Dena C. Lebo, V.M.D. Institutional Animal Care and CDoinrescutlotranotf,OVpeetreartiinoanrsy aPnadthColoomgpyl:iaWnc.eR:ayBaBrrboawrna,J.D.PVa.tMt.e,rsPohn,..B,.AA. CVP FINAL REPORT: AbecfoimnpalriezheednfsoillvoewidnrgafctofnisnualltraetpioorntwwiiltlhbteheprSeppoanrseodr.onThcoemrpelpeotritownilolfitnhcelusdteudtyheand will following: Summary and Conclusion. Experimental Design and Method. Evaluation of Appendices: Test Results. Figures, Summary and Individual Tables Summarizing the Above GDaLtPa,CPormoptloicoalncaendStAastseomceinatt,edReApmoertnsdmofenSutpspoarntdinDgevDiaattiao(nisf,apSptruodpyriDaitree)ctaonrd's QAU Statement. INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE STATEMENT: ITnhsetitpurtoiocneadluArneismdaelsCcrairbeedanidn tUhsisepCrootmomciotltheea.veAbllepernorceevdiuerweesddbesyctrhiebeTdesitnitnhgisFapcriolttoyc'osl that involve study animals will be conducted in a manner to avoid or minimize discomfort, distress or painto the animals. `nTehceesSspiotnysoforr'scosnidguncattiunrge tbheisloswtuddoycaunmdentthse ftahcetftahcatttthhaitsiinsfnoortmaatniounnnceocnecsesranriinlgythe dpurpolciecdatuirveesswteurdey mavaayilbabeleobftoarimneeedtfirnogmtthheesStpatoendsopru.rpNosoeaslotfemtahteivsteu(diyn.vitro) 0305 418-013:PAGE C-17 Protocol 4P1a8ge01137 REFERENCES: 1. CThersitsst.iaEn,nvMi.rSo.nmaenndtaVloyPtreokt,ecPtEio.n(A1g9e8n2c).y,IWnaVsihviongRteopnro,dDu.cCt.ivNeatainodnaMluTteacghenniiccailty Information Service, U.S. Department of Commerce, Springfield, VA 22161. 2. nCharlitsrteixaon,neM(.PSr.o(c1e9e8d4i)n.gsReopfrNoadlutcrteixvoenetoSxiycmitpyoasnidumt,erNateowloYgoyrekvaAlcuaatdieomnsyooff Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. Lang, P.L.(1988). Embryo and Fetal Developmental Toxicity (Teratology) Control Data in the Charles River Cr:CDER Rat. Charles River Laboratories, Inc... Wilmington, MA 01887-0630. (Data base provided by Argus Research Laboratories, Inc.) 4. Institute of Laboratory Animal Resources (1996). Guideforthe Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. Salewski, E. (1964). Frbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Arch. Pathol. Exp. Pharmakol. 247.367. C0030 PROTOCOLAPPROVAL: FOR THE TESTING FACILITY Ce Ndper--_ `Alan M. Hoberman, Ph.D., DABT Director of Research Jne OuWe Rayond G. Yor PHD. DABT ASsociate Director of Research Study Director arbara J. Pattersoh, (B.A. Use Commitee Chairperson, Institutidnal Animal Care and FOR THE SPONSOR Dp. Cue Marvin T. Case, D.V.M., Ph.D. `Study Monitor 418-013:PAGE C-18 Protocol 4p1i8t.013 9-nev-JS Date 09-198 Date 0-54 Date [ober 2g Date 0907 418-013:PAGE C-19 ATTACHMENT 1 SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE 6e303 ATTACHMENT 1 418-013:PAGE C-20 ProtocPoalg4e181.001123 STUDYSCHEMATIC PHARMACOKINETIC STUDY* Statof Disa Der LL Period Per . favs) | fi) Fede Fs Endof p Dosege Gestion Pad Caesarean Cen Sectioning Day2tol Presumed Gestalon Secfoning Daytsoi Day20of Presumed Presumed Gestain ~~Gestafon EEN a b. c. Dosage Period. For additional details see "Tests, Analyses and Measurements" section of the . Ds of corpora lutea, implantation sites and viable and nonviable embryos. Fetal evaluations (all fetuses - extemal examinations). 00309 ATTACHMENT 1 418-013:PAGE C-21 ProtocPolag41e82.00(123 SCHEDULE 10 NOV 98 16 NOV 98 - 21 JAN 99 27DEC98PM-01JAN99AM 28 DEC 98-01 JAN 99 12 JAN 99- 16 JAN 99 18 JAN 99- 22 JAN 99 26 MAY 99 Animals Arrive - Acclimation Begins. Dosage Period - Female Rats (42 daysprior to cohabitation until day 14 or 20 of presumed gestation). Cohabitation Period. Day 0ofPresumed Gestation. Caesarean-Sectioning presumed gestation). Period (Day 15 of Caesarean-Sectioning Period (Day 21 of presumed gestation). Draft Final Report. a. The study initiation ate is the day the Study Director signs the protocol. 0910 418-013:PAGE C-22 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 00311 418-013:PAGE C-23 MDAATTEARISAHLEESTAFETY -3 canter S5t5.144P-a1ul0,00Minnesota 1-800-364-3577 or (612) 737-8501 (24 hours) CALoLpyrriigghhtt,s 1r9e9s8e,rveMdi.nneCsooptyainMginianngd/oarnddoMwannluofaadcitnugrinofg Cthoimspany. 1isnfaolrlaoawteidonprfoovridtheed Tphuartp:ose of properly utilizing SM products 7) ptrheiorinafgorreseamteinotn iiss ocbotpaiiednedinfrfoumllSHw,ithandno changes unless 2) dniesittrhiebruttheed cWiotphy nthoer tihnetenotriiogninaofl eiasrnriensgalad oprrofoitthetrhweirsseon. TDRIAVDISEIOANE: :3M CHEMICALS 10FCN-U9M5BEFRL/UUO.RPA.DC.Brand Fluorochemical Surfactant 9988--00220171--00180838--75 0000--5511113355--0099038642--17 9988--00221017--30911064.-15 IS2SFU-E0D:002J-a1n0u4a4r-y129, 1.998 SDOUCPUEMRESNETD:ES:16N-o3v7e9m6b-e9r 05, 1987 0000.-5511113355.-0029301515--28 1. INGREDIENT C.AS. WO. PERCENT PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOOMNAATTEE............ 2378957.139.-939.6382 -- 88s PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE............ 2690422700-.4595-.35 23 --78 POTASSIUM PERFLUOROALKYL SULFOWATE...... 3872.28.11 -3 2. PHYSICAL DATA BVAOPIOLRINGPREPSOSIUNRTE::................................. NIA N/A EVAVPAOPRORADTIOENRANT.E.S:. I..I_I..TI...TY1:11] Wa N/A `SSPOELCUIBFIILCITGY RIAN VWETAY:T..[.E1.1..R1..1..:11.1 scal.igh0t.6 Water=t PERCENT VOLATILE:.............. 0 (%Bulk) VISCOSITY: oon NI(D0.1% Aqueous) MELTING POINT: 1L1IIIIIIIII Nin APPLEiAgRhAtNCcEolAorNeDd,ODOfRr:ee flowing powder. Abbreviations: N/D - Not Detersined N/A - Not Applicable 50912 CA - Approximately 418-013:PAGE C-24 JSaOnSu:aryFC2-99,5 1F9L9U8ORAD Brand Fluorochemical Surfactant Pace 2 3. FIRE AND EXPLOSION HAZARD DATA FFLLAARSUHAPBOLIENTL:I.N.I.T.S..+.L.E.L.:..1c1.1.1.... WNoInAe AFULTAOMIMGANBILTEIOLNINTITESMP- EURELA:T.U[R11E[1S1l.11 NNI/AA EXMTaItNeGrU,ISHCIaNrGbonMEDdIiAo:xide, Dry chemical, Foam SPWEeCaIArLfuFlIlREprFoItGeHcTtIiNvGePcRlOoCtEhDiUnRgE,S: including helast, self-contained, Positive pressure or pressure' dessnd breathing apparatus, bunker cost pirnodtepcatnitvs,e cboavnedrsinagrofunodr earxapso,sewdaiasrteasandofletghse, hfeaadc.e mask, and UN`USSeUeALMaFzIaRrEdoAuNsDDeEcXoPLaOpSoIsOiNtioHnAZAsReDcSt:ion for products of cosbustion. 4. REACTIVITY DATA STABILITY: Stable INNCoOtMPAaTppIlBiIcLaIbTlYe.- MATERIALS/CONDITIONS TO AVOID: WAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur. HAZCaArRbDoOnUSMoDnEoCxOiMdPeOSaInTdIONCarPbRoOnDUCDTiSo:xide, Oxides of Sulfur, Hydrogen Fluoride, Toxic Vapors, Gases or Particulates. 5. ENVIROMENTAL INFORMATION SPOIbLLserRvEeSPOpNrSaEc:autions from other sections. Vacuus, use Wet sweeping bceompaonunidgniortioantesrourtcoe.avoiCdleadnustuipngr.esiCdAuUeTIOwNi!thAwavtaecru.um cPllaecaenerincoaunld approved metal container. Seal the container. REDCoOMMnEotNDErDelDeIaSsePOStAoL:waterways or sewer. Do not use in products or p1/r1o0cesosfesthethaltowecsotuldECSr0esuolrtLCiSnOaqcuoancteinctrcaotnicoenn.tratIinocnisnergarteeateirn tanhan imantdeursitarli.al oCrombcuosmtmieornciaplrodfuacctislitWiyllininthceludperesHFe.nceDoifspoasaclombustible alternative: Dispose of waste product in a facility permitted to 00913 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately J4a0nSu:ary102-8,5 1F9L9U8ORAD Brand Fluorochenical Surfactant 5. ENVIRONMENTAL INFORMATION (continued) 418-013:PAGE C-25 ace 3 `accept chemical waste. ENSSVl6IuRMaOrgM.iElNlATaASuLuanttiDciATsAhF:(isLhepoLnCiSOs, suFcartohcehaidruWsi)n=n8osu(gPiisls,phRaaliensbporuoTssrlosust)(ss3a8lmmg/l, SO7aai:ran8e0r0i2y0en=tWimlg./L; 48-Hr. EGS0, Daphnia Magna = So wail; GOO--004 REVGoUlLaAtTiOlReY OIrNgFaOnRiMcATIGOoNs:pounds: N/A. Voc Less H20 & Exempt Solvents: WIA. Since regulations vary, consult applicable regulations or authorities before disposal. U.S. EPA Hazardous Waste Number = None (Not U.S. EPA Hazardous). TTshcias, pErIoNdEuCcSt, cGomOpSl,iesAICwSi,thMEtThIe acnhdemiKcoarela.registration requirements of ETrIcRaEn MwAaZAzRaD: cNuoss:PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC Yes &. SiscsteD FiRsT AID eveTacneodmiaactre:ly flush eyes ith large smounts of water for at lesst 15 minutes. Get inmediite medical Strention. SKITnanecdoiuTaAtCeTl:y flush skin with large amounts of water. Remove Ccoonnttaarmiinnaatteedd ccllootthhiinngg. beIfforierrirteautsei.on persists, call a physician. Wash INIHtALAsTiIgOnNs:symptoms occur, remove person to fresh air. If signs/sysptons continue, call a physician. IFDrsiuanLkLotwweoD:glasses of water. Call a physician. 7. PRECAUTIONARY INFORMATION exAevopirdoTesCyTeIOcNo:ntact. Wear vented goggles. 0911 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-013:PAGE C-26 MSDS: FC-95 FLUORAD Brand Fluorochesical Surfactant January 29, 1998 7. PRECAUTIONARY INFORMATION (continued) PAGE 4 SK`IANvoPiRdOsTkEiCnTIOcNo:ntact. Wear appropriate gloves when handling this mFaetceorsisaeln.ded:A pabiutrylof rugblboevre.s saUdsee ofnrsomorthemofroelloofwtihneg mfaotlelroiwailn(gs) are pcoevresroinnagl, pcroovteercatlilosn. itePmrsotaesctinveecegsasramreynttso p(roetvheenrtthsakninglcoovnetsa)ct:shouhledad bpeolymeatdheyloefnee/iptohleyrvionfyltihdeenfsollcohwlionrgidemat(eSrairaalnse:x). REUCsOeMMEwNitDhEDaVpEpNrToIpLrAiTaItOeN:local exhaust ventilation. Use in a wellveemnitsisliaotnesdbearleoaw. recPormomveinddeedsufefxipcoiseunrte vliemnittisl.atioInf texohamuasitntavienntilation is not adequate, use appropriate respiratory protection. RE`SAPvIoRiAdTObRrYeaPtRhOinTgECToIfONd:ust. Select one of the following NIOSH approved arcecsopridraatnocreswibtahseOdSHonA areigrubloartnieoncso:ncenhtarlaft-imoanskofdusctonatnadminmainsttsreasnpdiriantor, fhualllf--fsaacsek ssuupppplliieedd aaiirr rreessppiirraattoorr,. full-face dust and mist respirator, PRDEoVENnToItONeaOt,F AdCrCinIkDENoTrALsmoIkNeGESwThIeOnN:using this product. Wash exposed aberfeoarsetheaotrionugg.hly With soap and Water. Kash hands after handling and RECKOeMeMpENcDoEnDtaSiTneOrRAdGr:Ey. Keep container closed when not in use. FINRoEnfAlNaDsmEaXbPlLeO.SION AVOIDANCE: OTNHoERsmPoRkEiCnAgU:TISOmNoAkRiYngINWFhOiRMlAeTIuOsNi:ng this product can result in cofonttahneinhaatziaorndouosf tdheecomtpoobsaictcioonandp/roorducstmoskemenatnidonleedadinto stehcetiofnorm4atoifon this KSDS. MES WAZARD RATINGS: HPEEARLSTOHN:AL2PRFOLTEACMTMIAOBNI:LITXY:(S0ee RpErAeCcTaIuVtIiToYn:s, 0 section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT TYPE AUTH SKIN PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOORNAATTEE...... 00..11 MMGG//MM33 PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONRAATTEE...... 0.1 0.1 MG/M3 MG/M3: TM am TMA HA 3M 3M TM oo Y Y ~ YC00o15 Abbreviations: N/O - Not Determined N/A - Not Applicable GA - Approximately 418-013:PAGE C-27 JMuSnDSu:aryFC-299,5 1F9L8U8ORAD Brand Fluorocheaical Surfactant DPOSURE LIMITS (continued) ace 5 INGREDIENT VALUE UNIT TYE AUTH SKIN" POTASSTIN PERFLUOROALKYL SULFOWATE... 0.1 WG/KI TWA 3M Y IT+hheeS1KIaaNoitneNgnOtTimAauTlcIOoNuc:sontsreLsiibsbtrueatdnieosnuantbdsotateynheec,esoveeiirtnahdleiclratebexydpaoiwsriubtrohernb'eyY' torhu,endecmrourteaSnKeIpoNaurstriecfrueoluratretloy, AC diract Contact with the substance. Vehicles can alter skin absorption. `SSSOKUR:CE OFaNEXRPeOScUoRmEenLdIeMdITExDApToAs:ure Guidelines 5.HEALTH MAZARD DATA eveilcaontEyaec:irritation: signs/sysptoms can include redness, selling, pain, and tearing. SKIMSNiilgaCnOs/NSTskAyiCnpTt:iornrsitactainoninc(laufdteerrepdrnoelsosn,gedsweolrlirnegp,eataendd citocnhtiancgt.): m"arytonbdeedabstoimreb.ed through the skin and persist in the body for an TH`MMAaLyATbIeONh:aratul if inhal:ed. TMianye.be absorbed by inhalation and persist in the body for an extended Single overexposure, above recommended guidelines, may cause: SIorrreinteastsionof (tuhpepernosreesapnidrattohrryo)a:t, sicgonusg/hsiynsgptaonmdssnceaneziinngc.lude IFInSgAeLsLtOiWoEnD:is not a Likely route of exposure to this product. tIhlilsnesmsatemraiyalr.esult from a single swallowing of a moderate quantity of May be haraful if swallowed. MUTMAuGtEaNgIeCnIiTcYi:ty assays indicate the product is not mutagenic. . 060316 Abbreviations: NID - Not Determined H/A - Not Applicable CA - Approximately 418-013:PAGE C-28 MJSaDnSu:aryFC2-59,5 F1L99U8ORAD Brand Fluorocheaioal Surfactant ace "3. HEALTH HAZARD DATA (continued) REWPoRtODUtCeTrIaVtEo/gDeEnViEcLOiPnHEtNhTeALratTOXaItNSo:ral doses below ssternally toxic Levels. OTTHEhRisHEPArLoTduHctHAiZsARnDotINkFnOoRwMnATItOoN:contain any substances regulated under California Proposition 65. A Product Toxicity Sussary Sheet is available. SECTION CHANGE DATES HEADING SECTION CHANGED SINCE Novesber 05, 1957 ISSUE Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately TbehecoirnrfeocrtmatasionofitnhethdiasteMatiessruieadl. Sa3fMetMyAKDEatSaNOShWeAeRtRA(NMTSIDESS), isEXPbReElSiSeEvDedORto HIEMRPCLHIEADN,TABIINLCILTUDYINOGR,'FIBTUNTESNSOTFOLRIMAITPEADRTTIOC,ULAANRY PIUMRPPLOISEED HORARCROAUNRTSYEOFOF uPhEeRtFhOReMrANtChEe O3RM UprSoAdGuEctOFiTsRfADiEt.forUsearpairstirceuslpaornsipbulreposfeoranddetesruiatianbilneg for Guasnera'fsfemcetthtodheoufseuseandorapappplliiccaattiioonn.of aGi3veMnprtoheducvta,riestoymeofoffwahcitcohrsartehat tuhneiquuseelry ewviatlhuiantethetheuse3Hr-sprokdnuocwtledtgoedeatnedrmcionnterowlh,ethietrisitesisseTnittialforthaat particular purpose and suitable for user's method of use or application. D3Mueprtoovitdheesrienmoftoermaptoisosnibiilniteylectthartonieclecftorromniacs atrsaenrsvfiecrematyo hiatvsecursetsoumletresd. in errors, omissions or alterations in this inforsation, 34 makes no irenpfroersmeanttiaotnioonbstaiansedtofriotms acodmaptlaebtaesneessmayornoatccubreacays.curIrnenatddiatsiotnh,e information in the KSDS available directly from 3. 000917 418-013:PAGE C-29 ATTACHMENT 3 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE 00918 418-013:PAGE C-30 ATTACHMENT 3 Version: 41Pr8oto0.c5ol0N4O118V.903613) TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page 1013 Test Article: PFOS Vehicle: 0.5% Tween 80 in R.O. Deionized Water A. Purpose: TofhdeopsuargpeosseusopfetnhsisiopnrsocoefdPuFreOSis taondprtohveidveehaicmleetfhoordorfaolratdhmeinpirsetpraartaitoinonto rats on Argus Study 418-013. B. General Information: 1. Aslplecsiufsyptehnesipornotcoocnoltaniunmerbserw,illtebset laratbieclleedidaenntdifcioclaotriocn,odAerdg.usEbaacthchlabel will number, concentration, and storage conditions. dosage level, preparation date, expiration date 2a. _SuXs_penDsaiiolnys will be --prepareWde:ekly _For__daysofuse 2b. Vehicle wil be prepared: Daily X_ Weekly _For__daysofuse 3. Suspensions will be prepared at a final dosage volume of5 mL/kg 4. Safety X_ X Gloves, lab coat, goggles Dust:Mist Respirator or safety glasses and faceshield _ -- Half-Face Respirator Ful-Face Respirator/Positive Pressure Hood Z Tyvek SuivApron 5. --DosageYessuspensions_aXdj_ustNeod (fCoarlFcruelaetbioanssebaansded%oPnur1i0ty0%) __ FreeBase __ Purity 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 00319 418-013:PAGE C-31 ATTACHMENT 3 Version: 418.P0r1o3t(o0ca5lN4O18V.30613) TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page 2013 NOTE: Ttheestloarwtidcolesawgilel.beOnprceepathreedfianaslavoselruimalesdialurteioanchfireovmedt,hesthiirgbhardsosaargeetotobe added to the containers; administration. mixing should occur during sampling and/or C. PreparationofVehicle 1. Aladbdeltehdecroenqtuaiirneedr.aHmeoautntthoef wR.a0t.erdetoio5n0izCe,d w+5atC,etrao dadntahpeprreoqpuriiraetdely amount of Tween 80 and mix until uniform (See TEST ARTICLE CALCULATIONS) D. Test Article Suspension Preparation: 1. Tamoopurnetpaorfetetshtea0rt.i6c4lem(gSiemeL,TEGSroTuApRVTIsCusLpEenCsAiLonC,UaLdAdTIthOeNSre)quiinrteodan vaephpircolperiaantedlyhesaitzetdh,elmaibxetluerdectoont8ai0neCr.5QSC afodrtaopptrhoexriemqautierleyd 3a0momuinnuttweisth or unti the TAS dissolves. 2. Once the test article has dissolved; spin while the suspension cools. (Be msuarye btheeprereipsaarevidsitbhleedvaorytebxe,fotrhiesuwsiell.)achieve the desired emulsion. This 3. Toprepare the 0.32 mg/mL, `amount of stock suspension Group (Group IV suspension, V) (See TEST remove the ARTICLE required CALCULATIONS), QS ad with the vehicle and mix. 4. Toprepare the 0.08 mg/mL, amount of stock suspension Group (Group Ill suspension, remove the IV) (See TEST ARTICLE. required CALCULATIONS), QS ad with the vehicle and mix. 5. To prepare the 0.02 mg/mL. amount of stock suspension Group (Group Il suspension, remove the Il) (See TEST ARTICLE required CALCULATIONS), QS ad with the vehicle and mix. 009320 i 418-013:PAGE C-32 ATTACHMENT 3 Version: 418.P0r1o3to(c0a5lN4O1V8.9081)3 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page3ol3 6. Tveohipcrleeptaoreanthaepp0rompgr/imatLe,lyGrsoizuepd,| suspension, add labeled container required amount of (See TEST ARTICLE CALCULATIONS) and mix. Witten by: _L;::o Call Approved by; {zxfF /- Datef: a05 new -0F Clarification: _x No _ Yes (See attached clarification form.) Initials/Date : __*< 9-3-9 00921 PRIMEDICA 418-013:PAGE C-33 Sn--at PROTOCOL 418-013 Oral (Gavage) Pharmacokinetic Study of PFOS in Rats SPONSOR'S STUDY NUMBER: T-6295.12 Amendment 1 - 6 January 1999 1 CaesareOa bsen rva- tioS ns-e Dac y2t 1Prieso umendGi estn atig on (pages 12 and 13 of the protocol) and Necropsy (page 14 of the protocol): At Caesarean-sectioning on day 21 of presumed gestation, lung and liver samples will be collected from three fetuses per litter from five litters per dosage group. These collections will occur after weighing and examination for sex and gross external alterations. One half of the lungs and one lateral lobe of the liver will be individually retained in neutral (minimum buffered 10% formalin in scintillation vials. Prior to of two) sections (approximately 1 mm thick) of this fixation, several half of the lungs and this lobe of the McDowell-Trump's liver will Fixative be removed using a scalpel (stored under refrigeration in and will be scintillation retained in vials) for future evaluation. liver will be flash The other half of the lungs frozen in liquid nitrogen and and the other lateral lobe of the stored in heat-sealable pouches on dry ice. The procedure for collecting and flash freezing these tissues will be available in the raw data. The liver of each of the remaining fetuses, plus any remaining liver from the three selected fetuses, will be collected, pooled (per litter), frozen and stored (-70C or below) until shipment to the Sponsor for analysis as described on page 13 of the protocol. The lung and liver sections in McDowell-Trump's Fixative and the lung and liver samples in neutral buffered formalin will be shipped (on cold packs and ambient cliognhdtimtiiocnrso,scroepsyp,ectrievsepleyc)t,ivfeolryp,otsos:ible future evaluation by electron microscopy or 00922 418-013:PAGE C-34 ProAtomceonld4m1e8.n0t13 Fase? Jeanne deWard Pathology Associates International 4915 D Prospectus Drive Durham, North Carolina 27713 Telephone: (919) 544-5257 Telefax: (919) 544-3218 The frozen samples will be shipped (on dry ice) to Kris J. Hansen, Ph.D. atthe address cited in the protocol for possible future biochemical evaluation. All recipients will be notified in advanceofsample shipment. Reason for Change: previous studies The Sponsor has requested that these additional samples be retained for continued evaluation of the reasons for reduced pup survival observed in 2 ded CIPI He 06 F-99 Alan M. Hoberman, Ph.D., DABT Director of Research Date RaymondG. York, Ph. )ABT Associate Director of Resarch Date Study Director C 2tnollwle an 7 i CDheaniarpCe.rsLoenb.o,InVs.iMu.tDio.nal Animal CareDaantde MSatruvdiynMTo.niCtaosre, DVM. PhD. Date Use Committee 00923 APPENDIX D DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 00921 418-013:PAGE D-1 DOEPVEIRATAITOINNGFRPORMOCTEHDEUPRREOSTOOFCOTLHEANTEDSTTHIENGSTFAACNIDLAITRYD 1. rEeicgehitvyevdirtghien tfeesmtaalretircaltesfworefroertrya-ntdwoomdlayysaspsriiogrnteodctoohtahbietsattiuodny,arnadther tahsasnig1n2e0d vaisrgtihnefpeompaulleastiaosnsifgornetdhetostsutduyd.y, dosed, mated and then Female rats with confirmed date of mating were randomly selected for gestation day 15 (DG 15) Caesarean-sectioning. All remaining rats (Cwaietsharaenadn-wsitehcotuitoncionngf.irRmaetdsdwaittehs noof mcaotnifnigr)mewderdaetaeossfigmnaetdintgo wDeGre21 Caesarean-sectioned on their estimated DG 21. Tofhethseesdteuvdiyatbieocnasudsied nsouftfiacdiveenrtsrealtys awfefreectatvhaeiloaubtlecotomecoolrleicntttehrepreation pharmacokinetic data. All deviations are, umented in the raw data. ZA 2t-z00 R: ond G. York, of DABT Date Associate Director of Research and Study Director 00325 APPENDIX E TEMPERATURE AND RELATIVE HUMIDITY REPORTS 009Z ARGUS 418-013:PAGE E-1 Temperature and Relative Humidity Report Location: Room 04 Protocol Number: 418-013 Range of Dates: 10-Nov-1998 15:00 to 23-Nov-1998 15:49 Target Range: `Species: Rat `Total Number of Days: `Total Number of Hours: Total Number of Data Points: Temperature | Relative Humidity 64F 10 79F 30% 10 70% 14 14 312.75 31275 312 312 Mean( SD): Maximum: Median: Minimum: NumberofPoints in Range (%): NumberofPoints High (%): Number of Points Low (%): 691 (25) | 585 (1D) 764 644 683 586 66.8 536 M2 (1000) | 312 (1000) 0 .0) 0 0.0) 0 .0) 0 .0) Report Generated: 03-Feb-1999 at 13:44 COMMENTS: REVIEWED BY: ule \. DATE: _ghls3 Cumulative by Location (v04.01.97) 00927 ARGUS 418-013.PAGE E2 Temperature and Relative Humidity Report Location: Room 27 Protocol Number: 418-013 Range of Dates: 23-Nov-1998 15:49 to 22-Jan-1999 16:00 `TSapregceitesR:aRnagte: TToottaall NNuummbbeerr ooff HDoauyrss:: Total Number of Data Points: TSemFpleroaTtHurFe | Rola3t0i%vetHou7m0i%dity 143601.74 143o9t.74 1442 1442 | Mean ( 5D): MMeiMdnaiixaimnmuu:mm:: 07 @on| S41 82) 776902.880 6619.4.198 Number of Points in Range (%): Number of Points High (%): Number of Points Low (%): 1442 0 0 (100.0) 1418 (.0) 0 (0.0) | 24 (98.3) .0) an Report Generated: 03-Feb-1999 at 13:48 COMMENTS: REVIEWED BY: onal Sra Son DATE: _2/3/7) Cumulative by Location (v04.01.97) 40928 418-013:PAGE E-3 ARGUS s eme------------------rir Relative Humidity Location: DReovioamti2o7ns Report Protocol Number: 418-013 ES -- e eee e a mee Range of Dates: 23-Nov-1998 15:49 to 22-Jan-1999 16:00 HSpuemciideist:y RTaatrget Range: 30% to 70% OaDna1t9e99 T16i:m0e0 R2H81.L 0O56Jlaann1i9o9 200::0000 B2I64LL 0O6sJJaannitsseese 00120000 220313LL 0066JJaann119s9999 0034:0000 117864LL OO66JJaanni1oos% 00560000 11649900 OT6uaanniiseess 01170000 Hanis 200 227790LL 2910 fiiddaann11e9s8e 11340000 22884800 1T1iv3a0n1i9e8s9e 1165:0000 22867900 14Jan 1999 00:00 2960 taDna1teese T0i2m00e R2H8s. 114dJaann11e9s0e 00430000 22r78SlL 114eJdaann1199%999 0058:0000 2287301L 1144dJaantno1soe 00870000 225883L1 H= Value outofrRaHn.g=e R-eHliagthive HLum=idViatlyue(%o)utofrange - Low Report Generated: 03-Feb-1990 at 13:57 /These deviations did not adversely affect the outcome or interpretation of the study. The following deviation(s) impacted on the outcome of the study as described: Study Director: v ) Z-- Deviations by Location (v04.01.87) Date: \) = Fed 99 00929 APPENDIX F HISTORICAL CONTROL DATA 50930 418-013:PAGE F-1 SUMMARY OFCRDEPRRAOTDUCTIVE INDICES PERIOD JUNE 1996 - JUNE 1998 NUMBER OF STUDIES 105 NUMBER OF RATS: TESTED 23: FOPURNEDGDNEAANDT 206P5 DEALBIOVRETREEDD 00 NUMBER OF RATS PREGNANT AT `CAESAREAN-SECTIONING 282 N`UCMOBNECREPOTFUSRALITTSTWEIR:TH SINGLE RESORBLEvDE 61 ABORTED 0 % PREGNANT AVERAGE # CORPORA LUTEA AVERAGE # IMPLANTATIONS AVERAGE LITTER SIZE AVERAGE # LIVE FETUSES AVERAGE # DEAD FETUSES AVERAGE # RESORPTIONS AVERAGE # EARLY RESORPTIONS AVERAGE # LATERESORPTIONS MEANor% 528 169 153 1s 01 07 0.7 00 RAMNEGAEN/OSrT%UDY (64.0-100) (14.0210) (12.8180) (118169) 14) 20) 19) 01) 00931 418-013:PAGE F-2 SUMMARY OF REPRODUCTIVE INDICES CD RAT MEAN or % AVERAGE % DAMS WITH ANY RESORPTIONS 46.1 AVERAGE % DAMS WITH ALL CONCEPTUSES RESORBED 01 AVERAGE %DAMSWITH ONE OR MORE LIVE FETUSES 998 AVERAGE SEX RATIO, (% MALES/LITTER) 502 AVERAGE FETALBODYWEIGHT (G) ~~ 3.48 AVERAGE FOR MALES (G) 358 AVERAGE FOR FEMALES (G) 338 AVERAGE % DEAD OR RESORBED CONCEPTUSESILITTER 47 RANGE/STUDY MEAN or % (0-875) (04.5) (85.4-100) (42.857.0) (3.10-3.78) (3.17-3.90) (2.98-3.67) (0124) 00332 418-013:PAGE F-3 SUMMARY OF MATERNAL NECROPSY OBSERVATIONS CD RAT PERIOD JUNE 1996 - JUNE 1998 #STUDIES, 124 #RATS TESTED 2668 #RATS PREGNANT 2480 #RATS DIED Fad # RATS ABORTED 0 #RATS DELIVERED 431 #RATS WITH 100%RESORPTION 3 EXTERNAL OBSERVATIONS Red substance around noseand/ormouth White substance present in anterior chamber of oyes Red perivaginal substance MEAN RANGE /STUDY NO% ON % 2 007 01 (0125) 1004 01 (042) 1 0.04 0-1 (04.0) GROSS LESIONS ESOPHAGUS `Tom( attributed to an intubation accident) LUNGS Dark red (attributed to an intubation accident) THORACIC CAVITY Filled with light red or cloudy red fluid THYMUS Large AXILLA Mass present Lymph nodes enlarged on left side 1004 01 (040) 1004 01 (040) 2 007 1004 1004 1004 01 (040) 01 (040) 01 (040) 01 (042) * Pregnancy status for one dam could not be determined * Onewas a moribundsacrificeand one was attributedtoan intubation accident . 00933 418-013.PAGE F4 `SUMMARY OF MATERCNOARLANTECROPSY OBSERVATIONS GROSSLESIONS VER Adhesionbetweenleft laabtderoamlilnoablewaanldleflateral Rliogbhetslpraotrtaudneeddrtmheradoiualgnh diaphragm STOMACHINT`ESSTItNEaondminatesctinhes Gdeicstuemncdoendtwaiinhegdaasblack `Sstuobmsatcahnccoentained a dark red, granuar substance BACK `Spine, protrusion ofbone. on dorsalhoracicporton KINEY(S) Penis,sighimoderate duidilwitah otrwiothonut Peldvaitsi,onmarkediextreme LMaotrtgieed CoSrmtaelxpti Le,pelviscontained Byellloaw ftlpueiledvisrconataline,d numerouscalcull PRaiglhet indentation Lefct, somwairlthitnguhtm,eeernolxuasr.ged vipisncpoouisntyyeelllloowwafrlueiad sinand pelvis ADRENAL GLLaANrDg(eS) NOM%EAN RAONNGE /S%TUDY 100 100 01 (040) 01 (049) 100 100 1 004 01 (040) 01 (040) 04 (042) 1 004 01 (040) 16 08 17 00042 2100047 1004 100 111 000000 02 (0125 0011 ((004400) 0011 ((004400) ot (034) 01 (040) 0011 ((004400)) 01 (128) 1 004 100 01 (040) 01 (040) 0093% 418-013:PAGE F-5 `SUMMARYOF MATERCNOARLANTECROPSYOBSERVATIONS GROSS LESIONS ABDOMINAL CFAlVoIdwTiYnightredfid SPLEEN Twowhitareas on Lasregreo.salsurface BLADDER Wallthickandcontained one calculus URETERS Distended withclearfluid VAGINAICERCVoIrXvixcontaaidrnkeecd, Gogrevliaxtcionnoutsaisnuebdstgarneceen. VVaigsicnoaucsontuaidined brown, Viscousfluid uterus Contained resbrownfiid RRiiLgegfhhttthhohomo,mml,,uatmbreseenaandbtiskeent on cGsteenrdeevndd;wioivatcriiacnaefoanldrd, Right hom,clearmasses P`Scounbotsatinaorinfncgeetgfperlueitaseteriinnnotoeuhsnom prolapsed ovaries RiTgihdtcloendtabiunsedalaroesdt, NoMwEAN 1 004 210osw 1 oo 2 0.07 1 004 1 oo 1 ooo 1 004 11 os0.04 1 004 1 om 1 oom 1 004 RANNGEw/STUDY 01 (040) 0o1z ((004800)) 01 (40) 01 (04.0) 04 (040) 01 (040) 01 21 01 (040) 0o-t1 21 (0-40) 04 (040) 01 (040) 01 (040) 04 (040) 0035 418-013:PAGE F-6 SUMMARY OF FETAL GROSS EXTERNAL ALTERATIONS. CORAT PERIOD JUNE 19-9JUN6E 1998 ##SLITTUTDEIRESSEXINACMLIUNDEEDD 8 1652 #LIVE FETUSES EXAMINED 23689 ALTERATION HEAD Exencephaly Hematoma Microcephaly EYE(S) Eyobuiges depressed Eyelids open Microphthaimia EARS. Lowset snout Short TONGUE Absent Protrudes Fusedtolowermargn oforalopening No% FL 4 024 4 002 FL110000 FL 1100006 LF 99 005044 L202 F 2 001 F L 1100006 Lo1 006 F100 FL 2012 2 001 FL 1100006 F L 1100006 L 1 006 F100 RANGE NO 01 01 01 01 01 01 /STUDY % (042) (003) (022) (002 (040) (003) 02 (0-125) 02 (009) 01 01 (042) (003) 01 (040 01 (003) 01 (0125) O01 (009 01 (042) 01 (003) 01 (040) 01 (003) 01 (042) 01 (003) O-1 (04.8) 01 (004) L: LITTER INCIDENCE F: FETAL INCIDENCE 000936 . 418-013:PAGE F-7 `SUMMARY OF FETAL CoRAT GROSS EXTERNAL ALTERATIONS ALTERATION saws Micrognathia Agnathia cen Now Lo2om FF[o112o000m0m1 LFoi1o0m6 RANNGE w/STUDY 01 (048) 000111 ( (00:0093)) 0011 ((000448)) BODY Edema Umbilicalhemia Spinabifida Hematoma Conjoined tin HINDLIMESThidimbpresent ANUS Noopeningpresent TAL Threadiike Agenesis. Short Constricted L 2 012 F 2 001 LF 55 000302 L 1 006 FLFoz21 o00m020 LFo1i0o6m FL oi1 o0m06 FL o11 o0m05 LFos5 o0.m30 L 2 012 F 2 001 FL o11o000m6 LFo110o0m6 01 (043) 01 (00.3) 00-11 ((0040.33)) 01 (042) o00r11 (0(00040.303)) 0o1i ((000439) 0011 ((00-194)3) 0011 ((002082)) 0011 (og (0-125) 01 @1 ((004:.053)) 0o1f ((00-0224)) Oo0t1 ((002042)) LiFLEITTTAELRIINNCCIIDDEENNCCEE 60937 418-013:PAFG-E8 `SUMMARY OF FETAL SOFT TISSUE ALTERATIONS CDRAT PE#SRTIUODDIES INCLUDJEUDNE1996 -JUNE 199386 #LFIETTTUESRESSEEXXAAMMIINNEEDD E8x0 ALTERATION BRAN Latraiventrides, modersiadiation ~~ Lateral ventricles, markeddilation `Thirdventricle, mLaGreakse,tdaidmnioedlgtauhtriiiarordnavye|nlt. sihmaegpueaddyshaped eves Microphthaimia TONGUE Absent aw Micrognatria HEART `Septaldefect VESSELS Innominate, absent No% FL1102002 L 2 024 F 2 003 L 1 012 F 1 002 LFoi1 001m2 1 012 F 1 002 L303 F 3 005 LF1100012 L F1t02ooiz L 1 042 F 1 002 LFos6 0o7n2 RAONN GE/ST%UDY 0011 ((000483)) 01 (043) 01 (006) 01 (042) 01 (0-06) 0011 ((004020)) 04 (042) 01 (006) 01 (043) 01 (007) 0011 ((0048.0)) 0041 ((00400)) 01 (04.0) 01 (00.6) 0022 (004-283)) LF:: LFIETTTAELR IINNCCIIDDEENNCCEE 00938 418-013:PAGE F-9 `SUMMARYOF FETACLSDORFATTTISSUEALTERATIONS ALTERATION VESSELS (inCnOomNiTn)ate,arissson Uombfitlicalartery, urdiensacreynbdlsatodldeefrtof Situs inversus Mota,descendsto Puriimghotnaryartery, bedhesicnednadosritoaright LUNGS Rightapical,cardiac lanodbdeissapphpveaagrmaatsiocne inatbesremnedtiatslobe, BODY Edoma ABDOMIN`ASLitCusAiVInTvY eorfsivuers, Intestines, stomach, sKpildeneenyspancreasand KIDNEY(S) Polis,sightdiation Pelvis, moderatedilation ADRENALD(aS)rkred FL:: LFIETTTAELRIINNCCIIDDEENNCCEE N% FLo 11 000122 FL 1165 012818 L F 10 1 2 0@ L 1 012 Fo 1 002 LL 1 012 Fo 1 002 L 1 012 FL 11 001022 F 1 002 FLo 11 001m2 FLo 11 000122 FL 22 002043 LFo 1 0.42 1 002 LFo 22 0024 RAONN GE/ST%UDY 01 (040) 01 (008) 0022 ((0018.74)) 0011 ((000482)) 04 01 ((004008)) O01 (040) 01 (008) O01 (040) 0011 ((00:4006)) 01 (008) 0011 ((000480) 0014 ((000480)) 0022 ((008173) 0-1 (043) 04 (008) 0011 ((000483)) 00939 418-013:PAGE F-10 SUMMARY OF FETAL SKELETAL ALTERATIONS CO RAT PERIOD JUNE 19-J9UN6E 1988 #STUDIES INCLUDED 36 #LITTERS EXAMINED 816 #FETUSESEXAMINED 6151 ALTERATION SKULL Frontal(s): incompleteolyr L notossified F Pnaroiteotsasl(isf)i:edincompleteolry L F Nasai(s): short L F Sphenoid: incompletely L ossified F MaxillaeandPremaxillae: L short F `Skull: incompleotrenloyt L ossified F VERTEBRAE `Thoracic: Centrum,bifid L F + Centra, unilateral L ossification F : Centrum, incompletely L ornotossified F +Centra,fused L F + Avch,small L F Lumbar:Centrum,bifid L F : Centum, incompletely ~~ L or notossified F : Arches, incompleteloyr L notossified F +Centra, unilateral L ossification F N% 2 024 2 003 2 024 2 003 3037 3 005 1 012 1 002 3 037 3 005 1012 2 003 67 821 74 120 6 074 6 010 3 037 4 006 1012 1 002 1012 1 002 1012 1 002 2 024 3 005 12 147 17 028 2 024 2 003 RANGE/STUDY ON % 041042) 01(008) 041042) 01(008) 01(042) 041(008) 01(042) 01(008) 041(042) 01006) 0021(004122)) 05 (0-208) 06 (03.4) 02080) 02(010 02 (0:83) 0031004128)) 01005) 01(040) 041008) 01043) 01(006) 02 (0-83) 03018) 03 (0-12.0) 04 (021) 01(042) 01008) L: LITTER INCIDENCE F: FETAL INCIDENCE 009410 418-013:PAGE F-11 `SUMMARY OF FETcADLRSAKETLETAL ALTERATIONS ALTERATION RIBSCervicalRib(s)present Oneormore,wavy Oonsseiofiremda(rhs,yipnocpomlpalsetteoilrcyn)o,t Eoxsvsaifiibesd, 14th,unilateral Extra ibs, 14th,bilateral N% FLo223 024m2 FL 522 3m 08 FL 232 207%0 FL 44 000483 FLo11 00202 RAONNGE/S%TUDY 04 0174) 0054002393)) 08051) 00250(2098.1)) 0044(00012620)) 001100000480)) STEROnNeEoBrRmAoEreincompletely ossifiedornotossified Fused Asymmetric FL101690 1234346 FL1100012 FL110022 0-0192 ((003667.)0) 00110(00482)) 0011(0000452)) scaBpeunLtAE LFo1100202 0011000004825)) PELVPISublandslor(scehiusm(@)): ~~ Puibins(ceso):minpocolrmnpaoltetotesalsyilflyed ossified Pubis(es): notossified Iosscshiifuime(da): incompletelyornot FL 181106 1249232 L 9% 1176 F 147 233 FL 33 000357 FL 5389 043784 0-0176 ((00--9350)4) 07 (0304) 0-16 095) 0011(000462)) 00490(04-716).7) FORERLadIiMuBs(aSn)d Uina: Bent LF 1100022 010042) 01006) LF:: LFIETTTAELRIINNCCIIDDEENNCCEE 00911 418-013:PAGE F-12 SUMMARY OF FETAL OSSIFICATION SITES `SKELETAL AVERAGES CD RAT (CAESAREAN-SECTIONED DAY 20 GESTATION) PERIOD: JUNE 1996 - JUNE1998 #STUDIES INCLUDED 35 #LITTERS EXAMINED 793 #FETUSES EXAMINED 5961 SKELETAL AVERAGES HYOID VERTEBRAE CERVICAL THORACIC LUMBAR CSAAUCDRAALL RIBS (pairs) STERNUM MANUBRIUM STERNAL CENTERS XIPHOID FOREPAWS (Calculated as average per mb) CARPALS METACARPALS DIGITS PHALANGES HINDPAWS (Calculated as average per limb) TARSALS METATARSALS DIGITS PHALANGES FETUSLITTER MEAN RANGE/STUDY 086 (0.69-0.98) 7.00 - 13.04 (13.00-13.15) 595 (5.85-5.99) 3.00 (2963.01) 4.82 (4.35516) 13.03 (13.00-13.09) 1.00 (0.98101) 361 (3.26-375) 098 (0.94-1.00) 000 - 354 (3.33374) 5.00 = 5.04 (4.90527) 0.00 - 399 (3.93-4.03) 5.00 - 498 (4.82.5.08) 00942 418-013:PAGE F-13 FETAL OSSIFICATION SITES `SKELETCADL ARVAETRAGES (CAESAREAN-SECTIONED DAY 21 GESTATION) STUDY# 580 #LITTERS EXAMINED 23 . # FETUSES EXAMINED 190 HSYKOEILDETAL AVERAGES 096 VERTEBRAE TCHEORRVAICCIACL 137..0060 LUMBAR 594 SCAAUCDRAALL 370203 RIBS (pairs) 1305 MSATNERUNBURMIUM 1.00 XSITPEHRONIADL CENTERS a1.s0t0 FORaEvPeAraWgSe (pCearllciumlba)ted as CARPALS 0.00 DMIEGITTASCARPALS 53.9090 PHALANGES 75 HINDPAWS (Calculated as average per fimb) TARSALS 002 DIMGEITTASTARSALS 45.6020 PHALANGES 578 000943 APPENDIX G STATEMENT OF THE STUDY DIRECTOR 50944 PRIMEDICA 418-013:PAGE G-1 APe o baie mEaa PROTOCOL 418-013: ORAL (GAVAGE) PHARMACOKINETIC STUDY OF PFOS IN RATS SPONSOR'S STUDY NUMBER: T-6295.12 STATEMENT OF THE STUDY DIRECTOR This final report accurately reflects the raw data obtained during the performance of the study. No deviations from the U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations; Final Rule, the Japanese Ministry of Health and Welfare (MHW) Good Laboratory Practice Standard for Safety Studies on Drugs and the European Economic Community (EEC) Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice' occurred that affected the quality or integrityof the study. 4J iz LL Sopa 2-dun-g Raymond G. Yofk, Fh.D.. DABT Date Asstiate Direc gf Research and Study Directo a. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. b. Japanese Ministry of Health and Welfare (1988). Good Laboratory Practice Stanfd ora Sar fed ty Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. c. European Economic Community (1989). Council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratorypractice. Official Journal of the European Communities: Legislation. 32(No. L 315; 28 October): 1-17. 000915 APPENDIX H QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 20916 r>PRIME]DICA 418-013:PAGE H-1 Argu90s5ReSsheeaerhcyhDLraibvoer,atBouriiledsi.ngInA TelephoHnoe:r(s2h15a)Pm4A,43-189701404 Telefax: (215) 43.8587 QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT Study Director: Raymond G. York, Ph.D., DABT Executive Director of Research: Mildred S. Christian, Ph.D., Fellow, ATS Protocol 418-013: Oral (Gavage) Pharmacokinetic Study of PFOS in Rats Sponsor's Study Number: T-6295.12 `The draft protocol for this study was audited for adherence to U.S. Food aMinndisDtrryugofAdHmeianlitshtraantdioWnel(fFaDrAe)(GMoHoWd);LaGbooroadtoLrayboPrraatcotriycePrRaecgtuilcaetiSotnasn,dJaarpdafnoerse Safety Studies on Drugs, and European Economic Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles. of good laboratory practice on 18 OCT 98. and Critical phases of this study were inspected raw data were audited twice (see tables 1 and six times; study 2 for dates and information phases/data) The draft final report and the raw data for this study were compared and audited for accuracy, for adherence to protocol requirements, and for adherence to U.S. Food and Drug Administration (FDA) Good Laboratory Practice Regulations, Japanese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic CEuormompuenaintyEc(o1n98o9m)iccoCunocmimludneictisyioonf aonn O28ECJuDlyd1ec9i8s9ioonn/trheecoamcmceenpdtaatnicoenboynthe compliance with principles of good laboratory practice between 25 APR 99 and 26 MAY 99, and for revisions requested by the Sponsor 17 JUN and 18 JUN 99 and for finalization on 24 JUN 98. 00947 418-013:PAGE H-2 Adminis`tTrhaitsisotnud(yFDwAa)sGcooondduLcatbeodraatcocroyrdPirnagcttioceU.RSe.guFloaotdioansn,dJDarpuagnese Ministry of Health and Welfare (MHW); Good Laboratory Practice Standard for Safety Studies on Drugs, and European Economic Community (1989) council decision `on 28 July 1989 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice. Zari boas2unty ia, Loach.24Tume 99 Nancy J. Gonglievski Date isa A. Zaborowski, B.S. Date Quality Assurance Manager `Senior Quality Assurance Associate and Principal Auditor 0650918 TABLE 1 CRITICAL PHASES INSPECTED 418-013:PAGE H-3 TestAticlePreparation Dateof inspection: 19 NOV 98 Date results 20NOV 88 reported to the Study Director and Management Test Article Administration - Gavage Date of inspection: 19 NOV 98 Date results reported to the Study Director and Management: 20 NOV 98 Cohabitation Date of Inspection: 28 DEC 98 Date results reported to the Study Director and Management 31DEC 98 Blood Collection Date of inspection: 04 JAN 99 Date results 04 JAN 99 reported to the Study Director and Management: Urine and Fecal Collection Date of inspection: 04 JAN 99 Date results 04 JAN 99 reported to the Study Director and Management: Caesarean-Sectioning Date of inspection: 13 JAN 99 Date results 19 JAN 99 reported to the Study Director and Management: 00949 418-013:PAGE H4 TABLE 2 RAW DATA AUDIT(S) `The from 18 following study information MAR 99 to 25 MAR 99: and raw data were audited Protocol. Protocol amendment. ELrisrtorofcpoedressonannedlcaonddescofmopructiienricaolpesriagtnoorbcsoerdveast.ions. AInn-iimfealtrraencseaipctt,iornanrdeocomridz.ation, physical examination and acclimation. Feed consumption. Cohabitation. Caesarean-sectioning. Maternal gross observations. Fetal gross observations. Necropsy. Gravid uterine weights. Placental weights. Tissue packing lists. Male breeder colony records. General comments. `Study maintenance records. Temperature and relative Feed and water analyses. humidity reports. Edit requests. RDaonsdaogmeizvaotliuomness.. Deviations. Data review pages. Blood and liver collection Maternal packing lists for data and packing liverglands. lists. FPeotoalleldivleivrefrl,unpglatciesnstuae,plaicvkerinagndlisltsu.ng packing lists. Fecallurine collection data and packing lists. EFemtbarlyocalrpclaascsenptaackciolnlgecltisitosn. and packing lists. Amniotic fluid collection and packing lists. on The resultsofthis 26 MAR 99. audit were reported to the Study Director and Management 0950 418-013:PAGE H-5 The following study information and raw data were audited on 25 MAR 99: Vehicle/Control article receipt, preparation and use. Test article/substance receipt, preparation and use. Test article/substance packing lists. Test article/substance analysis. The results of this audit were reported to the Study Director and Management on 29 MAR 99. 000951