Document 91vVR0jeN7x2ZvXRqpDRZLOjp

EHR - 08 373 AR16-- 0967 ORIGINAL Cow 2 6 FINAL REPORT y PROTOCOL 418-010 ORAL (STOMACH TUBE) DEVELOPMENTAL TOXICITY STUDY OF N-EtFOSE IN RABBITS . SPONSOR'S STUDY NUMBER: 6316.8 FINAL REPOR' DATE: 11 JANUARY 1999 AEeRpDAaT-IoTS 003863 & 3. 7: LI PROTOCOL 418-010 ORAL (STOMACH TUBE) DEVELOPMENTAL TOXICITY STUDY OF N-EFOSE IN RABBITS SPONSOR'S STUDY NUMBER: 6316.8 TAOFB CONL TENE TS SUBJECT PAGE I SUMMARY AND CONCLUSION 1 A Methods 5} B. Results 1-2 C. Conclusion 3 Il. DESCRIPTION OF TEST PROCEDURES 11 A. Conduct of Study 1 A. Sponsor =] A2. Testing Facilty I] A3. Study Number 14 A4. Sponsor's Study Number I AS5. Purpose of the Study 1-1 AS. Study Design 1 AT. Regulatory Compliance I] AB. Ownership of the Study 12 A9. Study Monitor 2 . 003864 SUBJECT D.2. Analytical Results E Test System EA. Species E.2. Strain E.3. Supplier (Source) E4. Sex E.5. Rationale for Test System E.6. Test System Data E.7. Method of Randomization E.8. System of Identification F. Husbandry FA. Research Facility Registration F.2. Study Rooms F.3. Housing F4. Lighting F.5. Sanitization F.6. Feed F.7. Feed Analysis F.8. Water F.9. Water Analysis G. Methods G.1. Dosage Administration n 0 03865 PAGE 1-5 5 1-5 1-5 5 1-5 1-6 11-6 1-6 1-6 "7 7 I-7 7 7 n-7 7 7 1-8 1-8 1-8 1-8 SUBJECT PAGE G2. Rationale for Dosage Selection 8 G3. Route ofAdministration 19 G4. Rationale for Routeof Administration Io G5. Frequency of Administration 9 G6. Length of Study 19 G7. Method of Study Performance 110 G8. Gross Necropsy 110 G9 Statistical Analyses 1112 ll. RESULTS 1 A. Mortality, Abortions, Clinical and Necropsy Observations 1-1 AA. Mortality 1 A2. Abortions [5] A3. Clinical Observations 0-3 A4. Necropsy Observations 4 B. Matemal Body Weights and Body Weight Changes 4 C. Matemal Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values 4 D. Caesarean-Sectioning and Litter Observations ns E. Fetal Alterations 5 EA. Summary of Fetal Alterations [3 E2. Fetal Gross External Alterations 11-6 E3. Fetal Soft Tissue Alterations ne Ed. Fetal Skeletal Alterations , oases "8 SUBJECT PAGE F. Satelite Rabbits 11-10 REFERENCES 1-12 APPENDIX A - REPORT FIGURE Figure 1. Maternal Body Weights A APPENDIX B - REPORT TABLES Table 1. Clinical Observations - Summary B81 Table 2. Uterine Contents and Litter Data for Rabbits that Died or Aborted B3 Table 3. Necropsy Observations - Summary B85 Table 4. Maternal Body Weights - Summary . B6 Table 5. Maternal Body Weight Changes - Summary B88 Table 6. Matemal (glday) - SAubsmomlaurtey Feed Consumption Values Bo Table 7. Maternal Relative Feed Consumption Values (g/kglday) - Summary 8-10 Table 8. Caesarean-Sectioning Observations - Summary B11 Table 9. Litter Observations (Caesarean-Delivered Fetuses) - Summary B12 Table 10. Fetal Alterations - Summary B13 Table 11. Fetal Gross External Alterations - Summary B14 Table 12. Fetal Soft Tissue Alterations - Summary 8-15 Table 13. Fetal Skeletal Alterations - Summary 8-17 Table 14. Fetal Ossification Sites - Caesarean-Delivered Live Fetuses (Day 29 of Gestation) - Summary B22 Table 15. Clinical Observations - Individual Data B23 v 003867 SUBJECT PAGE Table 16. Necropsy Observations - Individual Data B-35 Table 17. Maternal Body Weights - Individual Data B41 Table 18. Matemal Feed Consumption Values - Individual Data B-56 Table 19. Caesarean-Sectioning Observations - Individual Data B-71 Table 20. Litter Observations (Caesarean-Delivered Fetuses) - Individual Data B77 Table 21. FIentdiavlidSueaxl, DVaittaal Status and Body Weight - B-83 Table 22. Fetal Alterations - Individual Data B-95 APPENDIX C - PROTOCOL AND AMENDMENT C-110C-31 APPENDIX D - DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY D1 APPENDIX E - TEMPERATURE AND RELATIVE HUMIDITY REPORTS AND DEVIATIONS REPORT E-1toE4 APPENDIX F - PILOT REPORT F-1toF-98 APPENDIX G - HISTORICAL CONTROL DATA G-110G-15 APPENDIX H- STATEMENT OF THE STUDY DIRECTOR APPENDIX |- QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT H-1 Fitol4 003868 418-010:PAGE 1-1 TITLE: ORAL (STOMACH TUBE) DEVELOPMENTAL TOXICITY STUDY OF N-EtFOSE IN RABBITS ARGUS RESEARCH LABORATORIES, INC. PSPROONTSOOCRO'LSNSUTMUBDEYR:NU4M1B8E-R01:0 6316.8 IL SUMMARY AND CONCLUSION A. Methods wTewernetays-stiwgoneNdetwo Zeeaaclhanodf fWihvietdeos[aHgrae:g(rNoZuWp)sSP(FG]rotuipmsed|-tphrreogungahntV).femNailneetreaebbnits additional female rabbits were assigned to one of five dosage groups for the satellite study (three, five, three, three and five rabbits assigned to Groups | through V, respectively). The test article, N-EtFOSE, or vehicle, 2% Tween 80 WinatReerv)e,rwsaesOasdmmoisniisstMereemdbroraanlley P(vrioacsetsosmeadcDhetiuobnei)zeodncWeatdeairly(Rt.oO.theDseeionnaitzureadllybred rabbits on days 7 through 20 of presumed gestation (DGs 7 through 20). Dosages of 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day were administered at a dosage volume of 5 ml/kg, adjusted daily on the basis of the individual body weights. The female rabbits were observed for viability at least twice each day of the study. The rabbits were also examined for clinical observations of effects of the test article, abortions, premature deliveries and deaths before and approximately 60 minutes after dosage and once daily during the postdosage period. Body `weights were recorded on DG 0, the day of arrival at the Testing Facility and on DthGesTe7sttihnrgouFgahci2i9t.y. Feed consumption values were recorded daily after arrival at On DG 21, toxicokinetic samples were collected from the satelite rabbits assigned to the toxicokinetic evaluation. Blood samples were collected from the inferior vena cava and centrifuged. The resulting serum was shipped to the a Detailed descriptionsofall procedures used in the conduct of this study are p(rPoRviOdTeOd CinOtLheANapDprAopMrEiNatDeMsEeNctTi)o.ns of this report and in APPENDIX C 003869 418-010:PAGE 1-2 Sponsor for analysis. The liver was excised, weighed and a sample was taken from the right lateral lobe and shipped to the Sponsor for analysis. Rabbits were Caesarean-sectioned and fetuses were examined grossly to the extent possible as described for rabbits assigned to the main study. Fetuses and placentae were pooled by liter shipped to the Sponsor for analysis. On DG 29, rabbits in the main study were sacrificed, Caesarean-sectioned and a gross necropsyofthe thoracic, abdominal and pelvic viscera was performed. The number of corpora lutea in each ovary was recorded. The uterus was excised and examined for pregnancy, number and distribution of implantations, early and late resorptions and live and dead fetuses. Each fetus was identified, weighed and examined for gross external alterations. All fetuses were examined internally to identify sex and visceral alterations; cavitated organs were evaluated by dissection; and the brain was cross-sectioned and examined in situ. All fetuses were examined for skeletal alterations after staining with alizarin reds. B. Results No compound-related deaths occurred during the study. One, two and five does. aborted and were sacrificed in the 0 (Vehicle), 2.5 and 3.75 mg/kg/day dosage groups, respectively. These abortions occurred at the end of or after the completion of the dosing period. The abortions in the 2.5 and 3.75 mg/kg/day dosage groups were considered related to the test article because they occurred at dosage-dependent incidences in the two highest dosage groups. All other rabbits survived until scheduled sacrifice on gestation day 29 (DG 29). Increased numbers of does in the 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups had observations of abnormal stool (no, scant, and soft or liquid feces). All other adverse clinical observations were considered unrelated to the test article. One 3.75 mg/kg/day dosage doe had a pale liver thatwas considered possibly related to the test article because it occurred in a high dosage group. Groups administered the 1.0, 2.5 and 3.75 mg/kg/day dosages of the test article had statistically significant reductions in body weight gain or body weight losses. on DGs 7 to 10 and 10 to 13. Reflecting these effects of the test article, body weight gains were significantly reduced in the 2.5 and 3.75 mg/kg/day dosage groups for the entire dosage period. The 2.5 and 3.75 mgkgiday dosage groups had significantly reduced body weights on DGs 14 through 25. 003870 418-010:PAGE I-3 Absolute and relative feed consumption values were significantly reduced at several tabulated intervals during the dosage period in the 2.5 and/or 33..7755 mmgg//kkgg//ddaayy ddoossaaggee ggrroouupps.haRdefsilgencitfiincganttlhyesreedefufceecdtsaobfsotlhuetteeastndartriecllae,titvhee feed consumption values for the entire dosage period. The litter averages for late resorptions were increased in the 2.5 and 3.75 mg/kg/day dosage groups. These increases in the number of late resorptions were considered treatment-related because they occurred at the two highest dosages. No gross eternal, soft tissue or skeletal fetal alterations. (malformations or variations) were caused by dosages of the test article as high as 3.75 mg/kg/day. C. Conclusion On the basis of these data, the maternal no-observable-effect-level (NOEL) of N-EtFOSE is 0.1 mg/kg/day (the 1.0 mg/kg/day and higher dosages caused statistically significant reductions in body weight gains or weight losses and the 2.5 and 3.75 mg/kg/day dosages also significantly reduced absolute and relative feed consumption values). The developmental NOEL is 1.0 mg/kg/day (the 2.5 and 3.75 mg/kg/day dosages caused increased incidences of late resorptions and abortions). Based on these data, N-EtFOSE should not be identified as a selective developmental toxicant; the compound was not found to be teratogenic in the rabbit. Mildred S. Christian, Ph.D., Fellow, ATS Executive Director of Research Date Qoob `Alan M. Hoberman, Ph.D., DABT Director of Research fr Date ond G. Yo Associate Dir Study Director ~oo I3ee9s .D., DABT Research and Date 003871 418-010:PAGE II-1 Il. DESCRIPTION OF TEST PROCEDURES A. Conduct of Study: A. Sponsor: 3M Corporate Toxicology, 3M Center Building 220-2E-02, St. Paul, Minnesota 55144-1000 A.2. TestingFacility: Argus Research Laboratories, Inc., 905 Sheehy Drive, Building A, Horsham, Pennsylvania 19044-1297 A3. Study Number: 418-010 A4. Sponsor'sStudyNumber: 6316.8 AS. Purpose of the Study: The purpose of this study was to detect adverse effects of N-EtFOSE on New Zealand White [Hra:(NZW)SPF] presumed pregnant female rabbits and development of the embryo and fetus consequent to exposure of the doe from implantation to closure of the hard palate. This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process in a nonrodent species. AS. Study Design: `The requirementsof the International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline" were used as the basis for study design. A7. RegulatoryCompliance: The study was conducted in compliance with Good Laboratory Practice (GLP) regulations of the U.S. Food and Drug Administration (FDA)?, the Japanese Ministry of Health and Welfare (MHW) and the European Economic Community (EEC). There were no deviations from the GLP regulations that affected the quality of integrity of the study. Quality Assurance Unit findings derived from the 003872 418-010:PAGE 11-2 inspections during the conduct of this study are documented and have been provided to the Study Director and the Testing Facility management. AS. Ownershipofthe Study: The Sponsor owns the study. All raw data, analyses, reports and preserved tissues are the property of the Sponsor. A9. Study Monitor: Marvin T. Case, D.V.M., Ph.D. A.10. AlteStrudnyMa onittoer: Andrew M. Seacat, Ph.D. A11. Study Director: Raymond G. York, Ph.D., DABT (Associate Director of Research) A12. Technical Performance: John F. Joseph Barnett, W. Lech, B.S. B.S. (Director of Laboratory Operations) (Team Leader - General Laboratory) Betsy J. Kems, B.S. (Laboratory Technician) A13. Report Preparation: 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) A14. ReportReview: Alan M. Hoberman, Ph.D., DABT (Director of Research) Mildred S. Christian, Ph.D., Fellow, ATS (Executive Director of Research) A15. Date Protocol Signed: 11 August 1998 003873 418-010:PAGE 11-3 A.16. Dates of Technical Performance: Rabbit Arrival Date Dosage Period [Days 7 through 20 of presumed gestation (DGs 7 through 20)] Toxicokinetic Sample Collection (DG 21) Caesarean-Sectioning Period (DG 29) 28AUG 98 30 AUG 98 - 16 SEP 98 17 SEP 98 21SEP 98- 25 SEP 98 AA7. Records Maintained: `aTrheeroertiagiinneald rienptorhte,arrachwidvaetsaofanAdrgruesseRrevseesaarmcphlLeasboorfatthoeriteess,t Ianrct.iclAenayndprveesheircvleed mtaiislsiunegs oafrethreetdarianftedfiinnalthreepaorrtc,hiavfetserowfhtihcehTteismteintgheFaScpilointysoforrwoinledeyceiadreafttheerirthfei.nal Fdaicsiploitsyi.tioUn.nuAsleldunbuulskedtetsetsatratrictlieclweilslubsepernestiuornnsedwteortehdeiSstcuadrydeMdonatittohre uTpesotning completion of all work with the test articie. B. TestArticle Information: B.A. Description: N-EtFOSE - a waxy solid B.2. Lot/Batch Number: FM-3929 (30035, 30037, 30039 (Expiration date: May 2000)] B.3. Date Received and Storage Conditions: The test article was received on 20 May 1998, and stored at room temperature. B.4. Special Handling Instructions: rSetsapnidraatrodr,ssaaffeettyypgroegcagulteisonosr s(aufseetyofglpraostseecstiavnedclaotfhaicneg-,shgileolvde)s,wedrusett-maiksetn when handling the bulk test article and prepared suspensions. B.5. AnaloyfPsuriitsy: Information regarding the identity, composition, the test article is on file with the Sponsor. strength, purity and stabily of 003874 418-010:PAGE Il4 C. Vehicle Information: C.1. Description: 2% Tween 80 in Reverse Osmosis Membrane Processed Deionized Water (R.O. Deionized Water). C.2. LotNumbers: MO3HOS and L06662 C.3. Date Received and Storage Conditions: `The Tween 80 was received on 22 May 1998 and 8 July 1998 (lot MO3HOS5) and 1 September 1998 (lot L06862), from J.T. Baker, Phillipsburg, New Jersey, and stored at room temperature. R.O. Deionized Water is available from a continuous source at the Testing Facility and is maintained at room temperature. C.4. Special Handling Instructions: Standard safety precautions (use of protective clothing, gloves, dust-mist respirator, safety goggles or safety glasses and a face-shield) were taken when handling the vehicle. C.5. Analysis of Purity: Neither the Sponsor nor the StudyDirector was aware of any potential `contaminants likely to be present in the vehicle that would interfere with the resultsof this study. D. TestArticlePreparation: Suspensions of N-EtFOSE were prepared daily at concentrations of 0, 0.02, 0.2, 0.5and 0.75 mg/mL. Prepared formulations were stored at room temperature. 003875 418-010:PAGE II-5 er = fe Te Te] DA. Sample Information: lCom ncannon [or [RaTeAG5|[Frrozeen re Jee a31A%G] o Velie r Resarvs|lTweer80 (lot MasHos) |N SL | Sl SP |Rooimtompierailre |= Testing Fi aciy |GTOaCdTSeEl Archives fo oss) Aries Eee Na.A DiuNpgoltpicasadpt1eiscahamipleSepsownesorreftoankeanyrsom.tThhfesetmaanidiLeasgtpsamrpleeswpaoaenrrtehteaadinateydpriatep0oarenTde.stOinng FsockaaosfSeoalcchksa.e twas b. FLiassttpprreppaarraatiioonn. Homogeneity and stabilityofprepared formulations are on file with the Sponsor. D.2. AnalyticalResults: Concentration samples (2 were prepared. Analyses mL) were taken were performed on by the 3M first and last days suspensions Environmental Technology and Safety Services. The the Testing Facilty. results of these analyses have not yet been forwarded to E. TestSystem: EA. Species: Rabbit E2. Strain: New Zealand White [Hra:(NZW)SPF] E.3. Supplier (Source): Covance Research Products Inc., Denver, Pennsylvania Ed. Sex: Timed-pregnant female 003876 418-010:PAGE II-6 ES. Rationale for Test System: The New Zealand System because: White 1) itis [Hra:(NZW)SPF] rabbit was one non-rodent mammalian selected species as the Test accepted and twoixdieclityy u(seemdbrtyhor-foeutgahlouttoxtihcietyilntdeursattroygefnoircintoyn)c;li2n)itchails sstturdaiinesooffradbebvietlhoapsmebneteanl demonstrated to experience exist be sensitive to developmental toxins; 3) at the Testing Facility"; and 4) the test historical article is. data and pharmacologically active in the species and strain. E68. TestSystemData: Number of Rabbits 129 Approximate Date of Birth ~~ 28 FEB 98, 07 MAR 98, 28 MAR 98 Approximate Age at Arrival Weight (kg) on DG 0 5-6 months 28-44 Weight (kg) at Arrival 29-42 E7. Method of Randomization: Upon arrival, rabbits computer-generated were assigned random units. to individual housing on the basis of Rabbits were assigned to oneoffive dosage groups (Groups | through V), 22 rabbits per the study. An additional 19 satelite rabbits dosage group, were assigned for for the main portion toxicokinetic of `(eGvralouuaptsionIl;afnidveVr)a,babintds twherreee arasbsbiigtnsewdetroeeaascshigonfetdhetoloewacahnodfhtihgeh rdeomsaaigneinggroups udsoisnaggae gcormopuuptse(rG-rgoenueprsa|t, elldl a(wnedigIhV)t.-oRradebrbeidt)s rwaenrdeomaiszsaitginoendptroodcoedsuargeebgarsoeudpson body weights recorded Inc.) on DG 0. by and at the Supplier (Covance Research Products, ES. System of Identification: Each Band rabbit was individually and Tag Co., Inc., No. identified with MSPT 20103) a Monel inscribed self-piercing ear with the rabbits tag (Gey designated puneirqmuaenepnetrmraabnbeinttnunmubmebre,r.seCx,agteesttaargtsicwlee riedemntairfikceadtiownitahntdhedosstaudgye nluevmebl.er, 003877 418-010:PAGE II.7 F. Husbandry: FA. ch Facility Registration: USDA ot seq. Registration No. 23-R-099 under the Animal Welfare Act, 7 U.S.C. 2131 F.2. StudyRooms: aThhealsltwuadyyarnodomisndweeprenedemnatilnytasiunpepdliuenddweirthcoandmiitinoinmsuomf opofstietnivcehaainrfgleosw rpeelrathioveurtoof r1o0o0ms%).freRshooaimr ttheamtpheraadtbureeenanpdashsuemdidtihtryowueghre99mo.n9i7t%orHeEdPcAonsfittaenrtsly(AtihroroCulgehaount trehleatsitvuedyh.umiRdoiotymwtaesmptearragteuterde awta3s0t%artgoet7e0d%.at S6e1eFAtPoP7E2NDFI(X16EC to 22C); (TEMPERATURE AND RELATIVE HUMIDITY REPORTS). F.3. Housing: Rabbits were individually housed. compliance with the Guidefor the Al cage sizes Care and Use and housing conditions of Laboratory Animals. are in F.4. Lighting: Alinghatu:t1o2m-ahtoiucraslldya-rck,onwtirtohlleeadcfhludoarrekscpeenrtioldighbtecgyicnlneinwgaast m1a9i0n0tahionuerdsaEtS1T2.-hours FS. Sanitization: Cage pan liners were changed approximately three times each week. were changed approximately everyotherweek. Cages F.6. Feed: Approximately 150 g of Certified Rabbit Chow #5322 (PMI Nutrition fIinrtsetrdnaatyioonfadl,osSatg.eL,ouaits,whMiicshsotuirmie) wapapsroaxviamilaatbelleyt1o8e0agchofratbhbeitseaamcehcderatyifuinedtifetheed. iwnadsiviodfufaelresdtationleeascshstreabebli"tJe-taycphe"dafye.edeTrhseactetraticfhieedd tfoeeedacwhascaagvea.ilable from F.7. Feed Analysis: lAenvaellysseexsceweedriengroutthienemlayxpiemrfuomrmceodncbeynttrhaetifoenedfosrucpeprltiiefri.ed Nfoeecdoonrtadmeviinaatnitosnsatfrom 003878 418-010:PAGE Il-8 expected nutritional requirements were detected by these analyses. Copies of the resultsof the feed analyses are available in the raw data. Neither the Sponsor nor the Study Director 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 rabbits ad libitum from an automatic watering system (individual sipper tubes). 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 for possible bacterial contamination (Analytical Laboratories, Inc., Chalfont, Pennsylvania). Copies of the results of the water analyses are available in the raw data. Neither the Sponsor nor the Study Director was aware of any agent present in the water that was known to interfere with the results of this study. G. Methods: GA. Dosage Administration: ero] i cosas| Nuroecr| usage |concentata| Dossoe Group| Rabbits | (mgkgiday) | (mg/mL) | Volume [TT [2+" Jowehce)| | &Ta672-8563 652-8604| [|2 | of 770 | 5 eeeq-s6is eee oon [mw |ze"| 70 |"02[5 6676 86or|abun-seoz| [VV |2 | 26 [770815 68% 8659]6695-6695| LThY e test a|rtic2le wa%s co| nsider3ed71500%|pure0for t7he pu| rposeo5f do[sa8ge6ca0lcu8l8at7io]ns86-6700] a. Rabbits assigned to toxicokinetic evaluation. G.2. RatfoirDo osan ge Sa elel ctie on: Dosages were selected on the basis of a dosage-range study [Argus Research Laboratories, Inc., Protocol 418-010P (see APPENDIX F)]. 003879 418-010:PAGE Il-9 In 50 the 418-010P study, severe maternal body weight and 75 mg/kg/day dosage groups; there were no loss occurred in surviving rabbits the 10, 25, in these. Cgraoeuspasr.eaAnb-osretcitoinosn oocbcsuerrrveadtiionntshere5veaanlded10inmcgr/ekags/eddalyatdeorseasgorepgtiroonuspsa.nd froeudnudceadt f1etmagl/bkogiddyawye;ihghotwsevaetr5, mtgh/ekrge/wdaays.noIndcerceraesaesseininemarelay nrelsiottreprtsiioznes. were G3. Route of Administration: Oral (stomach tube) G.4. RationaleforRouteofAdministration: The with oral the (stomach tube) route was selected dietary route, the exact dosage can for be use because: 1) in comparison accurately administered; and 2) it is one of the possible routes of human exposure. G.5. Freq ofu Adme inin strc atiy on: Appropriate dosages of the stomach tube) once daily to test article or vehicle were administered naturally-bred rabbits on DGs 7 through orally 20. (via aDdomsiangiestseorfed0a(tVeahidcolse)a,ge0.v1,ol1.u0m,e2.of5 5anmdLi3k.g7,5amdgj/uksgt/eddadyaiolfytohne tthesetbaarstiicsleofwtehree aitndaipvpidruoaxlimbaotdeylywetihgehtssamreectoirmdeedeabcehfodraey.intubation. The rabbits were intubated G.6. LenofgSttudhy: Approximately 4 weeks a. See APPENDIX D (DEVIATIONS FROM THE PROTOCOL AND THE STANDARD item 1 OPERATING PROCEDURES OF THE TESTING FACILITY), 003880 418-010:PAGE I1-10 G.7. Method of Stud rmance: Tsohuercfeemaanlde srtarbabiintsbewfeorree nsahtiuprmalelnytbtroetdhbeyTebsrteiendgerFamcialiltey.raTbbhietsraobfbtihtes swearmee mafatteredthoenlafsitvedcaoynosfecmuattiinvge.daTyhseadnadysohfipmapteidntgowtahesTceosntsiindgerFaecdiltiotyboenDtGhe0d. aAy casosmipguntetrh-egreanbebriatstetdo f(iwveeidgohts-aogredegrreodu)prsanbdaosmeidzaotniobnodpryowceeidguhrteswraescoursdeedd oton DG 0 and supplied by Covance Research Products, Inc. Aglelnerarbablitaspwpeeraeraonbcseeratveldeafsotrovniacbeilldtuyraitngleaacsctlitmwaitcieone.acAhdddiatyioonfaltheexasmtiundaytainodnsfor for clinical deliveries aonbdsedrevaatthisonwseorfeefmfaedctes of the test article, before each daily abortions, intubation premature (DGs 7 through 2T0h)esaendobaspeprrvoaxtiimoantselwyer6e0 amlisnoumteasdaeftoenrcientduabialtyiodnurdiunrgintghethpeosdtodsoasgaegepeprieordi.od (DG 21 through 29). BanoddyonweDiGghsts7wtehrreourgehc2o9r.dedFeoendDcGon0s,umthpetidoany voaflaurersivwaleratetrheecToersdteidngdaiFalcyialfittyer arrival at the Testing Facility. G.8. Gross Necropsy: G.8.a. Satellite Rabbits Assigned to Toxicokinetic Sample Collection: OconllDecGted21fr(othmetdhaeyrafbobliltoswiansgstihgeneldasttodtohseagteo)x,ictookxiinceotkiicneevtiaclusaatimopnl.esFowlelroewing waenersethcoelslieactweidthfrpoenmtotbhaerbiniftearli,orbvleoonda scaamvpaliensto(sapeprruomxismeaptaerlayto4rmtLubpeesrarnadbbit) coenntdrriyfuigceeda.ndThmeairnetsatiniendg fsreorzuenm ((-a7p0prCo)xiumnatitlelsyhi2pmmeLn)twtaosthiemmSepdoinastoerlyfofrrozen alantaelryaslisl.obeT,hferolziveenr was and excised, retained weighed, and at -70C until sahispammepnltetwoatshetSakpeonnsforromfotrhe right analysis. eRxatbebnittspowsesirbeleC,aeassadreesacnr-isbeecdtifoornerdababintds faestsuisgensewdetroetheexammaiinnesdtugdryo.sslFyettuostehse tahnedSpploancseonrtafeorweanraelypsoios.led by litter and retained frozen (-70C) until shipment to Asfatmeprlceosmpwleerteiosnhiopfpseadmp(flreozceonlloecntidorny, iscee)rutmo,3lMiveErnsveicrtoinomnes,ntfaeltalTeacnhdnopllaocgeyntaanld Safety Services, St. Paul, Minnesota. 003881 418-010:PAGE II-11 G.8.b. Scheduled Sacrifice: All surviving rabbits Special euthanasia were sacrificed solution on DG by 29. intravenous The rabbits injection of Beuthanasia-D. were Caesarean-sectioned and a gross performed. necropsy of the thoracic, abdominal and Gross lesions were preserved in neutral pelvic viscera buffered 10% was formalin for cpoosmsmibolne,fustpuorneteavnaelouautsiolnes(iwointsh itnhreabebxictes)p;tiaolln ootfheprartoivsasruieasnwceyrsetsd,iwschaircdheda.re e`Txhceisneudmabnedr eofxacmoripnoerdaflourtpeareignneaancchy,ovnaurmybwearsanrdecdoirsdterdi.butTihoneouftiemrpulsanwtaastions, early and appeared late resorptions and live and dead fetuses. Uteri nonpregnant were stained with 10% ammonium from does that sulfide to confirm the oarbgsaennocgeenoefsiimsplwanatsatniootngsriotsessly. eAvindeenatr.lyArelsaotreprteisoonrpwtaisondewfaisneddefaisneodneasinownheicinh dwehfiicnhedthaesoacctuerrrmenfecteusofthoartgarneosgpeonnedseidstwoamsecghraonssilcyalevsitdiemnulti.. A live fetus was Nonresponding fteetrumsfesetaunsdeslaartee rceosnosripdteiroensd atroebdeifdfeearednt(itahteerdebwyertheendoegdreeaedoffetauusteosl)y.sisDepraedsent; marked to extreme autolysis indicated that the fetus was a late resorption. Each Caesarean-delivered alterations and individually fetus was weighed, identified with a tag examined for gross extemal noting study number, litter innutmrbaeperr,itaonnedalutienrjiencetidoinstorfibBuetiuotnh.anLaivseiafet-uDseSspewceiarle. saAlclriffeitcuesdebsywearne examined binytedrinsaslleycttiooind"e;ntaifnydstehxeabnrdaivniwscaesraclraolstse-rsateicotniso;nceadvi(taastiendgloergcaronsss-wseercetieovnalwuaasted gmraodses lbeestiwoensenwetrhee pparreiseetarlveadndintnheeutfrraonltablufbfoenreesd)1a0n%dfeoxramamliinnefdorinpossitsui.blFeetfaulture evaluation ArleldfSet.usesSkweelretealexparmeipanreadtifoonrsskweelretealreatlatienreadtiionnsglayfcteerrinstwaiitnhintghwyimtohlaalidzdaeridn as a preservative. Late resorption were examined to the extent possible. Representative photographs of fetal alterations are available in the raw data. `Rwaebrbeitesxatmhaitnedidedfororcwaeursee osafcdreifaitchedonbetchaeudsaeyotfhaeboorbtsieornvaotriopnrewmaastumraeddee.livery dPerleigvneraendcypusptsatwuseraendexuatmeriinneedcotonttehnetsexwteenrte proescsoirbdleed,.usAibnogrttheedsfaetmuesemseatnhdo/dosr described for fetuses. 003882 418-010:PAGE Il-12 G9. StatisticalAnalyses: The following schematic represents the statistical analyses of the data: TyofpTeset" I. Parametric A. Bartlett's Test* II. Nonparametric A. Kruskal-Wallis Test (575% ties) Significant at ps0.05 emer Not Significant Analysis of Variance Significant atps0.05 | Dunn's Test Not Significant Significant atps0.05 Not Significant ore Test B. Fisher's Exact Test (>75% ties) Il. Test for Proportion Data Variance Test for Homogeneity of the Binomial Distribution a. b. UStsaetidstoincallylytosiagnnailfiyczaentdpartoabwaibtihlthioesmoagreenreeiptoyrotfedvaarsieaintchee.r ps0.05 or ps0.01. c. d. Proportion data are not Test for homogeneityof included in variance. this category. 003883 418-010:PAGE I1-13 Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution`. Ccoonntsiunmupotuisondavtaalu(ees.g.a,nmdaltitetemraalvebroadgyewsefiogrhtpse,rcbeondtymwaelieghfettcusheasn,gepse,rcfeenetd rosessiofribcaetdiocnonsicteepdtautsae)s,wefretealanbaoldyyzewediguhstisn,g fetal anomaly Bartlett's Test data and fetal of Homogeneity of VTeasrtiawnacsenso`t asnigdnitfhiecaAnnta(lpy>s0i.s05o)f].VaIrf itahencAnea,lywshiesnofaVpparroiparnicaetewa[is.e.s,iBganritfliectatn'ts i(npd<i0v.i0d5u)a,l Dgurnonueptst.'sIfTtehsetA"nawlayssiussoefdVatroiiadnencteifwyatshenosttataipsptricoaplrsiiagtneif[ii.ce.a,ncBaertolfettth'es Test than woraesqsuiaglnitfoic7a5nt%(ptsi0es.0w5e)]r,e tphreesKernuts;kawlh-WeanllmiosrTeetshta"n was 75% used, when less ties were present, sFtiasthiesrt'icsalElxyascitgnTiefiscta'n"t w(pa<s0.u0s5e)d,. DuInnnc'assMesetihnowdhiocfhMutlhteipKlreusCkoaml-pWaarlilissoTness"twwaass. used to identify the statistical significance of the individual groups. Count data procedures opbrteaviionuesdlyatdCeasecsriabreedanf-osretchteioKnriusnkgawl-eWraelleivsalTueastetd.using the 003884 418-010:PAGE lI-1 nl. RESULTS A. Mortal tions, Clinical and N (Summaries -Tables 1 and 3; Individual Observati Data -Tables 2, 15 and 16) Ad. Mortality No deaths were attributable control group doe, an event to EtFOSE. attributable The to an only death intubation occurred accident. in a vehicle Vehicle control group seventh daily doe 8587 was found dosage on gestation dead approximately day 13 (DG 13). No one hour after other adverse the clinical observations occurred in this doe. This doe lost weight after DG 9 and its feed `pecrofnosruamtpitoino(n0w.5acsmrexd0u.c1edcma)ftienr tDhGe r1i0g.ht Ndieacprhorpasgymaoftitcheludnogelorbeev;eaallledotaher tissues normal appeared normal. The for their developmental litter ages consisted of eight at gross external fetuses that examination. appeared A.2. Abortions One, two and five** does aborted at the endofor after the completion of the dosage period (on DGs 19, 20, 21, 23, 26 or 29) in the O (Vehicle), 2.5 and 3.75 3.75 mg/kg/day mg/kg/day dosage dosage groups, groups respectively. The abortions in were considered effects of the the test 2.5 and article because the incidences were dosage-dependent. 0 (Vehicle) malkgiday Doe 8581 aborted on DG No other adverse clinical 26, six days observations after the occurred last dosage in this doe, was and administered. its body weight ggarionssanledsifoenesd wceornesurmepvteialoendwbeyreneccormoppasryaobflethteo dooteh.erTchoentrliotlergrcoonuspidsoteesd.ofNonine late resorptions; autolysis of these specimens precluded further evaluation. 2.5 malkglday ADdodeiti8o6n4a7l aabdovretresdeocnlinDicGal2o1b,soernveadtiaoynsafitnerthtihsedloaestidnocslaudgeedwsacsanatdfmeicneisst(eDreGds. 14 to 21), soft or liquid feces (DGs 16 to 20) and red substance in cage pan (DG 21). This doe consumption after generally DG 7. No lost weight and had severely reduced feed gross lesions were revealed by necropsy of the doe. ATuhteollyisttiesr cpornescilsutdeeddoefvtalwuoatlaitoenroefsotrhpetiloantes,reasnodrpttiwoonsd.eaTdhaenldivneiannedlidvee afdetufseetsu.ses ** Significantly different from the vehicle control group value (p<0.01). 003885 418-010:PAGE Ill-2 aexpapmeianrateidonn.ormTaelnfoorf tthheeireldeevveenlofpemteunstesalhaadgensotatosgsriofsisedexptuebrensalaatnsdkelsoefttaltissue examination. ADdodeit8io6n5a2l aabdovretresdeocnlinDicGal2o6b,sseirxvadtaiyosnsafitnerthtihsedloaestidnocsluadgeedawarsedadpmeirniiorsatlered. fseucbesstainncceag(eDGpa8n) t(hDaGtswa12s,p2r2obaanbdly24a)s,sosocfitatoerdliwqiutihd faencienstu(bDaGtison13prtoob2l1e)m,,sncoant feces 1021). (DGs This 13 to doe 21, lost 23, 25 weight and and 26) had and ungroomed coat (DGs 14 to 15 severely reduced feed consumption and 20 after cDoGnsi10s.tedNoofgtrwoosslilveesfieotnussewse,reonreevdeeaaldedfebtyusneacnrdoposnye of the doe. The late resorption. litter The fetuses and the late resorption gross external, soft tissue and appeared normal for their skeletal examinations, developmental ages at 3.75 malka/day Doe 8660 Additional aborted adverse on DG clinical 20 before administration observations in this doe of the 14th daily included soft or dosage. liquid feces (DGs 80 10), ungroomed coat 16), no feces in cage pan (DGs (DGs 8 15 and to 17 11), scant feces (DGs 11 to 14 to 19), tan perianal substance and a(nDdG h1a9d) asenvderreeldysruebdsutcaendcefeien dcacgoenspuamnpt(iDoGn 2a0f)t.er TDhGis7d.oNeogegnreorsaslllyesliosotnswewiegrhet Arlelvfeeatluesdesbyapnpeceraorpesdy.noTrmhaelliftotrertcheoinrsidsetveedloofpmtehnrteaeldaegaedsaantdgrsoesvsenexltievernfaeltuses. examination. Soft tissue the early developmental and skeletal examinations agesof the fetuses. were not performed due to ADdodeit8io6n6a1l aabdovretresdeoclninDicGal2o3b,stehrrveaetidoanyssinafttheirstdhoeelaisntcdluodseadgseofwtaosraldiqmuiindifsetceersed t(hDeGusnd1e0rtsoid1e2),(DsGcsan1t1fetoce2s3)(,DnGosf1e1cetso i1n5caangde 2p1anto(2D3G),s l1o6catloiz2e0d),aluonpgercoiaomoend cloosattw(eiDgGhst2a0ndtoh2a3d) saenvderreeldysruebdsutcaendcefeiendccaognespumapnt(ioDnG a2f3t)e.r DThGis7.doNeogegnreorsaslly lesions were fetuses, one dreeavedalfeetdubsyanndectrwoopslyatoefrtehseordpoteio.nsT.heOnliettefrectuosnswiastsedeodfesmiaxtoliuves. at gross external developmental eaxgaemsinaattigorno;ssalelxottehrenralfeetxuasmeisnaatpipoena.reAldl normal for their fetuses appeared normal at soft tissue fetuses also examination. had split ribs Al fetuses had not ossified at skeletal examination. pubes and one of these Doe 8663 Additional aborted adverse on DG clinical 26, six days after the last observations in this doe dosage included was soft administered or liquid feces (DGs (DGs 11.and 13 to 13 to 15 and 15), 25) scant feces (DGs 11 and no feces in cage to 19, 21 pan (DGs and 25), 20 and ungroomed coat 22 to 24). This 003886 418-010:PAGE III-3 doe DG generally lost weight and 7. No gross lesions were had severely reduced revealed by necropsy feed consumption. After of the doe. The litter ecvoanlsuiasttieodnooffntihnee ldaeteadrefseotrputsieosn.anAdlloanbeorltateedrfeestourpsteisonw.erAeutpaorltyisailslypcraencnliubdaeldized bauttg,rwoistshienxttehrenalilmiatnsdofseovfaltuiastsiuoene,xaampipneaatrieondsn.orSmeavlefnorotfhetihredfeevteulsoespmheandtanlotages ossified pubes examination. and one of these fetuses also had spit ribs at skeletal Doe 8667 Additional aborted adverse on DG clinical 1o9b,saefrtvearttihoens13itnhthdiasildyodeoisnacgleudwedasscaadnmtinfiescteesre(dD.Gs 12 waenidgh1t4atfote1r9)DaGn7d, raenddsiutbssfteaendcceoinnscumapgteipoannw(aDsGse19v)e.reTlhyirseddouecegdenaefrtearllDyGlos1t0. fNoourgfreotsusselsestihoantsawpepreearreevdenaolerdmablyfnoerctrheoiprsdyeovfelthoepmdeone.talTahgeelsittaetr gcroonsssisetxetderonfal examination. Soft tissue and the early developmental ages skeletal examinations of the fetuses. were not performed due to Doe 8669 Additional aborted adverse on DG clinical 29, nine days observations after the last dosage was in this doe included scant administered. feces (DGs 11 t0 in 17,20 and 23 to 26), cage pan (DGs 18 to soft or 19, 21 liquid to 22 feces (DGs 13 and 27 to 28). and This 15 to 16) and doe generally no feces lost weight DG 9. tNhorogurgohsosultetshieonsstwuedyreanrdevfeeaeldedcobnysnuemcprtoiposyn.waTsheselviettreerlcyonrseidsutceeddoafftteenr late resorptions that were too autolyzed for further evaluation. A3. Clinical Observations T0.h1e,1i.n0c,i2d.5encaensdof3.s7c5anmt,g/okrgs/odfatyordolisqaugidefgertouuspess;wtehreeiinnccirdeeansceedoifnsdcoanets fiencetsh.e was significant (ps0.01) in these four dosage groups. No feces in the cage pan adlossoaogcecugrrroeudpsi,n roensep,ecotinveelayn.dAsdidxi"tidonoaelsaidnvtehrese1.0c,li2ni.c5alaonbdse3r.v7a5tmiogn/skg/day astutbrsitbuatnacbeleintothtehectaenstpaarnticilneooncecudroreedininthdeoe2.s5tmhga/t kagb/odrateydaannddthirneceluddoeedsreind the 3.75 3.75 mg/kg/day mg/kg/day dosage dosage group group. and a tan perianal substance in one doe in the Aalrltioctlehebreacdavuesresethceliinniccaildeonbcseesrvwaetrioennsowtedroesacgoen-sdiedpeerneddenutnr.elTatheedsteo otbhseetrevsattions iunncdleurdseiddea, ruendgpreoroiomreadl scuoabts,talnaccer,imlaotciaolniazenddaalospceacbiaoornmathsesloinmbst,henehicnkdpaanwds/.or ** Significantly different from the vehicle control value (p<0.01). 003887 A.4. NecropsyObservations 418-010:PAGE lll-4 Oconnesi3d.e7r5edmgp/oksgsi/bdlayyrdeolastaegdetodotehe(t8e6s6t2a)rttihcalte hbaedcaauspaeliet olicvceurrtrheadt winaas high dosage group diaphragmatic rlaubnbgit.lobCeonatsrotlherarbebsiutlt85of87anhaindtuabapteirofnoreartrioorn, in the right as previously described. B. ralBody Weights and B ight Changes (Figure 1; `Summaries - Tables 4 and ; Individual Data - Table 17) Groups administered the 1.0, 2.5 and 3.75 mg/kg/day dosages of the test article had statistically significant (p<0.05 or p<0.01) reductions in body weight gains or absodcyomwepiagrhetdltoosstehse oconntDrGolsg7rotuop10vaalnueds.10Rteofl1e3ct(i2n.g5tahnedse3.e7f5femctgs/okfg/tdhaeyteosntly), article, body weight gains were significantly reduced (p<0.01) in the 2.5 and 3to.2715).mg/kg/day dosage groups for the entire dosage period (calculated as DGs 7 The 2.5 Ps0.01) and 3.75 mg/kg/day dosage groups had body weights on DGs 14 through 25, as significantly reduced (p<0.05 or compared with the control group values. Body weights and body weight gains were unaffected by the 0.1 mg/kg/day dosage of the test article. Cc. VMaatleurensal(SAubmsmoalruitees (-g/Tdaayb)leasn6d aRnedla7t;ivIend(igv/ikdgu/adlayD)atFaee-dTaCbolnes1u8m] ption Adubrsionlguttehe(gd/odsaay)geanpderrieoldatiinveth(eg/2k.g5/daanyd)ofree3d.7c5onmsgu/mkpgt/idoany vdaolsuaegsewgerroeuprse.duced These reductions were significant (p<0.05orp<0.01) for absolute feed cgroonuspumapntdioDnGvsal7u1e0s on 10, DGs 10 to 13 10 to 13 and and 13 to 13 to 16 in 16 in the the 3.75 2m.g5/mkgg//kdagy/ddaoysdaogseage group. Significant reductions (ps0.05 or ps0.01) in relative feed consumption vgraoluupe.s occurred on DGs 10 to 13 and 13 to 16 in the 3.75 mg/kg/day dosage Reflecting these effects of the test article, the 3.75 mg/kg/day dosage group had stihgeniefnitciarnetldyosreadguecepder(ipod<0(.c0a1l)cualbastoeldutaes aDnGd re7lattoiv21e).feed consumption values for Feed consumption as 1.0 mg/kglday. values were unafiected by dosages of the test article as high 003888 418-010:PAGE lll-5 D. Caesarean-Sectioning and Litter Observations (Summaries - Tables 8 and 9; Individual Data - Tables 19 through 21 Pregnancy occurred in 18 (81.8%), 20 (90.9%), 20 (80.9%), 21 (95.4%) and 21 (95.4%) rabbits in each dosage group. Caesarean-sectioning observations on DG 29 were based on 16, 20, 20, 19 and 16 pregnant rabbits in each of the five respective dosage groups. One, two and five does aborted in the 0 (Vehicle), 2.5 and 3.75 mg/kg/day dosage groups, respectively, and one vehicle control group rabbit was found dead on DG 13. The litter averages for late resorptions were increased in the 2.5 and 3.75 mg/kg/day dosage groups. - These increases in the number of late resorptions were considered treatment-related because they occurred at the two highest dosages. No biologically important or statistically significant differences occurred in the litter averages for corpora lutea, implantations, live fetuses, total or early resorptions and fetal body weights. There were no dead fetuses, no does with all conceptuses resorbed. All placentae appeared normal. E. Fetal Alterations (Summaries - Tables 10 through 14; Individual Data - Table 22] Fetal alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); and 2) variations (common findings in this species/strain, and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluationof the degree of fetal ossification. Fetal evaluations were based on 133, 167, 165, 141 and 129 DG 29 Caesareandelivered live fetuses in 16, 20, 20, 19 and 16 litters in the 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mglkg/day dosage groups, respectively. Each fetus was examined faovrergargoesss.external, soft tissue and skeletal alterations and fetal ossification site EA. Summary of Fetal Alterations (Summary - Table 10; Individual Data Table 22) Combination of malformations and variations resulted in the following incidences for fetal alterations. In the 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups, respectively, 10 (62.5%), 11 (55.0%), 10 (50.0%), 12 (63.2%) and 9 (56.2%) litters had fetuses with one or more alterations observed. In these same respective dosage groups, the total numbers of fetuses with any identified alterations were 23 (17.3%), 22 (13.2%), 29 (17.6%), 23 (16.3%) and 14 (10.8%). One or more alterations occurred in averages of 17.0%, 13.6%, 003889 418-010:PAGE ll 16.5%, 16.1% and 11.7% of the fetuses per litter in the five respective dosage groups. No gross external, soft tissue or skeletal fetal alterations (malformations or variations) were caused by dosagesof the test article as high as 3.75 mg/kg/day. Tihnceirdeenwceerseonfoandyosgargoes-sdeexpteernndael,ntsoofrt stiigsnsiufeicoarntskdeilfeftearlenacletesraitnitonhse. litter or fetal E.2. Fetal Gross External Alterations (Summary - Table 11; individual Data - Table 22) E.2.a. Malformations eCoxnatmrionlatgiroonu.p Sfoefttusti8s5s8u6e-e8xahmaidnaatsihoonrtretvreuanlkeadnfdusaebds,enditstpalilacatedgraonsds semxatlelrnal Kidneys and situs inversus. Skeletal examination of this fetus revealed fused 1st through 4th sternal centra, fusion of the xiphoid and 4th sternal centrum, only six thoracic vertebrae present, no ossification of the centra and arches of the 5th aatntdac6hthedthtoortahceic6tvherrtiebbsraaen,d faubsseedn4cteh oafnadll5ltuhmlbeaftr,risbsa,craanl eaxntdracaosusdiaflicvaetritoenbsriatee. One fetus in the 2.5 mg/kg/day dosage group (8644-5) had a short snout with a protruding tongue as the only alteration. High dosage group (3.75 mg/kg/day) fetus 8665-8 had an edematous neck, a sehxoarmtinsantoiuotn.witShofat ptriostsruuedeinxgamtionnagtuieonanrdevsemaalleld enaorsadadtitgiroonsasl eaxltteerrantailons. aSkneglueltaatleedxaalmaienaotfitohne ohfyotihdi.s fetus revealed fused 3rd and 4th sternal centra and E.2.b. Variations - One 0.1 mg/kg/day dosage group fetus (8600-11) had a distended abdomen. Soft tissue and skeletal examinations revealed no other additional alterations. E.3. Fetal Soft Tissue Alterations (Summary - Table 12; Individual Data Table 22) E3.a. Malformations Externally malformed control group fetus 8586-8 had fused, displaced and small kidneys and situs inversus. This fetus had a short trunk and absent tail at gross described. external examination and associated skeletal malformations, as previously 003830 418-010:PAGE IIl-7 `Small and fused lung lobes occurred in one 2.5 mg/kg/day dosage group fetus (8646-1). This fetus had common truncus arterious as the only other alteration. All other soft tissue malformations have been previously described. E3.b. Variations E3.b.1. Eyes One control group fetus (8579-4), one 0.1 mg/kg/day dosage group fetus (8597-8) and one 2.5 mg/kg/day dosage group fetus (8646-2) had a circumcorneal hemorrhage of one or both eyes, a variation generally attributable to trauma during processing. Fetus 8597-8 also had absenceof the intermediate lobe of the lungs. No other alterations occurred in these fetuses. E.3.b.2. Vessels Common truncus arteriosus occurred in one 2.5 mg/kg/day dosage group fetus (8646-1). This fetus also had lung malformations, as previously described. E3.b3. Lungs Absence of the intermediate lobe of the lungs occurred in 2, 5, 1, 1 and 4 fetuses from 2, 4, 1, 1 and 4 litters in the 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups, respectively. One of the 0.1 mg/kg/day dosage group fetuses. (8597-8) also had circumcorneal hemorrhage of the right eye, as previously described. One of the 3.75 mg/kg/day dosage group fetuses (8672-6) also had a variation in skull ossification (internasal), fused sternal centra and misaligned caudal vertebrae. E3.b4. Kidneys The left kidney was displaced caudally in 12, 1, 17 and 5 fetuses from 2, 1, 2, and 2 litters in the (Vehicle), 0.1, 1.0 and 2.5 mg/kg/day dosage groups. The eKxitdenrenyasl,lyasmaplrfeoviromuesdlyvedheiscclreibceodn.troAlnogtrhoeurp vfeehtiusclaelscoonhtraodl fgursoeudpafnedtussm(a8l5l90-8) also had a variation in skull ossification (internasal) E.3.b.5. Hindlimb The skin was constricted on the right hindlimb of one 3.75 mg/kg/day dosage group fetus (8673-3). No additional alterations occurred in this fetus. 003891 418-010:PAGE Ill-8 Ed. Fetal Skeletal Alterations (Summaries - Tables 13 and 14; Individual Data - Table 2: E.4.a. Malformations E.4.a1. VertebraelRibs/Sternum Externally malformed vehicle control group fetus 8586-8 had skeletal malformationsofthe sternum, vertebrae and ribs related to the observations ofa short trunk and absent tail at gross external examination. The 1st through 4th stemal centra were fused, fusion of the xiphoid and 4th sternal centrum, only six thoracic vertebrae present, absent ossificationof the centra and arches of the. 5th and 6th thoracic vertebrae, fused 4th and 5th left ribs, an extra ossification site attached to 6th ribs and absence of all lumbar, sacral and caudal vertebrae. E.4.a.2.Thoracic Vertebrae/Ribs Interrelated vertebraliib malformation or malformations of the thoracic vertebrae and ribs occurred in one 0 (Vehicle) mg/kg/day, two 1.0 mg/kg/day and one 3.75 mg/kg/day dosage group fetuses. These types of vertebralirib `malformations are relatively common at matemnally toxic dosages in rabbits and generally considered to be secondary to maternal stress". Each of these fetuses is described below. Fetus 8582-8 (vehicle control group) had a small left arch and unilateral ossification (left) of the centrum of the 10th thoracic vertebra and fusion of the 10th and 11th ribs Fetus 8625-5 (1.0 mg/kg/day dosage group) had fused centra of the 8th and 9th thoracic vertebrae, a bifid centrum in the 9th thoracic vertebra and fused th and Sth right ribs. Fetus 8628-8 (1.0 mg/kg/day dosage group) had only 11 thoracic vertebrae and 11 ribs present; the 7th right rib and the 8th left rib were split Fetus 8670-1 (3.75 mg/kg/day dosage group) had a right hemivertebra present as the 12th thoracic vertebra and a split 10th left ib. E.4.a.3. Lumbar Vertebrae One 0.1 mg/kg/day dosage group fetus (8612-6) had a right hemivertebra, present between the 6th and 7th lumbar vertebrae, as the only alteration. 003892 418-010:PAGE Ill-9 E.4.a4. Caudal Vertebrae Misaligned caudal vertebrae occurred in three 0.1 mg/kg/day dosage group fetuses and 1 3.75 mg/kg/day dosage group fetus. One 0.1 mg/kg/day dosage group fetus and the 3.75 mg/kg/day dosage group fetus had additional skeletal alterations, as previously described. E.4b. Variations E4b.A. Skull Common small irregularities in ossificationof the skull the presence of small ossification sites within the sutures or calvaria (nasal, frontal or parietal bones) and/or irregular shaping or fusionof the bones] occurred in 5, 4, 4, 5 and 2fetuses in 5, 3, 4, 4 and2 litters in the O (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups, respectively. Irregular ossification of the nasal bones (midiine suture displaced or internasal ossification site) were the most commonof these small irregularities in ossification pattems, occurring in 3, 4, 3, 4and 2 fetuses in 3, 3, 3, 3 and 2 litters in the five respective dosage groups. One of the 2.5 mg/kg/day dosage group fetuses (8648-18) also had fused stemal centra and one 3.5 mglkglday dosage group fetus also had fused steal centra and misaligned caudal vertebrae. No other skeletal alterations occurred in these fetuses E.4b.2. Hyoid One or both alae of the hyoid were angulated in 3, 4, 1, 2 and 6 fetuses in 2, 4, 1,1and 4 litters in the 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups, respectively. One 0.1 mg/kg/day dosage group fetus (8596-1) and two 3.75 mglkglday dosage group fetuses (8665-8; 8678-7) also had fused steal centra. Another 0.1 mg/kg/day dosage group fetus (8614-4) also had short hyoid : alae and a misaligned caudal vertebra. E.4b.3. Vertebrae One 0.1 mg/kg/day dosage group fetus (8601-5) had unilateral ossification of the centrum of the 13th thoracic vertebra as the only alteration. `The centrum of the 12th thoracic vertebra was fused to the centrumof the 1st lumbar vertebra; the centrumof the 1st lumbar vertebra was bifid in one 1.0 mg/kg/day dosage group fetus (8628-6). 003893 418-010:PAGE I1l-10 E.4b.4. Sternum Fused 3rd and 4th sternal centra occurred in 1, 4, 3, 5 and 4 fetuses from 1,4, 1, 3 and 3 liters in the five respective dosage groups. Additional alterations in these fetuses were described previously. E4b.5. Pelvis Three fetuses from two 2.5 mg/kg/day dosage group litters had unossified pubes as the only alteration. The significant increase (p<0.01) in the fetal incidence of this alteration was considered unrelated to the test article because it was not dosage-dependent. E.4.b.6. FOetasl sificSiatetAvieroagnes The average numbers of ossification sites in the hyoid, vertebrae (cervical, thoracic, lumbar, sacral and caudal), ribs, sternum (manubrium, sternal centers and xiphoid), forelimbs (carpals, metacarpals and phalanges) and hindlimbs. (tarsals, metatarsals and phalanges) occurred at similar incidences in litters in all dosage groups and did not significantly differ. F. its (Individual Data -- Tables 1 20 One satelite doe in the 1.0 mg/kg/day dosage group and another satelite doe in the 3.75 mg/kg/day dosage group aborted and were sacrificed. Satellite doe 8691 aborted on DG 18 after the 12th daily dosage was administered. No other adverse clinical observations occurred in this doe. This doe lost weight after DG 16; its feed consumption values were unremarkable. No gross lesions were revealed by necropsyof the doe. The litter consisted of three early resorptions. Satelite doe 8700 aborted on DG 19 after 12 daily dosages had been administered. Additional adverse clinical observations in this doe included scant feces (DGs 7, 11, 13 and 17 to 18), no feces in cage pan (DGs 12 and 14 to 16), localized alopecia on the underside (DGs 12 to 18) and soft or liquid feces. (DG 13). Body weight loss occurred in this doe after DG 8, and its feed consumption was severely reduced after DG 10. No gross lesions were revealed by necropsyof the doe. The litter consistedof four dead fetuses and two conceptuses that were presumed cannibalized. Adverse clinical observations in the satellite groups were similar to those in the main study groups. Observations of scant feces and soft or liquid feces occurred inthe 0.1, 1, 2.5 and/or 3.75 mg/kg/day dosage groups. One doe in the 3.75 mg/kg/day dosage group had observations of no feces in cage pan. 003894 418-010:PAGE Ill-11 Patterns of body weight gain and feed consumption were generally comparable 10 the rabbits in the main study at the same dosage levels. Only one rabbit in the 2.5 mgkglday dosage group was not pregnant at Caesarean-sectioning on fDiGve 2d1.osaCgaeesgarroeuapns-.seOcntieondionegianntdheli0t.te1r mpga/rkagm/edtaeyrsdwoesraegecogmrpoaurpahbalde balmaocnkgantdhe brown mottling of the lungs at necropsy; all other does appeared normal. Average liver weights for pregnant does on DG 21 were 134.4 10.0, 128.9 4.2, 106.6 + 42.2, 117.8 35.5 and 109.0 37.5 in the 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day dosage groups, respectively. The data for rabbits assigned to the satellite portion of the study are provided in individual tables only. 003895 418-010:PAGE IIl-12 REFERENCES 1. U.S. Food and Drug Administration (1994). International Conference on Harmonisation; Guideline on detection of toxicity to reproduction medicinal products. FederalRegister, September 22, 1994, Vol. 59, No. 183. 2. UR.eSg.ulFatoioodnsa;ndFinDarlugRuAldem.in2i1strCaFtiRonP.artGo58o.d Laboratory Practice 3. Japanese Ministry of Health and Welfare (1997). Good Laboratory Practice Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. 4. E1u9r8o9poenanthEecaocncoempitcanCcoemmbyuntihteyEu(1r9o8p9e)a.nCEocuonnciolmideccCisoimomnuonnit2y8oJfulayn OECD decision/recommendation on compliance with principlesofgood laboratory practice. Official Journal of the European Communities: Legislation. 32(No. L 315; 28 October): 1-17. 5. Christian, M.S., Hoberman, AM. and Smith, T.H.F. (1982). Dosage-range study of the teratogenic potential of suspensionsoftrnitrofluorenone (TNF) administered orally to New Zealand White rabbits. Toxicologist 2(1):40 (#143). 6. Cnharlitsrteixaonn,eM(.PSr.oc(1e9e8d4i)n.gsReofprNoadlutcrteixvoenetoSxiycmiptyoasinudmt,erNateowloYgoyrekvaAlcuaatdieomnsy of of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 7. FCheruissstniaenr,,ME.SL., (L1i9g9h2t)k.epA, Gd.eEc.a,dHeenofnersabyb,itRf.eArt,iliHtyodbaetra:manS,tuAdMy.ofahnidstorical control animals. Teratology 46(4):349-365 8. Institute of Laboratory Animal Resources (1996). Guide for the Care and Useof Laboratory Animals. National Academy Press, Washington, D.C. 8. SImaplleawnstkait,ioEn.ss(t19e6l4l)e.n aFmarUbteemreutshdoedreRaztuem. maArkcrho.skPoatphioslc.hEexnp.NaPchhawrmeaiksolv.on 247:367. 10. fSettaupsleess., RT.eEr.at(o1l9o74g)y. 9(D3e)t:eAc37t-i3o8n.of visceral alterations in mammalian 11. Staples, R.E. and Schnell, V.L. (1964). Refinement in rapid clearing technique in the KOH-alizarin red S method for fetal bone. Stain Technol. 39:61-63. 003896 418-010:PAGE Ill-13 12. Snedecor, GW. and Cochran, W.G. (1967). Variance test for homogeneity of the binomial distribution. Statistical Methods, 6th Edition, lowa State University Press, Ames, pp. 240-241. 13. Sokal, RR. and Rohlf, F.J. (1969). Barlletts test of homogeneity of variances. Biometry, W.H. Freeman and Co., San Francisco, pp. 370- 37 14. Snedecor, GW. and Cochran, W.G. (1967). Analysis of Variance. S2t5a8t-i2s7t5ic.al Methods, 6th Edition, lowa State University Press, Ames, pp. 15. Dunnett, CW. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1126. 16. Sokal, RR. and Rohlf, F.J. (1969). Kruskal-Wallis Test. Biometry, W.H. Freeman and Co., San Francisco, pp. 388-389. 17. Siegel, S. (1956). Nonparametric Statisticsfor the Behavioral Sciences, McGraw-Hill, New York, pp. 96-104. 18. Dunn, 0.J. (1964). Multiple comparisons using rank sums. Technometrics 6(3):241-252. 19. Khera, K.S. (1985). Maternal toxicity: Apossibleetiologicalfactor in .Teemrbartyool-ofgeyta3l1d:e1a2t9h-s15a3nd fetal malformations of rodent-rabbit species. 20. Christian, M.S., McCarty, R.J., Cox-Sica, D.K. and Cao, C.P. (1987). vReehciecnlet cionnctrreoalsNesewin ZtehaelianncdidWehnictees orafbsbkiutlsl., lJ.unAgmearn.d Criobl.alTtoexriactoilo.ns in 6(4):562 (#126). : 003897 APPENDIX A REPORT FIGURE 00389g "I i' )- ; iul ) Ae MATERNAL BODY WEIGHTS } Figur1e e Sais geyree A vdsre A > ok er | Ee Sodofcediaton # BR BA ae APPENDIX B REPORT TABLES 003900 EL -- Teetn me TT wera eer soem asm awn Bh ~ . a ww WA AS A = woroc--omemn--conet s jp wnLorsawwmlors eoul EE 0 e 19 aes u wr nawt un 2 RL Bot Ti oe rh Setadn tay af sentarion PLEEmBmeshaiisEsE 33 SRSR EAS ns you vot ios ann rosa emer josy om so Lene. ss se . we we we we Re TR Tck She+TARBITD[RASS01 ARISEEOIN FE S00 ON eer tates 0 okprotasa povscion & nEmmmaTLns omar 8 DEond mnaemun a4di3as mua ns 3T5% OE1S6dEorRteEs aRadby 3001 penavion. SEER HR elEVEN: coneret aren vate (ps0.00. 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B o=n Ir,idAaThEREXE IEER iA i i & g EB MO mu SALIELRARL s BE eamsu OS EEBR E E a ms ENE Ean our rman ro Sr oh TU mersimei sor cava g g RWEOIT am a om ttomhom we wry RSE SLE we ab atom om SSmum WIo E mim wom mom 8Q Bn m R h TRI I E A D WONRasoh ee IIIT he ah TREeb a Si nswa 2 , SR IErIas Lh Se Wh nah lh BR wma vn pl oe Whakenanh : 8 E we Um RI Rw G8, gm hat AEE E wu pmomomin neon um 8gmwn wn ASAR S ve pred Mpa), , PRIN RLAE L ERET- RE g 3 38 g mm ETT rt I I 5 mi 8 mln s| i -- in ail i ots we nh a. g mune - y . nie ie By Ed wh Ee ale g I ad 2 wr es orchron Rl E HfA Be $;i  wuld Sikh hh g mma LL 8 - " : ole lie Reed we wie "reat gf 3 g  mo dddday 8 A anti ats ws d ebe 0k 0.67 awe mm 38 wP es G E Ts A TA TA PAaTA itA/ i WM m T i W SHR Be Rahy ETT g g WEme ww ws SUTINOI TROD ne mw mind id Ra an : 5  pe =TEoR PRRaRbd2RdIhCIFZh hab we TIIILIIII, Ej R adA d ehh a a: a Chive EL DanyRESOR+PLATTEREISOROPTINON + moresmosivion ocanis i " or S2 i DE E|EE EE APA 2 & % Tn TN wn oesennrion g 8 wn omscasrrion wn omscarrrion Bae - swam wr : wr ImBewsorwits ary mTeissprow. : & og 8gE2 o EEErRe = Eins, rose, 1thana rn a (ine ikesesaepeions 4 taro on day 26 of seston; aksiyals precluded . g 3g i & BR eereoeir gg i mm . g8 N ETS SE BE ssi ---- IE AElE a a a ram " we 3 A een smssssosns 33 EERrRCESeSSt 5TEAeSELSTAeB peeAsee gB ene Bi EE me an pi r mo e mmm |W miee . me Rr, 28 B reu, 88 BTElam, kam, eae sTide Cosi g3 WW +WBE OF SPECIMENS WITH ALTENTIONS/WNEROFSPECINENSELANINED Tr z wn Ew en mews ee wn mn ws 8 "Ee" TY man nee RAE ems Win ERoF SPRCINERS WITHATEANTIONSWOESOFSPECIMENS EOIN Ce g & % mr Be Zgz2Son wes wn Sux wn wr owen ws pp wn omen i or _--_-- " vorg canverresAE wr wewsa a pp -------- ws EE f oo 2 2 8 BE ve me [ET g 282 E --Eb--ra E -- autolyais precioded res 1 we mm wr we se a =om SD, 3rdand4th ig $Egos om or : ; we sem ws on oak AssenT E a3 N ight " - I os os 3 rn PIII i S we ama ws 22 Sas ws wen em SI SE ST Wrogms m f ---- a PLeE.tersemerare om M TROL y Ame, e-- ens SREpee Win W0a RCN VETATI RARERO OFSTRN INGS EONINED Co pidotsriacis, g - i z 8 em g2 EBR n, g3 IrEosui, - man we we Emr we \ 22 F[J Wwa eso I oe g oe or ne ws vr Fr Sl eo -- ---------------- -- Se ossssssssssssstnson : 8 g 8Sa [ ne RR an Emsl mn - rs Fray iss. 11 ewes, Fass i 8 BFE en < 2 S [-- Set Ridney, coviatiy rm, pBrha--n_, TO wn wn ewerimn Beam, g rr 8 I A Se So g Boa = -82$ @ FO HE i0yean-- Rly .. -- ES -- i i ---- IE 23 3 82 ER ne MTV an ossenimon go$ Ew wen wr ------------------------ wn omscarmion wn osscnsrrion TW emFreeHemypch,ant wr wn WN EnOF SPRCINENSWITH ALTERKTIOHSWBE OFSvcsEwe -- E g g z 8 22S [reslrkeycrapmiroeenies SQ we ama we oe wa aces ee oe ia or Sac WYNTAN ION ABER0SECIS EANIRED Si, Wi ve omen Tw . vem A 8 & 3 in THR ees war Wane wn seme or sme ER CC 222Sun wan ws B [PI I Ee ve pm oliB : r -- mm ve Wi eenorShecivks WITH ALriaTicuswasnor src wise [ or a g 3S & 2 8 wer saoermocnone anor caomaion FE i Sma MTV wn owonmon wn oescnrrmon wn oesearrmon 8 r teva n precios B3Boowaes sew sem we we Cw www ws we we we we wr ves os Ter doer: ensiatiy 3 re | EB g Cgmow mTTe. 2 ow wm or eeeoeeneneemen no us upri hua tn. wn vs em ws uv ve mre . we ws g TLWE mimpe wa sotpi, ee 2 8 ma sem we mDeawtssun. we oe 2a3 rn whiescion pee ainiepractises : RE SARL 2 TM ODB xamianatit ocn.f"ohrtAhIeuLsraetiaisaitneiinogndfe1tuatsp hees scpl persiee d normalA 48 iors T exterF nr al ee xse minar tis ce ftor SeEverlRopaes)mtaldsgt.mAIpLafretaultes sppesced g3S iedaeseseArLLireosi norePALATE CMBR, Teeth de. Hr eee arresedmoral a Ths ivie, puta not oaieies BERGREAD at Teensha Fon SIE ION heh meatSon}a ite emsineion 2 BRE g - $ oemeson. 3 LI o Aeneaspracticed lin woo met po pa. mee a rss JR SOO. NOTRE reosterassona paises estes nt sssssirn oe gg i nT 8e oTsi, J 2 HR ~ wero Wwe ers vewss w----. ve pg os wn ve me vs mesa JE rn g ET iEte (ot SE mybop SA BLA . te lan wd Fo te Tetorptions 10 state on dey 20 of gestation: sutelysia precluded 8 2 | mE me enna JEEh $e 2 @ wn eosam ee nme Oe f Eyenere Bh. uoree pp etee an R 3 R. uore BL ------------ 3 7 2 wm RE ie $ 3 uwme swewmw uwme ovme mBew DM NI WONRES R OFD SPRCIS NENSSWITHALTERATIONSsWiMSsOtF sSPRCINDNSEoin wn en owms : " Lt g & g APPENDIX C PROTOCOL AND AMENDMENT 004029 418-010:PAGE C-1 r,PRIMED]ICA Argus30R5esSehaorcaHhohrLsDarhbioavrmea,.toBPriAues1,i96In4nc&4. TelTeoplhootnaxe:: ((221155))444433--88578170 PROTOCOL 418-010 SPONSOR'S STUDY NUMBER: 6316.8 STUDY TITLE: PURPOSE: TESTINGFACILITY: STUDYDIRECTOR: `SPONSOR: STUDYMONITOR: ALTERNATIVE STUDYMONITOR: Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits NTh-eEtpFuOrSpoEsoeonfNtehiwsZsetauldaynids tWohidtetee[cHtraad:v(eNrZsWe)eSfPfFec]ts of presumed pregnant female rabbits and development of the eimmpblrayntoatainodn fteotculsocsounresoefqutehnethtaordexppaolsatuer.eoTfhtishestduodey from evaluates ICH Harmonised Tripartite Guideline stages C and D of the reproductive process in a nonodent species. Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A| Horsham, Pennsylvania 19044-1297 Telephone: (215) 443-8710 Telefax: (215) 43-8587 ARsasyomcioantdeDGi.reYcotrokr,oPfh.RDe.s,eaDrAcBhT 33MM TCoexnitceorlBoguyidSienrgvi2c0e-s26-02 St. Paul, Minnesota 55144-1000 TMealrevpihnoTn.e:Case(6,51D)V.7M3.3,-5P1h8.0D. Telefax: (651) 733-1773 ATenlderpehwonMe.: Se(a6c5a1t), 5P7h.5D-.3161 Telefax: (651) 733-1773 004021 418-010:PAGE C-2 Protocol 41P8a.g0e120 REGULATORYCITATIONS HU.aSr.moFnoisoadtiaonnd; DGruuigdeAldimnieniosntrdaettieocnti(o1n99o4f)t.oxiIcnitteyrntaotrieopnarlodCuoctnifonerfoernmcoedneicinal products. Federal Register, September 22, 1984, Vol. 59, No. 183. 2U1.S.CFFRooPdaratnd58.Drug Administration. Good Laboratory Practice Regulations; Final Rule. JfoarpSaanfeestey MSitnuidsitersyoonf HDeraulgtsh, aMnHd WWelOfradriena(n1c98e7)N.umGboeord21L,abMoarracthor2y6P,ra19c9t7i.ce Standard EacucreoppteaannceEcboyntohmeicEuCroompmeuanniEtcyo(n1o9m89i)c. CoCmoumnucniiltdyeocfisainonOoEnC2D8 Jduelcyis1i9o8n9/roencotmh-e tmheendEautrioopneaonn Ccoommmpulniiatniceesw:itLhepgirsilnactiipolne.sof32g(oNoo.dlLab3o1r5a;to2r8ypOrcatcotbiecre).: O1f-f1i7c.ial Journal of REGULATORY COMPLIANCE: "rTehgiuslsattiuodnyswicliltebdeacboonvdeu.cted in compliance with the Good Laboratory Practice (GLP) ADlilrecchtaonrgaensdotrhereSvpisoinosnosro,fdtahtisedpraontodcomlaisnhtalalinbeeddwoicthumtehnetperdo,tocsoilg.nedbythe Study Tanhde wQiulalliintsypAecstsucrriatinccalepUhnaitse(sQAofU)thweilsltauuddyitinthaeccporortdoacnocle, wtihtehrathwedSattaanadnadrdthOeperreapotritn,g Proceduresof Argus Research Laboratories, Inc. `aTchceurfaintaellyrerpeofrltecwtislltihnecrluadwe daatsataotbetmaeinntedsidgunreidngbtyhtehpeeSrtfuodrymaDnicreecotforththeastttuhdey raenpdortthat aslilgnaipfpilciacnatbdleevGiaLtPionrsegfurloamtiGonLsPwerergeulfaotliloonwsedocicnutrh,eecaocnhdwuicltl bofetdheescstruidbye.d iSnhdoeutlad, together with how the deviation might affect the qualityor integrity of the study. `SCHEMATICOFSTUDYDESIGNANDSTUDY SCHEDULE: `See ATTACHMENT 1 to the protocol. 004022 418-010:PAGE C-3 Protocol 4P1a8g0e130 TA ESTRTIANC DVEL HICE LE: Identification: TestArticle: Name: N-EtFOSE. Physical Description: ~~ Waxy solid. LotBatch Number: FM-3929(30035, 30037, 30039). Specific Gravity: -1.7. Purity: 90.1%. Expiration Date: May, 2000. Information on the identity, composition, strength and purity of the test article is on file with the Sponsor. Vehicle: 29% Tween 80 in Deionized Water). Reverse Osmosis Membrane Supplier and lot identification Processed Deionized Water (R.O. of Tween 80 to be documented in the raw data. tNoeibteheprrtehseenStpionntshoervneohrictlheetShattudwyouDlidreicnttoerrfiesraewwairtehotfhearneysuplottseonftitahliscosntutdaym.inTahnetrsefloikreel,y no analysesotherthan those mentioned in this protocol will be conducted. SafetyPrecautions: Gloves, mask, appropriate eye protection and a uniformilab coat are to be wom during formulation preparation and administration. The Material Safety Data Sheet (MSDS) is attached to the protocol (see ATTACHMENT 2). Storage: Bulk Test Article: Vehicle Components: Prepared Vehicle: Prepared Formulations: Room Temperature. Room Temperature. Room Temperature. Room temperature (samples to be frozen). All test article shipmentstothe Testing Facility should be addressed to the attention of Julian Gulbinski, Manager of Formulations,atthe previously cited address and telephone number. Shipments should include information concerning storage conditions and shipping cartons should be labeled appropriately. The recipient should be notified in advance of shipment. 004023 418-010:PAGE C4 Protocol 41P8a-g0e140 [EORMULATION: Freqou fPreepn arac tioyn: Formulations (suspensions) will be prepared dalyatthe Testing Facil. Detailed preparation procedures are attachedtothis protocol (ATTACHMENT 3). AdjustfomrPeurnitty: `The test article will be considered 100% pure for the purpose of dosage calculations. TF estia ng ciRel seri veSt amply es: `The Testing Facillty will reserve a sample (1 g) of each lot of bulk test article and a sample (5 mL)of each lotofvehicle used during the courseofthe study. Samples will be stored under the previously cited conditions. ANALYSES `Samples additional to those described below may be takenif deemed necessary during the course of the study. BulkTestArticle Sampling: No analysesof the bulk test article will be conducted during the courseofthis study. Information on the stability ofthe bulk test article is on fil With the Sponsor. AnalysesofPreparedFormulations: Homogeneity and stability of prepared formulations on file with the Sponsor. However, records will be maintained to document how the test article formulations were prepared. c fon of Test Adicle F one: Concentrationofthe prepared formulations will be verified during the courseofthis study. Duplicate samples (2 mL each) will be taken from the first and last preparation orenmtahienidnagysparmeppalreesd.willObneerseatamipnleedoaftetahcehTessettiwniglFbaceilsthyipapsedbafcorkuapnaslyasmipsl;etsh.e Backup `samples will be stored under the previously cited conditions and discarded at the Testing Facility upon request of the Sponsor. 004 024 418-010:PAGE C-5 Protocol 418-010 Pages ShippingInstructions: Samples to be analyzed will be shipped (frozen on dry ice) to: K3rMisEJn.vHiarnosnmeenn,taPlh.TDe.chnology and Safety Services 935 Bush Avenue SBtu.ilPdaiunlg,2M-i3n-ne0s9ota 55133-3331 Telephone: Telefax: (612) 778-6018 (612) 778-6176 The recipient will be notified in advance of sample shipment DISPOSITION: Parrteicplaerweidllfboermrueltautrinoends twoilthbeeSdtiusdcyarMdoenditaotrathtetTheestpirnegviFoaucsilltyy.citAeldlardedmraeisnsinugpbounlk test completion of all work with the test article. JESTSYSTEM: TbehceauNseew: Z1e)ailtainsdoWnheintoen-[rHordae:n(tNZmWa)mSmPaFl]iraanbbsiptewcaiesssaeclceectpetdedasantdhewTiedseltySuyssetdem tthoxriocuigthyofuertatthoegeinnidcuistty)ry2f)orthniosnsctlrianiincoaflrsatbubdiiteshaofsdbeeveenlodpemmeonntsatlratotxeidcittyo (beembsreynos-ifteivtealto daenvde4l)optmheentteaslt atrotxiicnlse;is3)phhiasrtmoarcicoallogdiactaalalnydacetxipveeriinetnhcee exist at species tahnedTsetsrtaiinn.g Faciity*; NumberandSex: Population evaluated: Population selected for toxicokinetic evaluation: 110 timed-pregnant female rabbits (22 per dosage group). l1e9veslasteplliutse tfhermeaelaetrtahbebiottsh(efrivdeoastethleevellosw).and high dose BW odyeiganhdAgte: `The individual body weightsofthefemale rabbits will range from 2.5 kg to 5.5 kg; the arsabsbiigtnsmweinlt.beAacptuparloxbiomdatyewleyifgihvtestoresceovrednedmoatntrhecseoifptaagnedaatttshteutdiymaesosfisgtnumdeynt will be documented in the raw data, and the weight range will be included in the final report. 004025 418-010:PAGE C6 Protocol 418.010 Pages Source Covance Research Products, Inc. SDewnavmeprb,rPiedngnesyRlovaadn,iaBo1x77521070 "PTrhoedurcatbsbi,tsInwci.l,lDbeenvsehri,ppPeednnisnyflivtaenriead,ctaorttohnesbTyesttriuncgkFfacriolmty.Covance Research Identification: RTaabgbiCtos.arInec.p,eNrom.anMeSntPlTy identified 20103). using Monel Female rabbits saerfet-gpiiverecninugnieqauretpagesrm(aGneeyntBand and igdeesnttaitfiicoantiboondnyuwmebieghrtss.when assigned to the study on the basisofday 0 of presumed ANIMAL HUSBANDRY: All cage sizes are in compliance with the Guideforthe Care and UseofLaboratory Animals. Housing: "nTehsetirnagbbmiattsewriilallbsewiilnldibveidsuuaplpllyiheodubseecdauinseuntithseoffesmiaxlteoreaibgbhittsstwaiilnbleesssasctreieflicceadgebse.forNeo parturition is expected. RT ooe mAimr,peraatndu Humr idie ty: fTrheeshanaiirmtahlatrohoams biseienndeppaesnsdeednttlhyrosuugphpl9i9ed.8w7it%hHatEPleAasftitteerns.chRaongoemstpeemrpehroautruorfe1wi0ll0%be maintained will also be at 61F (16C) to 72F (22C) and monitored constantly. monitored constantly and maintained at 30% to 70%. Room humidity Light: Amaninatuationmeadt.icaElalychcodnatrrkolpleerdi1od2-whilolurbelgiignhta:t121-9h0ou0r dark fluorescent hours EST. light cycle will be Diet: Approximately 150 g of Certified Rabbit Chow #5322 (PMI Nutrition International) wil be available to each rabbit each day until the first day of dosage, at which time aTphperocexritmiafiteedlyfe1e8d0wiglol fbtehaevasialambelecefrrtoimfieidndfieveidduwailllstbaeinolfefsesresdteteole"aJ-cthyprea"bbfieteedaercsh day. attached to each cage. 00402 418-010:PAGE C-7 Protocal 418.010 Page? Water: Water will be available adlibitumfrom individual bottles attached to the cages of from an automatic watering access system. All water will be from a local source and passed through a reverse osmosis membrane before use. Chlorine will be added to the processed water as a bacteriostat; processed water is expected to contain no more than 1.2 ppm chlorineat the time of analysis. Water is analyzed monthly for possible bacterial contamination and twice annually for possible chemical contamination. Contaminants: Neither the Sponsor nor the Study Director is awareof any potential contaminants likely to be present in the certified diet or in the drinking water at levels that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier or those mentioned in this protocol will be conducted. MAANT D RAI NDOMN IZATG ION: `The female rabbits will be naturally bredatthe Supplier by breeder male rabbits of the same source and strain before shipment to the Testing Faciity. The day mating occurs wil be designated day 0ofpresumed gestation. The rabbits will be mated on five consecutive days and shipped to the Testing Facility after the last mating day to arrive on day 1,2, 3, 4 or 5 of presumed gestation. Before shipment of the rabbits, the Supplier will forward breeding records and day 0 of presumed gestation body weights. A computer-generated (weight-ordered) randomization procedure will be used to assign the rabbits to dosage groups based on this information. ADMIN N: RR outee andasfoo rChon ice: The oral (stomach tube) route was selected for use because: 1) in comparison with the dietary route, the exact dosage can be accurately administered; and 2) it is oneofthe possible routes of human exposure. MeatndFhreqouend cy: pFreemsaulmeerdabgbeisttsatwiiloln.beDgoisvaengetshewitlelstbeartaidcjluesotnedcedadlaylfyoornboddayyswe7itghhrtocughhan2g0esofand given at approximately the same time each day. Ratfoir DoosangeSaelelctieon: Dosages were selected on the basis of a dosage-range study (Argus Research Laboratories, Inc.,Protocol 418-010P). 004027; DC osaogenLevcelse. ntratai ndVooln umess: 418-010:PAGE C-8 Protocol 418-010 Pages CTorTove [oT5Toovonmpnmne|n [ov {zo [| 26 | os | |eusowoommmmm|n LTTRISoysTtSoerwwgoia[ cS ocnida tre ed 10s0| %apreeforothmpupso[fd|osoagec[alastaensw. oncommnnv|n TA ESTSN , ALANY DMES ASURE EMENS TS: Viability: Al Periods: Atleast twice day. CO linib cal serv and/aorGt enerialAo ppeanrans ce: Predosage Period: Atleast once. Dosage Period: Twice daily. Priorto dosage administration and once approximately one hour postdosage. Postdosage Period: Once daly Clinical observations may be recorded more frequently than cited above,if deemed appropriate by the Study Director and/or Study Monitor. BodyWeights: Predosage Period: Day 0of presumed gestation and on the day of arrival atthe Testing Facilty Dosage Period: Daily. Postdosage Period: Daily. [FeedConsumptionValues: Predosage Period Dosage Period: Recorded daily after arrival at the Testing Facilty (values not tabulated). Recorded daily. 004028 418-010:PAGE C-9 Protocal 418.010 Pages Postdosage Period: Recorded daily. Fineteerdvaclosnassumbpotdiyonweviaglhutesevdaulruiantgiotnhse.dosing period will be tabulatedforthe same Caesarean-SectioningObservations: bRaebbrietmsovwielldbferoCmaetshaeruetaenr-usseacntdiopnleadceodn idnaiynd2i9voidfuaplrceosntuamiendersg.estTathieonr.abTbihtes fwieltlubsee.s wil examined for number and distribution of: Corpora Lutea Implantation Sites [Placentae appearance (size, color or shapeif abnormal) 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 daetaedrmfefteutsuessthdaetmodnosetsrantoitnrgemsapornkdedtotosteimxutlrieamnedautthaotlyissinsoatrmeacroknesdildyeraeudtotloyzbeed; late resorptions.) Early and Late Resorptions. (A conceptus is defined as a late resorptionif tis grossly evident that organogenesis has occurred;if ths is not the case, the conceptus is identified as an early resorption.) FetalObservations: `BW odyeig and Ih dentit ficats ion: `The body weightofeach fetuswill be recorded. Only body weights of live fetuses will bideenutsifeidcattoiodnentoetrimnignsetluidtyernfuemtablebro,dlyitwteeringuhmtbaevreraangdesu.terFienteudsiesstriwiblultiboen.tagged with GrossExternalAlterations: All fetuses will be examined for gross external alterations. Late resorptions and dead fseutcuhseosbsaelrsvoawtiilolnbseweilxlanmoitnbeedifnocrlgurdoesdsinexetietrhnearldaalttaersautmimonasritozatthieoenxtoernsttaptoisstsiicballe but analyses. 004029 418-010:PAGE C-10 `Sof Tisue Examinati Sex Protocol 4P1a8g-e01100 All fetuses wil be examined internally to determine sex. Cavitated organs will be evaluated in all the parietal and fetuses frontal by dissection. bones, and the brAaisniwniglllebceroesxsa-mseicnteidoninwisliltub.e made between `Skeletal Examination: ASlklefleettaulsepsrewpilalrbateioenxsamwiilnlebdefroertsakienleedtailn aglltyecreartiinownistahfttehrysmtoalinaidndgewditahsalaizparreisnerrveadtiSve.. tRaekperne.sentative photographsoffetal gross, soft tissue and skeletal alterations will be MET OFSH ACRO IFID CE: sBaecuitfhiacenarsaibbait-sD(vSipaeciinatlra(vmeannouufsaicntjuecrteidonb)yaSncdhelirvienfge-tPulsoeusgh(viHaeailntthra)pweirliltboneeaulseindjetcotion) NECROPSY: Gross lesions will be retained in neutral buffered 10% formalin for possible future egvraoluupataitotnh(ecdoirsrcerseptioonndionfgtthiesSstuuesdywilDlirbeectorre)t.ain(eEdxcferpotmiorna:bbPitasroivnatrhieanvechyisctlse wciolnltbroel dciitsecdarbdeeldo;w,thaellseotahreerctiosmsmueosnw,ilslpboendtiasnceaorduesd.lesions in rabbits.) Unless specifically `SatelliteRabbitsAssignedtoToxicokineticSampleCollection: `Osnamdpalyes21wilofbperceoslulmecetdedgefsrtoamtitohne (rtahbebidtasyafsoslilgonweindgttohtehelatsotxdiocsoakignee)t,ictoexviacloukaitnieotni.c: wFiollllboewicnogllaencetsetdhfersoima tohfepeinntfeorbiaorrbviteanla, cbalvooadinstaomspelreusm(aspeppraorxaitmoartteulbye4s manLdpceerntrraibbfiutg)ed. `mTahientraesiunletdinfgrsozeernum(-(7a0ppCr)ouxnitmilatsehliyp2memnLt)towiltlhebeSpiomnmseodiraftoerlaynaflryosziesn.oTnhderyliivceer awinlldbe excised, weighed, and a sample wil be taken from the right lateral lobe, frozen and retained at -70C until shipment to the Sponsor for analysis. Rpoasbsbiibtlsewailsl dbeescCraiebseadreaabno-vseefcotriroanbebditasndasfseitgunseeds twiollthbeemeaxianmsitnueddy.grFoesstluysetso tahnedextent placentae will be pooled per ter and retained frozen (-70C) until shipment to the `Sponsor for analysis. After completion of sample collection, serum, liver sections, fetal and placental samples will be shipped (frozen on dry ice) to Kris J. Hansen, Ph.D., at the previously cited 004030 418-010:PAGE C11 Protocol 4P1a8g.e01110 address for analysis. Both the recipient and the Study Monitor will be notified in advance of sample shipment. ScheduledSacrifice: On day 29 of presumed gestation, female rabbits will be Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera will be performed. Uteri of `apparently nonpregnant does wil be stained with 10% ammonium sulfide to confirm the `absence of implantation sites". RabbitsFoundDeadorMoribund: Rabbits that die or are sacrificed becauseof moribund condition, abortion or premature doeblsievrevraytiwiolnl bisemeaxdaem.inPerdefgonratnhceycsatuasteusoafnddeauttheroirnemocornitbeunntdscwoilnldibteiornecoonrdtehde.daAybotrhteed fetuses and/or delivered pups will be examined to the extent possible, using the same methods described for fetuses. Uteriof apparently nonpregnant does will be stained with 10% ammonium sulfide to confirm the absenceof implantation sites". 004031 418-010:PAGE C-12 Protocol 4Pa1g8e01102 PROPOSEDSTATISTICALMETHODSTM*: Aapvperroapgrieastea.ndAdpdeirtcieonnatlagpersocweildlubreescaalncdu/loarteadn.alLyitsteesr vmaalyuebsewpilerbfeorumseedd, wifhaeprpreopriate. TyofpTeset" I. Parametric A. Bartlett's Test Il. Nonparametric A. Kruskal-Wallis Test (75% ties) 7 atps0.05 Not TT" 7 atps0.05 Not Significant Nonparametric as Variance Dunn's Test Significant at ps0.05 | Dunnett's Test Not Significant B. Fisher's Exact Test (>75% ties) Il. TPesrtfoorportiDoatna Variance Test for Homogeneity of the Binomial Distribution a. Statistically significant probabilities are reported as either ps0.05 or ps0.01 b. Proportion data are not included in this category. c. Used only to analyze data with homogeneityofvariance. d. Test for homogeneity of variance. 004032 418-010:PAGE C-13 Protocol 4P1a8g.e01103 DATA ACQUISITION,VERIFICATION AND STORAGE: Data will be hand- and/or computer-recorded. Records will be reviewed by the Study Director and/or appropriate management personnel within 21 days after generation. All original records will be stored in the archives of the Testing Facilty. All original data will breequbeostu.ndParnedseirnvdeedxetdi.sAsuescowiplyl obfeaslltorraewd daatttahewilTlesbteinsgupFpalciieidtytoatthneoScphoanrsgoerfourpoonne ytoeadretaeftremrimnaeitlhinegdiosfptohseitdiroanfotffitnhaelsreepomratt,eraifatlesr.which time the Sponsor will be contacted RECTOO BE MR AIND TAINS ED: Protocol and Amendments. Test Article, Vehicle and/or Reagent Receipt, Preparation and Use. Animal Acquisition. Randomization Schedules. Veterinarian Examination. Mating History. TGerneeartamlenCto(mimpernetsscr.ibed by Staff Veterinarian). Clinical Observations andor General Appearance. Blood Sample Collection, Processing and Shipment. BFeoeddy WCeoingshutmsp.tion Values. Caesarean-Sectioning and Fetal Observations. Gross Necropsy Observations. Organ Weights. Photographs (f required). Study Maintenance (room and environmental records). Feed and Water Analyses. Packing andor Shipment Lists. KPEEYRSONNEL: Executive Director of Research: Midred S. Christian, Ph.D., Fellow, ATS Director of Research: Alan M. Hoberman, Ph.0., DABT ADsirseocctioarteofDLiarbeoctroartoorfyROepseeraarticohnsa:ndJSothundyF.DiBraemcettort:, BR.aS.ymond G. York, Ph.D..DABT Manager of Study Coordination: Valerie A. Sharper, M.S. Managerof Animal Operations and Member, Institutional Animal Care and Use Committee: Dena C. Lebo, V.M.D. Manager of Regulatory Compliance: Kathleen A. Moran, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP 004033 418-010:PAGE C-14 Protocol 4P1a8g.e01104 FINALREPORT: `A comprehensive draft final report will be prepared on completionofthe study and will be finalized following consultation with the Sponsor. The report will include the following: `Summary and Conclusion. Experimental Design and Method. Evaluation of Test Results. Appendices: Figures, Summary and Individual Tables Summarizing the Above Data, Protocol and Associated Amendments and Deviations, Study Director's GLP Compliance Statement, Reportsof Supporting Data (if appropriate) and QAU Statement. INSTITUTIONALANIMALCAREANDUSECOMMITTEESTATEMENT: "The procedures described in this protocol have been reviewed by the Testing Facility's Institutional Animal Care and Use Committee. All procedures described in this protocol that involve study animals will be conducted in a mannetro avoid or minimize discomfort, distress or pain to the animals. The Sponsor's signature below documents the fact that information concerning the necessity for conducting this study and the fact that this is not an unnecessarily duplicative study may be obtained from the Sponsor. No alternative (in vitro) procedures were available for meeting the stated purposesofthe study. REFERENCES: 1. Christian, M.S., Hoberman, A.M. and Smith, T.H.F. (1882). Dosage-range study of the teratogenic potential of suspensions of trinitrofluorenone (TNF) administeredorallyto New Zealand White rabbits. Toxicologist 2(1):40 (#143). 2. Christian, M.S. (1984). Reproductive toxicity and teratology evaluations of naltrexone (Proceedings of Naltrexone Symposium, New York Academy of Sciences, November 7, 1983), J. Clin. Psychiat. 45(9):7-10. 3. Feussner, E.L., Lightkep, G.E., Hennesy, R.A., Hoberman, A.M. and Christian, M.S. (1992). A decade of rabbit fertility data: Study of historical control animals. Teratology 46(4):349-365. 4. Institute of Laboratory Animal Resources (1996). Guideforthe Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. Staples, R.E. (1974). Detectionofvisceral alterations in mammalian fetuses. Teratology 9(3):A37-38. 004034 418-010:PAGE C-15 Protocol 4P1ag8e01105 6. Staples, RE. and Schnell, V.L. (1964). Refinement in rapid clearing technique in the KOH-alizarin red S method for fetal bone. Stain Technol, 3:61-63. 7. SImaplleawnstkait,ioEn.ss(t19e6l4l)e.n aFmarUbteemruestdheordReazttuem.maArkcrho.skPoatphiosl.chEexnp.NaPchhawremiaskovlo.n 247:367. 8. tShneedbeicnoomri,alGWdi.straibnudtioCno.chrStaant,isWti.cGal.M(e1t9h6o7d).s,V6atrhiaEdnicteiotne,stlfoowrahSotmaotgeeUnneiivteyrsoifty Press, Ames, pp. 240-241. 9. Sokal, RR. and Biometry, W.H. Rohl, F.J. (1969). Freeman and Co., SaBanrtFlreatnctitsecsot,opfp.ho3m7o0g-e3n7e1ityof variances. 10. Snedecor, G.W. and Cochran, W.G. (1967). Analysis of Variance. Statistical Methods, 6th Edition, lowa State University Press, Ames, pp. 258-275. 11. Dunnett, CW. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1129. 12. WS.oHka.l,FRrRe.emaanndanRdohClo,.,F.SJ.an(1F9r6a9n)c.isKcrou,skpapl.-W3a8l8l-i3s89T.est. Biometry, 13. Dunn, O.J. (1964). 8(3):241-252. Multiple comparisons using rank sums. Technometrics 14. Siegel, S. (1956). McGraw-Hill New Nonparametric Statisticsfor York, pp. 96-104. the Behavioral Sciences, 004035 418-010:PAGE C-16 Protocol 4P1a8g.e01106 PROTOCOLAPPROVAL: FOR THE TESTING FACILITY Ge je E/Dearlove, Ph.D., DABT Associate Director of Research RAsssadiatde GD.irYeocrtokr, oPfhRiels,eaDrAcBhT Study Director -- Loa So i Dena C. Lebo, V.M.D. Member, Institutional Animal Care and Use Committee VAT Date =A 9 Date 4 Lg 38 Date FOR THE SPONSOR Pan, TGoac_ Marvin T. Case, D.V.M., Ph.D. Study Monitor 004036 2 Ay wr Date 418-010:PAGE C-17 ATTACHMENT 1 SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE 004037 ATTACHMENT 1 418-010:PAGE C-18 ProtocPola4g1e8.10o1r02 STUDY SCHEMATIC RABBIT DEVELOPMENTAL TOXICITY STUDY* Timed Pregnant SDaorstaogre Rabbis DPGareyess0tuiomoefnd DPGaresyetsau7tmoieofnd DEonsdaogfe CSaeecstaorneianng. DPGreasetsyaut2mieoo0ndf PGDeraseytsa2ut9mi0oefnd wm Dosage Period a For additional details, see "Tests, Analyses and Measurements" section of the protocol. b. Fetal evaluations (external soft tissue and skeletal). 004038 ATTACHMENT 1 418-010:PAGE C-19 ProtocPoal g4e182.00f120 28AUG 98 30 AUG 96 - 16 SEP 98 13 SEP 98-17 SEP 98 21 SEP 98- 25 SEP 98 22DEC 98 SCHEDULE* Animals Arive - Acclimation Begins. pDroessaugmeePdergeisotdat(iDona)y.s 7through 20 of Toxicokinetic Sample Collection (Day 21 of presumed gestation). Caesarean-Sectioning Period (Day 29 of presumed gestation). Draft Final Report. a The study initiation date is the date the Study Director signs the protocol. 004039 418-010:PAGE C-20 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 004040 418-010:PAGE C-21 MDAATTEARISAHLEESTAFETY 33MH Center N-E+FOSE s5t5.144P-au1l0,00Minnesota 1-800-364-3577 or (612) 737-6501 (24 hours) CA{oLnpfLyorrriaigaghthtit,sonr1f9e9os8re,rvteMhdie.nnpeusrCopotopasyeinMgiofniannpgdr/ooparenrdldyoMwannulutofiaaldciitznuigrnignogfSHtChopimrspoadnuyc.ts * i1s) apthrleiloorwiendafgoprrremoeavmteiindotendiissthacotob:ptiaeidnedinfrfuolmlIMw,ithandno changes unless. 2) Geiistthreirbuttheed Wciotphy tnhoer ithnetenotriiogninaofl iesarnriensgolda oprrofoitthertwhiesreeon. TDRIAV'FDICSE-I1ON0NA:MFEL:U3OMRACDHBErMaInCdALSFluorochemical Alcohol 109NB-U0M2B1E1R./1U1.1P3.-C75. 00-51135-00495-2 95.0211.1575.7 00.51135-02145.3 9986-.00221111-.16168230--06 ISZSUFE.Do:o02J.a0n5u7a2r.y228, 1- 998 + - SDOUCPUEMRESNETD:ES:10N-o3v7e7m8b-e7r 05, 1997 0000--5511113355--1009454329--32 1. INGREDIENT C.A.S. NO. PERCENT PPEERRFFLLUUOORROOOHCETXAANNEESSUULLFFOONNAAMMIIDDOOAALLCCOOHHOOLL............ PERFLUOROHEPTANESULFONAMIDO ALCOHOL... 314649515--9093--23 68555-73-7 38.00.0 2.0 -- 79.00.0 - 6.0 PPEERRFFLLUUOORROOPBEUNTTAANNEESSUULLFFOONNAAMMIIDDOOALAGLOCHOOHL.O.L....... 364845459--8792-63 21..00 -= 63..00 2. PHYSICAL DATA BOILING POINT:....evveenvesne 621. m1m18CHo VAPOR PRESSURE:.........0cress <Ca1l0cMOHD20 C VAPORDENSITY:........veeesenes >Ca1.l0c Adr2s0t C. SEOVLAUPBOIRALTIITOYN IRNATWEA:T.E.R.:..............e.e..... SPECIFIC GRAVITY:.............. neg1l.i0g.BUDAGE1 ca. 1.7 Watersi PERCENT VOLATILE:.....vveenvees 0(%of melt) VIBSHCiOcSeeIeTuYn:aansnLniiL inssL inneenns NNIIDA MELTING POIN.T.LE. 0.000.00.00 NID 004041 Abbreviations: N/D . Not Determined N/A - Not Applicable CA - Approximately MSDS: FC-10 FLUORAD Brand Fluorochesical Alcohol January 29, 1998 418-010:PAGE C-22 PAGE 2 2. PHYSICAL DATA (continued) APPAEmAbReArNCwEaxAyNDsoOlDiOdR: 3. FIRE AND EXPLOSION HAZARD DATA FFLLAASMHMABPLOEINLTI:M.I.T.S..-..L.E.L.ic.e.e.e.e.e.n.e. >N/A148 C Setaflash AFULTAOMIMGANBILTEIOLNIMITTESMPE- RAUTEULR:E.:............. N/A N/A EXWTaItNeGrU,ISHCIarNbGonMEDdIiAo:xide, Dry chemical, Foam SPEWpCeoIasAriLtifvuFelIlREpprFreIosGtsHeuTcrIteNiGvoerPRpcOlrCoeEtsDhsUiuRnrEgeS,:dienmcalnuddibnrgeathhelimnegt,appsaerlaft-ucso,ntabiunnekde,r coat and pants, protective bcaonvdesrinagroufnodr aerxmpso,sedWaiasrteasandofletghse, hfeaadc.e mask, and UNUSSeUeALHazFIaRrEdoAuNsDDeEcXoPmLpOoSsIiOtNioHnAZAsReDcSt:ion for products of combustion. 4. REACTIVITY DATA STABILITY: Stable INCOMPATIBILITY - MATERIALS/CONDITIONS TO AVOID: Not applicable. HAZARDOUS POLYMERIZATION: Hazardous polymerization Will not occur. HACZaARrDbOoUnSMoDnEoCxOiMdPeOSIanTdIONCarPbRoOnDUCDTiSo:xide, Oxides of Nitrogen, Oxides of Sulfur, Hydrogen Fluoride, Toxic Vapors, Gases or Particulates. 5. ENVIRONMENTAL INFORMATION SPILL RESPONSE: Refer to other sections of this MSDS for physical and health hazards, respiratory information protection, regarding ventilation, anfyn4042 personal residue. pPrloatceectiivne a eqU.uSi.pseDnOtT.-appCroolvleedctcosnptialilneedr.material. Clean up Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately MSDS: FC-10 FLUORAD Brand Fluorochemical Alcohol January 29, 1998 5. ENVIRONMENTAL INFORMATION (continued) 418-010:PAGE C-23 PAGE 3 RECoIfOnMcMiaEnNecDroEamDtbeuDsIitSniPbOalSeApLme:artmeirtitaeld. haCzoamrdbouusstiwoanstperodiuncctisnerwaitlolrininclutdhee HpFr.esence Dispose of waste product in a facility permitted to accept chemical : waste. ENVLIaRbOoNrMaEtNoTrAyL DATA: tests showed (Pinephales promelas) - no No mbioordteaglriatdyataitonw.ate9r6-sHart.uraLtOiSoOn.FatNheoad Minnow sJetnagttihstiicnal3l0ydasyignFiaftihceaadntMiefnfneocwt eogng of FG-10 into % hatch, % fry study. suLravbivtaels,tswesihgohwte,d 2a0nd0 muscle fillets of channel catfish. fold bioconcentration REGVUoLlAaTtOiRlYe OINrFgOaRnMiAcTICOoNm:pounds: N/A. VOC Less H20 & Exempt Solvents: N/A. This product complies With the chemical registration requirements of TSCA, EINECS, CDSL, AICS and Korea. EPFCIRREA HHAAZZAARRDD: CLNAoSS:PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 6. SUGGESTED FIRST AID EYE CONTACT: Immediately flush eyes with large amounts of water. Get immediate medical attention. SKIIcNnomnCetOdaNimTaiAtnCeaTlt:yedwacslhothsiknign. wiItfh ssioganps/asnydmpltaormgseocacmuoru,ntscaOlfl water. Remove a physician. Wash contaminated clothing before reuse and dispose of contaminated snoes. INHALATION: If signs/synptoms occur, remove person to fresh air. If signs/symptons continue, call a physician. IFCiSamWlmAleLdLaiOaWtpEehDly:ysicaisandirIeMcMtEeDdIATbEyLYm.ediIcfalswaplelroswoendn,el.indNuecveervogmivietinagnything by mouth To an unconscious person. Abbrevi 004043 N/D . Not Determined N/A - Not Applicable CA - Approximately MSDS: FC-10 FLUORAD Brand Fluorochesical Alcohol January 29, 1998 7. PRECAUTIONARY INFORMATION 418-010:PAGE C-24 PAGE 4 EYEAvoPiRdOTEeCyTeIOcNo:ntact. Wear safety glasses with side shields. SKI`ANvoPiRdOTsEkCiTnIOcNo:ntact. material. A pair of Wgelaorvesapmpardoeprifartoem tglhoevefsollwohweninghanmdaltienrigalt(hsi)s are Tpeecrosmomneanldepdr:otecbtuitoynl irutbebmesr.as nUseecesonsearyortomorperevoefnttheskfionllcoowntiancgt: coveralls. REC6VOeMnMtEwiNilDtaEhtDiaopVnEpNrtTooIpLmrAiaTaiItnOetNa:ilnocaelmisesxihoanusst bveelnotwilraetcioomnm.endePdroveixdpeosusrueffilciimeitnst. T exhaust ventilation is not adequate, use appropriate respiratory protection. RESaNPvIIoORiSAdHTObaRrpYeparPtoRhvOienTdgECrToeIfsOpNia:riartboorrsnebamsateedrioanl.airSbeolrencetcoonnecenoftratthieonfoloflowing CFeosnptiarmaitnoarn,tsfaunldl-fianceaccsourpdpalniceed WaiitrhrOeSspHiArarteogru.lations: half-mask dust PREDVoENnToItONeatO,F AdrCiCnIkDENoTrALsmoIkNeGESWThIeOnN:using reas thoroughly With soap and water. thWiasshprhoadnudcst.aftWearshhaenxdploisnedg and before eating. RECSOtMoMrEeNDaEwDaySTfOrRoAmGEh:eat. Keep container closed when not in use. FIRNEonfA1NaDmaEaXbPlLeO.SION AVOIDANCE: OTHN`EooRntsmaPomRkiEinCnaAgtU:iToInOSNmAooRfkYintghIeNwFhOtiRolMbeAaTcIcuOosNi:nagnd/tohris smporkoeducatndcalneadresTuoltthein formation of the hazardous decomposition products mentioned in section 4 of 004044 this MSDS. HMIS HAZARD RATINGS: HPEEARLSTOHN:AL1 PROFTLEACMTMIAOBINL:ITXY:(S1ee RpErAeCcTaIuVtIiToYn:s, 0section 7.) EXPOSURE LIMITS INGREDIENT VALUE UNIT PERFLUOROCTANESULFONAMIDO PERFLUOROHEXANESULFONAMIDO ALCOHOL... ALCOHOL... ~~ ~ 0.1 0.1 MG/M3 MG/M3 PERALFCLOUHOORLO.HEvPeTeAvNvEvSvUnLeFnOnNaAnMsInDeOeenenenees 01 HG/M3 Abbreviations: NID - Not Determined N/A - Not Applicable TYPE AUTH SKIN THA THA aM 3M v THA am v CA - Approximately 418-010:PAGE C-25 MSDS: FC-10 FLUGRAD Brand Fluorochesical Alcohol Jaruary 29, 1998 PAGE 5 EXPOSURE LIMITS (continued) INGREDIENT VALUE UNIT TYPE AUTH SKIN PERFLUOROBUTANESULFONAMIDO ALCOHOL... ~~ 0.1 MG/M3 HA aM Y PEARLFCLOUHOORLOPEvNeTsANeEeSnUsLnFnOnNsAnMnInDOneeeenenns Ou1 MG/M3 THA 3M +theSKpIoNteNnOtTiAaTIlONc:ontLriisbtuetdionsubtostatnhceesoveirnadlilcateexdpowsiutrhe 'Y' under SKIN refer to by the cutaneous route including by direct mcuocnotuasctmWeimtbhranteheansdubsetyea,ncee.itheVrehibycleasirbcoanrnealtore,r more skin particularly, absorption. SCOaUMR:CE OFoEnXPROeScUoRmEmenLdIeMdITExDApToAs:ure Guidelines 8. HEALTH HAZARD DATA EYE CONTACT: No adverse health effects are expected from eye contact. SKIPNroCdOuNcTtACiTs: not expected to be irritating to the skin. May be absorbed through the skin and persist in the body for an extended time. INHALATION: May be absorbed by inhalation and persist in the body for an extended Time. IF SWALLOWED: Ingestion is not a likely route of exposure to this product. Illness may occur after a single swallowing of relatively large quantities of this material. MUTAGENICITY: Not mutagenic in in-vitro assays. REPSRuObDsUtCaTnIcVeEw/aOsEVnEoLtOPMtEeNrTaAtLogeTnOiXcINSi:n the rat at doses as high as 30 milligrams per kilogram per day via oral route. OTHTEhRisHEpAroLdTuHctHAiZsARnDotINFkOnRoMwAnTItOoN:contain any substances regulated under California Proposition 65. A Product Toxicity Summary Sheet is available. 004045 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately MSDS: FC-10 FLUGRAD Brand Fluorochesical Alcohol January 29, 1888 418-010:PAGE C-26 PAGE SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 0S, 197 ISSUE Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately TbehecionrfroecrtmataisonofinthethidsateMatiesrsiuaedl.Saf3eMtMyAKDEatSaNOSheWeAtRRA(NMTSIDESS), isEXbPeRlEiSeSvEeDdOtRo MIEMRPLCIHEADN,TABIINLCILTUDYINOGR, FBIUTTNENSOSTFOLRIMAITEPDARTTOI,CUALNAYR IPMUPRLPIOESDE WORARRCAOUNRTSYEOFOF WPnEeRtFnOeRrMANtChEe O3RH UpSrAoGdEuctOFisTRATDiEt. forUsearpiasrtirceuslpaornsipbulreposfeoradndetesrumiitnaibnlge for cusaenr'asffmeectthotdheofuseuseandorapapplpiliccaattiioonn.of Gaiv3eHnptrhoeducvta,riestoymeofoffawchtiocrhsatrehat uthneiquuesleyr weivtahliunatethetheus3erM'sprokdnuocwtledtgoedaentdercmoinnterowlh,ethiterisitesissenftitialforthaat particular purpose and suitable for user's method of use or application. D3uMeptroovitdheesreimnoftoermpatoisosnibiinliteyletchtartonieclecftorromniacs tarasnesrfveircematyo hitasvecursetsoumletresd. iFneprerersoernst,atoimoinsssiaosnstooritsaltceormaptlieotnesnesisn otrhisacciunrfaocrym.atioInn, ad3dMitmiaokne,s no 004046 iInnffoorrmmaattiioonn ionbtatihenedMSDfSromavaaildaabtlaebasdeiremcatylyNotfrobme a3Hs. current as the 418-010:PAGE C-27 ATTACHMENT 3 TEST ARTICLE PREPARATION PROCEDURE 004047 418-010:PAGE C-28 ATTACHMENT 3 Version: 418.P0r1o0toc(o0lA84U1G80918)0 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page 1013 Test Article: N-EtFOSE Vehicle: 2% Tween 80 in R.O. Deionized Water A. Purpose: The purposeofthis procedure is to provide a method for the preparation ofdosage suspensions of N-EtFOSE and the vehicle for oral administration to rabbits on Argus Study 418-010. B. General Information: 1. All suspension containers will be labeled and color coded. Each label will specify the protocol number, test article identification, Argus batch naunmdbsetro,racgoencceonntdriattiioonns,. dosage level, preparation date, expiration date 2a. _SuXs_penDsaiiolnys will be prepared: __ Weeky _For__daysofuse 2b. Vehicle will be prepared: __ Daily X_ Weekly _For__daysofuse 3. Suspensions wil be prepared at a final dosage volume of5mig. 4. safety: X_ Gloves, lab coat, goggles or safety glasses and faceshield X__C HDuasltf--MFiascte RReessppiirraattoorr -- Ful-Face RespiratoriPositive Pressure Hood Tyvek SuivApron 5. DosageYessuspensions aXd_justNeod (fCoarlFcruelaetbioanssebaansded%oPnur1i0ty0.%) __ FreeBase __ Pury 6. Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 004048 418-010:PAGE C-29 ATTACHMENT 3 Version: 418-0Pr1o0t,o(c0o9lA4U1G809180) TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page20f3 NOTE: The test article will be prepared as a serial dilution from the high dosage to the low dosage. Once the final volumes are achieved, stir bars are to be added to the containers; mixing should occur during sampling and/or administration. C. Preparationof Vehicle 1. Add the required amountofR.O. deionized water to an appropriately labeled container. Heat thewaterto 50C +5C, then add the required amountof Tween 80 and mix until uniform (See TEST ARTICLE CALCULATIONS). D. Test Article Suspension Preparation: 1. To prepare the 0.75 mg/mL, Group V suspension, add the required `amount of test article (See TEST ARTICLE CALCULATIONS) into an appropriately sized, labeled container. Add the required amount of vehicle andheatthe mixture to 80C +5C for approximately 30 minutes or until the TAS dissolves. 2. Once the test article has dissolved; spin over night while the suspension cools. (Be sure there is a visible vortex, this will achieve the desired emulsion.) 3. Toprepare the 0.5 mg/mL, Group IV suspension, remove the required `amount of stock suspension (Group V) (See TEST ARTICLE CALCULATIONS), QS ad with the vehicle and mix 4. To prepare the 0.2 mg/mL, Group Ill suspension, remove the required `amount of stock suspension (Group IV) (See TEST ARTICLE CALCULATIONS), QS ad with the vehicle and mix 5. To prepare the 0.02 mg/mL, Group ll suspension, remove the required amount of stock suspension (Group Ill (See TEST ARTICLE CALCULATIONS), QS ad with the vehicle and mix 004049 418010:PAGE C-30 ATTACHMENT 3 Version: 418Pr-ot0oc1ol0A4.U1G(8-00818)0 Page 3of3 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE 6. Tveohipcrleeptaoreanthaepp0rmopgr/imaLte,lyGrsiozuepd,| slaubsepleendsicoonn,taaidnedrt(hSeereeqTuEiSreTdAaRmToIuCntLEof CALCULATIONS) and mix. Witten by: _ VY sk Approved by: Date: _/1-AG-3 Clarification: _X_No _ Ys (See attached clarification form.) Initials/Date : _H 10-5-% 004050 OPRIMEDICA 418.010.PAGE C-31 ArgusSReseamrch WorsshaemLabo,ratories, PrA 19e6i4n4c Telosplhootnaax: ((231185)) 444433-.08678175 PROTOCOL 418-010 ORAL (STOMACH OTFUBNE-)EDTEFVOSEELOIPN MREANBBTIATLSTOXICITY STUDY SPONSOR'S STUDY NUMBER: 6316.8 Amendment 1 - 22 December 1998 1. Sponsor (page 1 ofthe protocol): The Sponsor is 3M Corporate Toxicology, rather than 3M Toxicology Services. Rea forsCho angn e: This change was made at the requestofthe Sponsor. fon 7$ ) J OMe Met. 22-05590 MN ttn = A`sGasborcgiea'tEe.DDieraercltoovreo,fPRhe.sDe.a,rDcAhBT Date ARsasyomcbdisaltle GD.irYeocrtokr,oPfhR.(Desoeasrpsh Study Director 22-P0.9y Date JP Bemagta- van 2 fr Date Pts Manin T. Case, D(VMa. sPh.D_. 250D0ate0 ChaanidrdUefsseonC,omImnsittittueteional Animal Care Study Monitor 004051 APPENDIX D DEVIATIONS FROM THE PROTOCOL AND THE STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 004052 418-010:PAGE D-1 DEVIATIONS FROM THE PROTOCOL AND STANDARD OPERATING PROCEDURES OF THE TESTING FACILITY 3 On 2 September 1998 (Day 11ofpresumed gestation) one rabbit in Group V (3.75 mg/kg/day dosage group, rabbit 8675) was inadvertently not dosed. This deviation does not adversely affect the outcomeofthe study because this represents only a small lossofdata and was a single event. All deviations are documented in therawdata. 2 / G. York, Ph)D., DABT `Associate Direct `and Study Director search 11-300 Date 904053 APPENDIX E TEMPERATURE AND RELATIVE HUMIDITY REPORTS AND DEVIATIONS REPORT 004054 418-010:PAGE E-1 ARGUS Temperature and Relative Humidity Report Location: Room 07 Protocol Number: 418-010 Range of Dates: 28-Aug-1998 14:00 to 21-Sep-1998 13:53 STpaercgieetsR:aRnAgeB:BIT TToottaall NNuummbbeerr ooff HDoauyrss:: Total Number of Data Points: T8e1mpFe0raTtu2rFe | Rola3t0i%vetHou7m0i%dity 572574 15275 4 578 578 Mean(50): MMeadxiiamnu:m: Minimum: NNuummbbeerr ooff PPooiinnttss HInigRhan(%g)e:(%): NumobfPeoinrts Low (): 674 @o4| 4 (30) 6ea7s.4 807s 663 531 s0% (010900)|| 576 (1000)0) oo | 0 Report Generated: 28-Sep-1998 at 13:48 COMMENTS: REVIEWED BY4: LlanN ate: _To-F( Cumulative by Location (v04.01.97) 004055 ARGUS 418-010:PAGE E-2 Temperature and Relative Humidity Report Location: Room 08 Protocol Number: 418-010 Range of Dates: 28-Aug-1998 14:00 to 25-Sep-1998 11:50 TSapregceietsR:anRgAeB:BIT TTToootttaaalll NNNuuummmooobbbfffeDDHeaaeoytrursarr:sP:oints: TSemFpe0r7a2ur7s | Re3la0ti%vetoHu7m0i%dity 6602.75 70 6629.975 70 Moan ( SD): Maximum: MMeidniiamnu:m: NumobfPoeinrts in Range (4): `NNuumbemroobffPPoeoiinnrttss HLiogwh(%()%:): 70 on| e25 (3g 693 700 6665.68 a63.10 on ano| es @99 0c 0.90) | o1o 0.10) Report Generated: 26-Sep-1998 at 13:56 COMMENTS: _ REVIEWED BY: 5 TL = DATE: _F-t5" (4 Cumulativeby Location (v04 01.97) 004056 418010.PAGE E-3 ARGUS Temperature and Relative Humidity Report Location: Room 09 Protocol Number: 418-010 Range of Dates: 28-Aug-1998 14:00 to 25-Sep-1998 11:50 - STpaercgieetsR:aRnAgBe:BIT Total Number of Days: TToottaall NNuummbbeerr ooff DHoautrasP:oints: Temperature 81F 1072F 29 66697.075 Relative Humidity 30%to 70% 29 66790.75 Mean ( 5D): MMaedxiiamnu:m: Minimum: NNuummbbeerrooffPPooiinnttss HinigRhan(g)e: (%): Number of Points Low (): 67 09 | 637 (42) 666957 67471 67 ws 6700 (1o0n00)|| 810 (68555) 0 oo | o Report Generated: 28-Sep-1998 at 13:57 COMMENTS: Reviewep av: `5 TL DATE: 4-214 Cumulativeby Location (04.01.97) 004057 ARGUS 418-010:PAGE E4 Relative Humidity Deviations Report Location: Room 09 Protocol Number: 418-010 Range of Dates: 28-Aug-1998 14:00 to 25-Sep-1998 11:50 HSpuemciideist:y RTaabrbgiett Range: 30% to 70% 29ADuagt-e1988 T0i9m00e R7H0.H 3300--AAuugg--11999988 0001:0000 770022HH 30-Aug-1998 22:00 708 H 033121-SAAueugpg-111999989888 001818:000000 777102.802MH 02Sep1998 2000 706H 011444-SSSeeeppp-111o99%99888 211013:000000 0700642HHH 1155SSeepp11999988 11960000 70031HH 1156SS00pp11999988 20180000 770170HH 1176.5S0e1p1999988 20100000 77135TMH 17.5ep1998 1400 722H 7SeDpat1e998 T2i30m0e R7H0.2M 1188--SSeepp--11999888 1101:0000 T73019HH 18-Sep-1998 21:00 70.1H 211909-.SS5eeeppp-111999899888 221120:000000 777001423HHM 20Sep1998 1300 704H 2215-eSepp1-0199988 1100:0000 772084HH H=Valus out ofRrHan.g=e -ReHliagthiv_e LHum=idViatlyu(%o)utofrange- Low Report Generated: 03-Dec-1998 at 10:02 These deviations did not adversely affect the outcomeor Interpretation of tshtudy. The following deviation(s) impacted on the outcome ofthe study as described: `Study Director L : A / : ee Date: _ 03D' 9 Deviations by Location (404.01.97) 004058 APPENDIX F PILOT REPORT 004059 418-010:PAGE F-1 FINAL PILOT REPORT StudyTitle Oral (Stomach Tube) Dosage-Range Developmental Toxicity Study of N-EtFOSE in Rabbits SPONSOR'S STUDY NUMBER: T-6316.8 Author Raymond G. York, Ph.D., DABT (Study Director) `StudyCompleted On 6 January 1999 (Final Pilot Report) PerformingLaboratory Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, Pennsylvania 19044-1297 LaboratoryProjectID Argus Research Laboratories, Inc., Protocol Number: 418-010P 004060 418-010:PAGE F-2 PROTOCOL 418-010P 418.010P.PAGE 2 ORAL (STOMACH TUBE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF N-EtFOSE IN RABBITS SPONSOR'S STUDY NUMBER: T-6316.8 SUBJECT ABSTRACT Purpose TAOFB CONL TENE TS PAGE 4 6 n Methods 6 In. Results 7 IV. Conclusion 12 Figure 1. Maternal Body Weights 13 Table 1. Clinical Observations - Summary 14 Table 2. Necropsy Observations - Summary 18 Table 3. Table 4. Table 5. Table 6. Table 7. wUteerreinFeoCuonndteDnetasd,anMdorLiitbtuenr dDaStaacrfiofrictehde oIrndAibvoirdutaeldRabbits that 20 Matemal Body Weights - Summary 23 Maternal Body Weight Changes - Summary 2 Maternal Absolute Feed Consumption Values (g/day) - Summary 28 Maternal Relative Feed Consumption Values (g/kg/day--) Summary 30 Table 8. (Caesarean-Sectioning Observations - Summary 32 004061 418-010.PAGE F-3 Table 9. 418010PPAGE 3 Litter Observations (Caesarean-Delivered Fetuses) - Summary 34 Table 10. Fetal Gross External Alterations - Summary 36 Table 11. Clinical Observations - Individual Data 38 Table 12. Necropsy Observations - Individual Data a4 Table 13. Maternal Body Weights - Individual Data 48 Table 14. Matemal Feed Consumption Values - Individual Data 55 Table 15. Caesarean-Sectioning Observations - Individual Data 62 Table 16. Litter Observations (Caesarean-Delivered Fetuses) - Individual Data 64 Table 17. Fetal Sex, Vital Status and Body Weight - Individual Data 66 ATTACHMENT 1 - PROTOCOL AND AMENDMENT 6 002062 418-010:PAGE F-4 418-010PPAGE 4 TITLE: TOORXAILC(ISTYTOSMTAUCDHYTOUFBEN)-EDtOFSOASEGEI-N RRAANBBGIETDSEVELOPMENTAL APRRGOUTOSCROELSNEUAMRBCEHRL:AB4O18R-A0T1O0RPIES, INC., SPONSOR'S STUDY NUMBER: T-6316.8 ABSTRACT tTohebepuusrpeodsien otfhethdisevsetluodpymweantsatlotporxiocviitdye(ienmfborrymoa-tfieotnalfotroxtihceitysealnecdtitoenroaftogdeonsiacges NpoetewntZieaal)lasntdudWyhoifteN-[EHtrFa:O(SNEZaWd)mSiPnFi]stperreesduomrealdlyprvieagnsatnotmfaecmhatluebreabtboits. rThainrdtyo-mfliyveapsrseisgnuemdedtopsreevgneanndtoNseagweZgeraoluapnsd, Wfhivietpee[rHgrrao:up(.NZWS)uSsPpFe]nsriaobbnistsofwere N-EtFOSE were (DGs 7 to 20) at administered dosagesof 0 orally once daily on days (Vehicle), 1, 5, 10, 25, 50 7 through 20 of gestation and 75 mg/kg/day. The Wveahtiecrl.e wTahse 2do%saTgweeevonlum8e0 winarsev5emrLs/ekogs,maodsjiusstmeedmdbarilaynoenptrhoecbeasssiesdodfetihoenized individual body weights. Checks for twice daily viability were made twice during the dosage period daily. (once Clinical observations were recorded prior to dosage administration and t`ohnecpeoasptpdroosxaimgaetpeelryioodn.e Bhooduryawfetiegrhdtossaangde faedemidncisotnrsautmipont)ioanndvaolnucees wdaeirlyeduring recorded daily during the dosage and postdosage periods. All surviving rabbits were `examined for the number sacrificed on DG 29 of presumed and distribution of corpora lutea, gestation and implantation sites and uterine contents. was performed. FeAtgursoessswneercerwopesiyghoefdthaentdhoerxaacimci,neabddfoomrignraolssanedxtpeerlnvailc viscera alterations and sex. dSoesveargee mgartoeurpnsa;lthbeordeywweerieghntolsousrsviovcicnugrrreadbbiintsthien t1h0,es2e5,gr5o0upas.ndA7b5ormtgi/okngs/day occurred in 5 and 10 mg/kg/day dosage groups. Caesarean-section 004063 418-010:PAGE F-5 418.010P.PAGE 5 observations revealed increasing late resorptions and reduced fetal body weights at5 mg/kg/day. Increases in early resorptions were found at 1 mg/kg/day; however, there was no decrease in mean litter size. Based on the results of this dosage-range finding study, dosages of 0, 0.1, 1.0, 2.5 and 3.75 mg/kg/day were recommended for the full developmental toxicity study of N-EtFOSE in rabbits. 004064 418-010.PAGE F-6 IL Purpose: 418.010PPAGE 6 The purpose of this study was to provide information for the selection of dosages to be used in the developmental toxicity (embryo-fetal toxicity and teratogenic NpoetewntZieaal)lasntdudWyhoifteN-[HErtaF:O(SNEZWa)dSmPiFn]isptreeresdumoreadllpyrveigansatntomfaecmhalteubreabbtiots. Il. Methods" The test article, N-EtFOSE (lot FM-3932), an off-white wax, was received on 2(l0otM3aHy05)1,99a8,claenadr vsitsocroedusatlirquoiod,mwtaesmpreercaetiuvreed. oTnh2e2veMhaiycle1,9928%, aTnwdeestnore8d0at room temperature. The vehicle diluent, reverse osmosis membrane processed tdheeioTneiszteidngwaFtaecirli(tRy.Oa.ndDeisiomnaiiznetdaiWnaetdera)t risoaovmaitleambpleerfartuormea. continuous source at Thirty-five presumed pregnant New Zealand White [Hra:(NZW)SPF] rabbits were VrIaD]n.domSluyspaesnssiigonnesd toof sNe-vEetnFdOoSsEawgeergeraodumpisni[sfitveerpeedrogrraloluyp((sGtroomuapcsh t| utbher)ouognhce daily to these timed-pregnant rabbits on days 7 through 20 of gestation (DGs 7 wtoas20)2%at Tdwoesaegnes 8o0f 0in (rVeevheicrlsee),os1m, o5s,i1s0,m2e5m,b5r0anaendpr7o5cmegs/skegd/ddaeyi.oniTzheed vWeahtiecrle (R.O. Deionized Water). The dosage volume was 5 mLlkg, adjusted daily on the basis of the individual body weights recorded immediately before intubation of the test article. Checks for viability were made twice daily. Clinical observations were recorded twice daily during the dosage period (once prior to dosage administration and tohnecepoasptpdroosxaigmaetpeelryioodn.e hour Body after dosage weights and administration) an feed consumption d once values daily were during recorded daily during the dosage and postdosage periods. All surviving rabbits were sacrificed on DG 29 of presumed gestation and `uetexraimnienceodnftoernttsh.e nAumgrboesrs annedcrdoipstsryiobfuttihoneotfhocroarcpiocr,aalbudtoeam,iniamlplaanntdatpieolnviscivteisscaenrda was performed. Fetuses were weighed and examined for gross external alterations and sex. a. Dareetapirloevdiddeesdcriinptthieonaspopfraolplripartoecseedcutrieosnsusofedthiins trheepocrotnadnudctinofthtehiaststtaucdhyed pOrpoetroactoilnganPdroacmeednudrmeesntofs.theDTeevsitaitnigonFsacfirloitmy tahree ParvoatiolcaoblleainndtShetarnadwarddata. v04065 418-010:PAGE F-7 Wl. Results: 418-010PPAGE 7 A d bo Clini ecroj bservations (Summaries - : Individual Data - 11,12an3d) A. Mortality One, two and one rabbits were found dead in the 1, 50 and 75 mg/kg/day dosage groups, respectively. The remaining rabbits in the 50 and 75 mg/kg/day dosage groups and all five rabbits in the 25 mg/kg/day dosage group were moribund sacrificed. 1 malkgld Doe 8206 was found dead on DG 16, within an hour following dosage administration, after 10 dosages of the test article. The rabbit had no adverse clinical signs during the dosing period. The rabbit gained weight during the dosage period, but feed consumption values were reduced on DGs 12 through 15. The uterus contained eight fetuses, which appeared normal for their developmental ages. Viability of the fetuses could not be determined because of the death of the dam. All tissues appeared normal at necropsy. 0 malka/day Dosage Grou Doe 8226 was found dead on DG 14, within three hours following dosage administration, after nine dosages of the test article. The rabbit had soft or liquid feces and scant or no feces between DGs 9 and 14 and lost weight and had reduced feed consumption during the dosage period. The uterus contained six fetuses and one early resorption. Viability of the fetuses could not be. determined because of the death of the dam. Necropsy revealed the stomach mucosa contained black viscous material; all other tissues appeared normal. Doe 8229 was found dead before dosage administration on DG 12, after five dosages of the test article. The rabbit had soft or liquid feces or scant feces on DGs 9 to 12 and lost weight and had reduced feed consumption during the dosage period. The uterus contained 13 fetuses. Viability of the fetuses could not be determined because of the deathof the dam. Necropsy revealed greenish-gray areas on all lobesofthe liver and 30 mL of fluid in the thoracic cavity. All other tissues appeared normal for the slight degree of autolysis. 004066 418-010:PAGE F-8 418.010PPAGE 8 1yDosageGroup 35 found dead before dosage administration on DG15, after nine he test article. The rabbit had soft or liquid feces or scant feces on decreased motor activity on DGs 13 and 14 and excess salivation he rabbit lost weight and had reduced feed consumption during the 1d. The uterus contained nine fetuses and one early resorption e fetuses could not be determined because of the death of the dam. nal cavity contained approximately 18 mL of thin, brown fluid and mucosa contained a viscous, brown material. All other tissues mal. `undSacrificed the 25, 50 and 75 mg/kg/day dosage groups were moribund DG 15. Clinical observations in these rabbits included decreased +, impaired righting reflex, cold to touch, emaciation, red substance an, perioral substance, excess salivation, ungroomed coat, and es (scant, soft or liquid, absent). ed and Sacrificed in the 5 mg/kgiday dosages group and all five rabbits in the 7 dosages group aborted and were sacrificed. Dosage Group orted on DG 26. The rabbit had scant feces, soft or liquid feces or Gs 10 through 25. There was a red substance in the cage pan on rabbit lost weight and had reduced feed consumption during the d. There were one aborted late resorption and five late resorptions issues appeared normal at necropsy. orted on DG 24. The rabbit had scant feces or soft or liquid feces 23. There was a red substance in the cage pan on DG 20. The sight and had reduced feed consumption during the dosage period. 7 aborted late resorption. There three live fetuses and eight late 2 utero. All tissues appeared normal at necropsy. orted on DG 26. The rabbit had scant feces or no feces on DGs 17 was a red substance in the cage pan on DG 26. The rabbit lost the dosage period and had reduced feed consumption on DGs 10 004067 418-010:PAGE F-9 418010PPAGE 10.26. There were four implantations and three aborted dead fetuses. The fourth conceptus was presumed cannibalized. All tissues appeared normal at necropsy. 10 malkal e Doe 8216 aborted on DG 26. The rabbit had scant feces, soft or liquid feces or no feces on DGs 10 through 25 and dark orange urine on DG 24. There was a red substance in the cage pan on DG 26. The rabbit lost weight and had reduced feed consumption during the dosage period. There were eight aborted late resorptions. The liver had pale, tan areas on all lobes, ranging in size from 1.5cm x 1.0 cm necropsy. to 4.0 cm x 1.6 cm. All other tissues appeared normal at Doe 8217 aborted DG 22. The rabbit had scant feces or no feces on DGs 9 1021 and a red substance in the cage pan on DG 21. The rabbit lost weight and had reduced feed consumption during the dosage period. There were two aborted late resorptions and four early and four late resorption in utero. All tissues appeared normal at necropsy. Doe 8218 aborted no feces on DGs 9 on to DG 23. 24. The The rabbit rabbit lost had scant feces, soft or liquid weight and had reduced feed feces or consumption during the dosage period. There were eight aborted late resorptions and two late resorptions in utero. All tissues appeared normal at necropsy. Doe 8219 aborted on DG 22. The rabbit had scant feces or no feces on DGs 9 through 21 and a red substance in the cage pan and dark orange urine on DG 21. The rabbitlostweight and had reduced feed consumption during the dosage period. There were eight aborted early resorptions, four aborted late resorptions and one early resorption in utero. Necropsy revealed that all lobes of the liver were pale. All other tissues appeared normal. Doe 8220 aborted on DG 23. The rabbit had scant feces or soft or liquid feces. on DGs 9 to 22. The rabbit lost weight and had reduced feed consumption during the dosage period. There were three aborted late resorptions and seven live fetuses in utero. All tissues appeared normal at necropsy. A4. ClinicalObservations Clinical observations considered to be test article related included scant feces, soft or liquid feces or no feces in 5, 10, 25, 50 and/or 75 mg/kg/day dosage 004068 418-010:PAGE F-10 418.010P.PAGE 10 groups, and dark urine in the 10 mg/kg/day dosage group. Ungroomed coat, decreased motor activity, impaired righting reflex, cold to touch, lost righting trhefaltexw,eremeafcioautnidond,eapderioorramlorsiubbusntdanscaecrainfidceedxcinetshses2a5l,iva5t0ioanndo/cocrur7r5edmgi/n krga/bbdiatys dosage groups. Red substance in cage pan was correlated with abortion and/or moribund condition in the 5, 10 and 75 mg/kg/day dosage groups. Ablelcaotuhseer: cli1n)itcahleoibnsceirdveantcieosnswewreerenoctondsoisdaegree-ddeupnernedlaetnetd; taontdh/eorte2s)t tahrteiycloeccurred tinheonlliymbosneanrda/t.orTbhaecks.e observations included abrasion and localized alopecia on AS. Necropsy pNreecvriooupsslyyodbessecrrviabteido.nsOfnreom5 amllg/ukngs/cdhaeydudloesdadgeeagtrhosuopr dsoaceri(f7i3c8e5s)hahvaed bpaeleen iver lobes. The only other necropsy observations were confirmation of clinical observations of localized alopecia. B. B Weights htCh: igure 1; `Summaries - Tables 4 and 5; Individual Data - Table 13) Severe maternal body weight losses occurred in the 10, 25, 50 and 75 mg/kg/day dosage groups; there were no surviving rabbits in these groups after DGs 26, 15, 15 and 15, respectively. Maternal body weight losses occurred for the 5 mglkgiday dosage group for the e(nDtGirse 2t1hetodo2s9)agaendpetrhieoden(tciarlecguelsatteadtiaosn DpeGrsiod7 t(oD2G1s),0tthoe2p9o)stdosage period Maternal body weights and body weight gains for the 1 mg/kg/day dosage group were generally comparable periods (DGs 7 to 21). to control values during the dosage and postdosage C. Absolute (g/day) and Relative (g/kg/day) Feed Consumption Values (Summaries - Tables 6 and 7; Individual Data - Table 14) Absolute and relative feed consumption values were severely reduced in the 10, 25, 50 and 75 mg/kg/day dosage groups; there were no surviving rabbits in these groups after DGs 26, 15, 15 and 15, respectively. 004069 418-010:PAGE F-11 418.010PPAGE 11 Absolute and relative feed consumption values for the 5 mg/kg/day dosage group were severely reduced, compared to the control group, during the dosage and postdosage periods. Absolute and relative feed consumption values for the 1 mg/kg/day dosage group were generally comparable to control values over each interval tabulated. D. Caesarean-Sectioni ns(Summaries -Tabl through 10; Individual Data - Tables 15 through 17) Caesarean-sectioning observations were based on 5, 4 and 2 pregnant rabbits with live litters in the 0 (Vehicle), 1 and 5 mg/kg/day dosage groups. There were no surviving does in the 10, 25, 50 or 75 mg/kg/day dosage groups. Arenlaitinvceretaosteheinceoanrtlryolrgesrooruppt.ioTnhsioscocbusrerrevdatiniotnhewa1 smgc/oknsgi/ddearyeddoasnagefefegcrtouopf,the test article because it was outside the historical control range for the Testing Facility. An increase in late resorption (and a concomitant decrease in litter size) and relative to reduced fetal body the control group. wTehiegshetsoboscecruvrarteidonats twheer5e magls/okgc/odnasyiddeorseadgea,n effect of the test article because they were dosage-dependent and occurred at maternally toxic dosages (decreased body weight and feed consumption values) No dams had all resorbed conceptuses and there were no dead fetuses. Totals of 36, 34 and 13 live fetuses were evaluated for external gross alterations in the three respective dosage groups with litters. One fetus (8207-7) in the 1 mg/kgiday dosage group had a distended abdomen. One fetus (7385-1) in the 5 mg/kglday dosage group had open eyelids and protruding tongue and another in the same litter (7385-4) had downward flexed forepaws, cleft snout, absent incisors and a large right eye. This litter also contained three late resorptions and three normal fetuses. No fetal gross extemal atterations were observed in the vehicle control group fetuses. 004070 418-010:PAGE F-12 IV. Conclusion; "reot0mpAGE 12 Based on the results of this study, dosagesof 0 (Vehicle), 0.1, 1.0, 2.5 and 3.75 mg/kg/day study in rabbits of N-EtFOSE were (418-010). The 0.1 recommended for the mg/kg/day dosage is developmentaltoxicity expected to be a no-observable-effect-level (NOEL) for both maternal and embryo-fetal toxicity, and the 3.75 mg/kg/day dosage is expected to produce matemal toxicity (decreased matemal body weight and feed consumption values) and may produce minimal developmental toxicity (decreased fetal body weight and delayed ossification). LoMn rng Alan M. Hoberman, Ph.D., DABT Director qf Research Date Ra) ind G. York, Associate Director of and Study Director A. . DABT Research 06-Fw-99 Date 004071 MATERNAL BODY WEIGHT FIGUR1E w" ee en " I at axa gefe SE 0 N i . . NN NST " = PPE A NF | free de hh hdd kh dha hs i 3 FII 3 | ee 8T2 soorsmonLr aguro Fm eces mene--m-- s Tommie we we wr ome wLwose uwwlee anwea wwaa vs wn ponaar W hdAd tAitt BrEE Ho nie RETCEILI AINE I 1) to cnsemation for cutite chat ied ox stores ,i 3E ES 3: | 2gJR mwmewmn --_-- 2 om > #0sunsaRcE 1xCAGE ae uwee wwsn a Vweliuwee owws oe we wa -- FoEsR ETL srmpa Atti wo sev ren RR GhoupGi DAYS 3 THROUGH 39 00 PRESS GESTATION g 33 id THGTOCOL 418-9167, GAAL (STONACH TUBE) COSGE. ANGE DEVELOPMENTAL TOIICITY STUDY OF N-ELFOSE IN SABNITS (SPONSOR'S STUDY. poo Tk ye oui ows oui 2 PERE Ak POSSIBLE RCISERCE + (DAYS x AMRIT NARS OF RAMNIp TS. r fr > wtpe % es cme. . iE 3 32 Sg nou 32E FoESE=EESTE EE e ikia oY .8 E35 | | Wome Bo ME 3 an, . :: :. .: :: SRE EI IA fy SIRI SE mn Ss Tre i:Eg 81 J 82$ wTmuiimMmka .es x . . oo. , .. :: . 8 um i CR LR EE : IRAIALS etepe sosrestions nt ae 1h 07SENITILSOTTITEREI B TrS t :f giB3 8I 2.g wEmmRaT ttt tt 4 ten wma taT2 g&3 EB wegwgmmamgr cm tit tee eee on Ne aR J I RAs $5 [I g wu 28 J LY wan meee acne 5 6 a soem soe som oe ee opm tw tem we memes 3s m3 3 3 C SH aLa R EL vee wm eee 3 3 0 se ees . .g [HE] EI] $ on gmmenemen a a ac ae eae ,i 3$i B < or 8~ wens 0. json mses aay : Cweoresnen : seermn : nmsaets DttioomnoamminnoqJEL oWHummobuenmd F. f em rn i3 . mu g $ ul wa oan wean dain wie vane abe -- imioe enna. wero pnrteo asem EpaEnL aha TbEaEn DEE : 5 02 ii gggs Iu 2 on an" t rmi:ia n maa t:en aetoeee .nesses. as:ec :resem as: e DR min 5 Exciuten valoes for Tatbite that dled or vere moribud sucrificed. 3 . g 3 i& g8E 32 6 min me 2B moinne rmaenms.e LL amin mien eErmeinweorsmen nn us me aamosmenm sone Tw emeee aise A PI a t ERRER RR ra .t :3 58 g omen ma: LI> D DE: D I:n E A TE r 2 tn :2 Eg 3 23  mun man 3: over a oso. ime waza main snl os a nes Hi sass b EERE mr rer ,& ios :5s 33 53 a' : 32 TE. . moo wwss a : : . : iE at .8 3 i8 Bg Z2 gE mnie mmm In pm mm ew 3 Pox g8  w anl we oars13 36 ens. 0. ou one Wen . -- 3 ntes cn . 3 I8 Boz Eo mSmENeEEL. mee wl. wl. wel. . B rmsm nt. as aa wee on em wfvs--co--r -- . i : Tn ---------- vo, sr -- se Ei eed a 3 hr 38 pani FOgE 2 iB 8  iene mw Sheas San nan 5S$ F mmrEEL > DN reise 2 Wo 2 .-- . .if8 [I io acme wan 2S war BEM mss oT os. wana " awn an ease . ws ea wes ae : nas ca i ! rtp e p 20 of gestation. :& E3 33 38 3n & . 2 ,| gg i s REE di oo EOE om am Gomme nm ue Hh wa EE 13 yi .E bE Pgose i8 DO, Ae 2s. 8 . [I f8 3s g4 ] $ Hop SEmERS ,i il3 id  we BLA EpmELLE ga22 m EB[ipsrYoelhnt) LEAE Ta EBoRnREREo,ALEoL SnaSESnRIiAoSbnOPASNENT @ womb ora .2 E03 FH E [I Z 3 we E3S] E ERRRs SeSA ; :[I s wmm EfEomRn EEEEEERR 5 EgRnD EEEm m m < a Bathe BERET if [I BI 2g$ - my EF= BINelE Et E EasLn otE o icoria vwire T en 22 we IEOiN E BERE ET O 2 E5f3 Pol g sy EERE 8 mE ERs. gE mo Sr LI BUNT croR n I wore ,f i3 G iI i8 B@oOoEERR iPb6 EMuEmmiImmmaem Eo mown vw mmm ETL tos st Tate 1 so te retin tis sec Eo: 38 rok s i erarp H22a wh wn +n pTR eLmendT mie meme rb pene i wn mn mcmee wo ig T ge R -- :$B8 [I : Epa = AEs iba APeaARS Ro. Pon et tic bmonEr & cy A ii & S. nna Br mree reais sm 3828 smn ee Sm Iem HEmeaeLEen FH FB  E moR d OfB OHRRWEoRoRo EEoYo .f L I 33 : BB S o E B m EEHWo IT WofpIooVwoEmfr i BoooOhwn @ oad TES pettuscern (7 PO roU xAvenR icrs) aem een eB m em e am OdIR oomomoooo o ,f [gIE 3 8 0I% PERRO goood jE EOE pas OE ETHER ar 8 frmi a. Es i: LI  Tey S Eero Jal in i [a 3a aa 538 aeonsoushy36oraRsATIOn do am NSD NY LE aorE cemaion TN fd : & gE 35 82 | so C iWp e EEEEmBMEmIsmRiiziasaes I IN JI - - - Rr ALL ERCHE CE, Sg, Jere,Tepet nouriorcevneaaene4 (1 i. 3 2 gE # g8 Q HR BH ma EERISIRTRSLENE Ft | Bim i i waseSei m os src sn esr ms : : I2 Eos 5 2S oB h m HY mS EEEEmmRR EImNEmRS nEaIiuRA EiELauI . Laia. ' et &Ba fi ERS on or ma vaisSa ees rer rioanst - n E En n E SuEEme en +S I Eh nt a en 2 2 gE 335 FE g oP HOE $ BE OB RR BOE RW NR @ RR oS FEE RRR Bom EEE Re = Bo o@mo% OREOEOEY RRR - oe .f FE g8 3o BuIn EEAESEIEERERBR RB O% OZ A Ea r, : .8 E is g 4 oi ERLE EE wy BRR 3 pd 5 3 3 Raorreoouony 30or aeration * gaRm oPE e (Vhs hci Tho MiRAcEs) or - A . i o ERE B BSOBH EE ERR a eEm Tder E home e s neem ee ' .f :3 g4 g ILEEEER swe g EHR i.HI gf ig e EPH Pb heddeaiesbet a In- oh BaIy Em itR, N a n2 hLanha Wn Rs a ToDDAD Ou BAT 14 GFGESTATION wn BeHae - or or - - . i$gE g23 :8 $guEr mm mEmm ii s 8 wi S ageI niinSicoiubra1i4orcceeimarsiey 0 TART LS hE Pn ERIE - or , Boi 8 oe erie n " BOE PREP EE EEE EEE i BEEREEERE Riewhe curevrsrv Eos TB Bg I 331 Moniman SACRIFICED onORY 15 OFGRATION ia 1 LI . Bobb EEE Ew 1-3 `o0saGEGRoup 11 3 marae - 8e Bue ooa b GbeEn Ea EEam pno PU o4 rs a BO EEImisam oe Torre a 3 o oteeevior CL, EB ig l3 iid | | Wm EEEmmEsmieame | BE. JEumn a J CC) 8 BoB Eom EERE - ' ow -- in$ 2 25 28 8" sien LAMOB , 2 oh ah oS nome mn, EE 0Couron reafomat * "Fira shor Seton sme pocorn a 1 OTES sesoTrceomnix ,8 ii oom g TTR 8[o J .o oi o ion mm meemmteetm r--se -- Carona reonnss *Feia.sorBaswen seach 1haw Tienor conn E, g3f i3 B gm m msweernwommvconwesovceommer B N 3 RER SET wsWao i H] E]& FH 82 418-010:PAGE F-69 418.010P:PAGE 69 ATTACHMENT 1 PROTOCOL AND AMENDMENT 004128 418-010:PAGE F-70 418.010PPAGE 70 nesearcs, ArgusResearch Laboratories, ic. A [TL pr Ho9rf0s5hSaihme,ePrhaynnDesriyviev,haBnuiiavld1i9ne0g4A4 ---------------------------------------- PROTOCOL 418-010P `SPONSOR'S STUDY NUMBER: T-6316.8 STUDY TITLE: PURPOSE: TESTINGFACILITY: STUDY DIRECTOR: SPONSOR: STUDYMONITOR: ALTERNATIVE STUDYMONITOR: Oral (Stomach Tube) Dosage-Range Developmental Toxicity Study of N-EtFOSE in Rabbits The purposeofthis study is to provide information for the selection of dosages to be used in the developmental toxicity (embryo-fetal toxicity and teratogenic potential) study of N-EtFOSE administered orally via stomach tube to fNeemwalZeearalbabnidt.White [Hra:(NZW)SPF) presumed pregnant Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A THeolresphhaomn,e:Pen(n2s1y5l)v4an4i3a-671190044-1297 Telefax: (215) 443-8587 ARsasyomcioantde DG.irYeoctrokr, oPfh.RDe.s,eDarAcBhT 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, Minnesota 55144-1000 Marvin T. Case, DV.M., Ph.D. Telephone: (612) 733.5180 Telefax: (612) 733-1773 TAenlderpehwonMe.: Se(ac1a2t), 5P7h6.D-.3161 Telefax: (612) 733-1773 004129 418-010:PAGE F-71 418010P-PAGE 71 Protocol 41P8a.g0e1?0 REGULATORYCITATIONS: HUa.Sm.onFiosoadtiaonnd; DGruuigdeAldimnieniosntrdaettieocnti(o1n9o8f4)t.oxiIcnitteyrntaotrioenparlodCuocntfieonrefnorcemeodnicinal products. Federal Register, September22, 1984, Vol. 59, No. 183. U.S. Food and Drug Administration. Good Laboratory Practice Regulations; Final Rule. 21 CFR Part 58. Japanese Ministry of Health and Welfare (1997). GoodLaboratoryPractice Standard forSafety Studies on Drugs, MHWOrdinance Number 21, March 26, 1997. European Economic Community (1988). Councildecision on 28 July 1989 on the acceptance by the European Economic Communityof an OECD decision/recomnmendation on compliance with principlesofgoodlaboratorypractice. Official Journal of the European Communities: Legislation. 32 (No. L 315; 28 October): 1-17. REGULATORY COMPLIANCE: rTehgiuslsattuidonyswicliltebdeacboonvdeucitntehdatinthtehe spiorftihte Good Laboratory Testing Facility personnel will Practice (GLP) adhere to the Standard Operating Procedures for laboratory operations and data collection. The Testing Facility Quality Assurance Unit (QAU) will not audit the protocol, theraw data, the reports or the critical phasesofthe study. All changes or revisionsofthis protocol shall be documented, signed by the Study Director and the Sponsor, dated and maintained with the protocol. STUDYSCHEDULE: See ATTACHMENT 1 to the protocol. 004130 418-010:PAGE F-72 418-010PPAGE 72 Protocol 418.010P Page3 TA ESTRTIANC D VL EHICE LE: Identification: TestArticle: NaPhmyesi:cal Description: ~~ `LSopte/cBifaitcchGrNavuimtby:er: Purty: Expiration Date: WNa-ExtyFsOolSiEd.. -F1M7-.3929 (30035, 30037, 30039). 99.1%. May 2000, wIintfhortmhaetiSopnonosnort.he identity, composition, strength and purityofthe test article is on file Vehicle: 2% Tween 80 in Reverse Osmosis Membrane Processed Deionized Water (R.O. Deionized Water). the raw data. Supplier and lot identification of Tween 80 to be documented in tNoeibteheprrtehseenStpionntshoervneohirctlheetShattudwyouDlidreicnttoerrfiesraewwairthe tohfearneysuplottsenotfitahliscosnttuadym.inTahnetrsefloikreel,y no analyses other than those mentioned in this protocol will be conducted. Safety Precautions: fGolromvuelsa,timoanskpr,epaaprpartoiporniaatnedeyademipnriosttercattiioonn.anTdhae uMnaitfeorrima/llaSbafceotayt DaarteatoShbeeewt o(rMSdDuSr)inigs attached to the protocol (ATTACHMENT 2). Storage: VBeuhlikcTleesCtoAmrptiocnlee:nts: PPrreeppaarreedd VFeohrimcullea:tions: RRoooomm tteemmppeerraattuurree.. FRrooozmente(m-p20erCa)t.ure. JAlullitaenstGaurltbiicnlseksi,hMiapnamgeetrnototfhseFoTremsutliantgioFnasc,ilattythsehopurledviboeusaldydcrietsedseadddtrotehsse aatntdention of telephone number. cSahritpomnesnsthsouslhdoubled lianbcelluedde aipnpfroorpmraitaitoenlyc.onTcheerirnecgipsiteonrtasgheocuolnddibteionnostiafinedd sinhiapdpviangnce of shipment. 004131 418.010:PAGE F-73 41010PPAGE 73 Protocol 41P8a.g0e1s0 FORMULATION: FreqoufPreepnaractioyn: Formulations (suspensions) will be prepared daily at the Testing Facilty. Vehicle willbe prepared weekly at the Testing Facility. Detailed preparation procedures are attached to this protocol (ATTACHMENT 3). `AdjusftormPeurnitty The test article will be considered 100% pure for the purpose of dosage calculations. TestingFacility Reserve Samples: The Sponsor will reserve a sample (1 g) of each lot of thebulk test article used during the course of this study. The Testing Facility wil reserve a sample (5 mL) of each lot of the vehicle components used during the courseofthis study. Samples will be stored under the previously cited conditions. ANALYSES: Samples additional to those described below maybetakenif deemed necessary during the courseof the study. Bulk TestArticle Sampling: No analyses of the bulk test article will be conducted during the courseofthis study. Information on the stabilityofthe bulk test article is on file with the Sponsor. Analysesof PreparedFormulations: ActontdhuecrteeqduedsutroifngththeeScpoounrssoer,ofntoheasntauldysye.sHoofwperveepra,rerdectoerstdsarwtiilcllbeefomramiunltaatiinonesd wtioll be document how the test article formulations were prepared. DISPOSITION: Prepared formulations willbe discarded at the Testing Facity. All remaining bulk test article will be retuned to the Study Monitor at the previously cited address. 004132 418-010:PAGE F-74 418.010PPAGE 74 Protocal 41P8a.g0e1s0 TESTSYSTEM: `SpecianedRsea/soSnftorrSelaecitionn: TbehceauNseew: Z1e)alitainsdoWnheintoen-[rHordae:n(tNmZWa)mSmPaFl]iaranbbsiptewcaiesssaeclcecetpetdedasantdhewiTdeesltySuyssetdem ttohxriocuigthyohuertatthoegeinnidcuistty)ry2f)orthniosnsctirianincaolfsrtaubdbiiteshaofsdbeeveenlodpemmeonntsatlratotxeidcittyo (beembsreynos-ifteitvealto daenvde4l)optmheentteaslt atrotxiicnlse;i3s)phhaisrtmoarcicoallogdiactaalalnydacetxipveeriinetnhceesepxeicstiaetstahnedTsetsrtaiinn.g Facil"; NumabndeSerx: Population evaluated: 35 timed-pregnant female rabbits (5 per dosage group). BW odyeiganhdAt ge: `The individual body weights of the female rabbits will range from 2.5 kg to 5.5 kg; the rabbits will be approximately five to seven months of ageatthetimeofstudy assignment. Actual body weights recorded at receipt and at study assignment will be documented in the raw data. Source: Covance Research Products, Inc. `Swampbridge Road, Box 7200 Denver, Pennsylvania 17517 The rabbits will be shipped in fitered cartons by truck from Covance Research Products, Inc., Denver, Pennsylvania, to the Testing Facilty. Identification: Rabbits are permanently identified using Monelsel.piercing ear tags (Gey Band and Tag Co. Inc., No. MSPT 20103). Female rabbits are given unique permanent identification numbers when assigned to the studyonthe basis of day 0 of presumed gestation body weights. ANIMAL HUSBANDRY. All cage sizes are in compliance with the Guideforthe Care and UseofLaboratory Animals. 004133 418-010PAGE F-75 418010PPAGE 75 Protocol 41P8a.g0e1s0 Housing: `The rabbits wil be individually housed in units of six to eight stainless steel cages. No nesting materials will be supplied because the female rabbits will be sacrificed before parturition is expected RTooe m Aimr, peraat ndu Humr idie ty: `The animal room is independently supplied with at least ten changes perhourof 100% fresh air that has been passed through 99.97% HEPA fiers. Room temperature will be: mwialilnatlasionbeedamton6i1toFre(d16coCn)sttaont7l2yaFn(d2m2aiCn)taanindemdoantit3o0r%edtoco7ns0t%antly. Room humidity Light `An automatically controlled 12-hour light:12-hour dark fluorescent light cycle will be: maintained. Each dark period will begin at 1900 hours EST. Diet: Approximately 150g of Certified Rabbit Chow #5322 (PMI Nutrition International) wil be available to each rabbit each day until the first day of dosage, at which time approximately 180 g of the same certified feed will be offered to each rabbit each day. The certified feed will be available from individual, stainless steel, "J-type" feeders attached to each cage. Water: . W`aantaeurtowimlaltbiec awvaatielraibnlge aacdciebsistusmyfsrtoemm. iAnldlivwiadtuealrbwiolllbtes fartotmacaheldoctaol tshoeurccaegeasnodrpfarsosmed through a reverse osmosis membrane before use. Chlorine will be added to the tphraonce1s.s2epdpwmatcehrioarsinae baatcttehreiotsitmaet;ofparnoacleyssisse.d Wwaatteerriisseaxnpaelcytzeeddtmoocnotnhtlayinfonropomsosribele bacterial contamination and twice annually for possible chemical contamination. Contaminants: Neither the Sponsor nor the Study Directoirs aware of any potential contaminants likely wtoitbhethpererseesnutltisnotfhethicsersttiufdiye.d diTehteroerfiontreh,endoriannkailnygsweastoetrhaetrltehvaenlstthhoastewroouultdineilntyerfere performed by the feed supplier or those mentioned in this protocol wil be conducted. 004134 418-010:PAGE F-76 418010P:PAGE 76 Protocol 418P-a0g1e07? MRATAINNGDAONDMIZATION: The female rabbits will be naturally bred at the Supplier by breeder male rabbitsofthe w`islal mbeesdoeusricgenaatneddsdtraayin0obeffporreessuhmiepmdegnetsttaotitohne. TeTshtienrgaFbabciitlsitwyi.llTbheesdhaiyppmeadtitongthoeccurs Testing Facility after mating, to arrive on day 2of presumed gestation. Before shipment of the rabbits, the Supplier will forward breeding records and day 0of presumed gestation body weights. A computer-generated (weight-ordered) randomization ipnrfoocremdatuiroen.will be used toassignthe rabbits to dosage groups based on this ADMINISTRATION: RR outeeandasfoo rChon ice: dpTiohesetsaiorbrylaelro(ruostuett,oemtsahcoefhehxtauubcmeta)dnrooesuxatpgeoeswuaGrasen.sebleecatcecdurfaotreulyseadbmeicnaiustseer:ed1;)ainn dco2m)piatrisisoonne woiftthhtehe Meatnd Fhreqouendcy: Female rabbits will be given the test article once daily on days 7 through 20 of presumed gestation. Dosages will be adjusted for the most recently recorded body weight and given at approximately the same time each day. Ratfoir Do osan ge Sa elel ctie on: Dwiotshatgheestewsiltlabrteiclsee.lected by the Sponsor on the basisofprevious studies conducted 004135 418-010:PAGE F-77 418.010P-PAGE 77 Protocol 41P8a.g0e1s0 DCosaogenLecveles, ntrataindVoolnumess: || Dosage| Nuomtber Concentration| Volume I| Group| Ravbits | (mogiday gm) | (ming) gusBach Numeer | CTsTown| o |5[eumournonmennven] Lolso[+[ 02Ts[eumoursonmnnven] folsTs[+Ts [eusourcommomren [|v w [| 2 s [5 [esmwonroommonnran [v a [ 5 s [5 esmonrconmomven wsTo[ 0 | 5s [eawourrommommen] TChe ewst aricis wi 5s1Consid= ered 100|% pure o1rsthe pur|pose o5f GosJageeacanlcuoiautironscommomven] TESTS, ANALY: MENTS Viability: All Periods: Atleast twice daily. Clinical Observa Predosage Period: Atleast once. Dosage Period: Twice daily. Prtoidoosagre administration and once approximately on hour postdosage. Postdosage Period: Once daily. Clinical observations may be recorded more frequently than cited above, if deemed appropriate by the Study Director andlor Study Monitor. BodyWeights: Predosage Period: Dosage Period Postdosage Period: Day 0ofpresumed gestation and on the day of arrival atthe Testing Facilty. Daily. Daily. 004136 418-010.PAGE F-78 418010P:PAGE 78 Protocol 41P8a.g0e1s0 Fead ConsumptionValues: Predosage Period: (Rveaclouredsendotdatialbyulaaftteedr)a.rrival at the Testing Facility Dosage Period: Postdosage Period: Recorded daily. Recorded day. iFneteerdvaclosnassumbpotdiyonweviaglhutesedvualruiantgitonhse. dosage period will be tabulated for the same `Caesarean-Sectioning Observations: Rabbits will be Caesarean-sectioned on day 29 of presumed gestation. The fetuses will be removed from the uterus and placed in individual containers. The rabbits will be `examined for number and distribution of: ComporaLutea. Implantation Sites. data) [Placentaethat appear abnormal (size, color or shape) will be noted in the raw Live and Dead Fetuses. (A live fetus is defined as one that responds to stimuli; a dead fetus is defined as a term fetus that does not respond to stimuli and that is not markedly autolyzed; dlaetaedrefseotrupsteisondse.m)onstrating marked to extreme autolysis are considered to be Early and Late Resorptions. ao(Arngceaoanrnolcgyeeprnetesusosirpsitsihdoaens.f)ioncecdurarseda; lfattehirsesiosrnpottiotnhiefictasise,grtohseslcyoenvciedpetnutstihsatidentified as EotalObservations: BodyWeights: bTeheusbeoddytowdeeigthetromfineelaicthterffeettuaslwibloldbyewreeicgohrdteadv.eraOgnelsy.body weights of live fetuses will 004137 418.010:PAGE F-79 418010P:PAGE 70 Protocol 41P8a.g0e10100 GrossExternal Atrations: All fetuses will be examinedfor gross external alterations. Late resorptions and dead fSeutcuhseosbsaelrsvoawtiillonbseweixlanmoitnbeed ifnocrlgurdoesdsienxetietrhnearldaalttaersautmiomnasritozatthieoenxtoernsttaptoisstsiicballe but analyses. Fetuses with gross external alterations will be preserved in neutral buffered 10% formalin. All other fetuses will be discarded. Representative photographs of fetal gross alterations will be taken. Sex: All fetuses will be examined intemalltoy determine sex. MET OFSHACROIFIDCE: tBoeusatchraifniacseiraab-biDtsSp(evciiaali,ntmraanvuefnoaucstuirnjeedctbiyonS)cahnedrifngv-PfleotuusgehsA(nviiamailntHreaapletrhit,ownielallbe used injection). NECROPSY: Gross lesions will be retained in neutral buffered 10% formalin for possible future `evaluation (corresponding tissues will be retained from rabbits in the vehicle control group at the discretion of the Study Director). (Exception: Parovarian cysts will be discarded; these are common, spontaneous lesions in rabbits.) Unless specifically cited below, all other tissues will be discarded. Scheduled Sacrifice: On day 29 of presumed gestation, female rabbits will be Caesarean-sectioned, and a gross necropsyofthe thoracic, abdominal and pelvic viscera will be performed. Uteri of apparently nonpregnant does will be stained with 10% ammonium sulfide to confirm the absenceof implantation sites. RabbitsFoundDead or Moribund: Rabbits that die or are sacrificed because of moribund condition, abortion or premature delivery will be examinedforthe causeof death or moribund condition on the day the observation is made. Pregnancy status and uterine contents will be recorded. Aborted fetuses and/or delivered pups will be examined to the extent possible, using the same methods described for fetuses. Uteri of apparently nonpregnant does will be stained `with 10% ammonium sulfide to confirm the absence of implantation sites. 004138 418-010.PAGE F-80 418.010P:PAGE 80 Protocal 41P8a.g0e10110 `STATISTICAL EVALUATION: `Aavpeprroapgrieastea.ndAdpdeirtcieonnatlagpersocweildlubreescaalncdu/loarteadn.alLyistteesr vmaalyuebsewiplelrbfeorumseeddfwhdeereemed appropriate. DATAACQUISITION, VERIFICATIONANDSTORAGE: DDiarteactwoilrlabned/hoarnadp-praonpdr/ioartceommapuntaegre-mreecnotrdpeedr.soRnneeclowridtshiwinll21bedraeyvsiaefwteedr gbeynetrhaetiSotnu.dyAll boreigbionaulnrdeaconrddsinwdiellxebde. stAorceodpiyn otfheallarracwhivdeastoafwtihllebTeesstuipnpgiiFeadcitliotyt.heASllpoornisgoinralupdoatna will ryeeqauresatf.terPmraeisleirngvoefdtthiessdureasftwiflilnbalersetpoorrte,d aaftetrhewhTiecshtitnigmeFatcihleitSypaotnnsoorcwhialrgbeefcoornotnaec.ted to determine the dispositionofthese materials. RECTOO BE MR AINTDAINS ED: Protocol and Amendments. Test Article, Vehicle and/or Reagent Receipt, Preparation and Use. ARnainmdaolmiAzcaqutiisointiSocn.hedules. VMeatteirnignaHriistaonryE.xamination. GTerneeartamlenCto(mifmpernestcsribed by StaffVeterinarian). Clinical Observations and/or General Appearance. Body Weights. FCaeeesdarCeoanns-uSmepcttiioonniVnagluaensd.Fetal Observations. OGrrgosasn WNeeicgrhotpssy( Obrseequrivraetdi).ons. Photographs (if required). `Study Maintenance (room and environmental records). Feed and Water Analyses. Packing and/or Shipment Lists. 004139 418-010:PAGE F-81 418:010P:PAGE 81 Protocol 418-0100 Page 12 KPEEYRSONNEL: Executive Director of Research: Mildred S. Christian, Ph.D., ATS Director of Research: Alan M. Hoberman, Ph.D., DABT Associate Director of Research and Study Director: Raymond G. York, Ph.D., DABT MD Maanniaaggr eerore oofffLcaASbntt oiurmdao aytloCr orOopyredOripanetaritaiotoninso:nasn:VdaJlMoeehmrnbiAeeF..r,BShaIamnresptteftru,t,iBoM.n.SaS.l. Animal Care and Use Committee: Dena C. Lebo, V.M.D. Managerof Regulatory Compliance: Kathleen A. Moran, M.S. Consultant, Veterinary Pathology: W. Ray Brown, D.V.M., Ph.D., ACVP REPORT. Aimlmeetdtieratreeployrtffoollrotwihnegpcuormppolseetioofndoofstahgeeisne-ilfeectpihoanfsoe.rthe full study will be prepared A summary report will be prepared including: abstract, summaries of the methods, arensdulitnsdaivnidducaolntcalbulseiso;na;ntdabrleepoofrtcsonotfesnutpsp;ocrtoipnygodfatthae(ipfraoptopcroolp;riaamtee)n.dmTehnetsre;posrutmwmilalrbye tihnecldurdaefdt raespoarnt aapnpdentdwioxctoopitehseoffutllhsetufdinyalrerpeoprotr.t The Sponsor will receive one copy of INSTITUTIONALANIMALCAREANDUSECOMMITTEESTATEMENT: The procedures described in this protocol have been reviewed by the Testing Facility's tIhnsattitiuntvioolnvale AsntuidmyalanCiamraelsanwidllUbseecCoonmdmuicttteede.in aAllmaprnonceerdtuoreasvodiedscorrimbiendiimnitzheis protocol discomfort, distress or paintothe animals. TnehceesSspiotnysfoorr'scosnidguncattiunrge tbheils oswtuddoycaunmdentthesftahcet ftahcatttthhaits iinsfnoortmaatniounnnceocnecsesranriinlgythe duplicative study may be obtained from the Sponsor. No alternative (in vitro) procedures were available for meeting the stated purposes of the study. 004140 418-010:PAGE F-82 418-010PPAGE 82 Protocol 41P8a.g0e10183 REFERENCES: 1. oCfhrtihsetitaenr,aMt.oSg.e,niHcopboeternmtainal,oAfMs.usapenndsSimointsh,ofTt.rHi.nFi.tr(o1f9i8u2o)r.enoDnoes(aTgNeF-)range study administered orally to New Zealand White rabbits. Toxicologist 2(1):40 (#143). 2. nCharlitsrteixaonn,eM(.PSr.oc(1e9e8d4i).ngsRoefprNoadlutcrteixvoenetoSxiycmiptyosainudmt,erNateowloYgoyrekvaAlcuaatdieomnsyooff Sciences, November 7, 1883), J. Clin. Psychiat. 45(9):7-10. 3. MFeSu.ss(n1e9r5,2)E.LA, dLiegchatdkeepo,fGr.aEb.b,itHfeenrtnielstyyd,atRa.:A.,StHuodbyeromfahnis,toAriMc.al aconndtrCohlriasntiimaanl,s. Teratology 46(4):349-365. 4. InstituteofLaboratory Animal Resources (1996). Guideforthe Care and Use of Laboratory Animals. National Academy Press, Washington, D.C. 5. Salewski, E. (1964). Implantationsstellen aFmarUbteemruesthdoedreRaztutem.. mAarkcrho.skPoatphioslc.hEexnp.NaPchhawremiaskovlon 247:367. 004141 418-010.PAGE F-83 418.010PPAGE 83 Protocal 41P8a-g0e10174 PROTOCOLAPPROVAL: FOR THE TESTING FACILITY /l ep <<), oboe George E/Dearlove, Ph.D., DABT Associate Director of Research RA eYagh3 G. York, Png,EDAVET ASstsuodcyiaDitreDeictroerctorof Reserch Ln Rls Dena C. Lebo, V.M.D. Member, Institutional Animal Care and Use Committee 14-Jun-78 Date Date fe an2 Date FOR THE SPONSOR Kone: 7Ca SMtaurvdiynMTo.niCtaosre, D.V.M., Ph.D. 19 Juni Date. 1275 004142 418-010:PAGE F-84 418.0107PAGE 84 ATTACHMENT 1 SCHEMATIC OF STUDY DESIGN AND STUDY SCHEDULE 004143 ATTACHMENT 1 418-010:PAGE F-85 Proto4co1l84-1081-00P1-0PAGE 85 Page 102 STUDYSCHEMATIC DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY" TimedPregnant Rabbits Start of Dosage Caesarean Sectioning End of Dosage Day 0of Presumed Gestation Day 7of Presumed Gestation Day20of Presumed Gestation Day 29 of Presumed Gestation WE a. Dosage Period. For additional details see "Tests, Analyses and Measurements" section of the b. protocol. Fetal evaluations (all fetuses - external examinations). 004144 ATTACHMENT 1 418-010:PAGE F-86 Proto4c1ol84-1081-00P1:0PAGE 86 Page20f2 26 JUN 98 01JUL98-14JUL98 23JUL98 30JUL 98 06 OCT 88 SCHEDULE" Animals Arrive - Acclimation Begins. Dosage Period (D7athyrousgh 20ofpresumed gestation). Caesarean-Sectioning Period (Day 28 of presumed gestation). Letter Report. Summary Report. a. The study initiation date is the date the Study Director signs the protocol. 004145 418-010:PAGE F-87 418.010P:PAGE 87 ATTACHMENT 2 MATERIAL SAFETY DATA SHEET 004146 418-010:PAGE F-88 MATERIAL SAFETY DATA SHEET OM N-E+FOSE 41g.010p-pacE88 S53tM5.14C4eP-anu1tl0e,0r0Minnesota 1-800-364-3877 or (812) 737-8501 (26 hours) ASCofpoyrriaiosgnthtit,son1rf9e9os8er,rvtehMdei.nnpeusrCopotopasyeinMgiofnianpngrdo/poaernrdldyoMwannUlutofisaldciiztniugnrginofgSHCtohpmirspoadnuyc.ts : i33s atPhrleioowriendfaogrprmreaoetvmiieondntediistshacotob:ptiaeidnedinrfousllSHw,ithandno changes unless 2) hGeakctrhiebruttehde wciotphy tnohre tihnetenotriiogninaolf iesarnriensgolda porrofoitthetrhweirseeon. TDRIAV0DI-SE1I0ONNA:HFEL:UO3RMADCHEBMrIanCdALSFluorochesical Alcohol 10 8NU0M2B0E4R/1U1.1P3..C7. 00-51135-00495-2 Sa011 1575.7 00.51135.02145.3 9988.-00221111--61612803..60 SISUSZPUFEER0DS0:E0D2EJS-a:n0u8aN7ro2yv8em29b,er1o 0959,8 19e 87 DOCUMENT: 10-3778.7 0000.-5511113355.-1009453492--23 37 tnaneptet Cs. HO. pencenT PPPEEERRRFFFLLLUUUODORRROOOOHHCEETPXATANANENESESUSULLUFFLOOFNNOAANMNATIMDDIDDDOAALALCLCOCOROORHLOO.LL............. 314649515--909-32-3 88565.73.7 38.00.0 2.0 -- 78.00.0 - 6.0 PPEERRFFLLUUDORROOBPUETNATNAENSEUSLUFLOFNOANNAIMDIODAOLCALOCHOOWLO.L.......... 3640484595-.8792..96 21..00 -- 63..00 2. PRvSIOAL DATA BOILINGPOINT: ........eoeenss VAPOR PRESSURE: .............. VAPOR DENSITY: ........c.connn. ESVOALPUOBRIALTIITOYNIRNATWEAST:.E.R.:..0.1...0.1........ SPECIFIC GRAVITY: ...... 0... PB ERCENTVo OLATILEt : ......s .cevnes VMEiLsTcIoNsGitPyO:II NT..I . 0e.I 00eues G2. 1 1o1m8 CHg <Ca1l0oaug20 >Ca1i.c0 A@tr2=0tC. <ne1g.l0i.BuOAG=} Gal,ot 1n.e7rtW)ater=t N0I%A wNInD 004147 Abbreviations: NID - Net Determined N/A - Not Applicable GA Approximately 418-010:PAGE F-89 JWaSnSu:aryFG-291,0 1F9L9U8DRAD Brand Fluorochemical Alcohol sascoeop2pacE se 2. PHYSICAL DATA (continued) APPnEeAvReArNCwEaryMDsood0a0R: 3 Fine Ai EXPLOSION HAZARD DATA FFFLmAaaSHanlPeOeINTLL:iiv.i.sT.s.C.LE .LG..EcIuoLc1Le1:es wW> Aa148 CSetaflash RROTGNITION TEMPERATURE: 111. WIA exTRIavteGru,ISHGIaNrGsonMEDGIiAo:xide, Dry chemical, Foun SPBERoeCoaIacAriLhvtauFemlIsRsEpprrbFeoIontGdseHsTc.eItNiaGvroaeruPnRpcdOlr.CoeaEtsrDhsaiUoenRr,gEe,S:sdAei1mnsactnlduaibnnrgeaLtehgheeil,anegtf,adcpepsaermlaafst-kuc,so,ntaanbidunnekde,r coat Srathetive covering Tor exposed areas of the hesd. UNUCSoUALarFiIrRaEosAsNDDeEcXoPmLpOeSsIiOtNioHnAZAsReDcSt:ion for products of combustion. 4. REACTIVITYDATA STABILITY: stable INCoOtMPAsTpIpBiIiLcIaTiYe.- MATERIALS/CONDITIONS TO AVOID: VAZAROOUS POLYMERIZATION: Hazardous polymerization will not occur. HAZCaAamRrrDoeOnrU,SWHDoyEnoCerOiaMegPeeOnSIaTnFIdlOuNoCrairPdbReoO,DnUCTDTioSoc:xiicdev,apoOrxsi,desGaosfesNiotrroPgaernt,icuOlxaitdeess. of pp ------------" spiDBRLeiLfeearenmesst]roonorastnaehat:eermcotssiievocentioewaqnsusaicpoeefse,ntth.irsesGHpoSitDrlSaetcfotorryspipirnlofltoeercdstaitmoianot,enrivraeelng.tairldaCitlniegoann, upand Peetoue, "lace in a 0.5. 0OTapproved container o 04148 Abbreviations: WD Not Deterained N/A - Not Applicable Gh - Approximately 418-010:PAGE F-90 MSDS: FC-10 FLUORAD Brand Fluorochemical Alcohol January 29, 1998 4P1A8G-E0103P:PAGE 80 5. ENVIRONMENTAL INFORMATION (continued) RECOMMENDED DISPOSAL: Incinerate in a permitted hazardous Waste incinerator in the presence Dofispaosceomobfuswtaisbtlee mpartoedruicatl.in aCofmabucsitliiotny ppreordmuictttsedwitollaicnccepltudecheHFm.ical waste. : ENVIRONMENTAL DATA: Laboratory tests showed no biodegradation. 96-Hr. LDSO Fathead Minnow s(tPaitmiespthiaclaelslyprsomieglnaisf)ica-ntNoefmfoercttalionty%athatwcaht,er%ssautruvriatviaoln,. weiNgoht, and floelndgthbioincon30cendatyratFiaotnheaodf Minnow egg FC-10 into mfursyclsetudfyi.lletLsaboftescthsannsehlowecdatf2i0s0h. REGULATORY Volatile OINrFgOaRnMiAcTICOoNm:pounds: N/A. VOC Less H20 & Exempt Solvents: N/A. This product complies with the chemical registration requirements of TSCA, EINECS, COSL, AICS and Korea. EPCRA HAZARD CLASS: FIRE HAZARD: No PRESSURE: No REACTIVITY: No ACUTE: Yes CHRONIC: Yes 6. SUGGESTED FIRST AID EYE CONTACT: Immediately flush eyes with large amounts of water. Get immediate medical attention. SKIN CONTACT: Inmediately wash skin With soap and large amounts of water. Remove Wcaosnhtamcionnattaemdinactleodthicnlgo.thiInfgsibgefnosr/esymrpetusoems anodccudri,spocsaellofa physician. contaminated shoes. INHALATION: If signs/symptoms occur, remove person to fresh air. If signs/synptons continue, call a physician. IF SWALLOWED: Call a physician IMMEDIATELY. If swallowed, induce vomiting immediately as directed by medical personnel. Never give anything by mouth to an unconscious person. 004149 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately 418-010:PAGE F-91 SMSaDnSu:iryFC-251,0 F1L3U98ORAD Brand Fluorochemical Alcohol Pace 4 418-010P:PAGE 91 7. PRECATIONARY INFORMATION evoe iPRdOTEaCyTeIOcNo:ntact. Wear safety glasses with side shields. sa`nAvopidrotsekcitniocno:ntact. Wear appropriate gloves when handling this CPmTaoeatvrceesorronianmadaallnl.si.apdr:oAtepca"btiuirtoynlofirtguelbmoesvre.sasmanUdseeeceofsnrseoamryorthfeosorfpeorlelvoofewnittnhgeskifmnaotlelcrooinwatilanc(gts:) are REUVCseOenMtEiwNliDatEthDioanpVpEtrNooTpILrmAiaTaiItnOetNa:ilnocaelaisesxihoanusstbevleontwilraetcioomne.ndePdroveixdpeosusruertiLciimeitnst. pIfroteexchtaiuosnt. ventilation is not adequate, use sppropriste respiratory RESoNPIiIORSdAHTOabRprpYeraotPvhReiOdTn.EgCrTeoIfsOpNis:riartboorrsnebamssetderiosnl.airbSoerlneectcoonneceonftratthieonfoolfloking FCeosnptiarsaitnoarn,tsfualnld-fianceacssourppdlainecde awiitrhrOesSpWiArarteogru.lations: half mask dust PREaSVorEeNanTsoItOtNheaotOr,FouGAgrChiClnIykDEKNEoTtrhALsasooIkaNepGESsaThnIedOnNW:autseirn.g tHhaisshphraodnudcst.aftKearshhaenxdploisnegd and Setore eating. RECSOtoMrMeENDaEwaDySTfOrRoAnGEh:eat. Keep container closed When not in use. FIRNEontAlNaDsnEaXbPiLeO.SION AVOIDANCE: OTHCNEooRntsamPnoRkiEinCnsAgtU:iToInSOaNoAofkRiYntghIeNNFnOtiRolMbeAaTcIcuOosNi:nagnd/tohrissphrooeduc4tndcaLneadresTuoltheinformation TofhistheHDhsa.zardous decomposition products mentioned in section & of HMIS WAZARD RATINGS: PWEEARLSTOHN:AL1 PROFTLEACWTMIBOINL:ITXY:(Se1e pRErAeCcTaIuVtIiToYn:s,0section 7.) EXeOSURE LIMITS INGREDIENT VALUE UNIT PPEERRFFLLUUOORROOOICETKAMNEESSUULLFFOONNAANNIIODDD AALLCCOOHOWLO.L... 00..11 MMOoMkS TYE Tak M ATH Yski0ns04150 TWA BMY PERALFOLOUMODRLO.HEPTANoESoULoFONANIDO 0 WOMD TWA MY Aobreviations: RI Not Decermined WIA - Not Applicable Gh - Approximately 418-010:PAGE F-92 JMSaDrSu:aryFC-21,0 1F9L9U8ORAD Brand Fluorocheaoal Alcohol 41P8A.G0E10P5PAGE 52 EXPOSURE LIMITS (continued) INGREDIENT VALUE UNIT TYPE AUTH SKIN PPEERRFFLLUUOORROOPBEUNTTAANNEESSUULLFFOONNAAMMIIODOOALCOHOL... 0.1 Wa/M3 ALCOHOL. vvvnnnneennnsnisesess 01 WGI TWA 3 TWA 3M v Y t+heSKIpNoteNnOtTiATaIlONc:ontrLiisbtuetdionsubtsotatnhceesovienradlilcateexdpoWsiutrhe b'yY' thuendecrutaSnKeIoNusrefreoruteto biynclduirdeicntg chuocnotuasctmweimtbhranteneansdubsstyas,ncee.itheVrehibcyleasirbcoarnnealtore,r smkoirne apabrstoircputliaornl.y, S"OaUMR:CE OFoHEXRPOeScUoRmEmenLdIeMdITExDApToAs:ure Guidelines 5. HEALTH HAZARD DATA EYENoCOaNdTvAeCrTs:e health effects are expected from eye contact. SKIPNrodCuONcTtACiTs: not expected to be irritating to the skin. eMaxytenbdeedabtsionreb.ed through the skin and persist in the body for an INHMAaLyATbIeONa:bsorbed by inhalation and persist in the body for an extended tine. IFISnWgAeLsLtOiWoEnD:is not a likely route of exposure to this product. qIulalnnteistsiemsayofoctchurisafmatteerriaals.ingle swallowing of relatively large MUTNAoGtENmIuCtIaTgYe:nic in in-vitro assays. REPSRuObDsUtCaTnIcVeEw/aDsEVnEoLtOPtMeErNaTtAoLgeTnOiXcINSi:n the rat at doses as high as 30 milligrans per kilogram per day via oral route. OTHTEhiRsHEpArLoTdHuctHAiZsARDnotINFkOnRoMwAnTItOoN:Contain any substances regulated under California Proposition 65. A Product Toxicity Sussary Sheet is available. 004151 Abbreviations: N/D . Not Determined N/A - Not Applicable CA - Approximately 418-010:PAGE F-83 MSDS: FC-10 FLUORAD Brand Fluorochesical Alcohol January 29, 1998 SECTION CHANGE DATES HEADING SECTION CHANGED SINCE November 05, 1997 ISSUE #15-010PPAGE 93 PAGE 6 Abbreviations: N/D - Not Determined N/A - Not Applicable CA - Approximately Tbhee cionrfroecrtmataisonof inthethidsateMatiessruieadl. Saf3eMtyMAKDEatSaNOShWeAsRtRA(NMTSIDESS), isEXbPeRlEiSeSvEeDd OtRo MIEMRPCLIHEADN,TABIINLCILTUYDINOGR, FBIUTTNESNSOTFOLRIMIATEPDARTTOI,CULAANRY IMPLIED PURPOSE WARRANTY OF OR COURSE OF wUPhsEeeRtrFn'OesRrMAmNetChtEehoOd3RM ofUpSrAuoGsdEuectOoFr isTaRpAfpDilEti.caftoirUosnea.rpaisrGtiivrceeunslpaotrnhesipbuvlraeproisfeeotryaodnfedtefsraumciittnoairbnslgetfhoatr cuanniquafefleyctwitthhein utsheeanudsera'psplikcnaotwiloendgeof aand3Mconptrroodlu,ct,itsoimseeosfsewnhtiicahl are that the user evaluate the 3M product to particular purpose and suitable for determine whether it user's method of use is or fit for a application. D3iMuneperrtrooovritsdh,eesormieinmsoftsoeiromnapstoisoosnribiainlltieterylaettcihtoartnosnieclinecftotrhroimsniacsintfaorrasmneasrtfvieiorcne,maty3o4himtaasvkeecsursetnsooumletresd.(QQg4. irnefporremsaetnitoantioonbstaiasnedtofritosm acodmaptlaebtaesneessmayornoatccubreacays. curIrnenatddiatsiotnh,e 152 Information in the MSDS available directly from 3M. 418-010:PAGE F-94 418-010P-PAGE 04 ATTACHMENT 3 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE 004153 418-010:PAGE F-95 418010pPAGE 95 ATTACHMENTS Protocol 418.0108 Version: 418-01P0aPJss_UN(t9o18t2)s TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Test Article: N-EtFOSE Vehicle: 2% Tween 80, in R.O. Water A. Purpose: The purposeofthis procedureisto provide a methodforthe preparation of dosage suspensionsof N-EtFOSE andthevehicle for oral `administration to rabbits on Argus Study 418-010P. 8. General Information: 1. All suspension containers will be labeled and color coded. Each label will `specify the protocol number, test article identification, Argus batch number, concentration, dosage level, preparation date, expiration date and storage conditions. 2a. Suspensions will be prepared: _X_ Daily -- Weekly __For___ daysof use 2b. Vehicle will be prepared: -- Daily X_ Weekly __For___ days of use 3. `Suspensions will be prepared at a final dosage volume of mL/kg. 4. Safety: X_ Gloves, lab coat, gogglesorsafety glasses and faceshield X_ Dust-Mist Respirator -- Half-Face Respirator -- Full-Face Respirator/Positive Pressure Hood -- Tyvek Suit/Apron 5. Dosage solutions adjusted for Freebase and% Purity. -- Yes X__ No (Calculations based on 100%) -- FreeBase __ Purity 6. `Sampling requirements: Cited in protocol. 7. Storage: Cited in protocol. 004154 418-010:PAGE F-96 418-010P.PAGE 36 ATTACHMENT 3 Version: 41P8r.o0t1o0cPo1l241J8U.N019080) Page20f3 TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE NOTE: The test article will be prepared as a serial dilution from the high dosage 0the low dosage. Once the final volumes are achieved, stir bars are to be addedtothe containers; mixing should occur during sampling andlor `administration. C. Preparation of Vehicle 1. Add the required amountofR.O. deionizedwaterto an appropriately labeled container. Heatthe water to 50C, 5C, add the required amountofTween 80 and mix until uniform (See TEST ARTICLE CALCULATIONS). D. Test Article Suspension Preparation: 1. To prepare the 15-mgimL, Group VII suspension, add the required `amount of test article (See TEST ARTICLE CALCULATIONS) into an appropriately sized, labeled container. Add the required amountof vehicle and heat the mixtureto 80C, 25C for approximately 30 minutes. 2. Once the test article has dissolved: spin over night while the solution cools. (Be sure there is a visible vortex, this will achieve the desired emulsion.) 3. Toprepare the 10-mg/mL, Group VI suspension, remove the required amount of stock suspension (Group VI) (See TEST ARTICLE CALCULATIONS), add the required amount of vehicle and mix. 4. Toprepare the 5-mg/mL. GrouVp suspension, remove the required `amount of stock suspension (Group VI) (See TEST ARTICLE CALCULATIONS), add the required amountofvehicle and mix. 5. Toprepare the 2-mg/mL, Group IV suspension, remove the required `amount of stock suspension (Group V) (See TEST ARTICLE CALCULATIONS), add the required amountof vehicle and mix. 004155 418-010:PAGE F-97 418.010PPAGE 97 ATTACHMENT 3 Version: 41Pr8oto.c1o1l02411J8U.0N019P089) TEST ARTICLE AND VEHICLE PREPARATION PROCEDURE Page3or3 6. To prepare the 1-mg/mL, Group Ill suspension, remove the required `amount of stock suspension (Group IV) (See TEST ARTICLE CALCULATIONS), add the required amount of vehicle and mix. 7. Topreparethe 0.2-mg/mL, Group ll suspension, remove the required amount of stock suspension (Group Ii) (See TEST ARTICLE CALCULATIONS), add the required amountofvehicle and mix. wien by: [ Approved by; Gill. A Date: r2-a09u5 Clarification: X._ No _Yes (See attached clarification form.) * intiatsate : (LoT.LponldTM 004156 418-010:PAGE F-98 418010P:PAGE 98 . [T] pn Argus Research Laboratories, inc. --------------------sc--nr--on--es------------------------Hor--Tshaa--mr,e--sPaa--rrna--eyi--vaar--riae--_ao1--9s0--44 PROTOCOL 418-010P ORAL (STOMACH TUBE)DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF N-EtFOSE IN RABBITS `SPONSOR'S STUDY NUMBER: T-6316.8 Amendment 1 - June 25, 1998 1 ClO inicb al serv and/a orGt enei ralAo ppen arans ce (page 8 of the protocol): `The postdosage observation will be made approximately one hour postdosage. ReaforsChoangne: This change corrects a typographical error. fet,orl Sitnns Sil >y [9% A GAesosrocgieateDireacrtloorvoef, PRhe.sDe.a,rDcAhBT Date ARsasyotcoinatde/GD.iYroerkc,otPfoh.r| Rseh3rchT Date `and Study Director Cra 0 Ll 5 ner Dena C. Lebo, V.M.D. Date MeUmsbeerC,omImnisttietuet,ional Animal Care and Pn 7%. [bul 98 Marvin T. Case, D.V.M., Ph.D. Date Study Monitor 004157 APPENDIX G HISTORICAL CONTROL DATA 004158 418-010:PAGE G-1 SUMMARY OF REPRODUCTIVE INDICES NZW RABBIT PERIOD: JANUARY 1896 -JANUARY 1998 NUMBER OF STUDIES: [3 NUMBER OF RABBITS: TESTED 901 FOPURNEDGNDAEANDT 86&1 DELIVERED PREAMBATOURRTEELDY 24 NUMBER OF RABBITS PREGNANT AT CDAAEYS2A9ROEAFNG-ESSETCATTIIOONN:ING ON 825 NUMBER OF RABBITS WITH SINGLE CONCEPTUS LITTER: LIVE 1 RAESBOORRBTEEDD. 21 MEANOr% % PREGNANT %0 AVERAGE # CORPORA LUTEA 7 AVERAGE # IMPLANTATIONS 89 AVERAGE LITTER SIZE AVERAGE # LIVE FETUSES 84 AVERAGE # DEAD FETUSES 00 AVERAGE # RESORPTIONS 04 AVERAGE # EARLY RESORPTIONS 03 AVERAGE #LATE RESORPTIONS or *aTthtrriebeutweedtroe amnoriinbtuunbdatsiaocnriafcicciedse,notne was RMAENAGNE/oSrT%UDY (750-100) (any) (38108) (32104) (01) (032) (28) (012) 004159 418-010:PAGE G-2 `SUMMARY OF REPRODUCTIVE INDICES NZW RABBIT MEAN or % 'RANGE/STUDY MEAN or % AVERAGE % DOES WITH ANY RESORPTIONS AVERAGE % DOES WITH ALL CONCEPTUSES RESORBED "AVERAGE %DOESWITH ONE OR MORE LIVE FETUSES 26.8 06 09.4 (0-100) (0-20.0) (80.0-100) AVERAGE SEX RATIO, (% MALES/LITTER) AVERAGE FETAL BODY WEIGHT (G) AVERAGE FOR MALES (G) AVERAGE FOR FEMALES (G) 51.2 4374 44.26 43.08 (31.461.0) (31.85-55.74) (29.55-56.97) (32.25-53.76) AVERAGE % DEAD OR RESORBED CONCEPTUSESALITTER 44 (0-18.8) 004160 418-010:PAGE G3 SUMMARY OF MANTZEWRNRAABLBNITESCROPSY OBSERVATIONS TPOETRAILOD# STUDIJEASNUARY 19 -JA9 NUA6RY 106978 T#OPTRAELG#NADNOTES 82176% OF PREGNANT ##DAIBEODRTED & 05 2 24 ##DDOEELSIVWEIRTEHD 1P0R0E%MARTEUSROERLPYTIONS 4 04 5 0s EXTERNAL OBSERVATIONS Fecal material in Lopcearilainzaeld raelgoipoencia Lettear, tom GROSS LESIONS THYMUS Small LUNGS Discolored Tepahrraignmraitgihtcdlioab-e Multiple lesions THORACIC CAVITY `Containedredfluid LIVER Pale andor discolored Acpcaetntteumaotnedlloobbeusl)ar BACK Break presentin lumbar region of spine RANGE /STUDY N% NO % 2101012 01 04 (050) (050 1 011 01 (050) 1011 01 (050) 6 065 04 (0200) 1100111 0011 ((004402)) 1011 01 (040) 7 076 04 (0-160) 1011 01 (50) 101 04 (050) * aTthtrreibeutweedrteo maonriinbtuunbdatsiaocnriafcicciedse,notne was 004161 418-010:PAGE G4 SUMMARY OF MNAZTWERRNAABLBINTESCROPSY OBSERVATIONS GROSS LESIONS BACK (CONDoTr)salmuscles,three hemorrhagic areas. in lumbarregion STOMACH MTurciocshaob,eezrooadred in areas SPLEEN Small Large KIDNEY(S) small Ricgahutd,adlilsyplaced ADRENALRGigLhAtN,Dabsent UTERUS Rihomgcoh ntaitned Hdoamrsk cbornotwanfilnueidda Viscous,green-brown Vassucublsatrainzcaetion Pblayceatnhtiacks,uyrerloluownded substance OVARIES Parovariancyst(s) N % RNAN/SGT0%UEDY 1011 01 (1285) 2 02 01 (043) 1011 01. (042) 3 032 04 (0200) 1011 04 (040) 10m 01 (0200) 1011 ot (050) 1 011 01 (050) 10m 01 (167) 1100111 01 04 (040) (043) 10m 01 (050) 0 326 05 (0250) 004162 418-010:PAGE G-5 `SUMMAROFY FETAL GROSS EXTERNAL ALTERATIONS NZW RABBITS PERIOD JANUARY 199-6 JANUARY 1998 #OFSTUDIES 65 #LITTERSEXAMINED 816 # LIVEFETUSES EXAMINED (DAY28) 6929 SKIN ALTERATION `Absent area HEAD Meningocele Cyclops Upperjawintwo. `segments Nares absent Fleshyprotrusion EYES Bugodepressed Eyelids open SNOUT Short TONGUE protrudes BODY UmbilicalHemia Edema L: LITTER INCIDENCE F: FETAL INCIDENCE RANGE / STUDY N% N % L F 1 012 01 1 001 01 (0-53) (006) L 2 024 01 " 3 004 02 L 1 012 01 F 1 001 01 L 1 012 01 F 1 001 041 LFoo11 000112 0011 FLo 1 o001021 0011 FL 33 003074 0O011 FL1100201 0011 (016.7) (037) (0-59) (007) (059) (0:07) ((000583)) ((000683)) ((000583)) ((000579) L 2 024 01 F 2 003 01 L102 04 F 1 001 04 (059) (00.7) (053) (0-08) L 9 110 02 F 10 014 03 LF41 000112 0011 (0-500) (0-120) ((00-0563)) 004163 418-010:PAGE G6 SUMMARY OF FETAL GROSS EXTERNAL ALTERATIONS NZW RABBITS ALTERATION BODY (CONT.) `Skin discolored purple `Spina bifida Hemorrhagic area(s) Meningocele Hematoma Dark red areas N L1 e3 L2 F2 L2 PB 2 L2 F2 LFoo11 L1 F1 RANGE / STUDY %N % 012 01 004 03 024 01 003 01 024 01 003 01 024 041 003 000112 01 0011 012 01 001 01 (053) (0-19) (0-200) (0-19) (050) (0-06) (0-167) (((000-5022980))) (0-56) (0-06) FORELIMBS AND/OR HINDLIMBS Paw(s): Flexed/ L Rotated P Paw: Short digits L F Limb(s): Rotated L F Limb(s): Absent L F 2 024 01 2 003 01 1 012 01 1 001 01 2 024 01 2 003 01 1 012 041 1 001 01 (056) (0-08) (056) (007) (0167) (0-19) (053) (0-06) TAL Short L 7 086 01 (0-200) F 10 014 04 (0-23) L: LITTER INCIDENCE F: FETAL INCIDENCE 004164 418-010:PAGE G7 SUMMARY OF FETAL SOFT TISSUE ALTERATIONS NZWRABBITS PERIOD #STUDIES JA1N996U- JA ANUAR RY19Y9388 ##LFIETTTUESRESSEXEAXAMMIINNEEDD 5677349 ALTERATION BRAIN Dilatedlateralventricles L (Moderate) (Grade2) ~F EE(S) Circumcomealhemorthage L F Microphthalmia LF HEART Large L F Threeventricles L F VESSELPSersistent truncus L airntneormiionsaitse, absent F L F Twopuimonary arteries LF LUNGS Oneormorelobes,partial L oRricogmphltetaeapnadgiecncaersadiislac F L lobesfused F DIAPHRHAeGmMia L F N% RANNGE/S0T%UDY 2 030 0-1 (0-50) 2 003 0-1 (0-08) 2210 023967 0032 (0-167) (019) 11 001052 01 04 (050) (008) 1 1 015 04 (059) 002 01 (008) 1 1 000125 0014 ((0005896)) 1 1 015 002 0014 ((000580)) 11 001052 04 04 (053) (007) 11 001052 0-1 04 (059) (006) 69 90 110.2547 0085 ((00-2570.)8) 1 1 0.45 002 041 01 (053) (008) 11 001052 01 04 (050) (0086) L: LITTER INCIDENCE F: FETAL INCIDENCE 004165 418-010:PAGG-E8 `SUMMARY OF FETAL SOFT TISSUE ALTERATIONS NZWRABBITS ALTERATION KIDNEY(S) Absent Displaced caudally SPLEEN Pale GonaDs Righttestisdisplaced caudally RANGE/STUDY N% N % L 1 015 01 (056) F 1 002 0-1 (0-08) L 1 045 01 (04.3) 1 002 01 (0-05) L 1 015 01 (07.1) F 1 002 041 (0-09) L 1 015 01 (059) 1 002 01 (0-08) - L: LITTER INCIDENCE F: FETAL INCIDENCE 00416 SUMMARYOFNFEETWARLASBKBEILTESTALALTERATIONS 418-010:PAGE G-9 Pe#RsTIuonDies JAN1996U-ANAUARRY 1Y66a87 ##FLETTTUESRESSEEXXUAMMINNEEDD soeann wor ALTERATIONS RANGE [STUDY sWtIoTkHes sku Nox ON % ATERATOV Sousmsmtacraztaotnoonfcfasllireguler LF1o8z5 2a78m0 Oo1w1o0e86s7) 35 ArrteerFsoretaneelde FLo 1 o0o1m8 0011000839 1 PosEntlaorFgeodt(anSsll)e (Grade1) Lt ooms orEsy 1 F 1 002 0-1 (0-06) Frornotsguesure Low 2m cspwn ow imeforsspesen PLoTw o2ms 00330001508) 13 Fusea LF'osu ooomm 0oz3o0wnms 4 Twosegments F{o7s ooims ooizesoy a Suture args [F4110om oc0i1e0s0)n 1 smal FLo o1 oootms 0o11000s9y Foot oom 01008 ParCsonia)inholes FLss ooms 0o1i000s8e 3 Fusedandsma F{oo1t oomm 0o1i0e0s8y inroaquratrysahaped LFo 11 0o0t2s o011000s5y) 1 pmeoopaorsseifsemd om LFo1i01oom 0011006479) 1 L FLEITTTAELRIWNCCIEOENNGGEE 004167 418-010:PAGE G-10 SUMMARYOFFEZTwALRSAKBEBLIETTSAL ALTERATIONS soTwroies SKULLA(LCTOENRTA)TIONS No% RANOGNE 1S%TUDYATEWRITAHTON Nasaitosgularsure L 4 om o2pwo 3 f-- Intanasals FLFoo2:4 03os0mt7 0o024c0(a02p19s2))e 1 Lt 200 oczpwen ow iDipacedatre Fused sma FLfoeus16 10223;8 000280(000145.092)0 M L 0 1m c2pw00 oe FL 11 0o1r9s 0o2i(00-s1.93) 1 F 1 002 0-1 (0-0.6) No esalFoao rnadlosrtmrio essalgred FL21318023 00320(401L9n)) Promasiis:used LFo1osom ooirpeosn 1 Pomaiae:rotossifed 1 015 01050 1 Vailas: used FLFo11 o0o0tm2s c00-1i100(0s00.y88) 1 Swroccptalsireguaty 1 015 04089 1 Es7h0aspoecdket mat FLz100o2m cies 0-1 (0-05) 2 Subemacsscatn FFL ol21 00o01o4sm 000-11100(0500.888) 1 Ho Ato.amas Lowe ter osE400) wu Sma oguiaryshaped FLFha137 2oo4ot1rs 0-oo184t(0e092s9ga) 1 Lo 1 ots oiesy 1 F 1 002 0-1 (0-06) L.uFTETTEALR INNGCIDDEENNGCEE 004168 418-010:PAGE G-11 `SUMMARY OFNFZETWARLASBKBEILTESTAL ALTERATIONS svtouroies ALTERATIONS No % RANOGNE/S%TUDYAERWAITTHION VCEoRnTeEsBRAE Ccousrtucmat,ounniawal FL2200oo 0011000878) 2 fmuesheedsandorConra, FL 33 0084s5 0011000578) 3 Hamverabra LFa3 o0s4ss o0i1@0s0s8) 3 Conmumasynmerc LFoot1 oo015 0011005087 1 Canta, bid Lo1oos c1Esy 1 F 1 002 04 (006) Tho"rHaomcveriobra Lon 10 c1esm `uAstehdesandorCons, FFL oe15 0o017n95 000222(00014.012)5 4 `Cotum,unialosl L 5 075 04053) 5 a``soCsysoimnfmiiceamttaiionnceormoe FFLo 51 00o01o95o 000-11100(005-8086)) 1 artrum,bid FL oo44 ooomr o02z0o12w)n 3 (Commrotossed FL t1 o01o5 0011004098 1 + Achabsont LFoor1 oorosm o01100s0e8 1 Aven, sma F Lo a3 o0s0ss 0c1i0s0e8) 3 LumbHaormiverobra L 2 om o1ose 2 `csCsotncattmoun nas FFLo1ot205o0o0m4 00011100(50000.88)) 1 Arch, mall LFoz2 oomo 0o1100s0n8) 2 `Contumnotossted UFL t1 0o1o5 0011005008 1 LiFLEITTTAELRIINNCCIIDDEENNCCEE 004169 418-010:PAGE G-12 SUMMAROFYFETALSKELETAL ALTERATIONS NZW RABBITS #OF STUDIES RANGE /STUDY WITH ALTERATIONS N % N % ALTERATION VERTEBRAE (CONT) Sacral +Archesopen L 1015 01083) 1 F 1002 010008) Caudal + Oneormore misaligned +Fused +11present +12 present +1310 14present +15 prosent +Imogularly shaped L 30 449 030187) 24 F 31 05 03020) L 8 120 041 (058) 8 F 10 018 03016) L 1015 01043) 1 F 1002 01(008) L 1015 04043) 1 F 2 004 020010) L 3 045 041 (056) 3 F 3 005 04008) L 1015 04 (043) 1 F 1002 04008) L 1015 04050) 1 F 1002 01008) VERTEBRAERRIB InterrelatedVertebral/Rb ~~ L malformations F RIBS Cervical Rib present L F Two or more, fused L F Bases proximate Lo F Oneormors, spit L F Onoormore,thickenedareas L F Fiat L F 3 045 03 (0158) 3 005 03018) 2 030 2 004 6 090 6 010 10 150 10 018 8 120 8 014 27 404 29 051 2 030 2 004 041056) 041 (008) 02 (0105) 02(012) 02091) 02011) 041 (056) 04 (006) 03 (0167) 03022 01088) 01008) 1 2 5 8 19 2 L: LITTER INCIDENCE F: FETAL INCIDENCE 004179 418-010:PAGE G-13 `SUMMARYOFNFZETWALRASBKBELIETTSAL ALTERATIONS #OF RIBS (ACLOTNETR)ATIONS Bm sma Broad Bont N % RANOGN E/S%TUDYATSEWTRUIADTITHEISO.N Lot oos sien 1 LFF z12 00oo2nn 00011100(00s058)) 2 L 1 015 041 (059) 1 FLo 11 0o0s2 0o110(0408s) 1 F 1002 04 (008) MANUBRIUM Ouplcated Fused Lot oos ciesy LF a100oe2s 0c12(000078) 2 F 3 005 02(011) STeRNEBRAE Twoormor,fused FLooun a1s3m o0s7o0e3z9s ow norma seymmec FL oe6 0o8n0 00220011025 5 oOrnnoootrcmsosrfs,andcomplosy FL oo1m1 oo18n5 00220044028 Ouplcatod FLoo 1 ooiosm ooi1e0s0sn 1 peLviS Piorsnosto)ssifnacodmplolsy F L4s08om 00120059m) 4 scapuLsE Ato: ineguary A`lshea:pewdavy Lo 4 om s20ws 3 LFFoo1t4 o0oo0os7; 00o21r(0E010s28y) a FL:IFTETTEARLINICNICDIEDNECNECE 004171 418-010:PAGE G-14 . Now Ragas `SUMMARYOFFETALSKELETALALTERATIONS wor SCAPULAALET(ECROATNITO)NS No% RANNGE/WSTUDYALTSWETRUHADTIEISON Miealgned sent LFoss ooomm o02z0e1u2n 4 F[o11 ooms ocio0smy 1 FORELIMB(S) +Prat present Lt oo 10s OPvagesprsant ~~ FL 11 000125 0011008087 1 [IC-- arpal,--atac-- apas,For: F 1002 01007) dotswdForages L 1 015 01083 1 absent 1 002 0-1 (0-06) LuFTETeARL INcCiIvDeENNCcEe 004172 418-010:PAGE G-15 SUMMARY OF FETAL OSSIFICATION SITES NZW RABBITS PERIOD #STUDIES JAN 1996U-JAANUAR RY1Y 998 37 ##LFIETTTUESRESSEEXXAAMMIINNEEDD 5666872 FETUS / LITTER SKELETALAVERAGES ~~ MEAN RANGE/STUDY HYOID 100 (0981.00) VERTEBRAE CERVICAL THORACIC LUMBAR SACRAL CAUDAL 7.00 1258 (1244-1272) 641 (627-657) 3.00 1696 (16.7517.13) RIBS (Pairs) 1251 (1236-1267) STERNUM MANUBRIUM STERNAL CENTERS XIPHOID 1.00 033882 ((039823:13.9090)) FOREPAWS (Calculated as average per limb) CARPALS' 0.00 METACARPALS DIGITS 499 5.00 PHALANGES 1391 = (4965.00) (13801399) HINDPAWS (Calculated es TARaSvAeLrSage per limb) 200 METATARSALS 4.00 DIGITS 400 PHALANGES 1200 (1.98200) - (11.87-12.00) 004173 APPENDIX H STATEMENT OF THE STUDY DIRECTOR 004174 r*,PRIMEDICA 418-010:PAGE H-1 ror p Beets Argus ResearchLaboratories, Inc. Telephone: (215) 443-8710 PROTOCOL 418-010: ORAL (STOMACH TUBE) DEVELOPMENTAL TOXICITY STUDY OF N-EtFOSE IN RABBITS `SPONSOR'S STUDY NUMBER: 6316-8 STATEMENT OF THE STUDY DIRECTOR This final report accurately reflects the raw data obtained during the performance ofthe study. No significant 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 principlesof good laboratory practice occurred that affected the quality or integrity of the study. Z "Cie n-gay 99 Raymond G. Yor .D., DABT Associate Director of Research and Study Director Date 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 Standard for Safety Studies on Drugs, MHW Ordinance Number 21, March 26, 1997. c. European Economic Community (1989). Council decision on 28 July 1988 on the acceptance by the European Economic Community of an OECD decision/recommendation on compliance with principles of good laboratory practice. Oficial Journalofthe European Communities: Legislation. 32(No. L 315; 28 October): 1-17. 004175 APPENDIX | QUALITY ASSURANCE UNIT FINAL REPORT STATEMENT 00417 '",2P. RIMED ICA 418-010:PAGE I-1 Argus30R5esSehaerecHhhoyrLsDarhbioavrmea:,toSPruiAieds1i,9n0Ign4cA4. TeleTeplheofnaex:: ((221155)) 444433--88751870 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 418-010: Oral (Stomach Tube) Developmental Toxicity Study of N-EtFOSE in Rabbits Sponsor's Study Number: 6316.8 The draft protocol for this study was audited 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 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 10 AUG 98. Critical phasesofthis study were inspected four times; study information and raw data were audited twice (see tables 1 and 2 for dates and 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 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 between 14 DEC 98 and 23 DEC 98, for revisions requested by the Sponsor 07 JAN 99, and for finalization on 11 JAN 99. 004177 418-010:PAGE I-2 This study was conducted according 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 Community (1989) council decision on 28 July 1989 on the acceptance by the European Economic Communityof an `OECD decision/recommendation on compliance with principlesof good laboratory practice. Barbara J. rson, BA. Director of Operations and Compliance Date Heather L. Rabuttino, M.S. Quality Assurance Supervisor and Principal Auditor Date 004178 ' TABLE 1 CRITICAL PHASES INSPECTED 418-010:PAGE |-3 TestArticlePreparation Date of inspection: 31 AUG 98 Date results reported to the Study Director and Management: 31 AUG 98 Test Article Administration - Gavage Date of inspection: 01 SEP 98 Date results reported to the Study Director and Management: 02 SEP 98 Blood Collection Date of inspection: 17 SEP 98 Date results reported to the Study Director and Management: 18 SEP 98 aesarean-Sectioning Date of inspection: 21 SEP 98 Date results reported to the Study Director and Management: 24 SEP 98 004179 TABLE 2 418-010:PAGE I4 RAW DATA AUDIT(S) `The following study information and raw data were audited on 110CT 98, 13 OC98Tto 17 OCT 98: Protocol List of personnel and computer operator codes. Error codes and codes for clinical sign observations. Animal receipt, randomization, and acclimation. Veterinary examination. In-lfe transaction record. Feed consumption. Caesarean-sectioning. Matemal gross observations. Fetal gross observations. Fetal fixative assignment. Fetal visceral examination. Fetal skeletal examination. Tissue packing lists. General comments. Study maintenance records. Temperature and relative humidity reports. Feed and water analyses. Edit requests. Dosage volumes. Data review page. Blood collection data and packing lists. Liver weights. Deviations. `The results of this audit were reported to the Study Director and Management on 19 OCT 98. The following study information and raw data were audited on 27 OCT 98: Vehicle receipt, preparation and use. Test article receipt, preparation and use. Test article packing lists. The results of this audit were Management on 29 OCT 98. reported to the Study Director and 004180