Document ZgMELnyzQRBjw5RdXKGaB8wp

Co AR 236-0327 Draft Report 5-Daily Dose Dermal Absorption/Toxicity Study of T-6684 in Rabbits -- a S---- soos: C5801 Sponsor: 3M St. Paul, Minnesota DRAFT FINAL REPORT : Study Title: 5-Daily Dose Dermal Absorption/Toxicity Study of T-6684 in Rabbits Author: F. Bud W. McDonald Study Completion Date: July 11,1997 `Testing Facility: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Testing Facility Project Identification: Covance 6329-200 Page 1of 52 = 005802 QUALITY ASSURANCE STATEMENT (To be provided in the final report) CCoovmancees6329-200 * 005303 -_-- cCoovamncee 6s329-200 STUDY IDENTIFICATION 5-Daily DoseoDferTm-a6l68A4bsionrRpatbiboint/sToxicity Study Test Material Sponsor Sponsor's Representative Study Director Testing Facility Study Timetable Study Initiation Date Experimental (n-life) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6684 3M Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St.Paul, MN 55133-3220 Roger 3M G. Perkins, PhD, DABT Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 (612) 733-3222 F. Bud W. McDonald Covance Laboratories Inc. P.0. Box 7545 Madison, WI 53707-7545 608.242.7901 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 April 18, 1997 May 5, 1997 June 2, 1997 (Date final report is signed) (Date final report is signed) 3 0C5804 -_-- cCoovmancme 6e329-200 KEY PERSONNEL Acute Studies F. Bud W. McDonald Study Director Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administiative Supervisor Laboratory Animal Medicine Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD `Anatomical Pathologist Toxicology Support . Kathy Myers Manager Deborah L. Pirkel/ JSuapcekrSveirsfoorrst Necropsy Calvin L. Horton Supervisor Quality Assurance Sherry R. W. Petsel Manager : " 0053505 - CONTENTS cCoovamncme 6e32n9200 IAEA. cevrcensernsserssersestscessmmmss msees --------------d `S10rage and RELENtiON....vvvssvvsssssersmntmssssssssssssssssmnsnonnn 10 Safty PIORIIONS...coucenmrmmsmmemmsmmmmmsesnsmmmmm------------------"ily A SSeHlUeAcYtiD on OfTest AE RIMALS........S ..uussussssssI smssssssssssssG ssssmessmssssN ssssssssssssss] sssesssesess1e Justification for Species Selection ........wuvmvrsuussessssssssssssmsmsmsssesssssssssosennec] 1 Preparation OfEXPOSUIE ATE........c.rweresnssnesmssmsssssssssssnsssmsssee 2. D038 AUSHEION c.ucnssuensersssummummmmmmsmirenenssssiesmmemmmmnmesmwnenese2 ne Reason for Route of AAMUNISIALON.........c.cvvversserssssssssssssssssssmsssmsssmsssssssssseseses 13 ODSErvations OfARIMAS .......o.vvussssmssmsssssssssssssessssssssssessssssssssssssssssnssn 13 BliooidsSsampD le Collectm ions/ShiPMeN--t...--...--v.vv--svv--esc--ssss--sss--ssss--srs--sss--nsss--sss--ssss--sss--sss--ssns--sn --13 ShipmentofBile and TISSUES ...vo.vvvuvvvscssrsssrmsssssssssmsssmssmsssssssssssssssssssssssssssss1ess Location ofRaw Data, Records, and Final REPOTt.........uwewsmsmmssmssssssrsssrsrerns 14 : 005806 cCoovanmcem632e920n0 BClOiGniYcaWlEOIBGLSSEIc VLONS. vv eerrssmrs esereeeoenssssoeososne 1513 DISCUSSION css16 TABLE 1 2 I1n0diivviidduuaall CaInndIMCaelanSIBoGdSy Wv eighe (8). ro 18 19 3 Individual Dermal IITitation SCOTES =.........ooevruvvemsserssssssssssssssssssmsssssssssesseess20 Group 1 (Control) - Dimethyl Sulfoxide (0.0 mg/Kg/day).......ovverrerrerrrrrrre2n0n. 4 InGdiOviPdua2.l DTe-r6m6a8l4I(F3F.i0taItiKoGn /SCAORTYES) rrr 21 =..........ovsuvmsmusesssssssrsmsssssssssssessseessssnsesnn 2] Individual Animal PathOlOgY Dat... 23 APPENDIX Bc.sovsssssssssmsmismssmsssssssssssmssesssmsmsmn35 PROMOCOI DEVIRHON .rrrrrnesmomsosetseeereeeeeeons 36 Amendment No. 1 TO The PrOtOCOL.......vuvvvrmsrmssssrsssssssssssssssmsesssssssssssssessssssssnens|S 6 005807 -_-- CCoovmamncee 6e32m9a20n0 SUMMARY "This study was done to assess the systemic absorption/toxicity and relative skin iritancy of T-6684 when applied to the skinofrabbits for five consecutive days. `The study was conducted using three male and three female acclimated rabbitsofthe Hra:(NZW)SPF strain for each treatment group. TT Group Comwol DoseLevel _ Numberof Animals_ Test Material (mg/kg/day) ~ Males Females 1 (Control) Dimethyl 0.0 3 3 Sulfoxide 2 T-6684 3.0" 3 3 a Administered ata dose volumeof0.1 mL/kg ofbody weight, The backofeach rabbit was clipped free of hair and a single dose of therespective control or test material was administered to the skin ofthe rabbits for 5 consecutivedays. `The treatment sites remained intact. The areaofapplication was covered with a gauze patch secured withpapertape around al edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing for each approximate 23-hour exposure period. Clinical observations were conducted predose on Day 1 only and at approximately1,2.5, a`nAdddi4tihoonuarlscalfitneircaelacobhsceornvtartoilonosrwteesrtemcaotenrdiuaclteaddmdianiilsytrtahteireoanfotenrDtahyrsoug1 hthDraoyug2h9.5. Mortality checks were conducted twice daily on Days 2 through 29 (a.m. mortality check only on Day 29). Body weights were determined on Day -S for randomization purposes, before the first contorr toeslt material administration (Day 1), and at the scheduled sacrifice interval (Day 29). The initial dermal irritation reading was made before the first controlortest material administration (recorded as the Day 1 reading). Subsequent readingsofdermal irritation were made after each patch removal on Days 2 through 6 and on Day 13. A blood sample (approximately 4 mL) was collected from each animal on 7 005808 Covance 6329-200 Days -3,8, 15, and 23. In addition, at the timeof necropsy (Day 29), approximately 20 to 40 mLofblood was obtained from each control animal and approximately 20 mL of blood was obtained from each Group 2 animal, All samples were centrifuged and separate samplesofserum and cellular fractionswere obtained and sent frozen on dry ice to the Sponsor. On Day 29, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated and necropsied. The whole liver, bile, an approximate 1-cm x 1-cm sectionofthe dermal application site, and both kidneys (collected as one sample) were collectedfromall animals, weighed (volume only determined for bile), and sent frozen to the Sponsor. Applicationof T-6684 did not result in any test material-related changes in body weight gein or macroscopicfindingsat necropsy. All animals appeared normal throughoutthe studywiththe exceptionof one Group 1 animal and two Grou2p animals which had decreased feed consumption for one to three days within the first nine daysofthe study. Slight dermal irritation was observed in one control animal while slight to moderate dermal irritation was observed in the other five. control animals treated with dimethyl sulfoxide (DMSO). In the animals treated with T-6684, slight dermal irritation was observed in one animal while slight to moderate dermal imitation was observed in the other five animals. No macroscopic lesions were seen in any of the animals at necropsy. 8 005809 -_ coCvovmanccee6m32s9a20m0 OBJECTIVE "The objective ofthis study was to assess the systemicabsorption/toxicity and relative skin iritancyoftestmaterials when applied to the skinofrabbits for five consecutive days. REGULATORY COMPLIANCE `Thisstudywas conducted in accordwaitnh cthee United States Food and Drug. Administration Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis ofthe test material mixtures for concentration, homogeneity/solubility, and stability was not conducted. All procedures usedinthis study were in compliance with the Animal Welfare Act Regulations.Inthe opinion ofthe Sponsor and study director, the study did not unnecessarily duplicate any previous work. All procedural times presented in this report fall within the acceptable ranges as specified in the Covance Laboratories Inc. (Covance) Standard Operating Procedures (SOPs). `TEST AND CONTROL MATERIALS Identification `The materials were identified and described as follows: Tdentification T6684 (test) DMSO (control) Physical Description off-white liquid Clear colorless liquid Dimethyl sulfoxide was manufactured by Mallinckrodt Chemical. 005810 _-- CcCoovvanmcee s63m2p92a0m0 Purity and Stability `The Sponsor assumes responsibility for test material purity and stability (including under test conditions). Analysisofthe test material mixtures determinations for concentration, homogeneity/solubility, and stability was not conducted. The purity and stabilityofthe control material were considered to be adequate for the purposesofthis study. Storage and Retention `The control and test materials were stored at room temperature. A reserve sampleof each the control and test materialswere taken and stored in afreezer set to maintaian temperatureof -20C Covance for one year. 10C. The control material reserve sam The test material reserve sample will be ple wil sent to l be the retained Sponsor. at Any `unused test material will be returned to the Sponsor. Any `remaining control material may be used for other testing and will not be discarded after issuance ofthe finalreport. Safety Precautions `The control and test material handling procedures were according to Covance SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbitsofthe Hra:(NZW)SPF strain were received from Covance Research Products Inc., Kalamazoo, Michigan on April 16, 1997. Housing After receipt, the animals were acclimated fora period of at least 7 days.During acclimation and throughout the study, the animals were individually housed in suspended stainless steel cages. Environmental controls for the animal room were set to maintain a 10 005811 -_-- coCovvamneces63m2e5.a20n0 temperature of16 t0 22C, arelative humidityof50% +20%, and a 12-hourlight/12-hour dark lighting cycle. In cases where variations from the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet `The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed. There were no known contaminants in the feed or water at levels that could be expected to interfere with or affect the resultsofthe study. Selection of Test Animals The animals were identified by animal number and correspondieanrg tag and were placed into study groups using a stratified body weight randomization program. The randomization body weights were obtained on Day -S. Study Design Animals weighing from 2,067 to 2,500 g and approximately 15 weeks ofage at initiation of treatment were placed into the following study groups: --GroGrwoupp 1(Control) 22 Control Dose Level DoseVolume _NumberofAnimals TeTsetsMtaMtateerriiaall _(m(gmg//kkgg//ddaayy)) (m_l(mLLi/kkgg//ddaayy))~MaMalleess FeFemmaalleess Dimethyl 00 Sulfoxide TTe66e84s 3300 01 3 3 e 01 1033 0033 Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animalofchoice because ofthe large amountofbackground information on this species. u 005312 PROCEDURES Covance 6329-200 Preparationof Exposure Area On the day before the first control or test material application, the back and, ifnecessary (to obtain unblemished skin), the flanksofeach rabbit was clipped freeofhair with an electric clipper. Theclipped area made up approximately 20%ofthe total body surface area. The test sites (intact skin) were inspected for interfering lesions, initation, or defects that would preclude the useofanyofthe animals. The animals were clipped as needed throughout the study (Days 3, 6, 13 and 29). Dose Administration All animals received five consecutive days of administrationofthe respective control or test material. The first dayoftreatment was designataesd Day 1. Group 1. An individual dose was calculated and measured based on each animal's body weight on the first dayoftreatment. The control material (dimethyl sulfoxide) was applied to the test iteat a dose volume of0.1 mL/kg in athin and uniform layer. Group 2. An individual doseofthe test material mixture (T-6684 and dimethyl sulfoxide)ataconcentrationof 30 mg/mL was calculated and measured based on _ ach animal's body weight on the first dayoftreatment. The test material mixture was applied to the test site at a dose volumeof0.1 mL/kgofbody weight in a thin `and uniform layer. Eachareaofapplicatiion Groups 1 and 2was covered with a4-ply 5-cm x S-cm gauze patch. Each gauze patch was secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape toprovidean occlusive dressing. Collars were, used torestrainthe animals during each approximate 23-hour exposure period. One Group 2 malewas found without its collar on at the a.m. mortality check on Day 3. The collar was placed back on the animal at this time (see protocol deviations page). Approximately 23 hours after each control or test material application, the restraining collars and patches were removed and any residual material removed from the application sites using tap water and disposable paper towels. 005813 Reason for Routeof Administration `The dermal route is a potential routeofexposure in humans. Covance 6329200 ObservationsofAnimals Clinical observations were conducted predose on Day 1 only and at and 4 hoursafereach control or test material application on Days 1 approximately through 5. 1, 2.5, Additional clinical observations were conducted daily thereafter `through Day 29. Mortality checks were conducted twice daily on Days 2 through 29 (a.m. `mortality check : only on Day 29). : Body weights control or test wmaetreeridaelteadrmmiinniesdtroantiDoany(D-5ayfor1),raannddomatiztahetisocnhpeudruploesdessa,crbieffiocreeitnhteerfviarlst (Day 29). The inital dermal iritation reading was made before the first control or test material application according to the Draize! technique (recorded as the Day 1 reading). Subsequent readingsof dermal irritation were made within 30 to 37 minutes after each patch removal (Days 2 through 6) and on Day 13. Blood Sample Collections/Shipment A blood sample (approximately 4 mL) was collected from a marginal ear veinofcach animal on Days 3, 8, 15, and 23. In addition, at the timeof necropsy (Day 29), `approximately 20 to 40 mL of blood was obtained from each control animal and approximately 20 mLofblood was obtained from each Group 2 and 3 animal via the `posterior vena cava. All samples were stored at room temperature until centrifuged. After centrifugation, separate samplesof serum and cellular fractions were obzined and stored in afreezerset to maintain a temperature of -20C + 10C until shipped to the Sponsor. The serum and cellular fraction samples obtained on Days -3, 8, and 15 were shipped frozen (on dryice)to the Sponsor (James D. Johnson, 3M E.T. & S, Bldg, 2-3E- 09, 935 Bush Avenue, St. Paul, MN, 55106) on Day 16. The serum and cellular fraction samples obtained on Days 23 and 29 were shipped to experimental (in-lfe) dateoftermination in the same the Sponsor the day after the mannears the samples obtained prior to Day 23. The Sponsor is responsible for the retention and dispositionofthe 3 005814 samples. Covance does not accept any responsibility for the analysisofthe samples collected in this study nor are these results presented in thisreport. Pathology At terminationofthe in-life experimental phase (Day 29), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, `exsanguinated, and necropsied in random order. The sites ofcontrol or test material applicationwerewashed with warm tap water before the necropsy procedure. All animals were subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. The whole liver, bile, an approximate 1-cm x 1-cm sectionofthe dermal application site, and both kidneys (collected as one sample) were collected from all animals, weighed (volume only determined for bile), and placed ina freezer set to maintain a temperature of -20C +10. After necropsy, the animals were discarded. Shipment ofBile and Tissues One week after in-life experimental termination, the tissues (whole livers, dermal application sites, and kidneys) and bile were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.T. & S, Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with documentationoftheir corresponding weights or volumes. The Sponsor is responsible for the retention and dispositionofthe samples. Covance does not `accept any responsibility for the analysisofthe samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyseswererequired bytheprotocol. Location of Raw Data, Records, and Final Report The raw data, records, andan original signed copy ofthefinal report will be retained in the archives of Covance in accordance with Covance SOP. ; 005315 RESULTS Body Weights cIonndtirvoilduaanlimaanld,meexhainbibtoeddyawweeiigghhtts laorsesinfrToambrlaen1d.omAilzlaatniiomnatlos,inwiittiahttiohne (eixnc-eipfte)iownhoifchone an be atributed to the acclimation ofthe animals to restraining collars conducted for 3 days (approximately 21-23 hours/day) before the inital control and test material administration. All animals gained weight from Day 1 to Day 29. Clinical Observations Individual clinical signs arein Table 2. All animals appeared normal throughout the study withtheexceptionof one Group 1 animal and two Grou2p animals which had decreased feed consumption for one to three days within the first nine daysofthe study. Dermal Irritation . Individual dermal irritation scores are in Tables 3 and 4. Slight erythema, edema and fissuring reactions were observed in one control animal while slight to moderate erythema and edema and slight desquamation reactions were observed in the other five control animals treated with dimethyl sulfoxide. OFthese five control animals, two animals also exhibited slight coriaceousness reactions. In the animals treated with T-6684, one exhibited slight erythema, edemaand fissuring reactions, one exhibited slighterythemaand slight to moderate edema reactions, one exhibited slight to moderate erythema and slight edemaand fissuring reactions, one exhibited slight to moderate: erythema and slight fissuring reactions, one exhibited slight to moderate erythema and edema and slight desquamation and coriaceousness reactions, and one exhibited slight to moderateerythema and edema and slight desquamation, coriaceousness, and fissuring reactions. is 005816 DISCUSSION Covance 625.200 `The acute systemic absorption/toxicity and relative skin irritancyof T-6684 was evaluated in consecutive male days. and female albino rabbits when administered Applicationofthis material did not result in dermally for five any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared normal throughout the studywiththe exceptionofone Group 1 animal and two Group 2 animals which had decreased feed consumption for one to three days within the first nine daysofthe study. Slight dermal irritation was observed in one control animal while slight to moderate dermal irritation was observed in the other five control animals treated `with dimethyl sulfoxide. In the animals treated with T-6684, slight dermal irritation was observed in one animal while slight to moderate dermal irritation was observed in the other five animals. Bs 005817 -_-- CCoovmamncce 6e32m9a20m0 SIGNATURE F. Bud W. McDonald Date: Study Director Acute Studies REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure), In: Appraisalof the Safety ofChemicals in Foods, Drugs andCosmetics - Dermal Toxicity, Association of Food and Drug Officialsofthe U.S, pp. 54-56 (1959). 1" 005818 -_-- Ccoovavnmcee6s32m9a-2m00 Tabl1e Individual and Mean Body Weights (g) Males `Animal Random Day NuNummbbeerr iezaattiionn 11 2299 Females -- "Animal Random Day NuNummbbeerr i-ziaztaitoonn ~T1 --2359 Group 1 (Control-) Dimethyl Sulfoxide (0.0 mg/kg/day) F601 2315 F63012 2430 F63013 2447 2277 2,529 2,500 2766 2388 2708 F017 2424 F63018 2380 F63019 2263 2333 2755 2277 2783 2244 2619 Mean 2397 2388 2668 Mean 2356 2285 2719 Group 2 - T-6684 (3.0 mg/kg/day) F63014 2404 F6301S 2485 F63016 2447 2402 2722 2444 2754 2390 2805 F63020 2262 F63021 2438 F602 2395 2,067 2,506 2429 2,804 2320 2735 Mean 2445 2412 2760 Mean 2365 2272 2,682 18 005819 -_-- CCoovvaanmccee6s3i299-a20000 Tabi IndividualCline Signs TT Animal owl Dwr bey owas How Wow ow Wow How Davy SNm mSbexOObewenrvrinon TTBE 4 ITNHTTITDBAITIHBTTTTHITTT ETTT o5n0T HeSs Group 1 (Con-tDirmeothly Sulfide (00 mkgiday) FOOL M Apetoomal SSS LLL SILLS sy, L consumpion d F012 M Appersioomsl 4 0 SSS L LLL ts ER ARR s s, E N, . FN F Appeadoomal 4 LLL L SLL SSS 0 sts s s, FOF Apesdoomil 40 SSL L LIL ts s s, FSO F Appeusinomal 4 LLL LLL LIL SIL Ls srs, Group2-T.(360me6rk8en4) A A RR FOS M Appessdromal 0 LLL S IL ss ss N N, s s, Demaedood Corsomption + ses sassy L LLL Epe RR R NN AA FOO F Apewtromal SSL L LL Ss ss ss, Dceowrmseumdpfiooond - ses so / Uy oy LL FON F Apeadoomal 40 SILLS LIS s sts ss ss r _F6--02 F Appessdromal LLL L Ly L LIL SI tr ss, 72 CCoonntiionnmtieeviddent. 19 005820 -_-- CCoovanmce e6329s-200 Table 3 Individual Dermal Irritation Scores Group 1 (Control) - Dimethyl Sulfoxide (0.0 mg/kg/day) -- DDemeemlaRleRaecatcioonn Males Study Day 1"1 22 33 44 55606 113 Animal No. F63011 Erythema 0001 22 2 Edema 0001 22 2 Atonia 0.000000 Desquamation 000000 1 Coriaceousness ~~ 0 0 0 0 0 0 0 Fissuring 0000000 Animal No. F63012 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 011111 0000 0 1 1 0000000 0000000 0000000 0000001 Animal No. F63013 Erythema 0111121 Edema 000 1 22 1 Atonia 000000 0 Desquamation 00000 01 Coriaceousness ~~ 0 0 0 0 0 0 0 Fissuring 0000000 Females StdyDay 11223 34 45 56 60 13 Animal No. F63017 001 22 21 0001 221 00000 0 0 00000 01 00001 00 0000000 Animal No. F63018 01111 2 2 0111 2.21 0000000 00000 01 00000 10 0000000 Animal No. F63019 0111 22 2 01 11122 0000000 00000 01 0000000 0000000 " 0 05821 -_-- coComvmanccee e63n29s-a20m0 Table 4 Individual Dermal Irritation Scores Group 2 - T-6684 (3.0 mg/kg/day) -- DeDmeramlaRleRaecacttiioonn Males TT StiyDay 112 2 33 44 55 66 113 Animal No. F63014 Erythema Edema 00 1 1111 001 1220 Atonia Desquamation 0000000 0000000 Coriaceousness ~~ 0 0 0 0 0 0 0 Fissuring 0000000 Animal No. F63015 Erythema 011111 Edema Atonia 00 1 1 111 0000000 Desquamation 0000000 Coriaccousness ~~ 0 0 0 0 0 0 0 Fissuting 000000 1 Animal No. F63016 Erythema Edema 01111 21 0000 1 1 1 Atonia Desquamation 0000000 00000 01 Coriaceousness ~~ 0 0 0 0 0 0 0 Fissuring 0000000 Females Study Day "T 12233 4455 661133 Animal No. F63020 001 22 21 000 1 221 0000000 00000 01 00000 10 0000010 Animal No. F63021 01 222 22 01 11 2 21 0.0000 00 00000 01 00000 10 0000000 Animal No. F63022 00 1 1 221 00 1 11 21 0000000 0000000 0.0000 00 0000001 n 005822 -_-- CoCvovnacncees6s39292-m2000 APPENDIX A Individual Animal Pathology Data 2 005823 es cone g $g a8 a82 ' sensi x [ep-- | aS 28a ropes Coven e300 aS 832 J divin sms strlen uta } SREB BERI R IEE or cu, BO ey PETE sh ontric 8 8 | | EEUmR Te en, 2 PRSi sae, a88g 88 || gg g -- | TT J . &g | | J -- Sndtviont anima achatogy uta g3 | s g | a F-- BEWERIT, EE en BS poneSage,ta sone, $ g & . - 0 cCoovmanmcee6m32a92m00 APPENDIX B PPrroottooccoollDTevPi6a7t9i8on `Amendment No. 1 To The Protocol 35 005536 Protocol Deviation Covance 6329-200 Protocol Actual Procedure Page 7, 7. Experimental Design, B. Dose Administration, (3) Dose Administration, Ninth Sentence: The rabbits will be collared during each approximate 23-hour application period. Animal No. F63016 (Group 2 Male) was found without its collar on at the a.m. mortality check on Day 3. The collar `was placed back on the aniamtathlis time. The paich appeared to have remained in place on the application site `This deviation study. is not considered to have had an adverseeffecton the outcomeofthe. 36 005837 : COVANCE Covance 6329200 costae [rover Sponsor: St.Paul,wMminnesota FroTOCOL TRE Study Ties 5Daily DoseDoeTa4l6kA4basRaernpsioToSvtey Date Api 18,1997 Teng Fay: C3o30nsKeinLsambaonratBooruieisvlacd. Madison, Wisconsin 53704 TastingFacily Projet entiation: Connee 29200 n 005838 -_ CoCovvaanmcee6e3299a-2m00 , Comncessezs2st0 ee STUDY IDENTIFICATION 5-Daily DoseDOefrTm-a6l68A4bsionrRpatbibointsToxicity Study Covance No. 629200 | Test Material Ton Sponsor TMoxicology Services 3MMedCiecnatlerD,eBpadr,tm2e2nt0.2502 SPt.0.PaBulo,xM33N22505133-3220 Sponsors Representative TMRoger G. Perkins, PAD, DABT ToMxeidcioclalogDyeSpearrvtimceenst P30M.CeBnotxer3,3B2l2d0g. 2202.02 St.Pau7l, MmNn$5133:3220 Study Director CF.ovBaundceW.LaMbocrDaotnoraileds Inc. PM.a0d.iBosxo7Wn5,I4553707-7545 082627901 Tesing Facility C33o0v1anKcienLsambaonraBtoourlieesvanrcd. Madison, WI $3704 PrEoxppoesreimdeSnttualdySTtiamneDtaatbele EDrxapfetriRmeepnotratlDTaetremination Date: WWeeeekkooffJMuanye 52,, 11999977 WeekofJuly 14,1997 38 005839 Covance 6329-200 ConTesa2ssn00 1 sSuaiyDoseDermal AbsorpTtoixoeny StinRabybis 2 PTteuoiaslnswthheenysptedsohaerpktoonfdoblitlsyotanvderceloinvseescitinevpnse yof1st . 3. "PRheigisulcsaeotdoRrewygiulClloimbopelnciotnSndtcuacentdeadrdi sGucideolirnenwwiithldheflcowsiongGhtodaLyibsrafroy {Teotbeecoindoitrsforconcntaion, luli, homoge, nd Subily wil [)0) 3C1 Coonsnt (dNDAo)uscedtsdy [) 40CFR160 (EPA-FIFRA) [] 40CFR792 (EPA-TSCA) 1[111)] C5N(9o8t1Nf)oc3ha0Siao(nFnNinNoaol.)a3(4O351EG3CD1)nda$70pMpAaFsnFe) MOH) + aAResiparniaocndsucr,esdInunetpihisocstppriontynotcpoorlfevrhrieeoiSnpwcyaoomnkpnl.iaanncse wdithdhreeAin,iilo dWeylsdoeAsctok 4 TAQhuaeilpitrynoAsssUurtsanicyesccoondr,nawdihhCeofwaelsepLoarbwoirliobneene.a(bCydotneeQeyuSdanydrd pertingPr(oSOPd aod ps oli. 5 TetMaerit A TMenhtncaton Of vhie l-- id 005849 Covance 6329-200 ---- TTraermt . TpouSpaornnsdoiSraatsbsiuymesresponsibilty forpur andsbily determinations (iwfnocrnlcuodnicnegputnidoenr,tebtomcoongdeitnieonisy)A. cSaim,poulnfdeessstamabterinall/yvshiscwleimtixttburee.(s) D. SRtooormagteemperare E. RReeseerrveeessaammplpe)sofcach bitch of cstmate willbs ake or is study. TCmhoiemnptaelisetnonttaeefrmptaerenastierfofps2ihm0pelCoefs+)h1ew0isleaCysu.tlorredeeCdofvoanhceeSipnonsfroereszerrsetfo F. Rneyeunniuosned est materi wil bereedttheSpansion G. ASasfeetqyuiPvreedcbvytoConvsance SOPs andpolicies 6 Control Material A DWiemnetitcSautolnfide (MSO) - B. PChiyes,iccaollDoesrcersipHtaiiond C. pTuorbiedoacnudmeSnatebdibyCovance nformasionfom he spp) D.. RoStoormagteemCponedriattieoens 40 005841 -_ cCoovvanmcee63m29a-20m0 Comme iT5ar2ei0s E. RReesseerrvvesSaammpplleess)ofeach btcof hconttol material wil be ake, "tTohme acointnrosl matteemrpiearlarteusreerovfe s2a0mpCle+s)10wiCll.be stored t Covance na fez set. F. RAentyensteimoninin control materialmaybe usd o tbe esting andwill notbe : iscaded fe sunofthe ia report. G. SAafreetqyuPirreedcabuytiCoonvsance SOPs and polices % Experimentt Design A Animals W SRapbebciites @ HSrta(inNsZSWoIurScPe Covance Research Products Ine. AAgdeust Initiation @ W2e0i0gh3t0aktsInitiation () mNuamlbeesrnadnd6Sfeemxales: INnednitvifdcuaaltinounmbered ear tag Husbindey @ HInoduisviindgual, in suspended ssnlss se cages a 005842 - cCoovnmcse e52n92m00 ComcnTenrseo (6) A{FFoomroosed,annco)n.aaTlcomoemvfpooonodefinLstseouxbinaev lryRnabo iiiebcyndHhaEe 4am5t 2l2i6esPnMIt dewraidriamlyfoamaynesofmorastpieccisfimyis,crSoeorrppshlensdweasesfe omen conan @) Contaminants. inihchs rty. a-- EEnmmviproeonrnmmseerrneatlofo19niC 0o23reC,istovohiiltbe osfSo05m2i05n, 0d 3tdeoeugfoh ifbopwrodskesy.e. The dk xlmybs (0 iAcclTimatdioans B eqSueonne keeseTrhosafAd nveboxiiysfsrweilght siccbriin ocCsorfeos SOPs. An rrroloagcreadimvsinmubivdsiiulgnsreoludypyssowoofien llysicaipilancve,dooyn westi.gThhnidomlmioisnlbs (9) iheJioousres.aetlc,iutffhoorfiSNpoeewccriaZegsetSneaildmeocoWtuaininottneoflbiackgroiundhinsfboremeantiohneonniial of o 005843 -_-- CoCovvamnceee63n2s9-a20m0 | Connce62T8re2s0s0 Fae? B. Dose Administration Test Groups Gp Convol TeaMaidl (DaosgehLeelvael) MuMamlbeesrofAFneimmaalless . (Con) DMSO oor 3 3 2 T684 300 3 3 * Tobe administered ata dose volumeof0.1 mL/kgofbody weight @ POrnetphaerdaatyionbeofforEextphoesnuraelAreesat and contol material pplication, the braabcbkitawnidl,libencelcipepsesdfa(er1ey oobftaaiin uwnblieamniestlheecdthrsiicn)cl,iptpheer.fTlahnkesosfhacvaecdh "aTrheeatwriellatcmoennsttiittutees a(pinptraoctxismkaint)elwyil20b%e ionfstpheectoetdalfobroidnytesruferrfiancgelaersicons, "iTtheiaonnim,aolrsdweiflelcbtes tchlaitppweodualsdpnreeecdleuddethtrhoeuguhsoeoutftahneysotfudtyh.e animals. . ) DAlolseaAndimilniisltlraetcieoinve iveconsecutdiavyesofadministrationofthe arpepslpieccattiivoencsoineteroxlcothedaty. mTahteertiaelstampptleireadltiovtehheiscaemmeixretsupreectwiivlel. be pberedpearseidgfneasthaesdcaDcahyday1.ofThademcinoinsttorlatmiaotne.riaTlhweiillsbtedaapypolfieedsuindmielnuttewdilatl awidtohseDMvoSlOumaeondfa0p.p1limeLd/akga.dTohsee Gvorlouumpe2otfe0s.t1mamtLe/rkiga.l iTlhebdeodsielsutfeodr ttahekeanniomnaDlsayin|bGerfoourpst1heanfdir2stwaidlmlibniesbtarsateidoonnntdheaapnpliimeadl'0btohdeayrweeaiogfht eaxnpd2os)uwriellinbaetchoimnplaentdeulnyicfoovremraeyderw.ithThae4-aprleya o.f0pp-l0imcaxt5io.n0(mGrgoauupzse | poavecrhw.raApllpepdatwcihtehs SwairlanbWersaepcu"raenddwiEtlhasptaoppelrastaptesaprotuonpdroavlieddegaens and oc2c3l-uhsouirveadpprleisciantgi.on pTehreiorda.bbTihsewpirllepbaerecdoltleasrtemdidxutruirnegseawiclhl baeppsroorxeidmaatte room temperature until administration. a 005844 Covance 325.200 Conseoacmso ThReesGoonmfaorfRo teo3fAdtmiensatrraotiionofcxposeinhas. (5) T(`RAee5pmmpocorvvohexaedilmswaoitsfenTnlgeyswc2tl3Mlhalotuerrsiipaalflerbreeoarcpohvtseetosracnnodentsro,sl mcaateelrsiaaml rap.aplicTahwteiiolsnlh,bees eeotnanDacyon2t3lpmoelthscploatloln wiilln pwrtohsewith or C. Observationof Animal 0CllientecsObsoaerosvrantaeishonrstmilloadnmissoionn,laoptpmlaye1o,n25, DuDaalyy'ysa1(maomr.sncmd piomar)tsoifnonlm,yodonlyDtiaySf2i9en. rgOootnirDaocyn3isfmscyo,nbaennixdnigec0ed hin dies he uy rn 2) DS(Reeicaodrohiennmhgeeo:nfadDi1etn.rgmAawalldnIeriorintoaettidoknndtleltcoodrninssdisDoayno1erbdieeemiamlndae -- SVtiudny--D3a0y10134.0nmvinitdsiaadseseclhrpaacihoonmeaanrs(iGsb2-6) osde00d fo (3) F(BoOodony1dW5oemiogivhzDtmsyio2n5,b0odrstte(lehstso ciolnvnlcmeat1 idup)lcion Placa Semple Collins @PeFmridenqssuina(eiayonnimoefDoemy 1.toonDfaosyd3sa.ys15f2o3r.edheaipleoisen termination (Day 29) " 005845 Covance 6329-200 ConnceeTsraets0s Pies (8) BMleotohdosdaomfpCleos l(slppcrotxiimoatne/l4yoNfmuALmnaibcmhea)lrwsillbe collected from marginal ear vein ofall animals st the intervals predose through Day22. bFeroombtheptostfaerroimiorcavncehnaaenicamdvaal, asppraoxicmaitrenlaymi2or0fimbuinodLfccboln.odeiotdidowinll : (fFrpoomessaicbhlceo,ntarpoplroaxniimmaatlelsyac2iftco04ed0omn DaoLfyb2l9.oo(dthevimlaxbiemoubmt.ained bvlooloudmewilplosbseibolbeiwaiillnbeedbfirnomeeda)ch,Graonduppr2oaxniimmaaltesalcyri2f0icmedLonof Days. "cTehnetbrilfouogdesd,amapnldetshwsielplabreastteosreerduamtarnodomcteelmuplearraftaucrteiaonnsdsttohreend in afafcrteieoznesrsoebtationmeaditnhtraoiung2h0DaCy +1150wiCl.lbTehseenstefrruomzeanndoncedlrlyuliacre to stheeruSmpanodnc0esluolatror fwaociwoeneskosbtpariineotdroafine-rifDeatyer1m5inwaitlilobne. sTehnet fterromziennatoinond.yTchee1S0thponsSoprosnsroerswpiantshiibnloenfhoe weeerketaeftnetrioinna-lnfde. dispositionofthe samples. "Theserum and cellular fraction samples will be hipped to: . JSaMmeEsTD..2JoShnson 9Bl3d5g,Bu2s3h.A0v9ene St.Paul, MN $5106 JShaimpemsenDt. Joofhthnesosnamoprlbeiss.altemate will be notified regarding the D. Pathology (1) `UAnnsycahneidmualleddySiancgrdiofriciensgtahned sDtedaythosr sacrificed ina moribund condition awiblnlobremsaulbjiesctweidltl0beanreacbobrrdeevdi.atAendigmraolsss innecarmoposryibeuxnadmicnoantdiiotnioannwdilalllbe a 005846 -_-- CoCovvamneces6m32v9-a20m0 . Connce 29T.o2m0s0 fiir} avneeisnt)hebtliezevdi witthhevseondaicuamvpae,ntaonbdaebxisaalng(uviinaiantfeedc.tiTohneinwthhleemairvgeirn,ablileer a(plpllvicaaitliaobnes)t,eaannpdpbrootxhikmiadtneey1s ccollxec1t-ecdmasseoncetsoiafmotphneledweirlmable cmoolrliebctuenddfcroonmdiltlioanniwmeailgshyeidn(gvodluurmiengonthleysdteutdeyrmoirnseadcfroificieldei,nasnd . `pAlfaecrednenctroop8syf,eetzheersaneitmafolmsawiinltlabienditsceamrpdeerda.tureof20C +10C. @ OScnheDdauyle29d,Stahcearinfiimcaels ill be anesthetized with sodium pentobasital (esxisaaningjueicntaioend,inatnhdesmuabrjgeicntaeldcarnveaib)e,vbilsedtveid ghroessvenneacrcoapvsay, `ecxoammpiuntaetri-ogne.neTrhateeadnriamnaldsomvinlulmbbeenrcsr)oapnsdiadl fsnbrroarnmdaolmteexsewil(lvibae rcecmordseedc.tiTonheofwthhoeldeelrimvaerl,baiplpeliacaltiaonvsiae,ianldaabntabhpplkrioedxni)emyas,te(cIol-leccxtmed abi5loen,easnadmppllaec)edwiilnltboe cforleleezcetreds,twteoimgahiendt(ainva teomopnellryadteuutreermomf2i0needC.or 210C. Afer necrothepansimyal,swillbediscarded. ) "TTihsessueamSpalmepl(eivSrh,ibpimle,ndtermal application st,and Kidneys) collected ~~ waittthhienoscnheedwueleedksaafctreirficcoellweitlilobne. sSeanmfprloezsecnoalnlertyediacu0nstchheeSdpuolnesdor smaacnrinfeirceassatnhdedseamaptlheis farpopmlitchaeblsec)hewdiulllebdessacernifwiciet.h sTnhdeisnamthpelessomaend thiesitceominesSepcotnidonin7.gCw.e4i)g(h6t)s.oThveoSlpuomnessoiril breessphoinpspiebldetfootrhteheperrestoenntion anddisposiion ofthe samples. E. SNtoatsisattiscialcaAlnaalnaylsyesses ae required " 005847 Covance 6:29:200 . [-- oenn 5 Reoporttro cin those ems sd low il be bite. DDeessecrppiioonnoofftehetotst asned cmontol materi FDoreesiofrceperimenal ion snd raion TTDaebseuelitatpitaoonnooofffmmaoenratyasltibtosydydcahtalbsy seexooansd idaonnside olGeonvel ove GIGrroonssdppiaittlaoogssy rTeopnrsewicghtundbleevlSumsDirt) 5. LOnorcaeytilondoiftnRagorwoDpapaee,heReescaotarn,ddsw,filRlresoeerlsveSpabmiplCecoo)f,vehaeendfneFltionousrR.esorWthaecning ninlluriengprthosceommspleis,ecdobnollotielable rraensdsnp)a,rchlilveosnogfCinol pveefsor TTroeonadtfoetroWeipyloleealCooaflglheodew.airgTcsoeimSnsogonroefdrhemyeFtdoacletspiril.vbOeenfseecymoa0hsSmptagresdof. + SopoeraCaorfettwei1Al1lrhSvpeconofsrooe3rdch6osem1 apveedCoovtancoe diesdposCeoovfheenmofltchsores DoPrsoteopcorl iansdoprnotoecoolnasmendments oBoidytweriigshs DoRsarekoimiinsitisornsidoana AOnovevlaionpsloy records SShiippiengcesltes SFiyreeaprpoorrtgnesnd ap) 005845 a -_ CoCovvaanmcee6s39292-20000 * Comme 62TPa5rg2ee0m0 a"Trhcehifvoeldlowwiitnhgtshuepspotrdtyindgatrse.cords will be retaineadt Covance butwill not be AWantiemraalnarleycesiipstraecccolridmsation records ARenfiimgaelraotoormantedmpferreaetzuerteeamnpderhautmuiredirteycorredcsords Instrument calibration andmaintenance records 005849 a PROTOCOL APPROVAL Conceerrn neh SCpolnsforsFirRceepiriee,stnPieDi,v,eABT 55 F7SueBdoytdDWi.scNDoenrDiorcablodnald CAocvaencSeuLeabsoratories Tc. . SReQparuebaseiintAantsiesveo7c UBnitons'] Coane Labrie FEZ2 . 4Ba-e 18-97 wir07 Bue 005850 Covance 6329200 tachment 1 ScoringSale for Acute Dermal Resctions s--T--ren Furie Eythe0m1sNogne 23 Msoowdeee Edema0 None: 231"..SeMSvioegdrheesriteel(dsyepmdeorrcseeppttrihobnliem|totmwlyeyll|dmefmi)ned by definite iio) Atonia 1. SNioingeh light impaioment of clic): 2. Modeste Glow eum nomal) 3 - Marked (no elasticity) Desqua0rmNtoionne 251.- "SMlMaiagrdhkteed(rsGlir(gohatnloseucsanlcaiendgd)fkaeksin)gwithdenuded ares) Corisc0e"oNuonenses 2.SMigohrttGeeertshenrptelxtusre)) 3 -Marked (toughand brittle) : Fisuri0n1g. NSoingeh dfn cacks in demi) 25. MMaordkeesdt(c(arcakckwiftnhdbeerstii)ng) 50 005854 COVANCE. cCoovamncme 6e329-200 comes ten AMENDMENT NO. 1 TO THE PROTOCOL PROTOCOL TPs 5 Daly Dose DoerrTmaa6l8A4bisnoRrapeinsToiety Sey Sponsor TTMoVniecodlogDy eSepraierst S70M,CBenotxer3,3B2i0g T2026.02 St PaulMN 55133-3220 Conse 9200 Testing Paci C5o30n1nKiLnasmbaonrBatloroifte.s MadisoWnI $3700 Sponsor's Representative Study Diecor et Roe ger Gr. Perkir ns, PAD-- , DABT-- . ------.--Dud W-- . MeDo-- reld -- Ths amends mci heFollowingpornsofbe proacl: Effective ay 20,1997 rTcohoenedaulbcsilaneamDnoacyeot2f2hMlDeaeedysr2abniDhasoytla1yn0,Dinmda2i.dllhyDeayFo2l2laowitnhgeoswosdss.ioTnosaodefasing 1 I. aCPoanlggleee8cr,io7n.sE,x6p)erFirmeeqnuetnacyDe.siMgon,diC.sOibsseervcaotinonwoifhAbneimFaoll,i(o0sDnooid iSanmple adePmmriienstisesiaoainnoy(tnDitamoyeD2ra9oym1)p. oon Dfaouyrsdy1s,2be3f.orantdhtepiedosie: n . st 00585, - OOOO cCoovmanmcee329-200 Amendment. 1 Conse 63T29e.r20e0 Pre 2 CPPoaalgglrteaispoh.nsiE,nx(hpi)esMisemctcihoootndlwoiDfteChosliolgee,cftCoi.olnlOoNwbuimnsbaeenrrdovforafAoAntianimpaioe.,nlc)hiBlnooed gSoamrple mBalrogoidnaslamcpalrevs (iapopraolxiamnaitmeallys4 mtLeeatche) wlill rbeocotllecpteedofrnoymDa.a 21. . PROTOCOL AMENDMENTAPPROVAL SRpoognesror's Rfeipnrdsniadi,veDAO wm . =Glo fio FBuhedciolorne) one) AScuutde DSiucdesr Covance Labor ne oi5e21-97 TeQpueasy Assurance Uni Covance Laboratories. Bie5.2007 . 005853