Document 8XjZ072Y6r7vLrbd7rbv58EB

. T2632) Ee RREPORT i L TTwweenn tyty---eeiigghhtt---ddaayy R Reeppeeaatted DDoossee O Orraall i TTooxxiicciitty y SSttuuddyy ooff SSaammppllee D D--1I iinn R Raattss q | (BMRI143C) Bio-Medical Research Laboratories Bio-Medical Research Laboratories CCoo.,.,LLttd d.. Ii) NECEIVEAl i Wisma ||! -- wi " 2797 Exhibit 2797 aw otssss? 3M MN01649987 22779977..00000011 REPORT T Tww enetnyt--ye-eigihh t--t--ddaayy RReeppeeaatteed d D Doossee OOrraall T Tooxxiicciitty y S Sttuuddyy ooff SSaammppllee D D--1I iinn RRaattss ((BBM MRR114433CC)) -~TTRRAAN NSSLLAATTIIO ONN-- | BME2,) -- Pure free F Feebbrruuaarryy 11866,, 11999933 Bio-Medical Research Laboratories CCoo..,, L Lttdd.., 22779977..00000022 3M MN01649988 PREFACE BY TRANSLATOR - PREFACE BY TRANSLATOR This fs tol temslasion of the crigiaal seport dn Japenese TLhanigsuagei.s aGn thtstotal ork, 1 have translation opfaidthecloosreigaitntaelntiroenportto tirnansJfaepranetshee floanntguafgaec.ts Onand ththise wcoorrk,recIt awn In have paid heclose teaxttt,entcioonntirtaointgrasnlsefnecrittihce sates to real facts tahned ortihegincaolrrseucttheme,antlhaeg sicnudythdeistuexctt,o Ke. Michi confirming Otsuke scientific matters to the original auther, the study director Mr. Mlchio Otsuka. sett 32, 109 April 23, 1997 remsacerf]pA a Yokoo Toten Makoto Tennlchl [-- General Manager Blo-tadtont Research Labortories Go. 14s. Rlo-Hedlcal Research Laboratories Co., Ltd. 22779977..00000033 S_MNotedssss 3M MN01649989 CCOONNTTEENNTTSS - sSrTaATTaErMEENNTT Oo~N GGLIP CcEeRRTTIIFPIICCAATTIIOONN sSItGoNmATTiURReE eGEmNEmRAaL MmArTrTeERRsS TTiittllee // SSttuuddyy NNuasmbbeerr // PPuurrppoossee // GGLLPP AAppppllliccaaittoonn // TTeessttiinnEg GGuuiiddeelliinneess // SSppoonnssoorr / / TTeessttiinngg FFaacciilliittiieess // RReessppoonnssiibbllee PPeerrssoonn / / DDaetteess iinn the the SSttuuddyy / / RReetteennttiioonn ooff RReeccoorrddss aanndd SSaammpplleess sSUoMmMnAaRyY MMAATTEERRIIAALLSS AANDD MMEETTHHOODDSS 1I.. TToesstt SSuubbssttaannccee 2. Preparation of the Desing Solution 2. Preparation of the Dosing Solution 3. 3. LLaabboorraattoorryy AAnnitmaaallss aasndd RReeaassoonn ffoorr CCh~ooiiccee 4. 4, AAniimnaal HHoouussiinngg 5. dentitization of Animals 5, Identification of Animals 6. ITdaemnmteiifsiccaattiioonn of CCaaggeess 6. of 77.. TToesstt PPrroocceedduurreess 7-1 Dose Lovels and reason for selection 7-i Dose levels and reason for selection 17--22 Humber Number of of snisals animals 17--33 RRoouuttee., ffrreeqquueennccyy,. eanndd dduurraattiioonn ooff aaddamiinniissttrraattiioonn 1-4 Mathod of administration 7-4 Method of administration 7-5 Grouping 7-5 GroupinE [3 22779977..00000044 33MM_MMNND011664499999900 8. Paraseters assessed - 8. Parameters assessed 81 General condition 8-1 General condition Clinical signs / Body weight / Food consuaption Clinical signs / Body weight / Food consumption 88-22 LLaabboorraattoorryy tteessttss Hematology / Biocheatstry / Urisalysis Henatology { Biochemistry / Urlnalys~s 8-3 Pathological exastnaiton 8-3 Pathological examinaiton Necropsy / Organ weight Necropsy / Organ weight 8-4 Histologicol examination 8-4 Histological examination 5. 9. Statistical Statistical AAnnaallyyssiiss Resurs RESULTS 1. Mortalsty 1. Hortallty 2. Clintenl sSiiggnnss 2. Clinical 3. Body Haight 3. Body Nei~ht 4. Food Consumption 4. Food Consumption 5. Hematological Findings 5. Hematological Findings 6. Biochestcal Findings 6. Biochemical Findings 7. etaalysts 7. Urinalysis 5. Pathological Findings 8, Pathological Findings 81 Organ weight 8-1 Organ weight 8-2 8-2 MNaaccrroosaccooppitco fflinnddlinnEgss sat% 53 Biatological findings 8-3 HlstoloElcal findings nmeaccrrooppseyy `DDIiSsCcUuSsSsIiOoNn aANpD CcOoNnCcLtuUsSiIoOnN FFIIGGUUREES AANDD TTAABBLLEESS ((GGrroouupp mmeeaann ddaattaa)) Figure 1. Changes in meas body weight Figure 1, Changes in mean body weight an 22779977..00000055 33MM_MMNNO011664499999911 Tabla 1. Group incidence of clinical signs - Table 1. Group incidence of clinical signs Table 2. Body weight Table 2. Body .eight Table 3. Food consumption Table 3. Food consumption Table 4. Hematology (in comnection with REC) Table 4, Hematology (in connection .ith RBC) Table 5. Hematology (in conection wih NEG) Table 5, Hematology (in connection with WBC} Table 6. Clinical chentstey Table 6, Clinical chemistry Teble Table 77.. UUrriinnaallyyssiiss Table 8. Organ weight (Absolute) Table 8. Organ .eight (Absolute) Teble 3. Organ weight (Relative) Table 9. Organ .eight (Relative) TTaabbllee 1100.. GGrroouupp ifnacciiddeennccee ooff macroscopic macroscopic ffiinnddiinnggss Teblo 11. Group tacidence of aicroscopic findings Table. 11. Group lncldence of microscopic flnd~ngs APPENDICES (Individual date) APPENDICES (Individual da~a) AAppppeennddiixx 11.. CClliinniiccaall signs signs Aopendix 2. Body weight Appendix 2. Body .e~ght AAppppeennddiixx 33.. FFoooodd ccoonnssuummppttiioonn Appendix 4. Hematology (in comnection with HSC) Appendix 4. Hematology (in connection .ith RBC) Appendix 5. Hematology (tn commection with HSC) Appendix 5. Hematology (in connection .ith WBC) Appendix 6. Clinical chemistry Appendix 6. Clinical chemistry AMppppeennddiixx 177.. UUrriinnaallyyssliss Appendix 8. Organ weight (Absolute) Appendix 8. Organ weight (Absolute) AAppppeennddiixx 99.. OOrrggaann .weeiigghhtt (Relative) (Relative) Appendix 10. Macroscopic indings Appendix 10. Macroscoplc findings Appendix 11. Microscoptc findioge Appendix 11, Microscopic findings Appendix 12. Microscopic findings (for the organs showed gross lesions) Appendlx 12, Microscopic findings (for the organs sho.ed gross lesions) PHOTCGRAPHS (Histological Eiadings) PHOTOGRAPHS (Histological findings) Pots 1-4 Photos 1-4 am (iii) 22779977..00000066 33MM_MMNND011664499999922 SSTTAATTEEM MEENNTT OONN G GLLPP - Tie Title : Tuenty-eight-day Repeated Dose Oral Toxicity Study of T~enty-etght-day Repeated Dose Oral Toxiclty Study of Semple 0-1 tn Bats Sample D-1 in Rats Study Nosber BMRLASC Study Number : BMR143C The said study has been conducted in cosplisace with the "GLP Standard The said study has been conducted in compliance .ith the "GLP Standard for Industrial Cheicals in Jopan (1984, amended 1988). for Industrial Chemicals" in Japan (1984, amended 1988). ((Ssiiggnnaattuurree)) ate) (Date) Signed Signed Fobeuney 16, 1083 February 16, 1993 (akoto Temnich) (~akoto Tennicht) General Honsger General Manager Bio-Medical Rasaarch Laboratories Co.. Ltd. Bio-Medtcsl Research Laboratories Co., Ltd. an(iv) 22779977..00000077 3IMM_MMNND011664499999933 CCEERRTTIIFFIICCA AT TIIOON N _ Tite Title : TTwweesnttyy--oeilgghhtt--ddaayy RReeppeeaatteedd DDoossee OOrraall TTooxxiicciittyy Study Study of of Saaple D1 in Rats Sample D-I in Rats Study Nusber : BNEMSC Study Number : BNR143C Hoviag oudited the final report, I, the undersigned, sssure that the Hsatvuidnyghaasudibteeedn ctohnedufcltneadl irnepcoormtp,liaI,ncethweituhndJesrpsaingonseod, "GaLsPsuSrteantdhaartd tfhoer sItnugduystrhiassl beeCnhemciocnadlusc"t.ed Janndcomapclcioamnpcleis.hIetdh Jiaptahneseth"eGLPprSottaoncdoalrd faoard oIpnpdruosptrriiaalte sCthaenmdlacradls"o,peraatnidng apcrcoocmepdluirsehsedof t.hIeth tostthieng pfraoctiolciotly, eaandd athpeproprreipaortte shtaasndarbdeenoperparetpianrgedproecxeadcutrleys obfastehde toenstitnhge faecsipleirtmye,staanld rtheesultrse.port has been prepared exactly based on the experimental results. ((Ssiiggnnaattuurree)) ate) (Date} StSgiganeedd FFeebbrruuaarryy 1166,, 11999833 (tutaka Kasbare) (Yutaka Kambara) Quality Assurance Unit QBuiao-lNiotdyicAaslsurRaensceearcUnhitLaboratortos Co.. Ltd Bio-Medical Research Laboratories Co.. Ltd. Dates of Inspection Dates of Inspection TT i Geer DDeavteens oof mention Inspection Object to Protocol Tew 6.08 tO Protocol 1as9s922.. 7. 3 6.23 11099922.. 77.. 73 TTeessttiinngg PPrroocceedduurreess | 160, 1992. 57.. 7 1992. 8. 4 Me19s9z2.. 88..1188 Records, Row Data 1682.12.17 1992.12.17 FRiercoar]dsR,epoRrat~ Data 1993. 2.18 Final Report 1993. 2.16 DaDtaetess ooff Study Director Study en Director 992.7. 5 1992. 6.23 1099922., 77.. 13 11999922,.87.. 47 1992. 8. 4 1199992.2. 88..2200 1992.22.01 11999932..122..1178 1993. 2.16 RReeppoorrtt to Mansgeaent Tos. 6.23 to Nanagement 192.7. 3 1992. 6.23 1109092.2. 77..73 1189592.2. 78.. 74 1992. 8. 4 11999922.. 88..2200 1952.12.17 1199593.2.122..1176 1993. 2.16 TM(v) 22779977..00000088 I3MM_MMNND011664499999944 SIGNATURE - SIGNATURE Tite, Tltle; Study No: Study No.: TT.ueennttyy--eeligghhtt--ddaoyy RReeppeeaatteedd DDoossee OOrraall TTooxxiicciittyy SSttuuddyy ooff SSeammppllee D0--11 t~nn RRaattss BMRMC BMR143C The said study has been conducted under the responsibility of the The said study .has been conducted under the responsibility of the undersigned, and this report hes been prepared so 8s to reflect Whole undersigned, and this report has been prepared so as to reflect ~hole study correctly. study correctly. (signature) (Signature) ate) (Date) signed Signed February February 1166., 11998933 Csschto Otsuka) (Michto Otsuka) Study Director Study Director Bio-Medical Research Laboratories Co. Ltd Bio-Hedlcal Research Laboratories Co., Ltd. wn 22779977..00000099 33MM_MMNND011664499999955 G GEENNEER RAALL M MAATTTTEERRSS - fey 1. Title Twenty-olght-day repeated doses oral toxicity study of Semple D-1 in TwentY-eight-day repeated dose oral toxicity study of Sample D-1 in rats 22.. SSttuuddyy NNuomsbteerr BBuHRRL1e43aCc 3. purpose 3. Purpose To iavestigate toxicological potential of the test substance, Sample To investigate toxicological potential of the test substance, Sample D1. through serie] oral adalaistration to rats for 28 days. It ws D-1, through serla! oral administration to rats for 28 days. It was iinntteennddeedd ttoo mmeeaett JJaappaanneessee legal legal regulation regulation for for Industrial industrial new he. chemical chemical substances. substances. 4. GP Application 4. GLP Application The study was conducted in compliance with the Japanese "GLP Standard The study was conducted in compliance wtt~ the Japanese "GLP Standard for Industrial ChesicalsTM (1384, asaonded 138) for Industrial Chemicals" (1984, ammended 1988). 5. Testing Guidelines 5. Testing Guidelines The testing methods conforssd to the Jspamess ""GGuuiiddeelliinneess for for The testing methods conformed to the Japanese Screening Toxicity Testings of Chontcals (1988). Sc~eeninE Toxicity Testln~s of Chemicals" (1988). 66.. SSppoonnssoorr Sunitomo 3 Co, Le Sumltomo 3M Co., Ltd. 3-0-6. Hinami-hashizoto, Segamihara, Kansgaws, Japs 3-8-8, Minaml-hashimoto, Sagamihara, Kanagawa, Japan ((RReessppoonnccltbbllee PPeerrssoonn)) NNoobbuusshhiiggee NHeurraakkaamait MMaannaaggeerr,. Technical DDeeppaarrttmneenntt.. Technical Chemicals Division Chemicals Division 1- -- 1 -- 22779977..00001100 33MM_MMNND011664499999966 7. Testing Facilities - 7. Testing Facilities Key ractisty Key Facility Bio-Medical Research Laboratories Go.. bad ~) Blo-Medical Research Laboratories Co., Ltd. 3075 Susugays. Kiyokowa-sura, Alkob-gan, Kaogawa. Japan 3079 Susugaya, Ktyokawa-mura. Aikoh-gun, Kanasawa, Japan Partial Contributor Partial Contributor (8100d Biochenteal Exantaation) (Blood Biochemical Examination) @ Xoshina Laboratory. Mitssbishi-Kasel Iastitute of Toxicological (~ Kash~ma Laboratory, Hitsublshi-Kasei Institute of end Savizommeatal Sciences (*) and Environmental Sciences (*) 10, Sunyons. Hasoki-mscht, Kashisa-gon, Ibaraki, Japon 14, Sunayama, Hasaki-machl, Kashlma-gun, Ibarakl, Japan +The *)The ccoommppaannyy nnaamsee hhaass aalltteerreedd ttoo N Milttseuubblisshhi] CChheeamtcicaall Institute. Institute. (Histopathologtoal Spectaens Preparation) (Histopathological Specimens Preparation) torn Pathological Resescch (~ Nara Pathological Research Kinonine. ayasa, Tugs-aurs. Yoasbo-gun, Nara. Kinoaine. Hayama. Tuge-murao Yamabe-gun, Nara, (ttstopsthologicsl Examination) (Histopathological Examination) toto Research Institute, Food and Drug Safety Center ~ Hatano Research Institute, Food and Drug Safety Center 720-5 Ochiat, Hodoso-city, Keasgans, Japan 729-50chlal, Hadano-city, Kanagawa, Japan SSaaffeettyy Jspan Japan 5. Responsible Personnel 8. Responsible Personnel (Circled figure sfter the nase indicates each belonging focility ited (Circled figure after the name indicates each belonging facility cited above.) above.) --p-- Makoto Tenateht. @ General Nanager Hakoto Tennlchl. Study Diractor Michio Otsuke, Study Director Hichio Otsuka, General Participents Bropuid 1it, General Participants Hlroyukl Ishi~, Naoto Tohikams, Naoto Ichikawa, som Temata, Osamu Tanaka, Mikiko Kavassbe. Hiklko Ka~anabe, QQuuaalliittyy A Assssuurraennccee Yutaka Kasbars. Yutaka ambara, --2= 22779977..00001111 3M_MNO1649907 3M MN01649997 Portial Participants - Partial Participants ((RReessppoonnssiibbllee to to biochentcal biochemical deteraination) determ~n~tion) Noto Toyota, @ Naoto Toyota, ~ ((RReessppoonnssiibbllee ttoo hhilssttooppaatthhoollooggilccaall speciaens specimens reparation) reparation) Yoshiat Tatswnt, @ Yoshlmi Tatsami, (~ (Director for histopathological exastnation) (Director for histopathologlcal examination) Shinsuko Yoshimura, @ Shtnsuke Yoshimura, (~ ((~Paarrttiicciippaanatt ttoo hbiissttooppaatthhoollooggiiccaali examication) examination) Funte Kavashiza, @ Fu~ie Ka~ashima, (~ 9. 9. DDaatteess iinn tthhee SSttuuddyy ((Iantiitaiattiioonn ooff tthhee ssttuucdyy)) JJuunnee 2233,, 11909822 (hntaals rocoption) (Animals reception) JJuullyy 11,, 11899822 (Msipistration period) (Administration period) "Halo- -Hale- JJuullyy 06 -- Ahuugg.. 22,, 21999922 Femslo- .-Female- July July 7 7 - ug.Aug. 33,, 1982 1992 ((TTaerrmaiinnaattiioonn ooff oobbsseerrvvaattiioonn,, bblZoooodd ssaasmpplliinngg aanndd nneeccrrooppssyy)) "Hato, main groups- -Hale, main groups- Anugg.. 3, 1952 3, 1992 "-FFeemmaallee.. amaiinn ggrroowuppss-- Ahuogg.. 44,.11099922 "-HHaollee,. rreeccoovveerryy groups- groups- ug. 17. 1982 Aug. 17, 1992 "-FFeemmaallee,, recovery recovery groupe 8roups- oAugg.. 10. 18. 1952 1992 (Gosplation of the study) (Completlon o~ the study) Feb. 18, 1089 Feb. 16, 1993 10. Rotation of Records sed Samples i0. Retention o Records and Samples M1 documentation records snd samples (including spaciuoms) of the All documentation records and samples (including specimens) o the ssttudyy aarree rreettaaiinneedd iinn tthhee oarrcchhiivveess ooff BBiioo-NMeeddiicceall RReesseeasrrcchh LLaabboorraattoorriieess CCoo.., LLttdd.. ffoorr 1100 yyeeaarrss aafftteerr tthhee ccoommpplleettiioonn ooff tthhee ssttuuddyy.. Ao to further rotention, Lt will bo decided on consultation with the As to further retention, it ~111 be decided on consultation ulth the ssppoonnssoorr aanndd tthhee ccoonnttrraaccttoorr.. -3- -- 3 -- 22779977..00001122 33MM_MMNND011664499999988 SUSUMMMMAARRYY. - 1. Semple D1 wee adaiaistorad to Both sexes of rats wich doses of 0.1, 1. I. Sample D-1 was 10, and 30 ng/kg administered for 20 days. to both A grows sexes of rats of rats gives with the v ehdiocslees of 0.1, I, (olive ofl) I0, ~nd 30 mg/kg for 28 days. A group of rats given the vehicle (olive oil) alone was included as the control. Sesides the main groups for nancecovery saxluotnye. waas saitnecllluidteed garsoutpheforcon1t4r-odla.y Breosciodmeesrythsetudmyaian sgrdoudpsd tofor snaocnhreocfovetrhey study, a satellite group for 14-day recovery study was added to each of the highest dos and the control groups highest dose and the control groups. 2. No dont occucsed throughout the adatatstration period. One ale of the 30 2. sfNtoe degartohu.occuhrorweedvert,hroudgiheodut Ctuhreingadmitnheistrraetcioovnersperpieordi.odO:ne male the of the death 30 was mcgo/nksgidergerodupt,o bohowceavuesre,d bydiethde tdeusrtingasbitthaencerecovery period: the death was considered to be caused by the test substance. 5. Ko sworsl clinical signs wera fowmd fn ell rats of the conceal 3. ( incNoludianbgnortmhaols e clinical for the sriegcnosverwyerestudfyo)undsn din th all e 10 rats of sa/ky ox the control lower dose (including those for the recovery study) and the 10 mg/kg or lower dose aroups throughout the whole of the observation period. In the 30 mg/h groups throughout the whole of the observation period. In the 30 mglkg Grow. however, duck sigss as the followings were observed during the agdroautpa,tstrhoawteivoenr,and~utchhe signs as. the recorery periods: reduced followings spwoenrteaneoobussersvoevdewdautr,ingtontihce caldomnitneistcroamtmiiosnioann,d thleyiorgecoovrerycropuecrhiiondgs.: rberdaudcyepdmessponoxtanceyoaunscamtosv,emesnatl,vattoinoinc- ccleodndiisch csoanlviuvlastiioonn, , blryionwgs uorrinec.roulcohsisngo,f bradypnea hair, snd coorntcysatnaoisniinsg,. salivation, These vere croendsdiidsehresdaltiovaStnionc,asabardowbny utrhienet,esltosssubsoftanhcaei.r, and coat staining. These were considered to be caused by the test substance. 4. Baty weight gain vas suppressed during the adainistration period in both 4. salBeosdyanwdeifgehntslgeasinofwatshe su3p0praegs/sAeyd during the Group fron administration the sacond eck period in snd afters both the msaulpepseasaanidonfecmoanlteisnuoefd tthheros3g0homugt/ktghegrToeucpowfereoym petrhieod.second week and after; the suppression continued throughout the recovery period. 5. Food comspticn was suppressed during the adaiaioteation period In bath 5. ablFeosodsdconfsounmsplteisonofwasthesu3p0pre5sg/skesd during the gro from tahdemisneicstornadtivoansk paenrdiodaftienrsbotIht cmaanldeesd toand rfeetmuarlnesdurofingthethe30se=cgo/vkegrygrpeoruipodf.rohmowtehveer.second week and after; Jt tended to return during the recovery period, however. 6. Homstolegical examination at the end of the sialaisteation period rovesled 6. Hematological examination at - the end =of the administration period revealed 22779977..00001133 _UNOI643999 3M MN01649999 decreased hemoglobin concentration fn sales of the 10 sad 30 mg/kg groups. decreased hemoglobin concentration in males of the 10 and 30 mE/k8 groups~ and reduced prothrosbin tise in fosales given 30 sg/Kg. Moreover, at the end and reduced prothrombin time in females given 30 mg/kg, Moreover, at the end of the recovery period. the followings xara still detected In the 30 ag/kg of the recovery period, the followlngs were stlll detected in the 30 mg/kg group: decrease of erythrocyte comt sd hemstosrit. shortening of group: decrease of erythrocyte count and hematocrlt, shortening of prothroabia tise. snd facresse of leukocyte count, fn melas: dscresse of prothrombln time, and increase of leukocyte count, in mates; decrease of hesoglobln concentration snd memn corpuscular hemoglobin concentration, hemoglobin concentration and mean corpuscular hemoglobin concentration, shortening of sctiveted partial thresboplastin tims, sad incense of mesn shortening of activated partial thromboplastin time, and increase of mean corpuscular voluse, fa fossles. corpuscular volume, in females. 7. Biochosicol exasintion revesled the the followings at the ond of the 7. Biochemical examination revealed the the followings at the end of the adaintstration period: deceease of GOT fn females of the 1 sg/kg and higher administration period: decrease of GOT in females of the 1 mg/kg and higher dose grows: increase of olbuain in females of the 10 mg/kg end higher dose dose groups; increase of albumin in females of the I0 mg/kg and higher dose | sroups; inccosse of GPF an chloride. aid decresse of total cholesterol fn groups; increase of GPT and chloride, and decrease of total cholesterol in both sexes, decrease of total protets and increase of alkaline phosphatase both sexes, decrease of total protein and increase of alkaline phosphatase and of A/G ratio in males, increase of ures nitrogen in females. all these and of A/G ratio in males, increase of urea nitrogen Jn females, all these in the 30 ng/g grow. in the 30 mg/kg group. At the ond of the recovery period. the followings wero besrved a cho 30 At the end of the recovery period, the followings were observed in the 30 ag/ke group: decresse of triglyceride sad increase of MG ratio, in males: mg/kg group: decrease o triglyceride and increase of A/G ratio, in males; Increase of urea mitogen, total protets, albumin, and calcium, and decrease increase of urea nitrogen, total protein, albumin, and calcium, and decrease of glucose asd triglycerides. in females of glucose and triglycerides, in females. Sestdes, reddish chasge of seus vas recognized in both sexes with the 10 Besides, reddlsh change o serum was recognized in both sexes ~ith the 10 aa/k oc higher dose main groups, and in the 30 mg/kg recovery Eau. mg/kg or higher dose main groups, and in the 30 mg/kg recovery group. 6. By urimalysis. acidic @hift in both sexes aud decrease of protefn fn 8. By urinalysis, acidic shift in both sexes and decrease of protein in females aro noted in the 30 mg/kg group during the administration period females were noted in the 30 mg/kg group during the administration period. Aeidio shift rosained in wales during the recovery period. Acldlo shift remained in males during the recovery period. 9. he for absolute orgs weight, the adalnistration resulted In increase of 9. As for absolute organ weight, the administration resulted in increase cf tthhee vlieverr wweeiigghhtt iinn bbootthh sseexxeess ooff tthhee 1I00 amgg//kkgg oorr hhiigghhearr ddoossee ggrroouwpss senndd docrasso tn the kidneys weight of sales given 0 sg/kg. Acar the recovery decrease in the kidneys weight of males given 30 mg/kg. After the recovery --5-- 22779977..00001144 33MM_MMNNOO1166550000D0O0 ppeerriioodd,, ilnaccrreeaassee iina tthhee lliivveerr of of both both sexes sexes sad and decrease decrease in in the the kidneys kidneys of of ffeemmaalleess .weerreo ssttiillll nnootteedd in In the the 30 30 xg/kg mg/kg grow. group, Relative organ weight increased in the liver of both sexes of the 10 mg/kg Relative organ weight increased in the liver of both sexes of the I0 mg/kg oorr hhiigghheerr ddoossee aaandd iinnccrreeaasseedd iinn tthhee kidneys kidneys in in females females of of the the 10 10 mg/kg mg/kg or or hhiigghheerr ddoossee.. AAfftteerr tthhee rreeccoovveerryy period, period, increase increase in in the the liver liver of of both both sexss sexes aanndd ifnnccrreeasssee i1nn tthhee kkiiddnneeyyss ooff ffeemmaalleess remained remained in in the the 30 30 mg/kg mg/kg group. group. 10. Autopsy for the male died during the recovery period revealed atrophy sad 10. Autopsy for the male died during the recovery period revealed atrophy and ddaarrkk rreeddddiisshh ppaattcchh iinn tthhee thyaus. thymus, hemorrhage hemorrhage sad and tarry tarry contents contents fn in the the ssttoommaacchh,, ttearrrryy ccoonntteennttss finn tthhee ssmmaallll~Iinntteessttiincee,. asnadd ggrraayyiisshh patch patch in in the the Liver. liver. In the scheduled necropsy at the end of the adainistration period, the In the scheduled necropsy at the end of the adatnistration period, the following findings were detected by sacroscopic examination: dark reddish follo.ing findings Here detected by macroscopic examination: dark reddish change in the liver of fomales receiving 30 =g/kg: grayish dot or patch and change in the liver of females receiving 30 mg/kg; grayish dot or patch and hypertrophy in the liver and blackish change in the kidneys in males given hypertrophy in the liver and blackish change in the kidneys in males given 3300 m mgg//kkgg:: ddoarrkk rreeddddiisshh cchhaannggee,, yyeelilloo~wiisshh change. change, grayish grayish patch. patch, snd and trophy in the liver of females receiving 30 mg/kg. trophy in the liver of females receiving 30 mg/kg. The exsatnation at the end of the recovery period revealed grayish The examination at the' end of the recovery period revealed grayish dark reddish change. and hypertrophy in the liver of both Sexes of dark reddish change, and hypertrophy in the liver of both ~exes of bypec- hyper- patch, patch, the 30 the 30 mmgg//kkgg ddoossee.. 1111.. HHiissttooppaatthhoollooggiiccaall eexxaammiinnaattiioonn at at the the end end of of tho the adainistration administration period period rreavveeaslleedd ddoossee--ddeeppeennddeanntt cchhaannggeess aass eosinophilic eosinophili degeneration degeneration in in centri centri- lobular hepatocytes and swelling in the liver in males of the 1 mg/kg or lobular hepatocytes and s.elllng in the liver in males of the 1 mg/kg or higher doses and in foaales of the 10 3g/kg or higher dose. Other findings: higher doses and in females of the 10 mg/kg or higher dose. Other findings: focal necrosis in hepatocytes ia both sexes given 30 mg/kg and fn males focal necrosis in hepatocytes in both sexes 81van 30 mg/kg and in males ggiivveenn 1100 xmgg//kkgg;: ssoommeewwhhaatt rreemmaarrkkaabbllee ffaattttyy degeneration degeneration in in peripheral peripheral lobule lobule iinn smaalleess receiving receiving 30 30 =g/ke. mg/kg. NNoo sabbunoorrsmaall cchhaannggee w.aass ddeetteecctteedd in in the the heart, heart, kidneys, kidneys, spleen, spleen, adrenals, adrenals, bbrraaiinn,, aanndd tteesstteess or or ovaries. ovaries. -- 22779977..00001155 33MM_MMNN0011665500000011 AAtt tthhee eenndd ooff tthhee rreeccoovveerryy ppeerriioodd,, eeoossilnnoopphhiilliicc ddeeggeenneerraattiioonn Iinn cceenmttreli-l IIcobbuullaarr hheeppaattooccyytteess ttuurrnneedd sslliigghhtteerr:; sswweelllliincgg ifnn tthhee lliivveerr,. hhoowweevveerr,. ddiidd nnoott rreeccoovveerr iian bbootthh sseexxeess ggiivveenn 3300 mmgg//kkgg,. aanndd ffooccaall nneeccrroossiiss aallssoo rreemmasiinneedd.. IInn ccaasseo ooff oonnee mmaallee ggiivveenn 3300 mgg//kkgg aanndd ddiieedd dduurriinngg tthhee rreeccoovveerryy ppaerriioodd,. iinn aaddddiittiioonn ttoo tthhee ssaemmee cchhaannggeess 8ass ffoouunndd iinn tthhee lliivveerr ooff tthhee mmaaiinn ggrroouuppss.. tthhee ffoolllloowwiinnggss wweerree ddeetteecctteedd:: hheemmoorrrrhhaaggiicc flooccii ilna tthhee bbrraaiinn,. uullcceerr aanndd hheesmoorrsrhhaaggee ifnn tthhee ggllaansdduullaarr ssttoommaacchh,, anndd ccoonnggeessttiioonn aanrdd hheemsoorrrchhaaggee finn tthhee thymus. : thymus. 1122.. JJuuddggiinngg ffrroomn tthhee sabboovvee,, tthhee ttooxxiiccoollooggiiccaall nnoo--oobbsseerrvvaedd--eeffffeecctt lleevveell ((NNOOEELL)) oOff tthhee tteesstt ssuubbssttaanncceo,, SSaammppllee DD-1l,, wwaass ccoonnssiiddeerreedd ttoo bbee 00..11 mxggl/kkgg ffoorr bbootthh ale and femsle rats. male and female rats.. --7= -- 7 -- 22779977..00001166 I3MM_MMNND011665500000022 MTERIALS AND METHODS - MTERIALS A~D METHODS 1. Test Substance 1. Test Substance 11 Haze 1-1 Name (Chemical Nose) (Chemical Name) ((Abbbrborveivaiattiioonn)) 11--22 SSttrruuccttuurraall ffoorrmmuullaa 2-2[-N[-NB-tEhtyhlyl--NN--ppeerrffiluuoorroosallkkyyll((CC--11--88))ssuullffoonnyyllaamniinnoo]] eetthhyyll aaccrryyllaattee SSaammppllee DD--11 cCy2 HHsy O 0 | u CCno FFa2une+1I8S0O;2 NN=-CCHH2y CCHH,200--CC---CCHH-=-CCHH,2 ((GCoonmgpoosstittiioonn)) I1--33 MMoolleeccuullaarr wweeiigghhtt 1i5-44 LLoott nnuummbbeerr 11-55 Adppppeeasrcaannccee 11--66 PPuurriittyy 11--77 SStteraaggee ccoonnddiittiioonnss ((na rraannggeess ffrroomn 11 to to 68)) aabboouutt 878X~ ffoorr nn==88 ccoommppoonneenntt,, aabboouutt 2211~% ffoorr nn=e11--77 ccoommppoonneennttss.. 662255 110011 ((ssuupppplliieedd bbyy tthhee ssppoonnssoorr)) Asber-colored waxy solid Amber-colored waxy solid. 9989~% oorr aabboovvee Stored at room tesperature Stored at room temperature 22.. PPrreoppaarraattiioonn ooff DDoossiinngg SSoolluuttiioonn 22--1I VVeehhitcellee oO1l1ivvee ootlti 22--22 PPrroocceedduurree. RReeqquuiirreedd saamoouunntt ooff tthhee tteesstt ssuubbssttaannccee vwaass wweeiigghheedd and and suspended suspended homogencously homogeneously iinn tthhee vveehhiiccllee m meennttiioonneedd aabboovvee.. TThhoe ddoossiinngg ssoolluuttiioonn wweass ddiivviiddeedd iinnttoo ddaaiillyy bbaattcchheess aanndd ssttoorreedd iinn aa rreefrritggeerrasttoorr aatt 441~CC uunnttiidl gjuusstt bbeeffoorree uussee.. 2-3 Frequency of preperation 2-3 Frequency of preparation TThhee ddoossiinngg ssoolluuttiioonn wwaass pprreeppaarreedd oonnccee aa wWoeoekk,, bbeeccoauussee tthhee ssttaabbiilliittyy ooff tthhee tteesstt ssuubbssttaanaccee iinn tthhee mmiixxttuurree ffoorr eight eight doys days was ~as ccoonnffiirrmaeedd iinn JJaappaann FFoooodd RReesseeoarrcchh LLaabboorraattoorriieess ((TTookkyyoo)) bbyy --8- 22779977..00001177 33MM_MMNNO011665500000033 our request. - our request. 3. Laboratory Aniaals and Resson for Chote 3. Laboratory Animals and Reason for Choice The aniaals used were SPF CeJ:CD(SD) rats of both sexos. They were purchased The animals used were SPF Crj:CD(SD) rats of both sexes. They were purchased fron Charles River Japan, Inc. (195 Shimo-furusawa, Atsugl. Kansgawa, Japan) from Charles River Japan,~ Inc. (795 Shimo-furusawa, Atsugi, Kanagawa, Japan) at four weeks of age en July 1, 1992. Mter five (sale) or six (Fesale) days at four weeks of age on July 1, 1992. After five (male) or six (female) days Of acclimatization, oniaals that apparently healthy sad showed morasl weight of acclimatization, animals that apparently healthy and showed normal weight 010 gain were were selected selected for for the the study study at at five five weoks weeks ooff aaggee. . TThheeyy wweerree ggrroouuppeedd ttoo be unifors for the meen body weight based on the last weighing during the be uniform for the mean body weight based on the last weighing during the aaccc~lliimmataitzizoatitioonn.. TThhee wweeligghhtt rraannggeedd ffrroomm 113322 ttoo 115511 gg ffoorr m maalleess asnndd 112211 ttoo 141 5 for females at the initiation of adinistration. 141 g for females at the initiation of administration. RReeaassoonn ffoorr cchhooiiccee:: HHeav~iiengg sapppprroopprriiaattee mnaattuurree ffoorr llaabboorraattoorryy amnaiim saalles sanndd being bred under good genetic and microbial control. this species and strain being bred under good genetic and microbial control, this species and strain are widely used in toxicological studles: our testing facility. besides. are widely used in toxicological studies; our testing facility, besides. Bas accusolated back ground data on this anisal. has accumulated back ground data on this animal. 44.. Antniumaall HHoouussiinngg Five or txo (durisg or after the acclimatization, respectively) animsls of Five or t.o (during or after the acclimatization, respectively) animals of tho same sex were housed together fn a stainless steel wire mesh cage of the ~he same sex were housed together in a stainless steel -ire mesh cage of the hheannggiinngg ttyyppee ((2266x3388x1188 ccam).), TThhee aanniimsaall ccaarree rrooec=a wweass aa bbaarrrriieerreedd ffaacciilliittyy t(hhaatt wweass aauuttoommataiticcaallllyy ccoonnttrroolllleedd ttoo kkeeeepp tthhee ffoolllloowwiinngg ccoonnddiittiioonnss:: Temperature: 2-28 T : Temperature : 22 - 26 =C Huntatty Humidity : so - Ton E : 30 - 70~ RH Vestilotion Ventilation + 13 tises/hour (all fresh ate) : 13 times/hour (all fresh air) Lighting Lighting Light dock cycle of 12 hours : Light /dark cycle of 12 hours (13ghts on 8:00 to 18:00) [lights on 6:00 to 18:00) A little dovistion of tespersture asd humidity occurred during tho animal A little deviation of temperature and humidity occurred during the animal Keeping: it wes, however, Judged mot to affect to the reliability of the keeping; it ~as, however, judged not to affect to the reliability of the study, study. Tho dist was MF palloted food for laboratory animals (Oriental Yeast Co.. The diet eas ~F pelleted food for laboratory animals (Oriental Yeast Co., --9- 22779977..00001188 I3MM_MMNND011665500000044 Ltd) snd deisking water as tap water sublected to filtration snd UY Ltd,) and drinking water was tap water subjected to filtration and UV iirrrraaddiiaattiioonn.. AAnoitmaaallss wweerree aalllloowweedd aacccceessss ttoo ffoooodd sanndd-waatteerr aadd llilbbiittuumm eexxcceepptt for fasting fron the evaning before necropsy. Aalysis of the ingredients for .fasting from the evening before necropsy. Analysls of the ingredients sd microbial contamination of the diet was perforsed by the mamufacturer, and microbial contamination of the diet was performed by the manufacturer, 2a1n0d ttooxxiicc ccoonnttasmainnsaanttss wweerree eexxaammiinneedd bbyy JJaappaann FFoooodd Teseerch Research Laboratories Laboratories ({TTookkyyoo)) aatt tthhee maamnuutfaaccttuucreerr''ss rfeessppoonnssiibbiilliittyy.. The The quality quality of of the the top tap water water wes poriodically examined by Atsugi Public Health Canter by our request. was periodically examined by Atsugl Public Health Center by our request. Theso data wore checked and stored by us These data were checked and stored by us. Cages wera roplaced with freshly autoclaved ones once a mack. Cages were replaced with freshly autoclaved ones once a week. 5. dentitication of Antmals 5. Identification o Animals Esch ontaal was dentiied by marking o musber on the tail with a saturated Each animal was identified by marking a number on the tat1 with a saturated alcoholic solution of picric acid. To identify the dose group to which each alcoholic solution of picric acid. To identify the dose group to which each snisal belonged, o colored mark wes painted on the fail with sn oil-based animal belonged, a colored mark was painted on the tall with an oil-based fert-uip pen. felt-tip pen. 6. laemtatication of Coges 6. Identification of Cages EBaecchh c~aaggee vwaass identified identified with with an an attached attached card card on on abbreviated name of the test substance, dosage. asd abbreviated name of the test substance, dosage, and were Listed. were listed. Which the which the individual individual study musbor, study number, amiss) musber animal number 7. Test Procedures Test Procedures The testicg methods conforasd to the Japanese The testing methods conformed to the Japanese "Guidelines for Screening =Guidelines for Screening Toxicity Tasting of Cheaicals * (amended on Dacerber 5. 1986) Toxicity Testing of Chemicals " (amended on Decenber 5, 1986). 7-1 Dose levels and reason for selection 7-1 Dose levels and reason for selection Prior to the principal study. prelisiasey dose-flodisg studies ranged Prior to the principal study, preliminary dose-flnding studies ranged betuasn 4 and 14 days in adainlstration period was pacforsed with such doses between 4 and 14 days in edminJstratlon period was performed with such doses as 20, 40, 50. 100, 200. 500. and 1000 mg/kg. each Ln a fon (theos to Five) as 20, 40, 50, 100, 200, 500, and 1000 mg/kg, each in a few (three to five) sale snd female rats. It resulted death of sore then Ralf musber of rats in male and female rats. It resulted death of more than half number of rats in ---- I100 ---- 22779977..00001199 3IMM_MMNND011665500000055 200 gig for 14 deg repmated siutuistration: pression of body weigh I000 mg/kg for 14 days repeated administration; suppression of body weight tn ws cared in 40 a ov. hugh sn of charges fond In 2 sx gain was observed in 40 mg/kg dose, though none of changes found in 20 mg/kg on. os. th bigest dose va dncdnd 520 gr 1s on social sty dose. Hence. the highest dose was decided at 30 mg/kg in the princlpal study en hn Tom doen so st 8% 10 mG. 3 RRS 5 0.4 sh for ots and the lower doses were set at 10 mglkg, I mg/kg, and 0.1 mglkg for both nen. gro of cate given the vehicle lon ve cine 3 th cosh sexes. A group of rats given the vehicle alone was included as the control. [ET or--. 7-2 Number of animals Forty-two rats of each sex were used. 18 tute, teensy. sot dation of siintention 7-3 Route, frequency, and duration of administration eaiseseon vas estoaed vin he ors vt, ane day fo 28 dy. Administration ,as performed via the oral route, once a day for 28 days, 4 tod of smistcion 7-4 Nethod of administration Ta osiog wna as stad dicey te he sions vin a pscic The dosing mixture was intubated directly into the stomach via a plastic rae tie. To slum initatored vs 1 2 pu 180g of body wight used gavage tube. The volume administered was 1 ml per I00 g of body weight based a to on snc es de on the most recent weight data. 5 Going 7-5 Grouping Tou don sup enticed ser and he contr gen er su. Bede, to Four dose groups mentioned above and the control group were set. Besides, to anton the roraiblicy of any (ois afta, swale gros wore aided monitor the r~versibility 0f any toxlc~ effects, satellite groups ~ere added FN to the control and the highest dose groups for a 14-day recovery study. Err El i Grouping by con rvs [eae teow vo of ii dose levels Dose (mg/kgl Main Groups(*l} Recovery Groups(*2) (Sex and Number of animals) ontrol totermttntet| owest dose ntermedlate-I 0 male & female, 6 rats each male & female, 6 rats each 3 | ssl 8 ene, cos oh 0.1 male & female, 6 rats each 1 male & female, 6 rats each ntermedlate-2 male & female. 5 rats each ighest dose 3O male & female, 6 rats each male & female, G rats each Total male & Ts female,30 rats each male & female,12 rats each 22779977..00002200 suunoiezo0ss 3M MN01650006 ((4'11)) AAnniimaaallss weerroe nneeccrrooppsslieedd oonn tthhee nneexxtt ddsayy ooff tthhee last last adaiaistratrion. admlnistratrlon. (+42) Antaals nore nocropsied immediately after the recovery period was over. (*2) Animals were necropsied immediately after the recovery period was over. NNoottee.. IInnddiivviidduusall sannilsmaall nmeummbbeerr.. EBaacchh aanniimmaall wvaass nnuummbbeerreedd wwiitthh ffoouurr--ddiiggiitt feiigguurree.. First digit, Dose groups: O0., the the ccoonnttrrooll ggrroouupp. First dlglt. Dose groups; 1. tthhee lloowweesstt ddoossee ggrroouupp. 21, tthhee iInntteenmeeddiiaattee--i1 ggrroouupp. 23. tthhee iinntteenmeeddiiaattee--22 ggrroouupp "3, tthhee hhiigghheesstt ddoossee ggrroouupp Second digit. Sexes: 4, 5 sales Second diglt. Sexes; 12, mfaelmeasles Third and fourth digits, 2, individual snimsflemoaflesmain of Fecovery groups: Third and fourth digits, indlvldu0a1-l06a,nimaslnisofalsmaiofn or recovery groups; the main groups 0071--1062.. aanntismaallss ooff tthhee mraeicnovegrryoupgsroups 07-12, animals of the recovery groups 88.. PPaarraammeetteerrss AAsssseesssseedd 88--I1 GGeenneerraall ccoonnddiittiioonn 8-1-1 Clinical signs 8-I-1 Clinical signs TThheo. a~ppppeeaarraannccee,, bbeehhaavviioorr,, aanndd eexxccrreottaa ooff eevveerryy aannlimsea)l .weerree oobbsseerrvveedd daily daily throughout the adaiuistration and the recovery periods. throughout the administration and the recovery periods. 8-1-2 Body weight 8-1-2 Body weight Every anisal wea weighed With an elostric balasce at the commencement of Every animal was weighed with an electric balance at the commencement of the odataistration, once a week thereafter. the admlnistration, once a week thereafter. 88--11--33 FFoooodd ccoonnssuummppttiioonn Leftover Leftover food food fron from each each ccaaggee wwaass wweeligghheedd oonn ddaayy 0 0 saandd oonnccee a a wweeeekk thereafter, thereafter, and and the the daily daily aovveerrasggee ccoonnssuummppttiioonn ppeerr aanniimmaall wwaess ccaallccuullaotteedd ffoorr eeaacchh ggrroouupp.. 8-2 Laborstory tests 8-2 Laboratory tests 8-2-1 Hematology : 8-2-1 Hematology BBlloooodd ssasmspplleess wweerree ccoolllleecctteedd fron from the the inferior inferior vena vena cava cava under under ether ether anesthesia anesthesia just just before before the the scheduled scheduled tine time of of macrifica. sacrifice. BBlloooodd sampling sampling nas was -- 12 -- -- 12 -- 22779977..00002211 33MM_MMNNO011665500000077 ddoonnee uunnddeerr nnoonn--ffaassttiinngg ccoonnddiittiioonn.. TThhee hheemmaattoollooggliccaall ppaarraammeetteerrss deteraized determined earree Lliisstteedd bbaoloen... TThhee aannttiiccoosagguullaanntt uusseedd wwaass oetthhy>lloenneeddiiaasmiinneettoettcraasaccoettlicc seid acid dipotaseius dipotaselum salt (BDTA-2K). except that 3.13X sodiva citrate was used to dotaraine the salt (EDTA-2K), except %hat 3.13~ sodium citrate -as used %0 determine the pprrootthhrroosmbbllan ttiismee aanndd tthhee aaccttiivvaatteedd ppaarrttilaall tthhrroosmbbooppllaassttiinneg ttiimee.. Parameters T(WI)) TLaevukkPooaccrayym rteeetecc~oauontunt @) Beythrocyte count ((@2)) EPrlayttehlreotcytceounctount ((34)) HPolsaotgelleobtincocuonntcenteation ((45)) HHeemmaotgolcorbiitn concentration ((58)) H Hoeamn actoocrrpiutscular volume ((60)) MMoerann ccoorrppuussccuullaarr bveoslucmgleobln ((78)) MMoeoann ccoorrppuussccuullaarr hheemmoogglloobbiinn (8) Mean concentration corpuscular hemoglobin (6) Dittorantial lcaouknocceyntteractioounnt (1(90)) D Prioftfherroemnbtiinal tiseleukocyte count ((1101)) PAcrottihvraotmedbinpartitmiael throsbo(11) Activated Plastin tine partial thrombe- plastin time W(WBBCC)) (RBC) ((BRLBTC)) (ah) (PLT) (et) (Hgb) oon (Hot) (KH) (MCV) (MCH) (NCHD) @D (AIT) (APTT) MHeotthhoodd EEileeccttrrliccaall rreessiissttaannccee mmeetthhoodd EElleeccttrriiccaall rreessiissttaannccee mmeetthhoodd Blectrical resistance method PEhloetcotreilceacltrriecsisctoalnocreiameteetyhod PPuhlosteo-deelteeccttriiconcmoelothriomd etry CPaullcseuladteetdectfiroonn m(2e)thoadne (5) CCaallccuullaatteedd ff~roomm ((22)) aanndd ((54)) Calculated from (2) and (4) Calculated from (8) and (5) Calculated from (4) and (5) [r------ Gtemsa-statned smear Quick's one-stage test Quick's one-stage test AAccttiivvaetteedd ccoeffaorroopplleassttitnn m seetthhoodd In((sID)t--u((r88u))ment:sAAuuttoommaattiicc cceollll ccoouunntteerr.. SSyyssmmeexx mmooddeell CCCC--1188004A.. TTOOAA MMeeddiiccaall EElleeccttrroonniiccss CCoo...LLttdd. @(9) : OOppttiiccaall mmiiccrroossccooppyy ((A100))--((1111)) :: BBlloooodd ccooaagguullaatteer,, MHooddeell KKCC--I1,, AAmseelluunngg CCoo..,,LLttdd.. Anong these data. PT asd APTT of one sale in the recovery growp (animal Among these data, PT and APTT of one male in the recovery group (animal no. OI) were missing owing fo cosgulation at blood sampling. On the no. 0111) were missing owing to coagulation at blood sampling. On the other hand, reticulocyte counting was added ln the main groups after other hand, rettcu/ocyte counting was added in the main groups after sequential staining with few methylene blue process and May-Gruemald- sequential staining .ith new methylene blue process and May-Gruen~ald- Gloasa process, because anemia was suspectad in aniasls of this group. Giemsa process, because anemia was suspected in animals of this group. 88-22--22 BBiioocchheemmiissttrryy The rosidusl blood sasple xas let stand at toon temperature for 30 min and The residual blood sample was let stand at room temperature for 30 min and -- 13 -- 22779977..00002222 33MM_MMNND011665500000088 tthheenn cceennttrriiffuuggeedd aatt 33000000 rrppma ffoorr l1O0 mmiinn ttoo sseeppaarraattee sseorruums.. TThhee sseerruuma., bbeeilno~g ffrrootzeenn ttoo -2200 ~CC., awass sseenatt ttoo KKaasshhilmmaa LLaabboorraattoorryy ooff MMiittssuubbiisshhii--KKaassee!i IInnssttiittuuttee ooff TTooxxiiccoollooggiiccaall aanndd Eanvviirroonmmmeenattaall SScciieenncceess ffoorr bblioocchheemsiiccaall oxantnation. Parazeters exasined vere as follows examination. Parameters examined were as follows: Paransters Method Parameters Tomiprotets ~ethod Blurers metbed (1) Total protein Biuret's method @) Albonin BBrroom nooccrreessooll ggrreeeenn smeetthhoodd @)(2) Albumin A/G rato Calculated fron (1) and (2) (3) A/G ratio Calculated from (1) and (2) @)(4) GGlluuccoossee EEanzzyysmsatiticc UUVV--ssppeeccttrroopphhoottoonmsettrryy ((HHeexxookkiinnaassee~-GG6o--PPDDHH)) ((58)) TTrritggllyycceerriiddeess EEanzzyymmaatiticc mmeetthhoodd ((LLPPLL-G-KG--GG33P0O--PPOODD)) (6) Total cholesterol Enzymatic method (CES-CO-POD) 1)(6) UTroetaal nicthcoolgeesnterol EEanzzyYsmstaiticc mUie-thsopdect(CsoEpSh-Cot0o-PmOoDtr) y (Urease-GLOK) (7) UCrreeaatinnlitrnoesen Jatte method Enzymatic UV-spectrophotometry (Ureaae-GLDH) (8) Creatin~ne Jaffe method Caletun oo--CCrreessoollpphhtthhm alleeiinn ccoom npplleexxoonnee m meetthhoodd (9) Calcium ((1100)) IInnoorrggaanniicc pphhoosspphhoorroouuss PPhhoosspphhoomsoollyybbddaattee--UUVY ssppeeccttrroopphhoottoomnsottrryy an cor (sm) UUV~ ssppeeccttrroopphhootts~mmstertryy--rraattee mmeetthhoodd ((M Mooddififiieedd SSSSCCC)) (a1z1)) oGeOrT wn (AST) UV spectrophotomstry-rate method" (Modified SSCC) a(1n2) GPpTea(ArLT) UV spectrophotometry-rate method~(Modifled SSCC) 7 -Glutenyl-p-nttroanilide substrate method . (13) T -GPT 7 -Glutamyl-p-nitroanilide substra(tMeodmifeitheoddSSC0) (14) Alkaline phosphatase (ALF) p-Nitrophenyl phosphate subs(tMroadtiefimedethSoSdCC) (14) Alkaline phosphatase (ALP) p-Nitrophenyl phosphate substrate method ((1155)) SSooddsiuusm. IToonn sseelleeccttiivvee eelleeccttrrooddeess zmoetthhoodd ((1166)) PPoottaassssiiuvma IToonn sseelleeccttiivvee eelleeccttrrooddeess mmeetthhoodd an caloride (17) Chloride IToonn sseelleeccttiivvee eelleeccttrrooddeess mmeetthhoodd IInnssttrruummeenntt:: AAuuttoommaattiicc aannaallyysseerr ((HHiittaacchhii 173366--1100 ttyyppee}) 88--22--33 UUrriinnaallyyssiiss FFrreosshh uurriinnee wwaass ccoolllleecctteedd ffrroomm aallll aanniimmaallss aatt ssiixx oorr sseevveenn ddaayyss bbeeffoorree tthhee ccoommpplleettiioonn ooff aaddmaiinniissttrraattiioonn,. aanndd ffoouurr oorr ffiivvee ddaayyss bbeeffoorres tthhee eenndd ooff tthhee rreoccoovveerryy ppeorriioodd.. ((IInn eeaacchh ooff ttwhoo ssuucccceassssiivvee ddaayyss,. tthhee ffoorrnmoerr aanndd tthhee llaatttteerr ddaayyss wweorree ffoorr mmaallee aanndd ffoorr ffeomaallse rreessppeeccttiivveellyy,.)) TThheessee wweerree tteesstteedd for the following parameters. for the following parameters. --- 1144 = -- 22779977..00002233 33MM_MMNN0011665500000099 Parameter pH, Occult blood, Protein, Glucose, Ketone bodies, Urobilinogen, Billrubin 8-3 Pathological exa~tnatton Method Paper test, MULTISTIX (~ (Miles-Sankyo Co.,Ltd.) 8-3-1 Necropsy and macroscopic examination On the next day of the last administration, all survived animals except those asslgned for the recovery test were sacrificed for necropsy after blood sampling; whereas for animals of the recovery groups, the necropsy was held on the scheduled day just after the recovery period was over. After careful Investigation for any macroscopic abnormalities, the following organs kere removed and fixed in 10~ neutral buffered formalin: brain, pituitary, eye ball. (wlth Hardertan glands), thyloid (with parathylold), heart, lungs, liver, kidneys, spleen, adrenals, stomach, tastes or ovaries, urinary b!adder, and right femur (with bone marro~). 8-3-2 Organ weight The following organs were weighed before fixation, then the relative organ weight (ratio of organ weight to body weight on the day of necropsy) was calculated: brain, liver, kidneys, adrenals, and tastes or ovaries. 8-4 Histological examination From the ollowing fixed organs of all rats in the control and the highest dose (30 mg/kg) groups, histological sections cf paraffin-embeded specimens were cut and subjected to hematoxylin-eosin staining, then histological changes were surveyed: heart, liver, spleen, kidneys, adrenals, brain, and testes or ovaries. (typical seven organs} The survey of the liver was expanded to anlmals of the other dose groups since some chages were found in case of 30 mg/kg dose group. -- 15 -- 22779977..00002244 3M MN01650010 additional histological survey wes conducted to the following cases fn which Additional histological survey was conducted to the fol2owing cases tn which any macroscopic changes were observed at the necropsy: lung in ons case any macroscopic changes were observed at the necropsy: lung in one case ((saaniimmaall nnoo.. 00110055)),, kkiidduneeyyss iinn ttwwoo ccaasseess ((aanniismaall mos. nos. 1103, 1103, 2102). 2102), and and testes testes in one cass (anisal so. 0111). in one case (anmal no. 0111). Concerning the ded aniasl which occurred during the recovery period in one Concerning the dead animal whlch occurred during the recovery period in one alo (saissl mo. 4109), histological survey was perforsed in the typical male (animal no. 410g), histological survey was performed in the typical seven organs and in thyss end stomach, the latter two were odditional seven organs and in thT=us and stomach, the latter two were additional because of the macroscopic changes found at the necropsy. In this animal. because of the macroscopic changes found at the necropsy. In this animal, tarry intestinal contonts were found betwesn duodenum aad flews st autopsy: tarry intestinal contents were found between duodenum and ileum st autopsy: hhoowweevveerr., pprreoppaarraattiioonn ooff tthhee ssppeecciimseenn aanndd hhiissttoollooggilccaall sexxaasmiminsattiioonn wweerree otnot pertorasd because of post-sorten autolysis - performed because o post-mortem autolysis AALlLl tthhrroouugghh tthhee hhiissttoo~paatthhoollooggyy., hhiissttool]ooggitccaall ssppaecciimmaennss wweerrae pprreappaarresdd iinn RNaarrae Pathological Resoarch. whereas the microscopic exmaination wes carried out Pathological Research, whereas the microscopic examination was carried out in Hatono Resesrch Institute of Food snd Drug Resescch Center, both at our in Ratano Research Institute of Food and Drug Research Center, both at our qaquueesstt. 5. Statistical Analysis 9. Statistical Analysis For metrical dats. homogensity of variance ssong all groups was first tested For metrical data, homogeneity of variance among all groups was first tested bbyy BBaarrttlleetttt''ss smeetthhoodd.. TThheenn tthhee hhoomsooggeenneeiittyy ooff aailll ggrroowupp mseeasnss wwaass tteesstteedd bbyy one-way layout analysis or Kruskal-allls H-test. dopending on whother the one-way layout anelysls or Kruska1-Wallls H-test, depending on whether the group varianco was hoacgensous of aot, respectively. When group memms were group variance was homogeneous or not, respectlvely. When group means were ssiiggnniiffiiccaannttllyy hhoetteerrooggeensecoouuss aonndd eeaacchh ggrroouupp hhaodd tthhee ssaammee nnuemsbbeerr ooff ddaattaa iStteemmss., ddiiffffeerreenncceess iinn mmeeaann vvaalluueess bbeettwweeeens oannyy ggrroouupp oanndd tthhee ccoonnttrrooll wweerree tested by Dumett's multiple range test or Dummett's rank sun test, tested by Dunnett's multiple range test or Dunnett's rank sum test, sccording to whether the group varlasce was homogeneous of ot. according to whether the group variance was homogeneous or not, respectively. Kien group seams were heterogencous and the musber of data respectively, When group means were heterogeneous and the number oF data itoas fn each group was unequal, the above-mentioned procedures were items in each group was unequal, the abovs-mentioned procedures were croeppllaacceedd bbyy SScchheeffffee''ss mmuullttiippllee Traannggee tteesstt oorr SSechheeffffee''ss rraannkk ssuuma tteesstt,, rreessppeeccttiivveellyy.. AArrmalittaaggee''ss cchhii--ssqquuaarreedd tteesstt ~waass uusseedd ttoo aesssseessss ddilssccrreettee ddaattae such as urinalysis findings. such as urinalysis findings. -- 16 -- 22779977..00002255 33MM_MMNNO011665500001111 RESULTS - RESULTS 11.. MMorotratatlsietyy ALL rats survived to the end of sdainistration period; during the recovery All rats survived to the end of administration period; during the recovery ppeerriioodd,, hhoowweevveerr,, oonnee mmaallee ggiivveenn 3300 xmgg//kkgg ddileedd oonn ddaayy 3322 ((vvlizz.. ddaayy 44 ooff rreeccoovveerryy 4). **)) EEaacchh ddaayy tthhrroouugghhoouutt tthheo ssttuuddyy i1ss iinnddiiccaatteedd xwiitthh ssuucchh wwaayy aass tthhee ddaayy ooff ccoommmmeenncceemmeenntt ooff tthheo asdmaiinnisstteraattiioonn 1iss nnsasmeedd ""ddaayy 00 *" ;; aonndd tthhee nneexxtt ddaayy oof tthhee llaesstt nsddmalinniisottrraottlioonn ((vvlise.. ddaayy 2280)),, tthhaatt iiss %thhee ffiirrsstt ddaayy ooff rreeccoovveerryy ppeerriioodd,, 1iss eexxpprreesssseedd aass ""ddoayy 0 of recovery . , 22.. CCllilnnilocaall SSiiggnnss ((TTaabblleo 11,, aanndd AAppppeennddiixx 1I)) 201 During the sdaintstration period 2-1 During the administration period NNoo sabbunoorrsmaall ssiiggnnss wweerroe nmootteedd ifnn aallll rfaattss ooff the ccoonnttrrooll asnndd tthhee 1I00 mmgg//kkgg the or lower dose grows. With rats receiving 20 mg/kg (inclusive of the or lower dose groups. With rats receiving 30 mg/kg (inclusive of the sroeccoovveerryy ggrroouupp)),. hhoowweevveerr,, tthhee ffoolllloowwiinngg fEinnddiinnggss wwoerree ddeetteecctteedd::" SSaallilvvaattilcoan-------aa ccoonnssiiddoerrsabbllee nnuumsbbeerr ooff sanniimmaallse ooff bbootthh sseexxeess,. ifnn tthhee llaatttteerr hhaallff ooff aaddmmiinniissttrraattiioonn ppeerriioodd;: aandd iinn oonnee mmaallee oonn ddaayy 00., bbeessiiddeess;: RReeddddiisshh ssaalliivvaattiioonn=-----iinn aa ffoeww smaalleess asnadd ffeemmaalleess,. ooccccaossiioonnaallllyy;: TToonnitcc--cclloonnitce ccoonnvvuullssiioonn,, llyyiinngg,. bbrraaddyyppnneesa--=---iian oonnee xmaallee oonn ddaayy 22S6:; Reduced spontaneous movement. clonic or tantc-clontc convulsion. lying Reduced spontaneous movement, clonic, or tonic-clonlc convulsion, lying oorc ccrroouucchhiinogg,, bbrraaddsyppnneeaa oorr ccyyaanmoossiiss-<-c--Iinn oonnee aanndd ttwwoo ffeenmsalleess oonn ddaayy 2200 0andd ffrroonm ddoayy 2255 ttoo 2271,, rreessppeeccttiivveellyy;: LLoossss ooff hhaaiirr aanndd ccooaatt ssttaeilnniinngg-e---iinn tthhrreeee saandd oonnee ffeammaslleess rreessppeeccttiivveellyy.. oonn ddaayy 1177 aanndd aafftteerr.. 2-2 Ducting tho ocovery pariod 2-2 During the recovery period MNoo aabbanoorrmsaall ssiiggnnss aappppeeaarreedd iinn bbootthh mmaalleess aanndd ffeemmaalleess ooff tthhee ccoonnttrrooll ggrroouupp aats wweellll asss iinn tthhee saddnmiinniissttrraattiioonn ppeerriioodd.. IInn ccaassee ooff tthhee mmaallse ggiivveenn 3300 -- 11 - -- 17 -- 22779977..00002266 I3MM_MMNND011665500001122 mmgg//kkgg oanndd ddiieedd dduurriinngg tthhee rreeccoovveerryy period, period, reduced reduced spontansous spontaneous movement. movement~ Llyyiinngg., bbrraaddyyppnoeena,, rreeddddiisshh ssaslliivvaattiioonn aanndd bbrroowmnaiisshh uurriinnee were were found found on on day day 3311 ((vvlizz.. ddaayy 3 3 ooff rreeccoovveerryy)),. tthheann died died on on the the next next day. day. Loss Loss of of hair hair found found iinn' 'ffeemmaalleess ooff tthhee 3300 mmgg//kkgg ggrroouwp dduurriinngg the the sdaintstration administration poriod period lasted lasted uunnttiill tthhee ttoerramiinnaattiioonn ooff tthhee rreeccoovveerryy period, period, whereas whereas the the coat coat staining staining in in oonnee fteemnaslleo ddiissaappppeaasrceodd aafftteerr day day 34 34 (viz. (vlz. day day 6 5 of of recovery) recovery). 3. Body Weight (Fig.1. Table 2. Appmodix 2) 3. Body Height (Fig.l, Table 2, Appendix 2) 3-1 During the adatatsteation period 3-1 During the administration period Body weight goin fa the 10 g/kg or lower down groups was siailer to that Body weight gain in the 10 mg/kg or ]ower dose groups was similar to that of the control throughout the adainlstration period. Significant suppres- of the control throughout the administration period. Significant suppres- ston of weight gain. bowsver. appeared in both males and females receiving sion of weight gain. however, appeared in both males and females receiving 30 ag/by (rats of the recovery group inclusive) fron week 2 and after. 30 mg/kg (rats of the recovery group inclusive) from week 2 and after. 3-2 During tho recovery period 3-2 During the recovery period The above-mentioned supprssion of weight goin during the adslaistration The above-mentioned suppr6ssion of weight gain during the administration ppeerriioodd llaasstteedd uunnttiill tho the ead end of of recovery recovery period period. 44.. FFoooodd CCoonnssuummppttiioonn C(TTaabbllee 33,, AAppeesnddilxx 33)) 4-1 During the adataistration period 4-i During the administration period FFoooodd ccoonnssuummppttiioonn ifnn tthhee 1100 mmgg//kkgg oorr lower lower dose dose groups groups was was siniler similar to to that that of the control throughout the edsiaistration period. Significant suppros- of t~e control throughout the administration period. Significant suppres- ston of the consumption, however, ses bserved in both sexes receiving 30 sion of the consumption, however, was observed in both sexes receiving 30 2mgg//kkgg ((rraattss ooff tthhee rreaccoovveercyy ggrroouupp iinncclluussiivvee)) from from week week 2 2 and and after. after. 4-2 During the recovery period 4-2 During the recovery period TThhee ssuupppprreessssiioonn iinn ffeemmsalleess of of the the 30 30 g/kg mglkg group group lasted lasted one one sore more week week after the finish of the adatnistration after the finish of the administration, -- 18 -- -- 18 -- 22779977..00002277 I3MM_MMNND011665500001133 5. Hematological Findings (Tsble 4 and 5. Appendix 4 and 5) Eematological Findings (Table 4 and 5, Appendix 4 and 5) 51 5-1 At At tho the esd end of of the the aaddamiinniissttrraattiioonn ppeerriioodd Hematological exsanation with onisals of the mals groups perforsed on the Hematological examination with animals of the main groups performed on the scheduled nocropsy-day revasled the following changes: decreased scheduled necropsy-day revealed the following changes: decreased hheemmoogglloobblinn ccoonncceennttrraattiioonn iinn mmaalleess ooff tthhee 1100 msgg//kkgg aanndd hhiigghheerr ddoossee ggrroouuppss;: reduced prothrosbin tise in fosales receiving 30 ag/ky. reduced prothrombin time in females receiving 30 mglkg. 5-2 At tho anid of the recovery peried 5-2 At the end of the recovery period Aftec-recovery exsanation with snisals of the 30 g/kg recovery grow After-recovery examination with animals of the 30 mg/kg recovery group revealed the folloving chasges: In males. dacresse of erythrocyte count. revealed-the folio.lag changes: in males, decrease of erythrocyte count, hemoglobin concentration, and hematocrit, shortening of prothrosbin tise. hemoglobin concentration, and hematocrit, shortening of prothrombin time, nd tncresse of leukocyte count; fn fosales, decrasse of hemoglobin and increase of leukocyte count; in females, decrease of hemoglobin Soncentration aad seen corpuscular hesoglobin concentration. shortening of concentration and mean corpuscular hemoglobin concentration, shortening of activated partial throsboplastia tise. eid increase of mewn corpusculsr activated partial thromboplastin tlme~ end increase of mean corpuscular vvoolluumsee.. 66.. BBiioocchheesmticcaall FFiinnddiinnggss ((TTaabbllee 66., AAppppeennddiixx 66)) 6-1 At the end of the sdaintstration period 6-1 At the end of the administration period The sxsainstion with smissle of the main groups resulted in decrease of The examination with animals of the main groups resulted in decrease of OGOTT iinn ffeemmaalleess ooff tthhee 1i mmgg//kkgg aanndd hhiigghheerr odossee ggrroouwpp,. aanndd iinnccrreesassee ooff aallbbuumsiino iinn aallssoo ffeemmaalleess ooff tthhee 1100 mmgg//kkgg aenndd hhiigghheerr ddoossee ggrroouuppss.. MMoorreeoovveerr.. In the 50 ng/kg group. the following changes were significant: increase of in the 30 mg/kg group, the following changes were significant: increase of GPT and of chloride, and docrosse of total cholesterol. all these in both GPT and of chloride, and decrease of total cholesterol, all these in both sseexxeess;: ddeeccrreeaassee ooff ttoottaall pprrootteelinn oanndd iinnccrreeaassee ooff sallkkealliinnee pphhoosspphhaattaessee aanndd Ooff MAIGG rraattiioo,, aallll tthheessee Iinn mmaalleess;; aanndd iinnccrreeaassee ooff uurreeaa nniittrrooggeenn iinn fceomnaalleess.. IInn aaddddiittiioonn,, rreeddddiisshh cchhaannggee ooff sseerruuam wwaass vviissuuaallllyy rreecccoggnniizzeedd iinn ttwroo mmaelleess ((aanniimmaall snooss.. 33110022,, 33110033)) aanndd iinn oonnee ffeemmallee ((aanniiamaall rnoo.. 33220058)) ggiivveenn 1100 msg//kksg., sanndd iinn ffiivvee smaalleess ((aasniismaall rnooss.. 44110011.. 44110022.. 44110033., 44110055.. 44110066)) maandd finn all females given 30 g/kg. all females given 30 mg/kg. ~ 1s -- 22779977..00002288 I3MM_MMNND011665500001144 66--22 AAtt tthhee e5n0dd of of the the recovery recovery period period SSiiggnniiffiiccaanntt cchhaannggeess ddoettaecctteedd ifnn tthhee 3300 mmsg//kksg rreeccoovveerryy ggrroouupp wmeerree aass ffoolllloo.wss:: lloowweerriinngg ooff ttrrilggllyyccoerrilddeess aanndd iinnccrreeaassee ooff AA//GG craartlioo finn mmaalleess:; iinnccrreeaassee ooff uurreesa nniltti~ooggeenn,. ttoottaall pprrootteeiinn,. aallbbuusmiinn,, ccaallcciiuuma,. iinnoorrggaanniicc Phosphorous asd sodium, aad decrease of glucose and triglycerides all phosphorous and sodium, and decrease of glucose and triglycerides. these in fonales these in females. AAnndd bbeessiiddeess,. rreeddddiisshh cchhaaonggee ooff sseerruums wwaass rreeccooggnniizzeedd ifnn ffoouurr mmaalleess ((aanniimmaall Bos. 4108, 4110, 4111. 4112) and fn threa females (animal nos. 6207. 4210, nos. 4108. 4110, 4111. 4112) and in three females (animal nos. 4207, 4210, am. 4211). 7. Ursnalyats (Table 7. Appendix T) 7. Urinalysis (Table 7, Appendix 7) 7-1 During the sdafnistration period 7-1 During the administration period TThhee eexxaamiinnaattiioonn ppeerrffoorrsmeedd wwiitthh rraattss ((iinncclluussiivvee ooff tthhee rreeccoovveerryy ggrroouupp)) oonnee .weeeekk bbeoffoorree tthhee eenndd ooff aadamiinniissttrraattiioonn ppeerriioodd rreessuulltteedd iinn tthhee ffoolllloowwiinngg changes: changes: ppHi sshhiifftt ttoo aallkkaalliinnee ssiiddee iinn mmealle~ss ooff tthhee I 1 mmgE//kkgs sanndd 1100 mmgg/lkkgg ggrroouuppss aanndd iinn ffeemmaalleess rreecceeiivviinngg 00..11 mmgs//kksg;; pPHH sShhiifftt ttoo aacciiddiicc ssiiddee finn bbootthh sseexxeess rreecceeiivviinngg 030 mmgE//kkgS:; ddoeccrraeanssee ooff ooccccuulltt bblloooodd iinn mmaalleess rreecceeiivviinngg 3300 sms//kkgs:: ddeeccrreenassee ooff pprrootteeiinn ilnn ffeemsaslleess rreecceeiivviinngg 3300 smgE//kkss.. 7-2 During the rocovery period 7-2 During the recovery period SShAiLfEt ooff ppHH ttoo ascciiddiicc ssiiddee .waass nnootteedd iinn mmaalleess ooff tthhee 3300 mmgg//kkg~ rreeccoovveerryy gsroouupp iinn tthhee eexxsaamiinnaattiioonn ccoonndduucctteedd oonnee e.eeekk bbeeffoorree tthhee eenndd of of the the period. period. 8. Pathological Findings 8. Pathological Findings 8-1 Organ weight (Table 8 and 3; Appendix 8 and 3) 8-1 Organ weight (Table 8 and 9; Appendix 8 and 9) 88-1I--11 AJbbssoolluuttee oorrggaann wweeiigghhtt 1) At the end of the administration period 1) At the end of the administration period Hith the antsals of the sain grows. liver Nelght increased in both males With the animals of the main groups, liver weight increased in both males ~ 20 -- 22779977..00002299 I3MM_MMNND011665500001155 and fomsles of the 10 mg/kg and higher dose groups: whoress weight and females of the 10 mg/kg and higher dose groups: whereas weisht ddeeccrreeaasseedd iinn kkiiddnneeyyss ooff mmaalleess saandd ccvvasrriieess oof~ ffeemmaalleess,, bbootthh iinn tthhee 3300 mmgE//kkgS group. grOuP. 2) At the ead of the recovery period 2) At the end of the recovery period CChhaannggeess nnootteedd iinn tthhee 3300 mmgg//kkgg rreeccoovveerryy ggrroouupp wweerree eaas ffoolllloowwss:: iInnccrreeaassee ooff tthhee lliivveerr wweeiigghhtt iinn bbootthh sseexxeess;; iinnccrreeaassee iinn tthhee tteesstteess ooff smaalleess;; ddeeccrreeaassee iinn tthhee kkiiddnseeyyss,, iinn tthhee aaddrreennaallss aanndd iinn tthhee oovvaarrileess,, aallll tthheessee ooff ffeemmaalleess.. 88--1I--22 RReellaattiivvee oorrggaann wweeiigghhtt 1I)) AAtt t%hhee eenndd ooff tthhee saddamiinniissttrraattiioonn ppeerriioodd TInnccrreesassee ooff lliivveerr wweeiigghhtt ffoouunsdd Iinn tthhee aabbssoolluuttee wweeiigghhtt wwiitthh tthhee 11O0 mmgE//kkgS aanndd hhiigghheerr ddoossee ggrroouuppss wwaass aallssoo ddeetteecctteedd iinn tthhee rreellaattiivvee wweedigghhtt iinn bbootthh wmaalleess aanndd ffoemmaalleess.. IInn aaddddiittiioonn,, tthhee ffoolllloowwiinnggss wweerree oobbsseerrvveedd:: iinnccrreaasssee iinn tthhee bbrraaiinn ooff bbootthh sseexxeess rreecceeiivviinngg 3300-mgg//kkgg:; iinnccrreeaassee iinn tthhee aaddrreennaallss eenndd tthhee tteesstteess ooff mmaazleess rreecceeiivviinngg 3300 mwgg//kkge:: ifnnccrreeaassea iinn tthhee kkiiddnneeyyss ooff feommaalleess gsiivveenn 1100 mmgg//kkgg.aanndd hhiigghheerr ddoossee.. 22)) AAtt tthheo eenndd ooff tthhee rreeccoovveerryy ppeerriioodd CChhaann~geess ddeetteecctteedd iinn tthhee 3300 mmgg//kkgg rreeccoovveerryy ggrroouupp wweerree aass ffooll]loowwss:: iinnccrreeaassee iinn tthhee lliivveerr aanndd tthhee bbrraaiinn ooff bbootthh sseexxeess:: ilnaccrreeaassee iInn tthhee tteesstteess ooff mmaalleess;: iinnccrreeaassee iinn tthhee kkiiddnneeyyss ooff ffeemmaalleess.. 88--22 MNaaccrroossccooppilcc ffiinnddiinnggss aott nneeccrrooppssyy ((TTaabbllee 1100;; AAppppeennddiixx I100 aanndd 1122)) 88-22--11 MMaaccrroossccoopptiec fiinnddiinnggss iinn the the ddeeaadd aanniimmaall AAuuttooppssyy oonn tthhee mmaallee ddiieedd dduurriinngg tthhee rreeccoovveerryy ppeerriioodd ((aanniimmaall nmoo.. 44110099)) rreevveeaalleedd tthhee ffoolllloowwiinngg cchhaansggeess:: aattrroopphhyy aanndd ddaarrkk rreeddddiisshh ppaattcchh iinn tthhee tthhyymsuuss;: hheemmoorrrrhhaaggee aanndd ttaarrrryy ccoonntteennttss ifnn tthhee ssttoommaacchh;: ttaarrrryy ccoonntteennttss iinn tthhee ssmaaal)l ifnatteessttiinnee;; ggrraayyiisshh ppaattcchh iinn tthheo lliivveerr.. 88--22--22 MMaaccrroossccooppiicc ffiinnddiinnggss aatt sscchheedduulleedd nneeccrrooppssyy - 21 - -- 21 -- 22779977..00003300 33MM_MMNN0011665500001166 Gross pathological findings observed were listed below. - Gross pathological findings observed were listed below. 1) At the end of the sdatnistration period l) At the end of the administration period Males of the 30 mg/kg growp---greylsh dot or patch in the liver mad Males of the 30 mg/kg group---greyish dot or patch in the lever and hhyyppeerrttrroopphhyy ooff tthhee lliivveerr,. iinn aallll ssiixx rraattss;: bbllaacckkiisshh cchhaannggee iinn tthhee Kkiiddnneeyyss sanndd ddaarrkk rreeddddiisshh cchhaannggeo iian tthheo aaddrreennaallss,. iinn oonnee rraatt.. FFeemmaslleess ooff tthheo 3300 magg//kkgg ggrroouupp------ddaarrkk rreeddddlisshh cchhaannggeo,. yyeelllloowwiisshh cchhaannggee aanadd ggrraayyiisshh ppaattcchh ifnn tthhee lliivveerr,, iinn ffoouurr,. oonnee oanndd oonnee rraattss rreessppeecottlivveellyy;: hbyyppeorrttrroopphhyy of of tthhoe lliivveerr,. iinn aallll ssiixx rraattse;: ppyyeelloeccttaasstiss iinn tthhee kkiiddnneeyyss ooff oonmee rraatt. FFeemsaslleoss ooff tthhee 1l0O mmgg/[kkgg ggrroouspp-------ddaarrkk rreeddddiisshh cchhaannggee finn tthhee lliivveerr iinn oonnee rat. OOtthheorrss-------ggrraayysshh ppaattcchh fin~ tthhee kkiiddnneeyyss oof oonnee mmaallee ooff tthhee 11 mmgg/lkkgg ggrroouupp;s pPyyeolleeccttaassiiss iinn tthhee kkiiddnneeyyss ooff oonnee mmaallee ooff tthhee 00..I1 mmgg/lkkgg ggrroouwpp:; ccyysstt iinn tthhee kkiiddnneeyyss aanndd ddaarrkk rreeddddiisshh ppaattcchh finn tthhee lluunngg., eeaacchh iinn oonnee mmaallee ooff tthhee ccoonnttrrooll group. group. 22)) AAtt tthhee eenndd ooff tthhee rreeccoovveerryy ppeerriioodd MMaalleess ooff tthhee 3300 smgg//kkgg ggrroouupp=-----ggrraayyilsshh ppaattcchh aanndd ddaarrkk rreeddddiisshh cchhaannggee., aanndd hhyyppeerrttrroopphhyy iinn tthhee lliivveerr,, aallll tthheessee iInn ttwroo rraattss.. FFeommaalleess ooff tthhee 3300 mmgg/lkkggggrroouspp--------ddaa~rkk rreeddddiisshh cchhaannggee aanndd ggrraayyiisshh ppaattcchh,, aanndd hbyyppeorrttrroopphhyy ifnn tthhee lliivveerr,, iinn ffiivvee,, oonnee,. aanadd oonnee rraatt rreessppeeccttiivveellyy.. OOtthhoerrss~-----aattrroopphhyy ooff tthhee tteesstteess iinn oonnee mmaalloe ooff tthhee ccoonnttrrooll.. : 88--33 HHiissttoollooggiiccaall ffiinnddiinnggss ((TTaabbllee 1I1I:; AAppppoensddiixx 1II1 aanndd 1122)) 88--33--11 HHiissttoollooggiiccaall ffiinnddiinnggss iinn the the dead dead aanniimmaall HHiissttooppaatthhoollooggilccaall eexxaammiinnaattiioonn wwiitthh tthhee mmaallee ddiieedd dduurriinngg tthhee rreeccoovveerryy ppeorrioodd ((aanniimmaall mnoo,.44110089)) rreevveeaalleedd tthhee ffoolllloowwiinngg ffilnnddiinnggss:: eeoossiinnoopphhiilliicc ddeeggeenneerraattiioonn iinn .cceennttrriilloobbuullaarr hheeppaattooccyytteess., ffooccaall nneeccrroossiiss aanndd ppeerriipphheerraall ffaattttyy cchhaannggee iinn tthhee lliivveerr';, ddiillaatteedd ttuubbuulleess iina rreessttrriicctteedd ppoarrtt ooff tthhee -- 22 -- 22779977..00003311 33MM_MMNNO011665500001177 Aitoores norehogte foi In the bra: ulcer sad hesorrhoge fn a. kidneys; hemorrhagic loci in the brain: ulcer end hemorrhage in theSlatuins stomach: congestion snd bomrshage In the thus. To sbeoraliey glandular stomach: congestion and hemorrhage in the thymus. No abnormality vi shearers tn the eure spine, adcats wu evees was obvserved an the heart, spleen, adrenals and testes. 02 Ristologtenl Endings o scbeduled ecropsy 8-3-2 Histological findings at scheduled necropsy Mistolopice findings chanerad are Listed Belo. Histological findings observed were lls~ed below. 1 At the ad of th sininistracion pict 1) At the end of the administration period iver (~ Liver lo contelotuine bepsonyten, cyapiass sppenced So be geansint sed In centrllobhlar hepatocytes, cytoplasm appeared tc be granular and conaaphic, sad swelling van recognized fn a1 wales wd fons of the eoslnophillc, and swelling was recognized in all males and females of the 50 wpe prow: In tn 10 ms gees, the sisiler chenges, thengh 30 mg/kg group: in the i0 mg/kg group, the slmilar changes, though hgh. wore tov fn a11 male: ad 1 fnales, sith tho sueilng or slightly, were found in all males; and in females, either the swelling or contaphili change ws hearvad in fou rats ave siightr incidence eoslnophillc change was observed in four rats even slighter in incidence 100 tensity then fn sale fn the 1 mp /kg grow, She statins change wes and intensity .than in male; in the i mg /kg group, the similar change was Shahn ta sensity and incidence than In 10 su/he group. (hots 1 and slighter in intensity and incidence than in 10 mg/kg group. (Photo I and .2). Focal secronia 1a hopaocytes wes found fn ive salen snd tuo females of Focal necrosis in hepatocytes was found in five males and two females of the 30 wy/he grop. snd fn tno mas of the 10 mt grow. The smerosis the 30 mg/kg group, and in two males of the i0 mg/kg group. The necrosis a oun 10 in on sala f the contol Was found also in one male of the control. Fatty change in peripheral Joule vas renssable in mses of the 10 seks Fatty change in peripheral lobule was remarkable in males of the 30 mg/kg fro (hots 0: fn fomlas, howsvr, he inckdonce nd intensity vas group (Photo 4) : in females, however, the incidence and intensity was stalar to thet In te control. Localized fatty change wes alse baerved similar to that in the control. Localized fatty change was also observed 1a cn sate of he control. in one male of the control. Nccegiamions sas found 1a a fon males sad fnsios of ALL groups. though ~licroglanuloma was found in a few males and females of all groups, though Close ditterence in ckdence nd inansity vas not noticed Bets each clear difference in incidence end intensity was not noticed between each [---- dose group and the control group. @ et ~) Heart No shmormaiitios were fond expt exteomoly localised mycardinnl No abnormalities were found except extremely localized myocardlnal togomeation in tw sale of the control grew degeneration in two male of the control group. J -- 23 -- 22779977..00003322 awotssoors 3M MN01650018 @ solosn - (~ Spleen No sbaorsslities were found except extrsasdullory hematopoiesis in three No abnormalities were found except extramedullary hematopoiest$ in three aslo of the control group. male of the control group. @ Kidoors (~ Eidneys RReeggeenneerraatteodd ttuubbuulleess wweerree ffoouunndd ilon bbootthh mmaalleess aanndd ffeemmaalleess ooff tthhee 3300 mmgg//kkgg asd the control grows though no differssce fn lscldence aad intensity and the control groups though no difference in incidence and intensity betueon two groups was soted. Other findings: dilated tubules in ons male between two groups was noted. Other findings: dilated tubules in one male ooff tthhee ccoonnttrrooll;; iinnffiillttrraattiioonn ooff llyymmpphhooccyytteess iinn tthhee iinntteerrssttiittiiaall ttiissssuuee Iinn one sale of the 30 xg/kg group ond three ale and female of the control: one male of.the 30 mg/kg group and three male and female of the control; dilation of pelvis in tro sales and fn one fessle of the 30 mg/kg group. dllation of pelvls in two males and in one female of the 30 mg/kg group. Arenal, brain and testes or ovaries ~) drenals, brain and testes or ovaries No sbnoraalities wero observed. No abnormalltles were observed. 2) At the end of the recovery period 2) At the end of the recovery period iver (~ Liver Contrilabular hepatocytes were eosinophilic and showed sellisg in all Centrilabular .hepatocytes were eosinophilic and sho.ed swelling in all males and females given 30 mg/kg. As compared to the maln group animals. males and females given 30 mg/kg. As compared to the main group animals, eostnophilic eoslnophilic intensity intensity turmed turned slighter slighter fn in both both sexes: sexes; hhoowweevveerr,. tthhee Satensity of swelling vas siatlar to that in the sain group. intensity of swelling was similar to that in the main group. Focal necrosis In hepatocytes wos found In three sales and two fosales of Focal necrosis in hepatocytes was found in three males and two females of the 30 xg/kg group. though none dn the control. the 30 mg/kg group, though none in the control. Other findings: fatty change in peripheral lobule aad wicroglansioss were Other findings: fatty change in peripheral lobule and microglannioma were observed, the former was somewhat definite in males gives 30 mg/kg. tho observed, the former was somewhat definite in males given 30 mg/kg, the latter. hoxever, no difference was fousd with the control. latter, however, no difference was found with the control. @ Hoare ~) Heart Very slight syocordial fibrosis wos found in one msle Of esch of the 30 ~/ery slight myocardial fibrosis was found in one male of each of the 30 o/kg and the control group, and myocerdial dogonoration wes found n one mg/kg and the control group, and myocardial degeneration was found in one feral of the cotrol. No other stnoras] fisdings were noted. female of the cotrol. No other abnormal findings were noted. @ spleen ~) Spleen No sbroraal findings were found except extramedullary hesatopetests in ore No abnormal findings were found except extramedullary hematopotests in one -- 24 -- -- 24 -- 22779977..00003333 33MM_MMNND011665500001199 aslo of each of the 30 3g/kg and control groups. - male of each of the 30 mg/kg and control groups. @ Kidneys (~) Kidneys Rogonerated tubules were found ln both sexes of every group: Infiltration Regenerated tubules ~ere found in both sexes of every group; infiltration of Iyaphocytes in interstitial tissue xas observerd ix one sale snd in to of lymphocytes in interstitial tissue was observerd in one male and in two fonsle of the 30 mg/kg and the control group, respectively: very slight female of the 30 mg/kg and the control group, respectively; very slight chronte nephropathy was found la one mele given 30 sg/kg. No other chronic nephropathy was found in one male given 30 ag/kg. No other abnoranalitios was found abnornmalities .es found. Testes (~) Testes No sboorsal findings were noticed except otrophy in one mele of the No abnormal findings were noticed except atrophy in one male o the control group. control group. Arenal, brain and ovaries (~ Adrenals, brain and ovaries No abnoraalities were observed. No abnormalities .ere observed. 3) Histological findings for gross pathologicel sbnormslities 3) Histological findings for gross pathological abnormalities SSuweelllliiongg ooff ccyyttooppllaassam ooff cceennttrriilloobbuullaarr hheeppaattooccyytteess aonndd ffooccaall nneeccrroossiiss wweerrea found corcesponding to the macroscople fladings of hypertrophy and grayish found corresponding to the macroscopic findings of hypertrophy and grayish ppaattecdh,, respactivaly. respectively. -- 25 -- -- 25 -- 22779977..00003344 I3MM_MMNND011665500002200 D DIISSCCU USSSSIIOON N AANNDD C COONNCCLLUUSSIIOON N - Major findings obtained through repeated oral administration of the test Rn an Cre thn rr substance to rats for 28 days were summarized below. Those changes were considered to be affected by the test substance. I, Examination Flndings m: male, f: female Troi [mmr [wos Period heDose ~ no " (mg/kg) Administration 0.1 1 10 30 Recovery 3O i wo em Clinical signs (*l)---reduced spontaneous movement bradypnea, anemia, salivation, convulsion; suppression of body weight gain and food consump- m : e es ese t oeprin ee tion ddeeaatthhooccccuurrrreedd ((nnuusmbbeerrooff tthhee sanaiimsaall)) CT Hematological findings---inclinatlon to anemia, abnormal change of blood coagulation .;:Te(1)m Biochemical flndings---Abnormal changes in liplds, protein, and in enzymes or in electolytes ,Urinalysis (*2)---acidic urine Organ .eight---change in absolute weight of the oeliver; c chanhgeiainn n rreellg aattiievveewweeiigghhttoofftthheelilivveerraannddkkiidd-- -neys Macroscopic findings---greyish dot or patch and hypertrophy in the ltver Histol~gical findings---such changes as eosinophil- ic s.elllng and focal necrosis in the liver. fatty change inperipheral lobule | fatty change in peripheral lobule m : f m m Eml ;f ---- (*i) Observation during each of the administration and the recovery fon period. (*2) Examination one week before each end of the administration and the hn recovery period. The other-examination was practised at each end of the administration th es cs and the recovery period, Cae -- 26 -- 22779977..00003355 3M MN01650021 [Changes suggesting relation with tho liver] to [Changes suggesting relation with the liver] Anong the changes detected, decrease of serua cholesterol vas remarkable. Among the changes detected, decrease of serum cholesterol was remarkable. Though the mochaniss of the resction fs sot clear. it is generally Th?ugh the mechanism of the reaction is not clear, it is generally accepted that cholesterol is produced through synthesis in the liver and accepted that cholesterol is produced through synthesis in the liver and absorption absorption ooff lliippiidd iinn tthhee iinntteessttiinnee aanndd iiss ccoonnttrroolllleedd tthhrroouugghh scousulation fn tissues, conversion fate energy aad excrotion from the accumulation in tissues, conversion into energy and excretion from the bbooddyy.. AAss ddeeccrreeaassee ooff ttoottaall pprrootteeiinn maandd iinnccrreeaassee ooff AA//GG rraattiioo iinn mmaalleess oorr increase of albumin in females wore found, considering the conection increase of albumin in females ware found, considering the connection asong protein aad ltpoproteln end cholesterol, influence of the test among protein and llpoproteln and cholesterol, influence of the test aubstonce on globulia or effect to decrease lipoprotein was sugested: substance on globulin or effect to decrease lipoprotein was suggested; hence, for the reduction of serus cholesterol, concern of the liver was hence, for the reduction of serum cholesterol, concern of the liver was ssuussppeecctteedd.. LLoow~eerrilnngg.ooff ttrriiggliyycceorrliddee ffoouunndd ilna bbootthh mmaalleess and and femsles females of of the the 30 30 mg/kg mg/kg recovery recovery ggrroouupp wwaass aallssoo considered considered ttoo. bbee aaffffeecctteedd bbyy tthhee tteesstt ssuubbssttaannccee;: because the same tendency was observed in the main group though because the same tendency was observed in the main group though statistically fnsignificant and the facts that the test substance was statistically insignificant and the facts ~hat the test substance was suspected to affect to 1ipids os mentioned above. suspected to affect to liplds as mentioned above. Moreover. changes in enzymes relating to the liver. increase of the liver Moreover. changes in enzymes relating to the liver, increase of the liver adght auch macroscopic findings in the necropsy as grayish dot/patch and weight such macroscopic findings in the necropsy as grayish dot/patch and hypertrophy. and histopathological pictures such as eosinophilic swelling hypertrophy, and hlstopathological pictures Fuch as eosinophilic sWelling sannddffooccaall nneeccrroossiiss iinn cceennttrriilloobbuullaarr hheeppaattooccyytteess wwhhiicchh wweerreo ccoonnssiiddeerreedd ttoo be comected with the macroscopic findings, all these changes Suggested be connected with the macroscopic findings, all these changes, suggested that the test substance affected to the liver parenchysa. that the test substance affected to the liver parenchyma. Additional changes suggesting relation with the liver were shortened Additional changes suggesting relation with the liver were shortened prothrosbin tise fn females of the 30 ag/kg main growp and Lo males of the prothrombin time in females of the 30 mg/kg main group and in males of the samme dose of the recovery group, shortened activated partial same dose of the recovery group, shortened actlvatad partial tthhrroosmbbooppllaassttiinnee ttiimmse iinn ffeemmaalleess ooff the the 30 30 mg/kg mg/kg recovery recovery grosp. group. Changes Changes of of calciun and glucose observed ia fomales of the 30 mg/kg recovery group calcium and glucose observed in females of the 30 mg/kg recovery group, though not found in the main group. were a little remarkable cosparing though not found in the main group, were a little remarkable comparing ith level of physiological variation. and connection With the administ ~ith level of physiological variation, and connection with the administ- -- 27 -- -- 27 -- 22779977..00003366 33MM_MMNNO011665500002222 arattiioonn oof~ tthhee tteesstt ssuubbssttaannccee waass pprreossuumseedd. _ [Changes suggesting ralation with other organs] [Changes suggesting relation with other organs] Ae" to the tncrasse of leukocyte count observed in the 30 mg/kg recovery As' to the increase of leukocyte count observed in the 30 ag/kg recovery. group. the mechaniss was unclear: the some tendency however, was moticed group, the mechanism was unclear; the same tendency, however, wasnoticed in the main grow though statistically insignificant. Therefore the in the main group though statlstlcally insignificant. Therefore the connection with the test substance was suspected connection with the test substance was suspected. For the tncrense of ures nitrogen found in females given 30 sg/kg in both For the increase of urea nitrogen found in females given 30 mg/kg In both the natn and the recovery grows. it as not clear Whether the change was the main end the recovery groups, it was not clear whether the change was caused by degradation of protein or by excretion disturbence fn the caused by degradation of. protein or by excretion disturbance in the Kidneys: however, considering such facts as the lscrease of chlorides in kidneys; however, considering such facts as the increase of chlorlde in bbootthh sseexxeess ooff tthhee 3300 smgg//kksg mmaaiinn ggrroouupp aanndd tthhee iinnccrreeaassee ooff tthhea Kkiiddnneeyyss aweiigghhtt iin ffeammaalleoss ooff tthhee 1100 maggl/kkgg oorr hhiigghheerr ddoossee mmaalian ggrroouuppss aanndd ooff tthhee 3300 gmg//kkgg rreeccoovveerryy ggrroouupp,. eeffffeecctt ooff tthhee tteesstt ssuubbssttaanaccee ttoo tthhee kKiiddsneeyyss wweass ssuussppeecctteedd,. tthhoouugghh nnoo hhiissttoollooggiiccaall cchhaannggeess wweerree ffoouunndd.. AAst ttoo rreeddddiisshh cchhaannggee ooff sseerruusm rreeccooggnniizzeedd vviissuuaallllyy finn bbootthh sseexxeess ooff tthhee 1100 and 0 mg/kg grows. sometimes 7-GTP activity does not change appareitly end 30 mg/kg groups, sometimes 7 -GTP activity does not change apparently when reddish serus was coused by hemolysis. In this case of the study, when reddish serum was caused by hemolysis. In this case of the study, However, the elation ssong the reddish serus ond hemolysis snd 7GTP was ho.ever, the relation among the reddish serum and hemolysis end 7 -GTP was unclear. unclear. [[CChhaannggeess ccoonnssiiddeerreedd, nnoott ttoo bbee rreellaatteedd ttoo tthhee tteesstt ssuubbssttaannccee]] TThhee ffoolllZoowwiinngg cchhaannggeess wweerree ccoonnssiiddeerreedd noott ttoo bbee rreellaatteedd ttoo tthhee eeffffeecctt ooff the test substance. the test substance. Changes of aorganic phosphorous and sodius observed fn females of the 30 Changes of inorganic phosphorous and sodium observed in females of the 30 s/h mg/kg rreeccoovveerryy ggrroouwpp wweerree uunnccoonnnneecctteedd ttoo tthhee test test substenc, substance, because because the the sagnitude of the changes wes within lait of physiological variation and magnitude of the changes was within limit of physiological variation and the changes wore not found in the ain group. the chan~es were not found in the main group. IInn uurriinsaallyyssiiss,, ssiiggnniiffiiccaanntt ddeeccrraesassee ooff ooccccuulltt bblloooodd wweass rreeccoorrddeedd ifnn smaalleess of the 30 mg/kg main group: It wes. Nowever, considered to ba apparent. of the 30 mg/kg main group; it was. however, considered to be apparent. -- 28 -- 22779977..00003377 I3MM_MMNND011665500002233 because ono case of occult blood occurred in the control sceldentally, because one case of occult blood occurred in the control accidentally, hence tho change vas detected statistically. hence the change was detected statistically. Urine pi shifted to alkaline side with the sdsinistration in males Urine pH shifted to alkaline side with the administration in males receiving 1 and 10 mg/kg ond In females socolving 0.1 mg/kg: the change, receiving 1 and 10 mg/kg and in females receiving 0.1 mg/kg; the change, boxsver. was not dose-dependent. and the change xas considered mot to be however, was not dose-dependent, and the change was considered not to be related to the test substance, therefore. related to the test substance, therefore. Voight lnccesse of the brain and the testes and decxesse of the ovaries Weight increase of the brain and the testes and decrease Of the ovaries exo observed in males or females gives 30 g/kg in both the main and the were observed in males or females glven 30 mg/kg in both the main and the recovery grows: ao histological findings. however. mere detected recovery groups; no histological flndlngs, however, were detected. Theratore. tho changes were considered to be apparent caused by decrense Therefore, the changes were considered to be apparent caused by decrease of tho body weight and to have no toxicological significance. of the body weight and to have no toxicological significance. bo for the mecroscoplc changes In the sdrenals found In soles or femsles As for the macroscopic changes in the adrenals found in males or females ggiivveenn 030 m mgg//kkgg., nnoo hhilssttoollooggilccaall tindings findings were were detected: detected; thoy they were were Judged judged to have Little toxicological significance, therefor. to have little toxlcologtcal significance, therefore. Concerning any other changes auch as gross pathological findings having so Concerning any other changes such as gross pathological findings having no dose dependency end no bistologocsl fisdings except sbove-entioned dose dependency and no histologocal findings except above-mentioned incidents, they wore Judgad to be uncomaected th the test substance incidents, they were judged to be unconnected ~ith the test substance. (Goncluaton] [Conclusion] Je `asntioned stove. sigaificast response was found fn the lver by Asdsai"nmiesnttoiorniendg the te above, st substance significant toresbpootnhsesexwsass offounrdotsinwitthhe1 lmigv/ekrg obry administering the test substance to both sexes of rate with 1 mg/kg or Migher dose. Whereas cone of response as observed in the 0.1 mg/kg group higher dose. Nhereas none of response was observed in the 0.1 mg/kg Eroup "1th both sale and femal. with both male and female. TThheerraeffoorree tthhee ttooxxiiccoollooggiiccaall anoo--oobbsseecrvveedd--aeffffeecctt lleevveell ((NOOBELL)) ooff tthhee tteesstt substance. Swapla 0-1. was concluded to be 0.1 xg/kg for both mala and substance, Sample D-I, was concluded to be 0.1 mg/kg for both male and tonal of rats. female of rats. ---- 29 29 -- 22779977..00003388 33MM_MMNNO011665500002244 FF II GG.UURREESS AA NN DD TT AA BB LL EE SS (GroupMean (Croup Data) Data) Naan 22779977..00003399 I3MM_MMNND011665500002255 SSttuugdyy NNoo.. BBuMRRLI443SCC @g rele 5Bs x= y= y W = pe ei en x ia pr 9 yo :nh 2 1 2 ST nnistation ------------ Rscoery---- ,,.I I ~ecovery TF N~ nu AdminisCration a & B & ye after comorcenmnt of rested FY ` [= BHE 4m 7 me t 15 i Fonts Female PCa ETE FTE em ye Administration I Recovery I 7 Dye afotner cansra ooountsof rested 5 2 Da~s after commencement of treated FFiigk.. 1 1 CSCffhhdeeaammmnnaaiggllneeeeisssirraraiitanntsstimmoeeddnaauunnrriionnfbbggooddSyya2288mpwvlddeeeaailyyggsshhbtt-1ooffooffaonodrrnmaaaallllee and and a1d0mi%ni:strraetcioovneryofpeSraimopdl:e D-I and GoOmmi~s +14 + Rime lok 4 Bk days recovery period. 0.I m~k~ o 1 ~s,'~ ~ tBm~/~ xX RWS 38m~'kg 1 22779977..00004400 au_no1650026 3M MN01650026 : | sl 51 | 3| il | 4il 3 i 2:: mm TZ:: Pod s =| T z --- -= aE a . ys HE] I i FE whe] | 5 182] I E22i )fc | | (| want Hy a 1 hI 1m 3 i 22779977..00004411 ; 3M MN01650027 (Fal = i~l || 14 = il E HlHs eEo :| = EEE PE: El z CT =i Hl | | HERR ERA | aE E [Ei c] | il 5 il 3HEHh 38k PE 3;i 22779977..00004422 3M MN01650028 $- | 0 TE E 0 $33 IF Nr oA | ole men ek | - BETH i" 22779977..00004433 3M MN01650029 il | 52% 52 53 83 2797.0044 3M MN01650030 Fs: |= | zB oo 22779977..00004455 W3M_MNMDN106156500003311 :oz| s5):8 i| 5 ==" Soe. | F.|28 33 ogni Le gz 2 : $3: |%.]38 38 gem oom oe 22779977..00004466 swnoresoos2 3M MN01650032 I 25:| mge Age sze sg age . 3233 - 2 3320 ome gee 38 eda 2$81|| 8o5go 8m2g gn&ge gnxae 8ae gx |3% 3% RE & aw 3 HEE EE - Bl Ze = gs z %| 83 s2 32 33 82 |. = Cpe S t v #5 s 22 E 233]EF 2 | 5038 $S5ze 5o3go 5gee 3 ge [F 255-3| |3=8 =33 z#3c 3s8egeol 5 = i 1 -5 | g3#332| BST 83 RET RIT ET z | BsumO5sWr ESum E fem aEmBO | 2]lg o32r F2 Z 43 | 22779977..00004477 awoteso0ss 3M MN01650033 ig L8E. R2ao =g3ow - S88 &3 i E238 &age = 38a R 3 EEL": $38 gg peek 330C : Ihigett | al pne awe |B: TrEEESLEB 2PB |EX Pa eadge 3Sg2 [: 24S3|| 2o8go 5o%ge 5 i cE wh| ATORT BE gli} 22779977..00004488 awmotesooss 3M MN01650034 g 5| m3 sge nge Age me | .i 32 933 gee oro ay e3|3r ar a i BRE mgr oa n $A 7 #5 "LSFSxU0E3 35s cg | 28 58 38 33 22 _ . 3%5|82 3ISa F8a3 0 aaron sf- Era gapi . ge =hSs O8a2 | R LE5 wy Bi| fT ET RT RI MT 2 | EEE | slg 2 og a LE EE Hi Z 22779977..00004499 3M MN01650035 i |i 2 25 | soe age : AE . 23 ~ % 383 5 EEE 4 > mgo ope ~ % 3 EEE REEL: 1 i gga| s38d. E5EEkk phia|ed mE : i LEH Fslil 2| gow gue t s |=|=2%|3% 3;: - : 8 22453 8 SST prth.adi dd i 833 | %R EE z E EEE | .2 H3EE 3 22779977..00005500 = swnoresooss 3M MN01650036  832| 3 "3 3 "3 x Bog | BEE [one wae sme pow ave| | 23 5% E 22779977..00005511 33M UMNNO011B65500003377  23d | age won i zLE EERE gd) fay) one an lt 22779977..00005522 3M MN01650038 2 k age sqs mum ane ave 2 52| 33s 83" osu osreoaze mI ff 22: 3% r- o..~ 22779977..00005533 aw unoresooss 3M MN01650039 *4,- ! 853 |= "2 53 3 22779977..00005844 3_MNO16S0040 3M MN01650040 gi 5835| aye 82pee Bggeee #2mee g8%ee i 54B5N | 93 wae ope wpe #Tae 15) sme aoe s20 see une 25E%N| s3= 92e g3= 33 Ige g | BeB| SEER EY EY IY) 3203]5232 a|FnEeTBEmTaeORRsTue REgTue DETee Bge - Si 2 goog | 5 [Tege ego ogo ogo ogo [ Spt Bs BIEBfE|]s 32| PsueEmoeRsEue RmaeADae ALBEo o~ v 2| soe gue gre wae gue BE2 5 5 |B 83 F3T 83 R2 is HH 837 837 ET ESTES IC ply Pid 82| oe Ss - 2 8 22779977..00005555 aw ness 3M MN01650041 g o | 2m ua are gr Eo i [=afm oer oor of| ee re are ene an 2| nee ge spe age ge g | HEE EEA ET EE L 2fzoig(o252P 38 E25T m38a 33 Le Ta UR, Dar TEE HE : PEI22E05 I2]Z MTpee gEaET ze sE soIzg, e EA HSH HHI = BEE EEE lz 3 22 gli Ii 22779977..00005566 au noresorz 3M MN01650042 g nL8e3ne|ewoe *ou=e - 5 eo gL (HEE EL . S38 Tot SEE i go fece eon tc TE Ce 3 55 [mg me BE < = 33 a g3 hd2 22779977..00005577 3M MN01650043 g So |= san HE 2 |e en go | H 3] E see sE un iad0: . 5a DLo OEopet ia [[32253)7550 50 f SF 5 |so | gEueEsew E s Pg & 5 r= |i3 om 8g 290 = 8| 7s "s g Hila 5 HEE FE 2 22779977..00005588 . -- 3M MN01650044 i 55| mze sze spe sge Rye & =88 i 5 Bae ge age sze Age $5 ee oe mum me ape i 5 23 28 58 23 28 Bs | age age g | B28 | 3% E57 :3 gE) 5 5 mane P20) g5 eg 53 _ dak LTeEnt"|8Bgg5||3oge ca3e cE tc 3 ie | ogo wage EFT EFT 55 83 os3e oq8s nm Soe ETT| : A82 Lis s so3e RB i ze 53~g| age eg x= 3xo3=o3: = : 2 | || &%3 f 32 3$ =3 sig || 22779977..00005599 awnotesooss 3M MN01650045 8: |S| Esgue =zg0eo 5g2as0sgue gp2ee i EE 2 3] 3" 530 re gee mae| i. flare maga fag?| : PEs s.z5c0 ||f5 35 5gso SzEeTe S2E2T= SnEgTe 3aEge [goLos H TEARN : LFBeogs | |8o%e | 8|-8 =Z@8 82%3 3E%g O82I E 2 1= 52802(5.5.58. s5 g8 S3ew3 Zsou s I 852239 45 4s Te | $ HEECREEIEE aJ maI ag BEE ET pli P11 2 . 2 S - 2 8 22779977..00006600 swnoresooss 3M MN01650046 3 95| axe ue E gs 0S 1~5a Em3ee i3|w AEi = EE i ize |a5|m mk . } D. oSgHs 530 eeks mit is B, oIEz sg2| ane gow 22 83g BERT 2 gil: 1! 22779977..00006611 3M MN01650047 g Ss se see 1 -3|w 23~ 8m EA . FE fg E8sg:3 | |2S3 4Z3ao frF 528 [25 ane Bee see | 3 8% 25 - aig 5 PLoEpLeEsH | E es E|uge ene E + 5a3S:e | 5 |=g.338 * =5 F282 (235 4 E 238325 || 338 <338 f2f E3 E S22 22779977..00006622 awwnotesooss 3M MN01650048 - | 7 Seen - g = J 3 oos $ dm" i fleece - EE | DA ile oooo f glo" i ll e ea on fgizl (pgfdlloccoecaiailiss os Tp IEEE TEE | 0 [ecoB E ss ik : ||E ff|zl|ae2eececcoemee fpsig2l 3odlizoci se q d|l=c=ooc=e oecoc2 FfFiEzE EEE BEl.s_cx 22779977..00006633 3M MN01650049 o|q eRe CoH 8 1 a1" = sg HEE IRE i hsi|-- 2 Allee g wo | oo i 3c 3 |e = gz: FI .g5h1 HSeE lecE Ded Mocks cSHfE . dEfIoFzSf HELE dc pil =i aog|cccEER q H oTh [EEfE 872 i2]| 22Ras = eeu gil. 22779977..00006644 3M MN01650050 , |e odio ` 2100s 5 eo o ofc TREE NI [ER 1s Is _ Sehyz wii g flol ecmooeoe alll= ' CEPE Ani Lo EERE#oddEE ri IiE coEd dlecoicil sellin iii #3: 22779977..00006655 3M MN01650051  J: 3 EEoo tl, 3 i aEH L5 $Es Lui3s: el a|pzsaloocl2 (3 s--el F Eg Eo E cslHz,"ozol~EFsE2 il |i = =[2 = |= B- i2 l. 22779977..00006666 5 3M MN01650052 U 3 3; |ege zz 3 38 8g" . | 28 ng s2= ase 23| o 22779977..00006677 SW_ND1650053 3M MN01650053 g Tir ag| uC 88| 5 22779977..00006688 an nore 3M MN01650054 i FETT 2ge 23 2ge fic |B |ace age 5go msc ssc| FI S00 NY (FORRES EEE RE 0 PO] || six sim sam sam sa B:o 0 22779977..00006699 3M MN01650055 22779977..00007700 I3MM_MMNND011665500005566 V 2 82 78 5 | =35 se spe ogo spe : Ip E3T BFC ET Ep" ES ; 22779977..00007711 3M MN01650057 8B zd |sgeez-| 22779977..00007722 M3M_NMoNt061s6050005588 3 foe geo get gn a 22 53 | &< Ea 8s Be 8 s a| HEI :3 sr WE HE | MH BOE EEE 22779977..00007733 3M MN01650059 3s Io 233235 z1 |=g 852 22779977..00007744 M3M_NMONT06156050006600 HE : 3L a w| oo mtuNO 0000 bk Zo eo ocoos oses o . pFL1e 1 SEN a3 he JB . m3 1 i : oz i, ii FCHEE E nnagl hE eldrE BEpEANn IE EL 22779977..00007755 3M MN01650061 El mss 3|7|1| oon 0 vo mores ERNE DY pp 2 | |2| e000 0 : 553] F253] | is 5 |2|s| 0000 0 0 oem oo 4: coon ~00~ oe 0 0 3 occ co coves 0 o|$ Lo: Fos i = gi=8: i : i PACT $82 3 rsli E| g RS Pcfta] 2: FAiRgEsRRERonRl,E,NE| 3m Hala awnotesaosz 3M MN01650062 22779977..00007766 g|&|2| ~0co mn 00 cooce 0 5 F|7|1| meen wo moons Ea] coo 0s fact Es ig gE $535[ [| i: PPER Sno moon oo 7 : =a < mi o loln lol} .or gcefld d ii ohio Bilin Elian. fl hahnant ar isi PegtEsisafiasgiis |g 22779977..00007777 sunoresoss 3M MN01650063 El Els] coco + 0 00 0 0000 0 0 J2 leB]le] MmNeOoHo cN o 0c0oo 0o0o0o0e0 ooo : elle heistomscetto Bs-0 SF| Flu| ~oon on = ~o0on 0 oo iE. | oven 0 wv v wavee www | E|#| oo00 co 0 sooo 0 0 2 FIR ens tmnnon | ~ Loe i Eo omen : B5ei Esisf Eizliireih E: 255023 28 2 gant a 2 |) HE Hie RH ER 3s HEHEHE 22779977..00007788 awotesaoss 3M MN01650064 8 g: 3 331 88 ZU El BY] i i ) cacoo cocoons cceece cemccaofd ; 2 2E.l.iyk :: i: gig5s .2f2l i3hd3azsrg azaa $ of Flip Ea HELE H|satrihaiiissnaiinagatege|f 22779977..00007799 33M UMNON10B165500005655 AAPP PP EENNDDII CCEESS Undividusl Data) (l.d~vidual Data) 22779977..00008800 I3MM_MMNND011665500008666 a goCl lll]] p bBRee rEsen e on ' . " Ch eBoaa 3d TL] . . . ||| CEB 0a3 a pa EE F "a ' . DBE 38 Fo lela 1 . . . CEE 033 Fa So" ff' ]' a fo F ladE on E a3 eee Cea |hyB i 18 CE 3 iz 3 cla eee 2 ETEET E|f e n 2a EE ann ng = lactone ns Jol lI en nd ! . F ijs-22E -82s5-8Es3S -g%sssvsezsS soessEsRze o iEs Bolz|~]|= 5 s : seosst | :Ei 5s i3 el 22779977..00008811 3M MN01650067 g2d 2.| vad Ed | oo) B Ed |. CofE ) i| NE 2 fi E3d8|5] 8 RE | zeseem. | 8331F Ce ~J col EiEf e2 e- 252 Bd.| l == o 2 2 =E i i iii3 == 22779977..00008822 -" awwnotesooss 3M MN01650068 I I ga]! g80 00] I I ! ! 2I I da Waiandi) E3000 He-m-mme B: a. )s|-|=]|3 : I BEoo 22779977..00008833 . 3M MN01650069 fae EEE ld sees oF 83 vi 8aL825 3338, #3 3 382,I (13 3 38Eex, Zex| .3.33 33 33828ER,E. : 3g 33 13333 38 a 85d.i3@3=ess.+ 155353353588 3.3 3338 33 3338 coe 113 = Eg I 2 zEBIed 33- 2: or 15 ga 3a aan Ge RE -H LE JE LE Cae | Te LEERY he arn i HBoofE a EE E FE [en coe fed - 38J0o0n Ccioain T[ReTEe. Ceeena I sd or | E3s8 555ae85m58s%3 ls8Ez E885558 5 8888= = . i "4 osx3 | Bi:> is Ii23 i a0 22779977..00008844 33MM_MMNNO011665500007700 [EEE gg. mma. 83. haa 3g mms i: d. mmm... k2 g 5 WEEE | lass... . i] mas Agi3zr.all | | 5 Eg0. d B223E Iiz . flm=pseE. .=e mame y) maze.| ls=se iz Emme.| , maz.| 2 2# H3e55.5oE5EE8Fs8!E5s5s8 2 8 fo] a : 8i= 2277977..00008855 - S_MNotesoort 3M MN01650071 3 $m omTeneesesnm ee | szasssesasas | z2aff 27297977..00008866 3M MN01650072 i 3 r|2 Fo: Frere |es3ses |e 3323% ||33 ~ =; 32 | E_.||535z3z8s3e3|8 | 5a;se 22| =E RE= EL2 22779977..00008877 awnoresoors 3M MN01650073 1 | | gesase| oe .53E | J | 3) EEEET 000000 22779977..00008888 3M MN01650074 2797.0089 3M MN01650075 I O~NN <Z 2797.0090 -4 3M MN01650076 g pageny 2 oo SSNRRNERRAIN qs" ""2Fe lg | sEn=z8zssss8yzsEsgsEsRe|| gaogo iE| S33REESENEET | AFECENks 22779977..00009911 awotsso0r? 3M MN01650077 -- -- Le |i | sesess $17 1%.)3%38ak) 8 35E |3 . "g2oo | Eo|| 3Rs23332i2z2|| B24ac 7253|| s3uRnEz3eEeR| s#a7z :[oFo ~ 22779977..00009922 aw wnoresoons 3M MN01650078 -- -- . Z azggne 388 | 2 - - | REERER| EC ER 34 |g .|%EEEEE | E2r =:: -Z 22779977..00009933 W3M_MNMDN106156500007709 E Z| 2 |3 | oozes HH FEE] LR TTT 25| 3zssss| ge Z5 | 888888 | 24 | 22779977..00009944 aMNO1650090 3M MN01650080 ge io: os gageue BEEEE | 53 | femmes | 5 _|=zzzssgesses| 83 < EEF | 2 = 3 |}| Zssssesssces | En FERE $5& |g _|ssa3zszIssss| sen 5| FSRERSNHG (0 | 22779977..00009955 aMnoreso0s1 3M MN01650081 FE 8 EG FE i| s i54l= s2 YZ IZ 22779977..00009966 S_MNotesoos2 3M MN01650082 32 $% . |i BE sss $8 | aoe| wge EE EE | E H | F sar 22779977..00009977 au uotesoosa 3M MN01650083 T | z 2797.0098 3M MN01650084 83 i - " N [5 sss et | 2 . CExg | Beds ed 2 0s x 8 1:2, a=| age { gg $ S28 | 2 3g%i || sm 22779977..00009999 S_MNotes008S 3M MN01650085 EN ER | | | ;3 .|f 2 N; Z = 22779977..00110000 3M MN01650086 Py 82 FE $2 | | a39gamse . 223 | a & g amwen= | 23 |: gg $ sl agoile)as : 22779977..00110011 SM.MNote50087 3M MN01650087 3 s 3% EEE Zee | J TEER] "EIrg : ai 2 i$i] EgcetzI2 z 22779977..00110022 3M MN01650088 8g I i3 2 yr |i . 3 LE 52 | enn| om[e3 || "| dee | 27 y E igtf | | 547 f|s 22779977..00110033 SMMNotes0088 3M MN01650089 8z | 5 |= 2 22779977..00110044 a]NO1650090 3M MN01650090 83 : E| S did 42 FEg PS. ume 0 - 222 | 8 25%2 5k= 22779977..00110055 awotes00s1 3M MN01650091 g 23| sa=nss| 83 . 23.3 i 2?| |3n2=3onswd--s~| w22oo = | 333338 | 3 - 2 i32:c834|| 2cge2ge3es8sa4an4s|| g2|g2e | x |d8sda8| 32 25 .s3z:3e annaga| ogo - E23 |p gad | EE 8 " Pk Pu == s | vosase| u3 = 22D|| w3aeicdsdnse | 2m8go 24B%S|| 2me3d3=3=3i2|| 2233 iL] g8T5es| EBERSE|| 83 if) | 88 22779977..00110066 awotssoos2 3M MN01650092  i : 5 oz 2 2 RR=288 /an . 23> 2i E 8a| 333338| &2 = | 233233 | 2 2 328| 8c| cgqmn8asn3sg|| ope 83 ? 2 |x| 8833 | A gI3o.33I22c opm [age LiF#d3} | 7 g| =RSR:R| RS 5 =. gHinz FR~ 858888| BI 3 ik 2 z = x| 333398| &F 245| 233383 | 3% 83si| B3BL2E| 85 Ig |emEEEE| uT| 22779977..00110077 S_MNotes0083 3M MN01650093 g 1 SR888K| 83 & 3S38 z 3| enesas| ase 8 = & g=esse | al | 33| om $28| cannon| mae z |i*E73 sd3dge 2 |x| 3dR8RA| &F $ zs - E2E2L5% | og o3| 233z5o2n4a2|| 8g2E 8 2g :TEEE I 23 E33s: |E%d| sss23s| 32- 2 | 33233| &3 243| 232282 | 22 i5g3 BBBEEEBRER| B5Z3 if| zzzses| 550 22779977..00110088 SW MNOTEs0054 3M MN01650094 : F3rf_| assass| nze i E8233 | 28 . = | 333338 | 37 :. | 3E3:2i842|| EmaZaaagngoaEa2se|| EsEpe %ei A 2|x2| I Rseerdoauns || gRETe } g ze - 383% |g g|3sEm| Em a - a Uys = 3 s 4= 5 |5 E3= BEES <33 EEE ~ |= | ggdaEe | I 243| 33333 | 3% i 832 BIERRE| BZ | B %5)Is8EEEL8EI|R 8 22779977..00110099 awotssooss 3M MN01650095 g HA =mN=sE| 82 3:2 $ iz E|9| e2e3n5e8a8=|| g8e ) = | 33m ane [33I0]| Soonon| oe z3 282| dessdd | 8c 8 | 8 ~x |Rss%d] | 8 $2: P g$8s0 |g og|FmaEaE aE g $F2f *Ei3ge les%| sess3e| 89= Pi = x| 338332| &F 245| 23332 | oe 53%| Eeense | 30 | io}f|| ssaepsss| i5a= 22779977..00111100 aw uotBs008S 3M MN01650096 gE 23 ARI| 82 258 &z% ~ 8 . Z 82288 2) 28 ~ 3 . |= 833888 35 )= B35d| maa2|| 882 2 "22 | 8|g|| dgagsadsse'e|| e2ee gd . 33H |p| zeae + Fees 33 HE si 5BRERS | B 5Pi5l]gz ~ : 2 2438|| 2=32322=323f || 3222F[8 21 3 33s| AREGEE | BF ; 3553|8s5s5se5=5s || 58 m 22779977.0.10111 W3M_MNMON106156500000977 g 25 =% A=8| =v F25o 2 a 3 E32 a | 8% | 35 333 | 5 , | 2288|| a3e8 c8e8s|| n48gw 2? 2 . i8a 8 xa| 33 33a5| 3w%w $e 3283531 zg a=nf 2y8on| gn 8 EK TEE BE . FE z | 38 sy | A3 = x33) 26353|| 5d2= 323832|| 538 23o%f 2g8 22888| EY3" -1e3 | seszzz | iT B 22779977..00111122 aW_MND1650098 3M MN01650098 g 35 azgzax| age 23 ji 3 3 EE EE 3:4 ~onnee ~g g Z g ze Pil ao 38 |g gamma n soapki |g 3i|eeseesenz| s=ee IE 243| d2233d| 2 433i| Beagsz| 2 | #4 smeese| 8" 22779977..00111133 aunorsso99 3M MN01650099 gE i23 x=8888| Az i E 3|2o2ua--3o5wsn| 2ozo e 3a ae EEE 4 --oon= me |g| ama izi= .I3i88] |g |3zamEzEy s i iE | meses| 53 Pes] 53si | ResEEE| 33 22779977..00111144 3M MN01650100 g 25 AgaI=E | 89 . 22.73% | 33m| BE I= 3 El . |g sme $ I= . - F3=o4e8s% |p| zanmaanEams | |ar ge | . SEs | : PE gt 25s3s8 | 83 Tih = 83am| . gic | mERERE| 83 22779977..00111155 3M MN01650101 g 53 RE8R2R| a5 g 2.3 . Ela ) 23am ay 3? > ERE EEE g zl - 2 smREEE aa -a 3881 gp) aEEm aw s : JE PR sxeass| age FEC z 2 3 = x| 335333| 3% e5s3|| 2f9o3d3a3s2s || 538 T 3c | gemmEg | 23 i| samme | 5 22779977..00111166 awotssoroz 3M MN01650102 o g zit A 2 3. | conmcn| ome SE | E-E3 00~00 s df | agp sesess "| gi3| 53g) 82 22 | 33Ezes 000000 | 34 22779977..00111177 a MNoTes103 3M MN01650103  sg] me [oa : Zs| eccsce| ogo | Zhe] swonen| vue ,..Or- LE| : of83 | sf = 32% | fy - : w Zeus | BEE | vecona| wae 53| 33suse 3 8| SssEss| 2 22779977..00111188 S_MNotes0108 3M MN01650104 y 3 Biz | ooo | op $ conse . Eg 5 drnmos | ee Pozi |GEr|esassz (sie if5) z538s3338888|| 5Is 22779977..00111199 aw_uotesoros 3M MN01650105 Eo: oz - || 2797.0120 3M MN01650106 H- i: 2797.0121 3M MN01650107 2 333| posse | 53 3 fi |sessse| 82 | 22 | 3353535 | 24 22779977..00112222 33MM_MMNNO0T1G6S500110088 2 : EEE"0 ~ 233 8 000000 22779977..00112233 Swmoreso 3M MN01650109 ' HEE 22779977..00112244 swunrsort0 3M MN01650110 HE na = EE C0] 7 22779977..00112255 swnotes 3M MN01650111 32 1 22779977..00112266 - || swunrsorez 3M MN01650112 : oc 3 zsssss| ae | : IEEE RS 22779977..00112277 3M MN01650113 TEE Iss fz 3 2 83 ie | 22779977..00112288 3UNOTBS0114 3M MN01650114 omE 2 oe LE [C7] | oe | seuss |e 22779977..00112299 M_Notes01ns 3M MN01650115 . m :_ 22779977..00113300 I3MM_MMNND011665500111166 35 2 Z=Z==EZ =z 33%| samesm| ae 22779977..00113311 swunorssor7 3M MN01650117 v 22779977..00113322 3M MN01650118 E T | ZR===e 22 |e |zsmmma a DoE gE] EeEE 22779977..00113333 anunorssns 3M MN01650119 28 sf mas i 22779977..00113344 aunoresozo 3M MN01650120 22779977..00113355 33MM_MMNNO011665500112211 Bro ff mmm 22779977..00113366 3M MN01650122  3 | 83832) | 28 *0 ~, . 0 22779977..00113377 '-" 3M MN01650123 f #3 883852 | 53 3 |ssmuE| ez RE REE 22779977..00113388 swunrsorzs 3M MN01650124 fis |, con 22779977..00113399 3M MN01650125 m L 22779977..00114400 I3MM_MMNND011665500112266 FES| sadads | SZ arorona 2797.0141 3M MN01650127  5| meena| 52 ES sessas BFE |sgomen |e v 22779977..00114422 swwmoresas 3M MN01650128 3 22779977..00114433 a3M_nMoN10e156050112209 gz os3 i 3 3c | 9e99d 99| ege . EEE e 5 | zasase 23 if) sEEEE | mT 22779977..00114444 W3M_MNMDN106156500113300 3 2 cacann| ozo 22779977..00114455 3M MN01650131 22779977..00114466 3M MN01650132 _% | zy |esssss ese 23.:580 || E5.E5E) | ceasnnnea| naexe 22779977..00114477 a unrsorss 3M MN01650133 DO im 2797.0148 3M MN01650134 2797.0149 3M MN01650135 9 g 5 | 229999%a9ee =i IIIIIIIIIIII 44- ai || i Illlllllllll ,4- -= 2 B8| 33333333 | . | 238 || ssz3s3g8s3s2s3s2sdessss: 22779977..00115500 awuNotBs0136 3M MN01650136 2 " IIIIII Ben | E3F| eemese |] 2 :..B | t oo 58%) 333555| ' sszass | 22779977..00115511 awnotesors? 3M MN01650137  3 | 23 0~0~00 FE i + + IIIIIt Lz 2 58| 33s| i | sgzIse 22779977..00115522 SuMNoteso1as 3M MN01650138 33 i | saPgFes Illlll + .1- Ei: | 25 < I E05 [2 ~ Ts FH : EERE] 3 I I!III 8: . | |... [1 I I I I I I 82 sam i 333288 22779977..00115533 awnores0iso 3M MN01650139 2 z woncocnanses 3 i dernnerrente 852 [3 3 viii | II IIIIIIIII 2- 4- 4- FFEE =..8 2 Teor |B Connon IIItlllllll'l Eo 3 [F llllllllllll z 58%| 3333333| O00000000.000 22 | qunzusoeseny 22 S35FTEIIIIET 22779977..00115544 W3M_MNMONT0E1S6500114400 g z phos 3 :| fssio se EE Eefl | ZCF7] I I I |0 : 03 | E53 Luieen | 2 : CzE..ef l12z Lae | 2: i |= : 422 ms 533222 22779977..00115555 Su_MNotesost 3M MN01650141 9 EE 3% SE II III FR Ea - Zuid po - 228% | = . z.-f |Z : CE 5 z oo | E 23g2:L Esye || o13zd8, | . gi 3888|| 33aoSsee a | i22 || 533327272327 22779977..00115566 W_MNDI6S0142 3M MN01650142 2 | 3 i Sess sBNmN NEN z3 z i | szs33888%8s} IIIIIIII:IIII Si | TT z..f [2 IIIIIIIIIIII i : 5| 5333335355| | i22f || 53535383588%3%8%8s8s8s8s 22779977..00115577 SW MNOTEsO143 3M MN01650143 o i 22 alesis i {EE ee]? IIIIII + + "Trg | 2 Coen | 2 lillll 5 o |& [EEL BE%| 33S) | 22 | szzass 22779977..00115588 awnoresorss 3M MN01650144 o g z Sarees : $3] : E| e8ug 88 HEE ? . Folze | IIIIII + + + Lad 2 TL |.llIll 1: ; - i B88| zzsaE| ~0000 22779977..00115599 W_MNDI65014S 3M MN01650145 3388 g 88| cama 22779977..00116600 3M MN01650146 g = | msmnimm 5 $3 es sss :z :EEC|EEEerasEiEiRsE Eig : llllllllllll + 4+ Geez | SEE| eriiiien | 2 I1~11111~111 .:.E 8| : I IIII II I 1II I $F:% | & |. E3 RLE 4- I I A BER{ wma | 527 | 833333588863 1 #% | szzzsssssscy 22779977..00116611 S_MNoteso14T 3M MN01650147 :i op |FEC| E EieEiis S EEF S E] llllll IIIIII IIIIII 3 0 |& (EES IB al Lz : 5287 | 888868 | 1 28) sgoazs 22779977..00116622 awnoresorss 3M MN01650148 Y ip |E| BEE gE | 53 | I I I I I I 4,' + Zee | EEE) en |B IIIIII z55 i . wid (2 : IIIIII EEz 3 2 3588%} | 3w533y 53)" ooooo0 2 | sssen: 22779977..00116633 W_MNDI65014S 3M MN01650149 & 2 2 coasne a3 $: 2 | 5f8g| a3diveiiseesd|| a2a Ir |Z. %sessz| ese EOF oF | EF] GNaSNS EDL Ig| BAEEER ECE 28) s528E2 iB 22779977..00116644 S_MNotes0150 3M MN01650150 - TTT 3 2 - o Syop | 37 eddses | 23 Eo EE | z|ezsaun age u 352f)| sgu8s2z2a8sc|| sa > ks | 22779977..00116655 aW_otes01S1 3M MN01650151 9 EAT | fg 33zRam) ast oDETTSyEgl | 3e 7 dR dddsd | aGlE E2 E8E 3el%| 222390| I23I g 22 5 8 s33s 338 || E148 2 22779977..00116666 aw otesors2 3M MN01650152 g | az 3E 8 S3EEET | 323 o SET o 27 |E%| 233 is| 3E3EEz | ECE . i5|3Z3zzsEs | L8 E 22779977..00116677 SuMNoteso1s3 3M MN01650153 EB it I3288 | 87 EER NER ene 2i5j| |asaazzazeeEz|| B54%R[3 12217 22779977..00116688 33MM_MMNNo011665500115544 g $F | fF sease | ad $283 = Doig PE PoEz EEE a [n2 un $k 5 a | seszze| ve Fi5T) | E5s3eT2smTs) | 5g407re 40fF 22779977..00116699 awnoresorss 3M MN01650155 a 23 N EE PIE AE Fo5E | EF) a8 dad)dag 22779977..00117700 3M MN01650156 z 3 53| szegsd| 32 a 8= 38 | fg) cssuse| el 5 = |& SE 3 351 | 8383838388 |i3 B o : 22779977..00117711 awotesors? 3M MN01650157 i 53| fener | 80 ETR c | 328| 2nsedasniss|| =8sge 1 |E,| ovanes| az- L3E: = 5: |E| AEA kL g i syezez| se ~ .Z 22779977..00117722 awotssorss 3M MN01650158  : : 8| dadadd | 45 } : iz 58| seddgs| 8 $ Iz | I menue| ug TOFL TD E VgpElwEmimiw oo BERET RE ~z 22779977..00117733 awnoresoiso 3M MN01650159 o 3z T$o3| 1ddod5dessds|| s3ee To || dfeEgd| gd zis | t~. 0 2%; if | SERRE (50 <Z 22779977..00117744 sw nores0ieD 3M MN01650160 o i 5%| gdgsez| 8 3. iE 838358| Be ry] 5 oo 23 | 3% sEEmme)ee >S|u| mBz3z3zRsEsE|| 82072 3 | 35| geozee| 54% [2 22779977..00117755 a3MW_MMNND01166S50011S611 2 i 3 =| = $F | Ig) seednd| 8d 135 | z. o Toph |p| 22779977..00117766 3M MN01650162 9 zI :2 i! 58| d2ddad| 2 |2 il Bunia 22a 2 LoZ.p | 53% Zasddd| ER E fog dEe|| asses ane 2: dg) 383E3R RF i- 25 | wus | 5 2 22779977..00117777 sw MNOTeso163 3M MN01650163 3 a2 2 c=dsss 2 3FoooF2 || E3E8Hp||iasnasainse|| inafge z 0 = 35 | zuzzse | sazfx i8 | 333882| 5C .Z 2If 22779977..00117788 S_MNotesoes 3M MN01650164 8 2 oa 53 sngeid | 24 PIE EES TOEF SE | Seeses |S Bi 5 2a E = 223%35|88a38e2o23z5e2s || 224% 125 22779977..00117799 aw otesorss 3M MN01650165 9 ag2, we 3 2 | EF enaenn| use 208 | Eg dsgsiz| 2a goa) fo sEEEE EE COEF | | eves fs EE AE|: | 333355 s3 LL 2 5 =2|s8 5 Zo| E3E388| ac 8 225]| 8cu8o8z3e8s2|| 554% i[a2 22779977..00118800 33M UMNONT0B1S60510616656  FF | Eb gnauin|oee 5 3% =R23I2% a. oofE S| 5 | SdadiR| SE fo 5 SEi) : | 33S a ok 5 Gu| BEBEEE | SIV = = = 3HE5E H0 s0 3E0 z0 Aa0 sR0 s| 347 22779977..00118811 SuMNotesorT 3M MN01650167 z 3 2: > 2 li saz| 2g2ue oan EE ogg = i 5 =|a cE 35 | 533388| 5472 22223333| 24 2 22779977..00118822 W_MND16501SE 3M MN01650168 32 2RF | 33| qoadnadess | sge as :i : PEALE | A i =; 22779977..00118833 aMNores0169 3M MN01650169 Eo PERE aE Hi ToaE|E mE EH 5R0 HEE|& [EceRssEes |e 35| szsazn | gam [2 22779977..00118844 3M MN01650170  oI) Z| eames ego 000000 FHFEREEE 0 ! < = |Seeess| LL iEi.E IE:k 22779977..00118855 3M MN01650171 IF 3 | dnssed | 8d So | 83 Sdssas| ad LE5| 5 | eedean| az zz dF): | ashen (eps 57 z i S38ss 2 lL - || E | i | FERRE | a= : 22779977..00118866 awotssorrz 3M MN01650172 fm 2797.0187 3M MN01650173 gi. FER EIEEERE 23 PoE CElE Ea = xe 2 } 1 EEEER 000000 TE 22779977..00118888 3M MN01650174 3 HE 3| sasnds| 83 2SO sFB || E3p| ena8adndiEls) | g8dst 3 Sle cE _ off hii 28 FP OFoFaE mgeeseess|2e2el LL & = ig:R|588s88 | o34"f| 22779977..00118899 a uNotBs017S 3M MN01650175 $1 i 2 OF |B HEBNI 2 #2 Zz 8828834 Sg TECH EEE 0 . i isms 255 2 22779977..00119900 swmwrssarrs 3M MN01650176 EiA: | | sdeEe | a EES 2% | 5%| 2030s 2g frag | P ogE sE r); |E ssEmsnaEgTe : S8 | SEAR ELE 22779977..00119911 3M MN01650177 gle] + gle EZlEl 0 le) Bolo i Lo lal 0 een LL cai ee 0 von I I t on Ils) + PLE eee ae aa he sable eI e EEE eIe ee aE E I R I . M: E Pere reer I I i PHs L: os s 3|i . 8$33085ozeIf: Eos :4 &8 FsI|P 3 fife Eitk.SP|E 2145 BEealss SEER if gR)s 22779977..00119922 3UN0TB50178 3M MN01650178 sla] + FslEzlLsl i lsF1 emer en I nn D Tara Hr Heh. seins pane dF ol-le] 0 nn coo en SiHz8% lEeE] 0 dalEl 0 ETEaTls0 i ;v veoe es oe oooo ie dnres seer iils. .i IEEE i: BED ozosd sfog: iNsRsE spE unsstE fsied fs 22779977..00119933 awnotesorrs 3M MN01650179 8) q= 0 Ele 5 Els] 0 0 i BIE IEE II I #0I I E5:els EEL 0 en II~,I I I~ ree I I I~., ve I I II.~ en I I~- oI o I aI I onI I I I I I I I em en ee IIII 0 IIII vo IIII IIII II.II IIII I I I I I I I I I I 0. a Bric gif 1 HN 2 sds o% FE i of 1 fh ing lf 3I|g:E PpRiRzEisEe:s ZBxEiEeEd iiEz g2%i)ss 22779977..00119944 sw MNOTeso1E0 3M MN01650180 8 i 3 i 5 zFe 3i ze Af3EEHhlE5H|A8 i3 aBHiliaHis iEELiEsE SEEEz:E3 3 i i 3 ; [lBaFd &of 2ot i zzi i 22779977..00119955 aW_MND16S0181 3M MN01650181 gl Tle| slrfl HERI Ee] ees e sn sein se emi + + :I: :I: I I I -t- .I I +.i- -H ~ n w EEi Ie a En R +I +I + I +1 + I I I I I +I +I + +1 I -HI I ~1 -I-I ~I -~I ~+' =I: + -I- -I, I ,I.I - ,I-I +I I FOIA w+ iI nI e IL [fz] I22EEE ES]le) F83EE* jszf] Folzlel ee +I I I-I i, wn +I I wenn 0 -H wri a +I c o onw vene oad +I I -I,I -I- I ~-I I + III laf wii wes -H "I'I I I I .I -H I +I+ +I,+ I I -H++I I 4,11 1 I I III I Jiis dLiies d 3: BE Egil i Piof gBiElEtmsitihny i: ifBl sa5i85i m3p0a 7li33f.ini-8i0ec2sa331l. Rhlcs2 22779977..00119966 a3M_MMDN10e1s6050118822 ols? MITRE EL HH I II I + RI E+I I I ! I II I M II I II I EFLeEE Ef a] Egle] LE] were os wire 0s wn EETls SE pz Toggle] Ela] wc oe oe, wee en -H I | wow ew I -i- 003 + I "H +I I I : Lf Fo i Lii i3fg:i2Ei81uiia3 s i3 53 pedi dn Bf mil 1] NgRf EERREE E EEn EEaE lt 3am gst En 22779977..00119977 awnotesorss 3M MN01650183 g zl errr =e in fl 1+14" ~ 4" I 4-111 I Ee] eee -Hill I 11 I I H+ I I 4" I 44 ~ I I I 4~ I I I +I I I I I +I -H I H I +I I I I I I -H 14- I I I 4--I- I-H ~ ~ I I I I I I,H t I = Zl +erl iI I ~E:~:oI I w-H w"H I I I 0I I 4"II l+ ::I: + I I I I I + I l l I SEL l s] wares em cn -I--'I- I + 4- 14-I I I I I I Fel [ls] vive eo +4,11 ~ I I I I I I I Belg .H I I I -H oe } + I I I I I I 4-I I I I I lane | I I I -H I I I +I I I "I I I I I I I I I ,I,,I I I I EJIi gLfFedfrFooe zi igl| sE IinEhb EEdBEnHIiiongnElLLld 22779977..00119988 swmoresorss 3M MN01650184 2 3 T HE E 3i $is3 (22 = Tel atlas l=3dt 3 5i TglSlesiiyzagiiil H PlifiE sEaEsRzR RaiRiRtE eRaRsEs os Psi1 311i: i i, N i i 22779977..00119999 3 swmoresorss 3M MN01650185 ia y i 2 = I iziic 3 HEHEEEEE] $3 BiiEii Toozagizosi: zis fgigi Eid dF leis aan. i ||z|35EsiHdiiEsiRidEslEelsGieElnYgs FR HE HEHHE H : 1: 1 Tadd % HEE Eo pay Isla ih Ghkkis i gif or: pial Hi 22779977..00220000 i aunoresores 3M MN01650186 8h Hy | Hl paakii 4583f3F fz 2 ale 3 | SEE R p pi i l na a g i ins piiiang PRE sE|SEB iEs MLE LRH sFRE E|B| RkEaBEiHniaitniaiiiiMiEiiTnrEesMy 5 FEELS 1d Bhi ogi ii 3edg|Eyle 3: kEEh: HCH Bs ir FEE i i :: i 22779977..00220011 i 3i E unrosre? 3M MN01650187 z I 3 : 2 Iw fh, mh: hE i3 | | h BRSeE. f3Pa.mm3T]R dafihhedl smii:nliYeilnleinl S| i EEE: Bg l EERE a Deli fi aig il fmf: dubabidah ola]AREEp dRHG Hn, EfHnana E|S|z Zai3e2siyiPaeeipyEy] ai siFlTsoEgEtievsyEtiagtiionatesty (FE ay MHELILIL, i IH i FBiEfEfS gLfF IFtlsd hLaaith: TG enh ig BNI POEOPER ffedos ous iozois l irs Boss d HEH Blur ir: PF EE i: i 22779977..00220022 a aunoresores 3M MN0i650188 PHOTOGRAPHS (gasopathelogical Pictures) 22779977..00220033 I3MM_MMNND011665500118899 Tk A 23 el FAYE: Hr Oe SSEYTE B GG nnR dHieadT rsoansniAin iE eanr oaen eS = TSR AS CroTAGT5R doa SRvsRSeEE EE T SEERA AT IRe ES GAs REAh HAS sown wert chr Photo.1 Liver of a male rat of control. (Animal No. 0103) Showing noemal centrilobular region. H.E.stain 180 fae eeEa B J LSE AE aGe] ISAERSEEC aRHIE BRFo ES eS Se la re Live of 2 ie 1 gis 50 mn, on Yo. 10 E GEEE SARA Eefaatn) TS CoRSA EBe lSCPIB Eo NSR RaRheAd] Photo.2 Llver of a male rat given 30 ag/kg. (Animal No. 4105) gostnophtltc change and swelling of centrilobular hepatocytes are noted. H.E.statn lEO CL - 22779977..0022004 an mmssiso 3M MN01650190 A RE aa IRA SelB 2: ER ERNE By EP] EAs oR RE EAR Ee A A A e7 gLEgAeSlTnG e ae ZP AOEEReA a SE It ERA ENg A E lT e s Sr meia a taRarEEheNE UA e rA Senei es e Geta? Toe 6a ete sit give Seah, test oe 0 Photo.3 Liver of a male rat given 30 mg/kg. (Animal No. 4105) Focal necrosis Is noted. H.E.staln 180 BT ---- FESha Se S SdRLSS egOlAiA Se RRA AS USERl R aS Sa a E el San ee S E Oe ES RR e E REEn A B aE e A f SEeSo Se Toto Liverof sale ut given 30 ge. Chote Bo. 4366 Photo.4 Liver of a male rat given 30 mg/kgo {Animal No. 4106) ertphorat foc chang tod Peripheral fatt~ change is found. R.E.stain 180 t= -- 2 -- 22779977..00220055 sw_moresoron 3M MN01650191