r 

### U.S. ENVIRONMENTAL PROTECTION AGENCY 

## PREMANUFACTURE NOTICE Dateofreceipt 

### AGENCY USEONLY

**~l'V-** 

### r '- ....... I 

# lllllllli/ll/11/lll//llilllll/i/ll/!!IIIIIllliiiJ!/1

# IiiiiII/I! II i/11111

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### / in the Premanufacture Notice 

# }~,b~ • Youmust provide all information requested in · Make reasonable estimates ifyou do nothave actual data. • Before you complete this form, you shouldread the "InstructionsManual forPrernanufacture Notification" (the InstructionsManual is available from the Toxic SubstancesControl Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603). • 1fa userfee has beenremitted for thisnotice (40 CFR 700.45), indicate in the boxes above the TS-userfee identification number you have generated. Remember, youruserfee ID numbermust also appear onyour correspondingfee remittance,

**~chis** sentto EPA, HQ Accounting Operations Branch (PM-226), P.O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSCAUser fee. Part I- GENERAL INFORMATION Youmust provide the CWTently correct Chemical Abstracts (CA)Name ofthe new chemical substance, even if you claimthe identity as confidential. You may authorize anotherpersonto! • ·· • • • ·• · ··· · "

**··--·~--you,** butyour submission will not be complete ar )ceives this information. A letter in support oJ

**~** 

### rs userfee identification number. You

**m~ ~**  his notice including all test data. 1f you clain ""' , gle sanitizedcopymust also be submitted.

**~**

•rt II- HUMAJ

**~** .tfthere are several sections A and B c Part III -LISTC Attach additiOnal ' each continuation · attachments, any I< notice. OPTIONAL INFO Youmay include ;u substance. Oupag pollution preventioJ substance. 

# 0 

# u

**~**



# ~

**~** 

# t: ·&RELEASE ns to be described inPart II, 1eded. era question fully. Label ing. InPart III, listthese nnation included in the
ider in evaluatingthe new i for you to described regardingthe new



# ~ So-called "binding" 

### r I\ oryou to indicate your willingness to be bo \1 

### ..J. ; section, such as use, productionvolume, intended to reduce delays that routinely accom _ •r Significant New Use Rules. Except in the

**ca.~e** ofexemption applications (such as TMEA, LVE, LOREX) where certain infonnation providedin such notification is binding on the submitterwhen the Agency approves the exemption application, checking a binding box in this notice does not by itselfprohibit the submitter :from later deviating :fromthe information (except chemical identity) reported in the form. CONFIDENTIALITY CLAIMS You may claim any information in this notice as confidential. To assert a claim on the form, mark (X)the confidential box next to the information that you claim as confidential. To assert a claim in an attachment. circle or bracket the information you

_ claim as confidential. Ifyou claim information in the notices as confidential you must lso vrovide a sanitizedversion ofthe notice. (including attachments). For additional 1Structions on claiming information as confidential, read the Instructions Manual. Mark (x) ifany information inthis notice is claimed as confidential. TEST DATAAND OTHERDATA You are required to submit all test data in yourpossession or control and to provide a description ofall otherdataknownto orreasonably ascertainable byyou, ifthese data are relatedto the health and environmental effects on themanufacture, processing, distribution in conunerce, use, or disposal ofthe newchemical substance. Standard literature citationsmaybe submitted for data in the open scientific literature. Complete test data (mitten in English), not summaries of data, mustbe submitted ifthey do not appear in the open literature. You should clearly identitywhethertest data is on the substance oron an analog. Also, the chemical composition ofthe tested material should be characterized. Following are examples oftest data and other data. Data should be submitted accordingto the requirements of§720.50 ofthePremanufacture Notification Rule (40 CFR Part 720). TestData (Check Below any included in this notice) Environmental fate data 

### 0 Yes Other data 0 Yes Health effects data 

### 181 Yes Risk assessments Environmental

**effect~** data 

### 0 Yes Structure/activityrelationships Physical/Chemical Properties* 

### 181 Yes Test data not inthe possession orcontrul ofthe submitter 

### * A physical and chemicalproperties worksheet islocated onthe lastpage ofthis form. TYPE OFNOTICE (Check Only Oue)



### ~ 

# 0 D 

# D D D D PMN (Premanufacture Notice) INTERMEDIATE PMN (submitted in sequence withfinal

**product~)** :;e:: SNUN (Significant New UseNotice) TMEA (Test MarketingExemption Application) LVE (Low Volume Exemption)@ <WCFR723.50(c)(1) N LOREX (LowRelease/Low Exposure Exemption)@ <WCFR

**723.SQ~)(2)** LVEModification LOREX Modification IS THISA CONSOLIDATED PMN? 

# D D Yes # ofchemicals (Prenotice Communication#required, enter # onpage 3) 

# I 

# f1

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### ll '""!",) ) ·-·-{ !)/-

### Public reporting burden for this collection ofinformation is estimated to average 110 hours perresponse, including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of { "ormation. Send comments regarding the burden estimate or any other aspect ofthis collection ofinformation, including suggestions for \

### '-1educing this burden, to Chief, InformationPolicyBranch, PM-223, U.S. Environmental ProtectionAgency, 401 M. St., S.W., Washington, D.C. 20460; and to the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

## CERTIFICATION I certifY that to the best ofmy knowledge andbelief: 

### 1. The company namedinPart I, section 

### A, subsection la ofthis notice form intends to manufacture or import for a commercial purpose, otherthan in small quantities solely for research and development, the substance identified in Part I, Section B. 2. All information provided in this notice is complete andtruthful as ofthe date ofsubmission. 3. I am submitting with this notice all test data in my possession or control anda description ofall other data known to or reasonably ascertainable by 

### me as requiredby 

### §720.50 ofthe Premanufacture Notification Rule. Additional Certification Statements: If

### you are submitting a PMN, Intennediate PMN, Consolidated PMN, or SNUN, check the following userfee certification statementthat applies: 

### [gl The Company named in Part I, Section A has remitted the fee of$2500 specified in40 CFR700.45(b), or 

# D The Company named in Part I, Section A has remittedthe fee of$1000 for an Intermediate PMN (defined@ 40 CFR 700.43) in accordance with 40 CFR 700.45(b), or 

# D The Company named inPart I Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of$100 in accordance with 40 CFR700.45(b). If

### you are submitting a low volume exemption (LVE) application in accordance with 40 CFR723.50(c)(l) or a Low release and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check thefollowing certification statements: 

# D The manufacturer submittingthis notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under the terms of40 CFR 723.50. 

# D The manufacturer is familiar with the terms ofthis section andwill comply with those terms; and 

# D The new chemical substance for whichthe notice is submittedmeets all applicable exemption conditions. 

# D If

### this application isfor an LVE 

### in accordance with 40 CFR 723.50(c)( 1 ), the manufacturer intends to commence manufacture ofthe exempted substance for commercial purposes within 1year ofthe date ofthe expiration ofthe 30 day review period. The accuracyofthe statementsyou make inthis notice should reflect your best prediction ofthe anticipated facts regarding the chemical substance described herein. Any knowilll! andwillful misinterpretation is sub·ectto criminal penaltv pursuantto 18 USC 1001. Signature and title ofAuthorized Official (Original Signature Required) Date Signature ofagent - (jfapplicable) Date Confidential 

### X X



## - **------------------------------------------------------~------------------------~----------~** EPAFORM 7710-25 (Rev. 5-95) Page2Person '\ Submitting

/ Notice (in U.S.)

b. Agent(if applicable)

Joint Submitter (if applicable)

2. Technical Contact (in U.S.) Company street) City, State, ZIP Code Name ofauthorized official Company Mailing and street) Code Name ofauthorized official Company street) City, State, ZIP Code Name ofauthorized official Company street) City, State, ZIP Code 3. If you havehad aprenotice communication (PC) concerningthis notice and EPA assigned a PC Numberto the notice, enter the number.

for the chemical substance coveredby this notice, enter the notice number assigned by EPA. I.

TypeofNotice - Mark (X) EPA FORM 7710-25 

# D Manufacture Only BindingOption Mark Telephone Position
Telephone Position
Telephone



# D (Rev. 5-95) Import Only Mark (X) ifnone Mark (X) ifnone Mark (X) ifnone BindingOption Mark I I 3. I 

# D X 

### X
Both Page3\ I. Class a. Class l or Class

c. d. Chenucal name correct Chemical Abstracts (CA) Namethat is consistent with TSCAInventory listingsfor similar substances. ForClass l substances a CAIndexNamemustbeprovided. ForClass 2 substances either a CAIndex Name orCAPreferredNamemust beprovided, which ever is appropriatebased on CA9CI nomenclaturerules and conventions). Please identify which method you usedtodevelop orobtainthe specified 

# D Method 1 (CAS InventoryExpert Service- a copy ofthe Identification report obtained from theCAS InventoryExpert Services mustbe CAS# identity reported in 

# D Method 2 (Other Source)

e. For a class l substance, provide a complete andcorrect chemical structure diagram. For a 2 substance- (l) List precursor substances with their respective CAS Registry Numbers. (2) Describethenature ofthereaction orprocess. (3) Indicatethe rangeofcomposition andthetypical composition (where appropriate). (4) Provide a correct representative orpartial chemical structure dia.rram. as comoJete as can he known. ifone can hereasonahlv ascertained.

this box ifyou attacl! a continuation sheet. EPAFORM 7710-25 (Rev. 5-95) '''"'!""'' on



### X

Page4\ a. Indicatethenumber-averageweight of the lowestmolecularweight composition of thepolymeryou intendtomanufacture.

c. Indicatemaximum weightpercent oflowmolecularweight species (not includingresidual monomers, reactants, or solvents)below 500 and below 1,000 absolute molecularweightofthat composition. Describethemethods ofmeasurement orthebasis for your estimates: GPC Other (Specify) i) lowestnumber averagemolecularweight: ii) maximumweight%below 500 molecularweight: iii) maximumweight %below 1000 molecularweight: attach a continuation sheet. You must make separate confidentiality claims for monomer or otherreactant identity, composition information, and residual information. Mark (X)the "Confidential" boxnextto anyitemyou claim as confidential (1) (2) - (3) - (4) - (5) - (6) - Providethe specific chemicalname and CAS Registry Number (ifa number exists) ofeach monomer or other reactant used in the manufactureof thepolymer. Mark (X)this column ifentry in column (I)is confidential. Indicatethetwicalweightpercent ofeach monomer or otherreactant inthepolymer. Mark (X)theidentity column ifyou want a monomer or other reactant used attwo weightpercentor less tobelisted aspartofthepolymer description on theTSCAChemical Substance Inventory. Mark (X)this column ifentries in columns (3) and (4)are confidential. Indicatethemaximumweightpercent ofeach monomer orother reactantthatmay bepresent as a reSidual inthepolymer as manufacturedfor conunercial purposes. this Conli-
% % % % % % % % % % Method I (CAS InventoryExpert Service- a copy oftheidentilication report obtained from CAS-Inventory Expert Servicemust be submitted as as attachmenttothisAbstracts (CA)namefor thepolymerthat is consistentwith TSCAInventory listings similarpolymers.

attach a continuation sheet. 

### X

EPAFORM 7710-25 (Rev. 5-95) PageS

.,

### .., **'['~~** --IdentifY each impuritythat maybereasonably anticipatedtobepresent in thechemical substance as manufacturedfor commercialpurpose. Provide the CAS Registry Number ifavailable. If there areunidentified enter "unidentified." Estimatethemaximum % ofeach If there estimatetheirtotal %.



# D Mark (X) this box ifyou attach a continuation sheet. 5. identification- Listtradenames for thenew chemical substance identified in subsection I or 2. 

# D Mark (X)thisbox ifyou attach a continuation sheet. 6. Generic chemical name- Ifyou claim chemical identifY as confidential, you mustprovide a genericnamefor your substancethat reveals thespecific chemical identity of thenew chemical substancetothemaximum extentpossible. Refertothe TSCAChemical Substancehwentory, 

### i985 Edition, Appendix B for guidance on developing genericnames. Maximum percent % % % % % % % dential 

### X

Confidential



### X

ofthenew chemtcal substance. Providethe CAS Registry

EPA FORM 7710-25 (Rev. 5-95) PageJ

## PartI -- GENERAL INFORMATION-- Continued Section C --PRODUCTION, IMPORT, AND USE INFORMATION: ' / Mark 

### (X) the "Confidential" box next to any item you claim as confidential. \ 

### Production volume-- Estimate the 

### maximum production volume during the first 12 months ofproduction. Also estimatethe maximum

-- production volume for any consecutive 12-monthperiod during the first three years ofproduction. Estimates should be on 100% newchemical substance basis. For a Low Volume Exemption application, ifyou choose to have your notice reviewed at a lower production volume than 10,000 kg/yr,

**s~ifY** the volume and mark (x) in the binding box. lfgranted, you are bound to this volume Maximum first 12-monthproduction (kg/yr) Maximum 12-monthproduction (kg/yr) Confi- Binding (100% new chemical substancebasis) (100% new chemical substance basis) dmtial Option Mark(x) 

### X 2. Use 

### Information -- You must make separate confidentiality claims for the description ofthe category ofuse, the percent ofproduction volume devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim as confidential. a. (I)-- Describe each intended category ofuse ofthe new chemical substance by function and application.. (2) -- Mark (X) this cohunn ifentry column (l) is confidential business information (CBI). (3) -- Indicate your willingness to have the information provided in column (l)binding. (4) -- Estimate the percent oftotal production for the first three years devoted to each category ofuse. (5)- Mark (X) this column ifentry in column (4) is confidential business information (CBI). (6)- Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7) -- Mark (X) this column ifentry in column (6) is confidential business infonnation (CBI). (8) -- Indicate% ofproduct volume expected for the listed "use" sectors. Mark more than one box ifappropriate. Mark (X) to indicate your willingness to have the use type provided in (8) binding. (9) -- Mark (X) this column ifentry(ies incolumn (8) is (are) confidential business infonnation(CBI). Category ofuse(1) CBI Binding Produc- CBI %in CBI % ofsubstance expectedperuse CBI Option tion% Form- (8) (by function and application i.e. a dispersive dye Mark(x) ulation Site- Con-* Indus- Com- Binding for finishingpolyester fibers) (2) (3) (4) (5) (6) (7) limited sumer trial mercia! Option (9)



## -- 

### X X X X %



## - X % 

### X X X X % 

### X X X % % % % % % % % 

### * lfyouhaveidmtified a "consumer"use, pleaseprovide on a continuation sheet a detailed description oftheuse(s) of this chemical substance in consumerproducts. In addition include estimates of the concentration of thenew chemical substance as expected in consumerproducts and describethe chemical reactionsbywhichthis substance loses its idmtity intheconsumerproduct. 

# DMark (X)thisbox ifyou attach a continuation sheet. b. Generic lfyou claim any category ofuse description in subsection 2a as confidmtial, mter a gmericdescription of that category. Readthe use Instructions Manual for examples ofgmericuse descriptions. description 

### A crosslinking agent for solvent based inks. A polymerization monomer. A water scavenger for anhydrous polymers. 

# D Mark (X)thisbox ifyou attach a continuation sheet. 3. Hazard Infonnation -- Include in thenotice a copy ofreasonable facsimile ofanyhazardwarningstatemmt, label, material safetydata sheet, or other Binding

information which will beprovidedto anyperson who isreasonably likelyto be exposedtothis substance regardingprotective equipmmt orpractices Option Mark(x) for the safehanding,transport, use, or disposal of thenew substance. List in part III hazard information you include.

- 

## ( { Mark (X)this box ifyou attach hazard information. M.S.D.S. 

# I EPAFORM 7710-25 (Rev_ 5-95) Pagef

# ( / 

## "-· nextto anyitemyou '1mplete sectionA for each type of pn>eessung, or use operation involving the new substance at industrial sites you

ntrol. hnporters do nothave to complete this for operations outside the U.S.; however, you may still have reporting requirements if there are further industrial · or use "'''.,."'t."''" · You describe these · See instructions manual
Site address (number and City, County, State, ZIP code the same operation occur at more than one site, enter the number ofsites. Identify the additional sites on a continuation sheet, and ifany ofthe sites have significantly different production rates or operations, include all the information requested in this section for those sites as attachments. b. I. Batch Mallillnutnkg/batch (100% new chemical 1 Hours/batch substance) 1 I 2. Continuous Process description Mark (X)to indicateyourwillingnesstohave yourprocess description binding. : Batches/year
1 Batches/year I (1) Diagramthe major unit operation steps and chemical conversions. Include interim storage andtransport containers (specifY- e.g. 5 gallonpails, 55 gallon drum, rail car,tanktruck, etc.). 

### Confidential

(2) Providetheidentity,the approximate weight (by kg/day orkg/batch on a 100%new chemical substance basis), and entry point ofall startingmaterials and feedstocks (includingreactants, solvents, catalysts, etc.), and ofall products, recycle streams, andwastes. Include cleaningchemicals (notefrequency if not used daily orperbatch.). (3) IdentifY bynumberthepoints ofrelease, including small orintermittent releases, totheenvironment ofthenew chemical substance.

box if you attach a continuation sheet. EPAFORM7710-25 (Rev. 5-95) Pagef



### .....

# , ....

**~·-**

## Partll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued Section A- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER- Continued 2. Occupational Exposure -- Youmustmakeseparate confidentiality claimsforthe description ofworker activity,physical form of thenew chemical substance, I number ofworks exposed, and duration ofactivity. Mark (X)the "Confidential"boxnextto any itemyou claim as confidential. I, . (I) -Describethe activities (i.e. bagdumping,totefilling, unloadingdrums, sampling, cleaning, etc.) in whidl workers maybeexposedtothe substance. (2) - Mark (X)this column if entryin column (I)is confidential business information (CBI). (3)- Describe anyprotectiveequipment and engineeringcontrols usedtoprotectworkers. (4) and (6) --Indicateyourwillingnesstohavetheinformation provided in column (3) or(5) binding. (5)- Indicatethephysical form(s) ofthenew chemical substance (e.g., solid: crystal, granule, powder, ordust) and%new chemical substance (ifpartofa mixture) atthetimeofexposure. (7)- Mark (X)this column ifentryin column (5)is confidential business information (CBI). (8)- Estimatethemaximumnumber ofworkers involved in each activity for all sites combined. (9) - Mark (X)this column ifentry in column (8)is confidential business information (CBI). (10) and (11)- Estimatethemaximum duration ofthe activity for any workerinhoursperday and daysperyear. (12) --Mark(X)this column ifentries in columns (10) and (11) are confidential business information CBI). Worker activity CBI ProtectiveEquipment/ Binding Physicalf01ms(s) Binding CBI #of CBI Maximum duration CBI (i.e., bagdumping, filling drums) EngineeringControls Option and%new Option Workers Hrs/day Days/yr Mark(x) substance Mark(x) Exposed (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) 

## NA 

# 0 Mark (X)this box ifyou attach a continuation sheet. 3. EnvironmentalRelease and Disposal- You must make separate confidentiality claims forthereleasenumber andtheamount of thenew chemical substance released and other release and disposal information. Mark (X)the "Confidential"boxnextto each itemyou claim as confidential. (1) --Enterthenumber ofeach releasepointidentifiedin theprocess description, partII, section A, subsection Id(3). (2) --Estimatethe amount of thenew substancereleased (a) directlytotheenvironment or (b) into control technology (in kg/day orkg/batch). (3) -- Mark (X)this column ifentries in columns (1) and (2) are confidential business information (CBI). (4) --Identifythemedia (stack air, fhgitive air (optional-see Instruction Manual), surface water, on-site oroff-site land or incineration, POTW, orother (specify))to whichthenew substancewill bereleasedfrom thatreleasepoint. (5) --a. Describe controltechnology, ifany, and control efficiencythatwillbeusedto limittherelease ofthenew substancetotheenvironment. Forreleases disposed ofon land, characterizethe disposal method and statewhether itis approvedfor disposal ofRCRAhazardouswaste, On a continuation sheet, for eadl sitedescribe any additional disposal methods thatwill beused andwhetherthewaste is subjectto secondary ortertiary on-sitetreatment. b. Estimate the amountreleasedtotheenvironment after controltechnology (in kg/day). (6)- Mark (X)this column ifentries in colmlllls (4) and (5) are confidential business information (CBI). (7) - Identifythe destination(s) ofreleasestowater. Please supply NPDES (National Pollutant DischargeElimination System)numbers for direct disdlarges or NPDES numbers ofthe POTW_(Publicly Owned TreatmentWorks). Mark (X) ifthe POTWname orNPDES #is confidential business information (CBI). Release !Unountofnewsubstance CBI Media of Control tedmology and efficiency (you maywish to optionally attach efficiency data) CBI Number released release

(1) (2a) (2b) e.g. stack air I Bin<ling (3) (4) (5a) I Mark (X) (5b) (6) I 1 I I I I I I I I I I I I I I I I I I I I (7) Mark (X) the 

# 0 POTWprovidename(s)below: CBI 

# 0 Navigable 

# 0 Other - Specify provide NPDES # CBI !---- destination(s) of waterway releasestowater. 

# ,L.J Mark (X)this box ifyou attadl a continuation sheet. EPAFORM7710-25 (Rev. 5-95) PageiO

## Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS _!::omplete section Bfortypical processing oruse operations involvingthenew cllemical substance at sites you do not control. Importers do nothaveto completethis ( 'ion for operations outsidetheU.S.; however, you mustreport anyprocessing oruse activities after import. SeetheInstructions Manual. Complete a separate , JonBfor each type of processing, or use operation involving the newchemicalsubstance. typical operation commontothese sites. Identifyadditional sites on a continuation sheet. If thesame operation is performed at morethan one site describethe 1. Operation Description - To claim information in this section as confidential, circle orbracketthe specific information thatyou claim as confidential. (I)-- Diagramthemajor unit operation steps and cllemical conversions, including interim storage andtransport containers (specifY- e.g. 5 gallon pails, 55 gallon drums, rail cars, tanktrucks, etc). Onthe diagram, identityby letter and briefly describe eacll worker activity. (2) -- Providethe identity, the approximateweight (bykg/day orkg'batcll, on an I00%new cllemical substance basis), and entrypoint ofall feedstocks (includingreactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaningcllemicals (note frequency if not used daily orperbatch). (3) --Identitybynumberthepoints ofrelease, including small or intermittent releases, tothe environment of thenew cllemical substance. (4) Please enterthe# ofsites (rememberto identifYthelocations of these sites on a continuation sheet):

- #ofsites

X Mark (X)this box ifyou attacll a continuation sheet. 2. Worker Exposure/EnvirorunentalRelease (I)-- Fromthediagram above, providethe letterfor eacll worker activity. Complete 2-8 for each worker activity described. (2) --Estimatethenumber ofworkers exposedfor all sites combined. (4) --Estimatethetypical duration ofexposureperworker in (a)hours per day and (b) days per year. (6) -- Describephysical form ofexposure and %new cllemical substance (ifin mixture), and anyprotective equipmart and atgineeringcontrols, ifany, usedto protectworkers. (7) --Estimatethepercent of thenew substance as formulated when packaged orused as a fmal product. (9)- Fromtheprocess diagram above, mterthenumber ofeacll releasepoint. Complete 9-13 for each releasepoint identified. (I0) -- Estimatethe amount ofthenew substancereleased (a) directlytothe mvironment or (b) into control technologytothe enviromnmt (in kg!day or kg'batcll). (12) -- Describe media ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surfacewater, on-site or off-site land or incineration, POTW, or other (specifY) and control technology, ifany, thatwill be usedto lintittherelease ofthe new substancetothe environmmt. (14) - Identity byproducts whicll may resuh from the operation.

(3), (5), (8), II), (13) and (15)- Mark (X this column ifany of theproceeding entries are confidential business information (CBI). Letter #of CBI Duration CBI ProtectiveEquip. I o/o in CBI Release Amount of CBI Media ofRelease CBI

of Workers of Engineering Controls/ Form- Number New &Control Act- Exposed Exposure Physical Form and% ulation Substance Technology ivity new substance Released (I) (2) (3) (4a) I _(4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (II) (12) (13) X I X X I X X I I I I I I X I X X I X X I I I I I I X I X X I X X I I I I I I X I X X I X X I I I I I I ( I - 14)- Byproducts: (15) X \ 

## -, X Mark (X)this box ifyou attacll a continuation sheet, EPAFORM7710-25 (Rev. 5-95) Page

**I~**

**''"''!~'~~** I

## Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued Section B -INDUSTRIAL SITES CONTROLLED BY OTHERS Completesection B fortypical processingoruseoperations involvingthenew dJ.emical substance at sites you do not control. hnporters do nothaveto completethis 'ionfor operations outsidetheU.S.; however, you mustreport anyprocessingoruse activities after import. Seethe Instructions Manual. Complete a separate , ionBfor each type of processing, or use operation involving the new chemicalsubstance.**~.ypical** operation commontothese sites. Identify additional sites on a continuation sheet. If thesame operation is performed at morethan onesite describethe 1. Operation Description - To claim information in this section as confidential, circle or bracket the specific information thatyou claim as confidential. (I)- Diagramthemajor unit operation steps and dJ.emical conversions, including interim storage andtransport containers (specifY- e.g. 5 gallonpails, 55 gallon drums, rail cars, tanktrucks, etc). Onthediagram, identifY by letter andbriefly describe eadJ. worker activity. (2) -- Providetheidentity, the approximate weight (bykg/day orkglbatdJ., on an 100%new chemical substancebasis), and entrypoint ofallfeedstocks (includingreactants, solvents and catalysts, etc) and all products, recyclestreams, and wastes. IncludecleaningdJ.emicals (notefrequency if not used daily orperbatdJ.). (3)- IdentifY bynumberthepoints ofrelease, including small orintermittentreleases, totheenvironment of thenew dJ.emical substance. (4) Please enterthe# ofsites (rememberto identifYthelocations of these sites on a continuation sheet):



### - #ofsites

X Mark (X)this box ifvou attadJ. a continuation sheet. 2. WorkerExposure/EnvironmentalRelease ( 1) -- Fromthe diagram above, providethe letterfor eadJ. worker activity. Complete 2-8 for eadJ. worker activity described. (2)- Estimatethenumber ofworkers exposed for all sites combined. (4) -- Estimatethetypical duration ofexposureperworker in (a)hoursperday and (b) days peryear. (6) --Describephysical form ofexposure and%new dJ.emical substance (ifin mixture), and anyprotectiveequipment and engineeringcontrols, ifany, usedto protect workers. (7)- Estimatethepercent ofthenew substance as formulated whenpackaged orused as a fmal product. (9) - Fromtheprocess diagram above, enterthenumber ofeadJ. releasepoint. Complete9-13 for eadJ. releasepoint identified. ( 1

0) - Estimatethe amount ofthenew substancereleased (a) directlytothe environmentor (b) into controltedJ.nologytothe environment (in kg/day orkglbatdJ.). (12) -Describemedia ofrelease i.e. stack air, fugitive air (optional-seeInstructions Manual), surfacewater, on-site or off-site land or incineration, POTW, or other (specifY) and control tedJ.nology, ifany, thatwill beusedto limittherelease ofthenew substancetothe environment. (14) --IdentifY byproducts whim may resuhfromtheoperation. .(3),(5), (8), 11),_(13) and (15) - Mark0 this column ifanv of theproceedingentries areconfidential business information (CBI). Letter #of CBI Duration CBI ProtectiveEquip. I %in CBI Release Amount of CBI Media ofRelease CBI

of Worl<ers of EngineeringControls! Form- Number New & Control Act- Exposed Exposure Physical Form and % ulation Substance Technology ivity new substance Released (1) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (11) (12) (13) 

### X I 

### X X I 

### X I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 14) -- Byproducts: (15) ( X



## l X Mark (X)this box ifyou attadJ. a continuation sheet, EPA FORM7710-25 (Rev. 5-95) Page**1~** 

## rrr( 

### \



# (

# (

(

## Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued Section B - INDUSTRIAL SITES CONTROLLED BY OmERS Complete section B fortypical processingoruse operations involvingthenew chemical substance at sites you donot control. hnporters do nothaveto completethis (" · 'ion for operations outsidethe U.S.; however, you mustreport anyprocessingoruse activities after import. SeetheInstructions Manual. Complete a separate ionBfor each type of processing, oruse operation involving the new chemical substance. Ifthe sameoperation isperformed atmorethan one site describethe 1:ypical operation commontothese sites. Ident.izy additional sites on a continuation sheet. 1. Operation Description-- To claim information in this section as confidential, circle orbracketthe specific information thatyou claim as confidential. (I)- Diagramthemajor unit operation steps and chemical conversions, includinginterim storage andtransportcontainers (specifY- e.g. 5 gallon pails, 55 gallon drums, rail cars,tanktrucks, etc). Onthe diagram, ident.izy byletter and briefly describe each worker activity. (2) --Providetheidentity,the approximateweight (bykg! day orkg/batch, on an I00%new chemical substancebasis), and entrypoint ofall feedstocks (includingreactants, solvents and catalysts, etc) and all products, recycle streams, andwastes. Include cleaningchemicals (note frequency ifnotused daily orperbatch). (3) -- Ident.izy bynumberthepoints ofrelease, including small orintermittent releases, tothe environment of thenew chemical substance. (4) Please enterthe# ofsites (remembertoident.izythelocations of these sites on a continuation sheet):



## - #ofsites

.- ){ Mark (X)this box ifyou attach a continuation sheet. 2. WorkerExposure!EnviromnentalRelease (I) -- Fromthe diagram above, providetheletter for each worker activity. Complete 2-8 for each worker activity described. (2) --Estimatethenumber ofworkers exposed for all sites combined. (4)- Estimatethetypical duration ofexposureperworker in (a)hoursperday and (b) days per year. (6) -- Describephysical form ofexposure and %newchemical substance (ifin mixture), and anyprotectiveequipment and engineering controls, ifany, usedto protectworkers. (7) -- Estimatethepercent of thenew substance as formulated when packaged orused as a fmal product. (9) - Fromtheprocess diagram above, enterthenumber ofeach releasepoint. Complete 9-13 for each releasepoint identified. (I0) -- Estimatethe amount ofthenew substancereleased (a) directlytothe environment or (b) into controltechnologytothe environment (in kg! day orkg/batch). ( 12) -- Describe media ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surfacewater, on-site or off-site land or incineration, POTW, or other (specifY) and control technology, ifany, thatwill be used tolimittherelease ofthenew substancetothe environment. (14) -IdentifYbyproductswhich mayresuhfromthe operation.

(3), (5), (8), 11), (13) and (15)- Mark 0\ this column ifany of theproceedingentries are confidential business information (CBI). Letter #of CBI Duration CBI ProtectiveEquip. I %in CBI Release Amount of CBI Media ofRelease CBI

of Wolkets of EngineeringControls/ Form- Number New & Control Act- Exposed Exposure Physical Form and% ulation Substance Technology ivity new substance Released (I) (2) (3) (4a). I (4b)_ (5) (6) (7) (8) (9) (lOa) I (JOb) (11) (12) (13) 

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## Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS £ompletesection B fortypicalprocessingoruseoperations involvingthenew chemical substance at sites you do not control. Importers donothaveto completethis 'on for operations outsidetheU.S.;however,you mustreport anyprocessingoruse activities after import. SeetheInstructions Manual. Complete a separate \ .onBfor each type of processing, oruseoperation involving the newchemical substance. ·typical operation commontothese sites. Identity additional sites on a continuation sheet. If the sameoperation isperformed atmorethan one site describethe 1. Operation Description- To claim information in this section as confidential, circle orbracketthe specific information thatyou claim as confidential. (1)- Diagramthemajor unitoperation steps and chemical conversions, includinginterim storage andtransport containers (specifY- e.g. 5 gallon pails, 55 gallon drums, rail cars,tanktrucks, etc). Onthediagram, identify byJetter and briefly describeeach worker activity. (2) -- Providethe identity,the approximate weight (bykg/day orkg/batch, on an 100%new chemical substancebasis), and entrypoint ofall feedstocks (includingreactants, solvents and catalysts, etc) and all products, recycle streams, andwastes. Include cleaningchemicals (note frequency ifnotused daily orperbatch). (3) -- IdentifY bynumberthepoints ofrelease, includingsmall orintermittent releases,totheenvironment of thenew chemical substance. (4) Pleaseenterthe# ofsites (rememberto identifythelocations of these sites on a continuation sheet):



### - #ofsites

- ( \"- X. Mark (X)this box if you attach a continuation sheet. 2. WorkerExposure/EnvironmentalRelease (1) --Fromthe diagram above, providetheletter for each worker activity. Complete 2-8 for each worker activity described. (2) -- Estimatethenumber ofworkers exposed for all sites combined. (4) - Estimatethetypical duration ofexposureperworker in (a)hours perday and (b) days peryear. (6) -- Describephysical form ofexposure and %new chemical substance (ifin mixture), and anyprotective equipment and engineeringcontrols, ifany, usedto protectworkers. (7) --Estimatethepercent ofthenew substance as formulated when packaged or used as a fmal product. (9) - Fromtheprocess diagram above, enterthenumber ofeach releasepoint. Complete 9-13 for each releasepoint identified. (10) - Estimatethe amount ofthenew substance released (a) directlytotheenvironment or (b) into controltechnologytotheenvironment (in kg/day orkg/batch). ( 12) - Describe media of release i.e. stackair, fugitive air (optional-see Instructions Manual), surfacewater, on-site or off-site land or incineration, POTW, or other (specifY) and controltechnology, ifany, that will be usedto limittherelease ofthenew substancetotheenvironment. (14) -- IdentifY byproductswhich mayresuhfrom the operation.

(3), (5), (8), 11), (13) and (15)- Mark (X this column ifany oftheproceedingentries are confidential business information (CBI). Letter #of CBI Duration CBI ProtectiveEquip. I %in CBI Relea•e Amount of CBI Media ofRelease CBI

of Workers of EngineeringControls/ Form· Number New &Control Act- Exposed Exposure Physical Form and % ulation Substance Technology ivity new substance Released (I) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (JOb) (11) (12) (13) 

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**~omplete** section B fortypical processingoruseoperations involvingthenew d:temical substance at sites you do notcontrol. Importers donothaveto completethis ( •ion for operations outsidetheU.S.; however, you mustreport anyprocessingoruse activities after import. SeetheInstructions Manual. Complete a separate ( ionBfor each type of processing, or use operation involving the newchemicalsubstance. If the sameoperation isperformed atmorethan one sitedescribethe

**~~.rpical** operation commontothese sites. IdentifY additional sites on a continuation sheet.

1. Operation Description-- To claim information in this section as confidential, circle or bracketthe specific information that you claim as confidential. (I)-- Diagramthemajor unit operation steps and d:temical conversions, includinginterim storage andtransport containers (specifY- e.g. 5 gallonpails, 55 gallon drums, rail cars, tanktrucks, etc). Onthe diagram, identifY byletter andbrieflydescribe ead:t worker activity. (2) - Providethe identity,the approximateweight (bykg/day orkglbatd:t, on an 100%new d:temical substancebasis), and entrypoint ofall feedstocks (includingreactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaningd:temicals (notefrequency if not used daily orperbatd:t). (3)- IdentifY bynumberthepoints ofrelease, includingsmall or intermittent releases, totheenvironment of thenew d:temical substance. (4) Please enterthe# ofsites (rememberto identifYthelocations of these sites on a continuation sheet):



### - #ofsites

" X Mark(X)this box ifyou attad:t a continuation sheet. 2. Worker Exposure/Environmental Release (I)-· Fromthe diagram above, providethe letter for ead:t worker activity. Complete2-8 for ead:t worker activity described. (2) -- Estimatethenumber ofworkers exposed for all sites combined. (4) ··Estimatethetypical duration ofexposureperworker in (a)hoursperday and (b) daysperyear. (6) •• Describephysical form ofexposure and %new d:temical substance (ifin mixture), and any protective equipment and engineeringcontrols, ifany, usedto protectworkers. (7)- Estimatethepercent ofthenew substance as formulated when packaged orused as a final product. (9) -- Fromtheprocess diagram above, enterthenumber ofead:t releasepoint. Complete 9-13 for ead:t releasepoint identified. (I0) •· Estimatethe amount ofthenew substance released (a) directlytotheenvironment or (b) into controlted:tnologytotheenvironment (in kg/dayorkglbatd:t). (12) ··Describemedia ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surfacewater, on-site or off-site landor incineration, POTW, or other (specifY) and controlted:tnology, ifany, thatwill beusedto limittherelease ofthenew substancetothe environment. (I4) -IdentifYbyproductswhid:t mayresuh from the operation.

(3), (5), (8), 11), (13) and (15) --Mark(); this column ifany oftheproceedingentries are confidential business infonnation (CBI). Letter #of CBJ Duration CBI Protective Equip. I %in CBI Release Amount of CBI Media ofRelease CBJ

of Workers of EngineeringControls/ Form- Number New &Control Act- Exposed Exposure Physical Fonn and % ulation Substance Technology ivity new substance Released (I) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (11) (12) (13) 

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## OPTIONAL POLLUTION PREVENTION INFORMATION To claim information inthis section as confidential circle orbracket the specific information thatyou claim as confidential. 

### ,r.,_ 

### this sectionyou may provide information not reported elsewhere in this form regarding your efforts to reduce ( minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal

(



## ( ofthe PMN substance. Please include new information pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due to the new chemical substance. Source reduction includes the reduction in the amount or toxicity ofchemical wastes bytechnological modification, process and procedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions of pollution prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance with existing regulatory requirements and can be either quantitative or qualitative. The EPAis interested in the information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting ofrisks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In addition, information on the relative cost or performance characteristics ofthe PMN substance to potential alternatives may be provided. 

### All information provided in this section will be taken into consideration during the review ofthis substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and examples. Describe the expected netbenefits, such as (1) an overall reduction in risk to human health orthe environment; (2) a reduction inthe volume manufactured; (3) a reduction inthe generation ofwaste materials through recycling, source reduction or other means; (4) a reduction inpotential toxicity orhuman exposure and/or environmental release; (5) an increase inproductperformance, a decrease in the cost ofproduction and/or improvedoperation efficiency ofthe new chemical substance in comparison to existing chemical substances used in similar application; or (6) the extent to whichthe new chemical substance may be a substitute for an existing substance thatposes a greater overall riskto human health orthe environment.



# l 

# 0 Mark (X) 

### this box ifyou attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) Pagepl-;l..



## - **-----~-'·r---- ·~-·-·-~~~--** 

## Part 

# III -- LIST OFATTACHMENTSt1ch continuation sheets for sections ofthe form and test data and other data (including physical/chemical properties and

**~ucture/activity** information), and optional information after this page. Clearly identify the attachment andthe section ofthe form to which itrelates, ifappropriate. Nwnber consecutively the pages ofthe attachments. In the colwnn below, enterthe inclusivepage nwnbers ofeach attachment. Mark(X)the "Confidential" boxnextto any attachment nameyou claim as confidential. ReadtheInstructions Manual for guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy ofthenotice form a sanitized version ofany attachment inwhich you claiminformation as confidential. Attachmentname Attachment pagenuml>erOO



### Index to the "Substance and Study Reports" 36



### - 1-Biodegradation Test ofV-OSH by Microorganisms 37-76 / I

**·-~-Biodegradation** 

### Test ofV-OSH in Carp 77-155

( \ 

### 3-Bacterial Reverse Mutation Test ofV-OSH 4-Chromosomal Aberration Test ofV-OSH Using Cultured Mammalian Cells 5-Twenty-eight-day Repeated-Dose Oral Toxicity Study ofV-OSH in Rats 6-Measurement of Molecular Weight Distribution of V-05H 7-Study on "Safety Evaluation Flow Scheme for Polymer" of V-OSH



# 0 Mark (X)thisbox ifyou attach a continuation sheet. Enterthe attachmentname and number. EPAFORM7710-25 (Rev. 5-95) 

### 156-178 179-203 204-289 290-305 306-340 Confidential



### X X X X X X X X

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### PHYSICAL AND CHEMICAL PROPERTffiS WORKSHEET To assistEPA's review of physical and chemicalproperties data, pleasecompletethe followingworksheetfor data you provide and include itinthenotice. IdmtifYthepropertymeasured, thepage of thenotice on whichtheproperty appears,the valueof the property,theunits in whichtheproperty is measured (asnecessary), andwhether ornottheproperty is claimedas confidential. The physical state of theneat substanceshouldbeprovided. Thesemeasuredproperties should befortheneat (100%pure) cl!emical substance. Propertiesthat are measuredfor mixtures or formulations should be so noted (% PMN substance in __). You arenot requiredto submitthis worksheet; however, EPA stronglyrecommendsthat you do so, as itwill simplifYreview and ensurethat confidential information is properlyprotected. You should submitthis worksheet as a supplemmtto your submission of test data. This worksheet is not a substitutefor submission of test data. Property Mark Page Value Measuredor Confi- (X) if number Estimate dential (a) provided (b) (c) Mark(X) _(1-!_orE) (d) 

### X 

### Physical state ofneat substance (s) X (I) 

### (g) Vaporpressure

_@_**Tem~rature** 

# oc Torr Density/relative density g/cm3 X X 

### Solubility @ 

### Temperature 20 

# oc

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### X 

### Solubility inwater_@_Tet11pe_rature 20 

# oc giL 

### Melting temperature 

# oc Boiling I 

### sublimation temperature@ torr _.pressure 

# oc X 

### Spectra GPC, Infra Red Dissociation constant Particle size distribution Octanol/waterpartition coefficient Henry's Law constant Volitalization from water Volitalization from soil _pH@_concentration X 

### Flammability Explodability Adsotption I 

### coefficient Other - Specify
ATTACHMENT 

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## \ Original copy ofinformation forthe substance and studyreports CDBiodegradation test ofV-05H by microorganisms Results Percentage biodegradationby BOD 0% Percentage biodegradationby HPLC 1% @Biodegraaatton test ofV-05H in carp Results 48-hour LC50 value >100mg/L Bioconcentration factors Level 1 < 12 Level2 <129 @Bacterial Reverse Mutation Test ofV-05H Results no reverse mutagenic potential . @Chromosomal Aberration Test of

**V~05H** 

## Using Cultured Mammalian Cells Results no chromosomal aberration @'1\venty-eight-day Repeated-Dose Oral Toxicity Study ofV-05H in Rats Results NOEL=200mg/kg/day ®Measurement ofmolecular weight distribution ofV-05H Results Mn=4120 Mw=16500 CVStudy on "Safety Evaluation Flow Scheme for Polymer" ofV-05H Results No difference was observed between those measured before the test and those measured after the test.



# --r...--

### Accepted number Test number $96-3013 13013 

# FINAL REPORT

Biodegradationtest ofV-OSH bymicroorganisms

March 31, 1997 Kurume Research Laboratories

Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan

### Sponsor Title Test number 

# STATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 

# U

Kurume Research Laboratories] Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan
Biodegradationtest ofV-OSH bymicroorganisms 13013 I, the undersigned, hereby declare that this report provides a correct English translation of the Final Report (Test No.13013, issued on March 31, 1997). However, sponsor's address, 11 

### 1-18-1 Nishiarai Sakaecho, Adachi-ku, Tokyo, 123, Japan 11 

### in the original; was changed to 11 

### 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan 11, 

### and CAS numberwas added to section 11.1 on the textby sponsor's request.
Date 

# J ClY\\At\Yf 

## i 

# 8", .J.-Vv 

## 2



# ~. _A<AclJ- Study director oshihtsa Sudo

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### Sponsor Title Test number 

# GLPSTATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan
Biodegradation test ofV-05Hby microorganisms

This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New Chemical Substances and Study on Harmful Effects ofDesignated Chemical Substances" (March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yak:uhatu No.229, Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry ofInternational Trade and Industry, Japan) and "OECD Principles ofGood Laboratory Practice" (May 12, 1981).
Date March 31, 1997 Management Signed in original Hiroshi Katsuura. '



### ( \.

( 

### Sponsor Title Testnumber 

# GLPSTATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection 

### & Testing Institute, Japan
Biodegradation test ofV-05Hby

**microorg~isms** 

### 13013 This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items ofthe Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" (March 31, 1984, RevisedNovember 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 K.ikyoku No.85, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles of Good Laboratory Practice" (May 12, 1981). This GLP statement was issued as supplement to the GLP statement issued on March 31, 1997 because ofthe amendment and addition ofthe Final Report. Date June 5, 1998 Study director Signed in original Hiroshi Tadokoro


### TlrT

### Sponsor Title 

# QUALITY ASSURANCE STATEMENT Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan NISSHINBO INDUSTRIES, INC. Biodegradationtest ofV-05Hby microorganisms Test number 13013 Study audits and inspections ofthis test were carried out and the results were reported to the management and the study directorby Quality Assurance Unit ofKurume Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & 

### Testing Institute, Japan as follows. Date ofaudit Date ofreport Date ofreport or inspection to management to study director January 31, 1997 January 31, 1997 January 31, 1997 February 12, 1997 February 14, 1997 February 14, 1997 March 17, 1997 March 17, 1997 March 17, 1997 February 26, 1997 March 19, 1997 March 19, 1997 March 12, 1997 March 19, 1997 March 19, 1997 March 17, 1997 March 19, 1997 March 19, 1997 March 18, 1997 March 19, 1997 March 19, 1997 March 31, 1997 March 31, 1997 March 31, 1997 It 

### has been assured that the final report accurately describes the test method used, that details in the report are in compliance with the protocol and Standard Operating Procedures and thatthe final report reflects the raw data. Date March 31, 1997 Quality Assurance Unit, Head Signed in original Susumu Uchitomi( \ 

### Sponsor Title 

# QUALITY ASSURANCE 

# STATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan
Biodegradation test ofV-05Hbymicroorganisms Testnumber 13013 Study audit of the corrected parts in the Final Report was carried out and it was confirmed that the correction has no problem. The result was reported to the management and study director as follows. Date ofaudit Date ofreport Date ofreport orinspection to management to study director

June 5, 1998 June 5, 1998 June 5, 1998 This statement was issued as supplement to the quality assurance statement issued on March 31, 1997. Date June 5, 1998 QualityAssurance Unit, Head Signed in original Susumu Uchitomi



# - ...--·..**~~**

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# CONTENTS ( 

## \ page SUMMARY 1 1. Title 2 2. Sponsor 2 3. Testing facility 2 4. Objective 2 

### 5. Testmethod 2 6. GLP Standardsto comply with 3 7. Testperiod 3 8. Personnel 3 9. Preparation offinal report 3 10. Approval offinal report 3 11. Test substance 4 12. Preparation ofactivated sludge 6 13. Performance ofbiodegradationtest 8 14. Validity oftest conditions 14 15. Results 14 16. Factors possibly affecting accuracy 16 17. Storage oftest substance, raw data, etc. 16 18.Remarks 16 19. Contents oftables and figures 17 Tables and figures 

# fr y;;
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# ( 

# I ! i 

# SUMMARY 1. Title Biodegradationtest ofV-05Hby microorganisms 2. Biodegradationtest

2.1 Conditions ofcultivation (1) Concentration oftest substance (2) Concentration ofactivated sludge 

### (as the concentration ofsuspended solid) (3) Volume oftest solution (4) Cultivation temperature (5) Cultivation duration 2.2 Measurement and analysis 100mg!L 30mg!L 300mL 25+1 

# oc 28 days (1) Measurement of biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus (2)

**Dete~ination** 

### oftest substance by means ofhigh performance liquid chromatography (HPLC) 3. Results (1) Percentage biodegradation by BOD (2) Percentage biodegradation byHPLC 4. Conclusion 0%, 2%, 0%, 0%, 0% 0% average 0% average 1% The test substance was not biodegraded by microorganisms under the present test conditions.

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## :nr

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# '" FINAL 

# REPORT Test No. 13013 1. Title Biodegradation test ofV-OSH by microorganisms 2. Sponsor
3. Testing facility

4. Objective Name Address Name Address Tel Management NISSHINBO INDUSTRIES, INC. 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 19-14, Chuomachi Kurume Fukuoka 830, Japan (0942) 34-1500 Hiroshi Katsuura This test was performed to evaluate the biodegradability ofV-OSH by microorganisms. 5. Test method This test was conducted according to the "Method for Testing the Biodegradability of Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry ofInternational Trade and Industry, Japan) and "Ready Biodegradability : 301C, Modified MITI Test (I)" (Revised July 17, 1992) in the OECD Guidelines for Testing ofChemicals. -2-(



# c 6. GLP Standards to comply with This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items ofthe Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances (hereafter referred to as "GLP standards")" (March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yak:uhatu No.229, Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare, and 59 Kikyok:u No.85, Basic Industries Bureau, Ministry ofInternational Trade and Industry, Japan) and "OECD Principles ofGood Laboratory Practice" (May 12, 1981). 7. Testperiod (1) Start oftest January 31, 1997 (2) Start ofcultivation with activated sludge February 12, 1997 (3) Termination ofcultivation with activated sludge March 12, 1997 (4) End oftest March 31, 1997 8. Personnel Study director Yoshihisa Sudo Test operator Hisako Haruguchi Stafffor cultivation ofactivated sludge Yoshiharu Ohshima Person in charge ofstorage oftest substance and raw data Akira Kobayashi 9. Preparation offmalreport Date March 31, 1997 Name Hisako Haruguchi 10. Approval offinal report Study director Date March 31, 1997 Name Signed in original Yoshihisa Sudo

- 3- 

# U1**'~'**

# ( 11.5 Confirmation oftest substance Two infrared (IR) spectra ofthe test substance provided by the sponsor and this laboratory were confirmed to be identical (see Fig.6 and Reference). 11.6 Physicochemical property*' Appearance Brown viscous substance 

# *1 Information supplied by the sponsor. 11.7 Storage and stability (1) Storage condition Cold storageplace. (2) Stability The test substance was stable during the cultivation, as shown by the fmding that IR spectra ofthe test substance before the start and after the termination ofthe cultivation were identical (see Fig.6).

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( 12. Preparation ofactivated sludge 12.1 Sludge sampling sites and date 

### (1) Sampling sites On-site sludge sampling was carried out atthe following 10 locations in Japan: Fushikogawa city sewageplant (Sapporo-shi Hokkaido) Kashima industry sewage plant (Kashima-gun Ibaraki) Nakahama city sewage plant (Osaka-shi Osaka) Ochiai city sewage plant (Shinjuku-ku Tokyo) Kitakami river (Ishinomaki-shi Miyagi) Shinano river (Nishikanbara-gunNiigata) Yoshino river (Tokushima-shi Tokushima) Lake Biwa (Otsu-shi Shiga) Hiroshima bay (Hiroshima-shi Hiroshima) Dookai bay (Kitakyushu-shi Fukuoka) (2) Date December, 1996 12.2 Sludge sampling method (1) City sewage Return sludges from sewage plants were collected. (2) Rivers, lake and sea Surface water and surface soil which are in contact with the atmosphere were collected. 12.3 Mixing offresh and old activated sludge The filtrate (5 L) ofthe supernatant ofthe activated sludge used at that time was mixed all together with each of the filtrate (500 mL) of the supernatant of a newly collected sludge (10 sources). The mixed filtrate (10 L) was aerated"2 

### after the pH value ofthe mixture was adjusted to 7.0+ 1.0. *2 Prefiltered open airwas used.

-6-

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### 12.4 Cultivation Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 113 ofthe whole volume was removed. Then an equal volume of dechlorinated water was added to the remaining portion. This mixture was aerated, and then a previously decided amount ofsynthetic sewage"3 

### was added to the mixture so that the concentration of the synthetic sewage was O.l(WIV)% in the volume of the dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 

## + 2 °C. *3 Synthetic sewage ' Glucose, peptone.and potassium dihydrogenphosphate were dissolved in dechlorinated water to obtain 5(W IV)% of the solution for each components. The pH ofthe solutionwas adjusted to 7.0+ 1.0 with sodium hydroxide. 12.5 Control and use During cultivating, the appearance of the supernatant, setting of the sludge,

. formation offlock, pH, dissolved oxygen concentration in the solution and temperature

were checked and adjusted if necessary. Microflora in the activated sludge was microscopically observed and the sludge with no abnormal symptoms was used for the test. 12.6 Inspection ofactivity and date ofinitiation ofuse ofactivated sludge (I)

### Inspection ofactivity Activity ofthe sludge was assessed using a reference substance and the relation between new and old activated sludge was taken into account. (2) Date ofinitiation ofuse January 21, 1997

-7- 

# ,·-·rrrr-I \ 

# "'- 13. Performance ofbiodegradation test 13.1 Preparation for test (1) Measurement ofconcentration ofsuspended solid Method In accordance with Japanese Industrial Standards (JIS) K 0102-1993 section 14.1 Date February 10, 1997 Result Concentration ofsuspended solid in the activated sludge was 4700 mg/L. (2) Preparation ofbasal culture medium Each 3 mL ofsolution A, B, C and D, which are prescribed in JIS K 0102-1993 section 21, were made up to 1000 mL 

### with purified water (Takasugi Seiyaku Co., Ltd.), and then the pHofthis solution was adjusted to 7.0. (3) Reference substance Aniline (reagent grade, Showa Chemicals Inc. Lot No. SF 2347B) was used as a reference substance. 13.2 Preparation oftest solutions The following test solutions were prepared and cultured under the conditions described in section 13.3.

(1) Addition oftest substance or aniline

(a) Test solution (water+test substance) (n=l,Vessel No.6) In one test vessel, 30 mg ofthe test substance was accurately weighed and added to 300 mL 

### ofpurified water, so that the concentration reached 100 mg/L. (b) 

### Test solution (sludge+ test substance) (n=3, Vessel No.2, 3 and 4) In each test vessel, 30 mg ofthe test substance was accurately weighed and added to the basal culture medium (300 mL - a volume ofthe activated sludge inoculated), so that the concentration reached 100 mg/L. (c) Test solution (sludge+ aniline) (n=1, Vessel No.5) In one test vessel, 29.5 flL [30.0 mg = 29.5 flL X 

### 1.022 g/cm3 

### (density)] of aniline was added into the basal culture medium (300 mL - 

### a volume of the activated sludge inoculated), so that the concentration reached 1 00 mg!L. (d) Test solution (control blank) (n=l, Vessel No.1) In one test vessel, nothing was added to the basal culture medium (300 mL a volume ofthe activated sludge inoculated). 

### -8- 

# r1 r1I \



### ( \ 

### (2) Inoculation ofactivated sludge The activated sludge cultivated under the conditions described in section 12., was added to each test vessel (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg!L. 13.3 Instruments and conditions ofcultivation (I) Instruments for cultivation Closed system oxygen consumption measuring apparatus (Coulometer: Ohk:ura Electric Co., Ltd.) (Data sampler: Asahi Techneion Co., Ltd.) Vessel 300 mL 

### in volume Absorbent for carbon dioxide Soda lime No.1 (for absorption ofcarbon dioxide, Wako Pure Chemical Industries, Ltd.) Stirring method Eachtest solution was stirred by a magnetic stirrer. (2) Conditions ofcultivation Cultivation temperature Cultivation duration .Room

-9- 

### 25+1

# oc 28 days Apparatus room No.5II

1 

### "Ti'f 

## r

### 13.4 Analysis oftest solution After the termination ofthe cultivation, the test substance in the test solutions was determined. 13.4.1 Pretreatmentoftest solutions for analysis After the termination ofthe cultivation, the test solution (water+ test substance), the test solutions (sludge + test substance) and the test solution (control blank) were pretreated for high-performance liquid chromatography (HPLC) analysis of the test substance as follows :


# I 

# l Test solution 300 mL**~chloroform** 

### 40 mL (graduated cylinder) • Irradiation with ultrasonic waves

(about 30 seconds)**~chloroform** 

### 50 

### mL (graduated cylinder)**~sodium** 

### chloride 90 g (even balance) • Shaking (about 10 minutes) 

# I Water layer 

# II Chloroform layer 

# I**~-------.--------~** 

### • Filtration and dehydration (IPS filter paper) • Filling up to 100 mL (chloroform,

volumetric flask) • Taking out 10 mL (transferpipet) • Dryness (rotary evaporator, about 40 °C,

purge with nitrogen)**~Tetrahydrofuran** 

### about 5mL (komagome pipet) • Irradiation with ultrasonic waves

(about 15 seconds) • Filling up to 10 mL (tetrahydrofuran,

volumetric flask) 

# I Sample for HPLC analysis 

# I

- 10-

### 13.4.2 

# (



# ( Quantitative analysis Detennination oftest substance The samples for HPLC analysis were analyzed underthe following conditions. The concentration of the test substance in the sample for HPLC analysis was proportionally calculated by comparing the peak area on the chromatogram of the sample for HPLC analysis with that on the chromatogram of300 mg!L ofthe standard solution (see Table-3 and Fig.4). The lowest detectable peak area ofthe test substance was regarded as 5000 J.l

### V ·sec considering the noise level, which corresponded to 3.2 mg/L ofthe test substance. (1) Analytical conditions Instrument Pump Detector Column Eluent Flowrate Measurement wave length Sample size Sensitivity Detector output (2) Preparation ofstandard solution High-perfonnance liquid chromatograph Hitachi, Ltd. type L-6000 Hitachi, Ltd. type L-4000 Shodex GPC KF-804L 30 emX 

### 8.0 mm I.D. stainless steel Tetrahydrofuran l.OmL/min 250 nm (see Fig.5) 20J!L 0.4 V/AU The standard solution to detennine the concentration ofthe test substance in the sample for HPLC was prepared as follows. 100 mg of the test substance was accurately weighed and dissolved in tetrahydrofuran to obtain 1000 mg!L solution. 300 mg/L standard solution was then prepared from this solution by dilution with tetrahydrofuran. (3) Calibration curve 75.0, 150 and 300 mg/L of standard solutions were prepared by the same method as described in (2).. These solutions were analyzed according to the analytical conditions described in (1). A calibration curve was drawn based on the relation between the peak area onthe chromatograms and the respective concentrations (see Fig.2).



### - 11-

### 13.4.3 Recovery test and blank test Each two test solutions (water+ test substance) and two test solutions (sludge+ test substance) for recovery test were prepared according to the methods described in 13.2. The test solutions were pretreated in accordance with the method described in 13.4.1, then analyzed according to the procedures and analytical conditions described in 13.4.2. A test solution for blank test was prepared according to the method described in 13.2. The test solution for blank test was analyzed in the same way as the recovery test. As for the blank test, no peak was appeared around the peak ofthe test substance on the chromatogram. Two individual recovery rates and their averages on the analytical procedure are shown below. The average recovery rates were used as correction factor, for the determination of the test substance in the analytical samples (see Table-2 and Fig.3). Recovery rate ofthe test solutions (water+test substance) 93.9 %, 97.2% average 95.6% Recovery rate ofthe test solutions (sludge+test substance) 98.1 %, 98.1 % average 98.1 %

- 12- I -:- II II

# ( 13.5 Calculation ofpercentage biodegradation The percentage biodegradation was calculated by the following equations and expressed in whole numbers.

(1) Percentagebiodegradation by BOD Percentage biodegradation (%) 

## = BOD-B TOD X 

### 100 BOD B TOD*4 Biochemical oxygen demand in thetest solution (sludge+ test substance) (experimental) (mg) Biochemical oxygen demand inthe control blank (experimental) (mg) Theoretical oxygen demlllld required when the test substance was completely oxidized (theoretical) (mg) *4 TOD was calculated withmolecularformula CI96Hz4oNJo02 (n

## = 14). 

### (2) Percentage biodegradation by HPLC Percentage biodegradation (%) 

## = Sw- Ss Sw X 

### 100 

### Ss Sw Residual amount ofthe test substance in the test solution (sludge+test substance) (experimental) (mg) Residual amount ofthe test substance in the test solution (water+ test substance) (experimental) (mg) 13.6 Treatment ofnumerical values Values were rounded offin accordance withJIS Z 8202-1985 reference 3 rule B.



### - 13-

,--.--...

**~-** ..

**~- ~**  ........- ,.._. ..-- ..........=----=- ...**~·--** -

### 14. Validity oftest conditions Percentage biodegradations ofaniline calculated by the BOD values was 64 

### % and 72 % 

### at the 7th and 14th day, respectively. It

### was concluded that this test conditions were valid (see Table-1 and Fig.I). 

### 15. Results

15.1 Appearances oftest solutions Appearances oftest media in cultivation vessels were as follows. Test solution Appearance Water + test substance Test substance was not dissolved. At the initiation ofcultivation Sludge + test substance Test substance was not dissolved. Water + test substance Test substance had notbeen dissolved. At the completion ofcultivation Test substance had not been dissolved. Sludge+ test substance Growth ofthe sludge was not observed.

- 14-

### ( 

### \ 

### 15.2 Analytic results oftest solutions Analytic results ofthe test solution after 28 days were as follows. (Water+ test (Sludge + test substance) Theoretical substance Table Fig. amount Vessel-6 Vessel-2 Vessel-3 Vessel-4 

### BOD*6 

### mg 0.7 0 0 0 89.7 

### 1 Residual amount and mg 30.6 29.8 30.7 31.2 30.0 percentage residue


## ----- -------------- ------------- .......................... 

### ------------- --------------- 3 oftest substance (HPLC) % 102 99 102 104 

## - *6 The value ofcontrol blank was subtracted from the values ofthe test solutions (sludge+ test substance). 15.3 Percentage biodegradation Percentage biodegradations after 28 days were as follows. Percentage biodegradation 

### (%) Method Table Vessel-2 Vessel-3 Vessel-4 Average 

### BOD 0 0 0 0 1 

### HPLC 2 0 0 1 

### 3 15.4 Conclusion The test substance was not biodegraded by microorganisms under the present test conditions.

- 15- 

### 1 4

### 16. Factors possibly affecting accuracy No adverse effects on the reliability ofthis test were noted. 17. Storage oftest substance, raw data, etc.

17.1 Test substance About 

### 5 g ofthe test substance is sealed in a store vessel and stored in a storage room in this laboratory forthe period designated by article 32 ofthe GLP standards. 17.2 Raw data and materials Raw data used for the preparation ofthe final report such as notebooks and results ofmeasurements and observations, protocol, a record ofthe request ofthe test from the sponsor, a record ofinformation about the test substance presented by the sponsor, etc. are stored with the final report in the same manner as the test substance. 18. Remarks

18.1 Instruments used for test

18.2 Fourier transform infrared spectrophotometer: Shimadzu Corporation type FTIR-8200PC Closed system oxygen consumption measuring apparatus : seepage 9 High-performance liquid chromatograph : see page 11 Electronic analytical balance : Sartorius type BP21OS pH meter: ToaElectronics Ltd. type HM-50S Ultraviolet and visible spectrophotometer : Reagents used for analysis Tetrahydrofuran (HPLC grade): Chloroform (reagent grade): Sodium chloride (reagent grade):

- 16- Shimadzu Corporation type UV-2200A 

### KANTO KAGAK.U Kishida Chemical Co., Ltd. MANAC Incorporated

**-..,.~~~.,..II ""~~"11---,.--!** ir:-!Tr -

### 19. Contents oftables and figures Contents oftables Table-1 Calculation table for percentage biodegradation by BOD Table-2 Calculation table for recovery rate by HPLC Table-3 Calculation table for percentage biodegradationby HPLC Contents offigures Fig. 1 Chart ofBOD Fig.2 Chromatograms ofHPLC analysis for calibration and calibration curve Fig. 3 Chromatograms ofHPLC analysis for recovery test Fig. 4 Chromatograms ofHPLC analysis for regular test Fig. 5 UVspectrum ( 

### Fig. 6-1 IR spectrum oftest substance measured before cultivation \ 

### Fig. 6-2 IR spectrum oftest substance measured after cultivation Reference IR spectrum supplied by sponsor



### - 17-

--- - ·· ..-

**·-~~- ~-**  

### ··- ,- ·

**~** 

### --'"'lr 

### I 'if"T( \

### "- 

### Table 1 Calculation table for percentage biodegradation by BOD. Test No. 13013 Cultivating Duration: 28 days 7th day 14th day 21st day 28th day Vessel Mean No. BOD Deg. BOD Deg. BOD Deg. BOD Deg. Deg. (mg) 

### (%) (mg) 

### (%) (mg) 

### (%) (mg) 

### (%) %



### ~ 

### 59.5 64 68.7 72 71.2 72 73.3 72 

# rn 1.6 

# - 3.9 

# - 6.3 

# - 8.0 

# -

**~** 

### 0.8 0 

### 2.8 0 4.7 0 6.1 0 0

**~** 

### 0.9 0 2.8 0 4.8 0 5.9 0 @ 

### 2.4 1 4.7 1 6.9 1 8.0 0 [§] 

### 0.0 

# - 0.2 

# - 0.7 

# - 0.7 

# - Deg. : 

### Percentage biodegradation Vessel no.

**~** : 

### Sludge + 

### Aniline Vessel no. 

# rn : 

### Control blank [B) Vessel no.

**~ ~@**  

### : 

### Sludge + 

### Test substance Vessel no. [§] : 

### Water + 

### Test substance



## - Test substance of 30 mg was added. Chart of BOD 

# . Fig. 1 

# . Deg. 

## = [ 

### BOD 

# - B ] 

## / [ 

### TOD ] X 

### 100 

### (%) TOD of test substance : 

### 89.7 (mg) CJ...eaH24oN3o02 + 

### 285 02- 196 C02 + 

### 120 H20 + 

### 30 N02 285 02 / CJ...aaH24oN3o02 = 

### 9119.66 

## / 3048.20 

## = 2.99 TOO = 

### 30.0 X 

### 2.99 = 

### 89.7 (mg) TOO of aniline : 

### 90.3 (mg) CaH7N + 

### 8. 75 02 - 6 C02 + 

### 3.5 H20 + 

### N02 8.75 02 / CaH7N = 

### 279.99 / 93.13 

## = 3.01 TOO = 

### 30.0 X 

### 3.01 = 

### 90.3 (mg) 199 7 . 03 . 12 ::..c..N



## a=m=e'---._(-~.-f ,--'f-1'--'---'ctr<..-·



## :...,sft-~~A_e-h_;___ _

### Table-2 Calculation table for recovery rate by HPLC Test No 13013 

### ( i Sample description A D E F Standard solution 300mg!L 516947 Water+ test substance -1 485506 28.2 93.9 Water + test substance -2 502551 29.2 97.2 95.6 Sludge+ test substance -1 507254 29.4 98.1 Sludge + test substance -2 507125 29.4 98.1 98.1 Control blank n.d. Amount oftest substance added : 30 (mg) A: Peak area (uV·sec) B: Final volume: 100 (mL) 

### C: Ratio ofportion used for analysis : 

### 3001300 D: Recovery amount (mg) D 

## =G x (A 

### I A(Standard)) x ( B 

### I C)

### I 1000 E: Recovery rate (%) E 

## =D 

### I 30 (mg) x 100 F: Average recovery rate(%) 

## - G: Concentration ofstandard solution : 300 (mg!L) See Fig. 3 March 19, 1997 Name

### Table-3 Calculation table for percentage biodegradation by HPLC Test No 13013 Sample description A E F G H Standard solution 300mg!L 476005 [6] Water+ test substance 463693 30.6 102 [2] Sludge + test substance 464259 29.8 99 2 [3] Sludge + test substance 477205 30.7 102 0 1 [4] Sludge+ test substance 485813 31.2 104 0 [1] Control blank n.d.

- 

### Amount oftest substance added : 30 (mg) A : Peak area (p.V·sec) B : Final volume : 100 (mL) 

# .. C: Ratio ofportion used for analysis : 300/300 

### D: Recovery rate: 95.6 (%)(Water+ test substance) 98.1 (%) (Sludge +test substance) E: Residual amount oftest substance (mg) E =I x (A 

### I A(Standard)) x ( B 

### I C) 

### I ( D 

### I 100) 

### I 1000 F: Percentage residue (%) F = E 

### I 30 (mg) x 100 G: Percentage biodegradation (%) G = {( 

### E(Water +test substance)- E(Sludge +test substance)) 

### I E(Water +test substance)} x 100 H: Average percentage biodegradation(%) I : 

### Concentration ofstandard solution : 300 (mg!L) See Fig. 4 .. 

### March 19, 1997

11 I I I

# ( Fig.1 Chart of BOD Test No. 13013 ( Test substance Apparatus · · · · · · · · · · · · · · · · · · · · · · · · · · · Cultivating conditions: Concentration Test substance · · · · · · · · · · · ·· · · ·· · Reference substance( aniline ) · · Activated sludge · · · · · · · · · ··· · · · · V- 

## ot-H No. .CM-22 100 (mg/.Q) 

### 100 (mg/.Q)



### 30 (rng/.Q) 

### Temperature · · · · · · · · · · · · · · · · · · · · ·· · Duration · · · · · · · · · · · · · · · · · · · · · · · · · · 25 

## ± 1' 

# c**28days(Feb.12~Mar.12,1997)** 

### Note:



# - t>O 

### 80- e .._, '0 c::**~** e <l) 0 

### 60- c:: <l) t>O >. >< 0 

# -**~** (,) 

### 40- 

# ·- e <l) .c:: (,) 0 

# ·- CQ 

### 20- 0 

### Regular condition



## III- 

## !ZJ-- []--- @1······**~-·­** 

### [§]-··-

f 

### I I 

# i I ..._ ....·····..:.:.:·.::,:·..······· .............· __ 

### 1

### @][2)~ 

## i**-..:.:.:;:--::··::··::··~~·**

## .. _-;.:.__**.::~.,**

## ..- .._.._.._.._..____..____________-[§] 7 I I I I 

### 1 1

### 4 I I I I I I 

### 21 28 Cultivation Time (**0~28thday)** 

### 1997.03.12 Name 

# /-1 ·**1-fttv--~ke.hi**

### Standard solution 300 mg/L Operating date : Mar. 13. 1997 PKNO Time Area Height Cone. 1 (min) (.uV ·sec) (.uV)' (%) I 7. 94 5212Q;;l 4090 100.00 Total 521603 100.00

0. 0 5. 0 10.0 15.0 Operator : Hisako Haruguchi 

### Standard solution !50 mg/L Operating date : Mar. 13. 1997 PKNO Time Area Height Cone. (min) (.uV·sec) (.uV) (%) 1 7. 89 244955 1954 100.00 Total 244955 100.00 1



# ~"--v 0. 0 5. 0 10. 0 15.0 Operator : Hisako Haruguehi 

### Standard solution 75. 0 mg/L Operating date : Mar. 13. 1997 PKNO Time Area Height Cone. (min) (.uV·sec) (.uV)' (%) 1 7. 89 132320 1057 100.00 Total 132320 100. 00 I



## ~ 0.0 5.0 10.0 15.0 Operator : Hisako Haruguehi 

### Solvent blank Operating date : Mar. 13. 1997 PKNO Time Area Height Cone. (min) (.uV·sec) (.uV) (%) Total 0 0. 00 Peak position 

### ...,,q.

**~** 0.0 5. 0 10. 0 15. 0 Operator : Hisako Haruguehi 

### Fig. 2 - I Chromatogram of HPLC analysis for calibration curve. Date : Mar. 13. 1997 Name
]"':" 

### :T ,IITest No. 13013



### Fig. 2- 2 Calibration curve and conditions ofHPLC analysis for V-OSH March 19, 1997 

### ( 

# .f( 

# 06I <:::H

**1~0.** I .:lU I ;;! 

### Standard solution 300 mg/L Operating date : Mar. 13, 1997 PKNO Time Area Height Cone. (min) (t.tV·see) (t.tV) (%) 1 7. 88 516947 4017 100. 00 1 Total 516947 100. 00 

# A.· -"--v- .. 0.0 5. 0 10.0 15. o(min) Operator -: Hisako Haruguchi 

### Water t 

### test substance- 1 Operating date : Mar. 13, 1997 PKNO Time Area Height Cone. (min) (t.tV·see) (t.tV) (%) I 7. 95 485506 3850 100.00 Total I 485506 100. 00



# ~-v- 0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguchi 

### Water t 

### test substance- 2 Operating date : Mar. 13, 1997 PKNO Time Area Height Cone. (min) (t.tV·see) (t.tvr (%) I 7. 93 502551 4006 100.00 I Total 502551 100. 00



# ~ 0. 0 5.0 10. 0 15. o(min) Operator : Hisako Haruguehi 

### Fig. 3 - l Chromatogram of HPLC analysis for recovery test. Date : Mar. 13. 1997 Name



### H-f-.1~~;

( 

# "- 

# f1 

# b7

-

**<--<~F** 0 • -•• • __ ,.._ ,..._- ..-_.,..--··· ..,...._.._--=--

**~--=·-~~** 0lest No. 13013 

### Sludge 

## +test substance - I Operating date : Mar. 13, 1997 PKNO Time Area Height Cone. (min) (p.V·see) (p.V) (%) 1 7. 91 507254 3990 100.00 Total 507254 100.00 1



# ~ ( \
0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguehi 

### Sludge 

## +test substance - 2 Operating date : Mar. 13, 1997 PKNO Time Area Height Cone. (min) (p.V·see) (p.V) (%) I 7.95 507125 3983 100.00 Total 507125 100.00 1



# - 

# A
0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguehi 

### Control blank Operating date : Mar. 13. 1997 PKNO Time Area Height Cone. (min) (p.V·see) (p.V). (%) Total 0 0. 00 Peak position 

# \/)\c.l'



# ~~ 0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguehi 

### Fig.3-2 Chromatogram of HPLC analysis for recovery test. Date : Mar. I3. I 997 Name


## (

### Standard solution 300 mg/L Operating date : Mar. 18, 1997 PKNO Time Area Height Cone. (min) (tJ.V·see) (tJ.V) (%) 7. 98 476005 3830 100.00 Total 476005 100.00

0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguchi 

### [ 6 ] 

### Water 

## + test substance Operating date : Mar. 18, 1997 PKNO Time Area Height Cone. (min) (tJ.V•see) (tJ.V) (%) 8. 06 463693 3771 100.00 Total 463693 100.00

0. 0 5. 0 10.0 15. o(min) Operator : Hisako Haruguehi 

### [ 2 ] 

### Sludge 

# +test substance Operating date : Mar. 18. 1997 PKNO Time Area Height Cone. (min) (tJ.V·see) 

### (tJ.vr (%> 7. 90 464259 3721 100.00 

# ( Total 464259 100.00

0. 0 5. 0 10. o 15. o(min) Operator : Hisako Haruguchi 

### Fig. 4 - l Chromatogram o.f HPLC analysis for regular test. Date : Mar. 19. 1997 Name( \

( 0. 0



### o. 0



### o. 0 

### [ 3 ]

**Slu~ge** 

## + test substance

5. 0 10,0 

## [ 4] Sludge 

## + test substance

5. 0 

### [ I ] 

### Control blank 10.0

Peak position V\' 

### d .

**~** I 'tV. I "'"'VI oJ Operating date : Mar. 18, 1997 PKNO Time Area Height Cone. (min) (t.t.V·sec) (t.t.V) {%) 7. 97 477205 3756 100. 00 Total 477205 100.00
15.o(min) Operator : Hisako Haruguehi Operating date : Mar. 18, 1997 PKNO Time (min) 7. 94 Total Area (t.t.V·sec) 485813 485813 Height Cone. (t.t.V) (%)

3798 100.00 100.00

15. o(min) Operator : Hisako Haruguehi Ope.rat ing date : Mar. 1B. 1997 PKNO Time Area Height Cone. (min) (t.t.V·sec) (t.t.Vl' (%) Total 0 0. 00

5.0 10.0 15.o(min) Operator Hisako Haruguehi 

### Fig. 4 - 2 Chromatogram of HPLC analysis for regular test. Date : Mar. 19, 1997 Name.; !


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### -o. 1 • 

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### sponsor( 

# Amendment and addition to Final Report

1. Title 2. Test number 

### 3. Content 

### 4. Reason 5. Content ofcorrection 6.Date 7. Approval . Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### TestingInstitute, Japan Biodegradation test ofV-05H bymicroorganisms 13013 

# CD 11.2 Chemical structure, etc.•, Molecularweight (page 4) 

# ® 11.2 Chemical structure, etc.•, Polymerization degree (page 4) 

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### ~leared. 

# CD "Molecularweight " Molecularweight 

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### 5, 1998 Date About 3000 "is corrected to About 30000 ".

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### 5, 1998 Study director Signed in original Yoshihisa Sudo



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### Accepted number Testnumber S97-3112 43112 

# FINAL REPORT

Bioaccumulation test ofV-05H in carp

February 6, 1998 Kurume Research Laboratories

Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan



# ~~ 

# 77 r--

### ( \

( 

# I Sponsor Title Test number 

# STATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & TestingInstitute, Japan Bioaccumulation test ofV-05Hin carp 43112 I, the undersigned, hereby declare that this report provides a correct English translation ofthe Final Report {TestNo.43112, issued onFebruary 6, 1998). However, sponsor's address, 11 

### 1-18-1 Nishiarai Sakaecho, Adachi-ku, Tokyo, 123-0843, Japan 11 

### in the original, was changed to 11 

### 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan 11 

### and CAS numberwas addedto section 10.1 onthe textby sponsor's request.

Date "3tti'IJW1 

## ;&, ..2 

# oo.l Study director



# r1J1~-vc Yasuko Matsunobu



# r·.•i 'I 

### I



### Sponsor Title Testnumber 

# GLP STATEMENT

NISSHINBO INDUSTRIES, INC. Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan
Bioaccumulationtest ofV-OSH in carp 43112 This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to 

### be observed bytesting facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items ofthe Test Relating to the New Chemical Substances and Study on Harmful Effects ofDesignated Chemical Substances" (March 31, 1984, RevisedNovember 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles of Good Laboratory Practice" (May 12, 1981).
Date February 6, 1998 Management Signed in original Hiroshi Katsuura

# (

( 

### Sponsor Title Test number 

# GLPSTATEMENT

NISSHINBO INDUSTRIES, INC. 

### Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan
Bioaccumulationtest ofV -OSH in carp 43112 This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items ofthe Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" (March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles of Good Laboratory Practice" (May 12, 1981). This GLP statement was issued as supplement to the GLP statement issued on February 6, 1998 because ofamendment and addition to Final Report. Date June 5, 1998 Management Signed in original Hiroshi Tadokoro

# - 

# QUALITY ASSURANCE 

# STATEMENT
Sponsor Title Test number NISSHINBO INDUSTRIES, INC. Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan BioaccumulationtestofV-05H in carp 43112 Study audits and inspections ofthis test were carried out and the results were reported to the management and the study director by ·Quality Assurance Unit of Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan as follows.

Item ofaudit Date ofaudit Date ofreport Date ofreport orinspection or inspection to management to study director Protocol November 14, 1997 November 14, 1997 November 14, 1997 November 18, 1997 November20, 1997 November20, 1997 December 16, 1997 December 24, 1997 December24, 1997 Test Conditions December 19, 1997 December 24, 1997 December24, 1997 January 8, 1998 January 13, 1998 January 13, 1998 January 9, 1998 January 13, 1998 January 13, 1998 Raw Data and February 6, 1998 February 6, 1998 February6, 1998. Final Report 

### It has been assured that the fmal report accurately describes the test method used, that details 

### in the report are in compliance with the protocol and Standard Operating Procedures andthatthe final reportreflects the raw data. Date February 6, 1998 QualityAssurance Unit, Head Signed in original Susumu Uchitomi

...**~!'""T** I**--·r--~---** I 1 ·;I I \

( 

# QUALITY ASSURANCE 

# STATEMENT
Sponsor Title Test number Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan NISSHINBO INDUSTRIES, INC. Bioaccumulationtest ofV-05H ester in carp 43112 

### Study audit of the corrected parts in the Final Report was carried out and it was confirmed that the correction has no problem. The result was reported to the management and study director as follows. Date ofaudit Date ofreport Date ofreport to management to study director June 5, 1998 June 5, 1998 June 5, 1998 This statement was 

### issued as supplement to the quality assurance statement issued on February 6, 1998. 

## • Date June 5, 1998 Quality AssuranceUnit, Head Signed in original SusumuUchitomi

# CONTENTS page SUMMARY 1 

### 1. Title 2 2. Sponsor 2 3. Testing facility 2 4. Objective 2 5. Testmethod 2 6. GLP Standards to complywith 3 7. Testperiod 3 8. Personnel 3 

## ( 

# " 9. Preparation offinal report 3 10. Test substance 4 11. Substance suppliedbythe sponsor 5 12. Performance ofacute toxicity test 

### 6 13. Performance ofbioaccumulation test 

### 9 14. Results 17 15. Factors possibly affecting accuracy 18 16. Storage oftest substance, raw data, etc. 18 17. Remarks 19 18. Contents oftables and figures 20 ( 

### Tables and figures \ 

# fr <61

# (

( \ 

# SUMMARY 1. Title Bioaccumulationtest ofV-05Hin carp 2. Test conditions 2.1 Acute toxicity test (1) Test fish (2) Duration ofexposure (3) Exposure method 2.2 Bioaccumulation test Orange-redkillifish (Ot:yzias latipes) 48 hrs. Semi static system (Renewal oftestwater, at every 

### 8 - 16 hrs.) (1) Test fish Carp (Cyprinus carpio) (2) Nominal concentrations oftest substance High exposure level (Level 1) 0.5 mg/L Lowexposure level (Level 2) 0.05 mg/L (3) DUration ofexposure 8 weeks ( 4) Exposure method Continuous flow system (5) Analytical method Highperformance liquid chromatography 3. Results (1) 

### 48-hourLC50 value (2) Bioconcentration factors 4. Stability oftest substance >100mg/L Levell Level2 

## < 12 <129 The test substance was confirmed to be stable under storage and test conditions.

- 1-

# FINAL REPORT

1. Title Bioaccumulationtest ofV-OSH in carp 2. Sponsor 3. Testing facility

4. Objective Name Address Name Address Phone Management TestNo. 43112

NISSHINBO INDUSTRIES, INC. 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 19-14, Chuomachi Kurume Fukuoka830,Japan (0942) 34 - 1500 Hiroshi Katsuura This testwasperformedto evaluatethe bioaccumulation potential ofV -OSH in carp. 5. Testmethod This test was conducted according to the "Method for Testing the Degree of Accumulation ofChemical Substances in Fish Body" stipulated in the "Testing Methods for New Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry of International Trade and Industry, Japan), and "Bioaccumulation : 305C, Degree of Bioconcentration in Fish" inthe OECD Guidelines for Testing ofChemicals (May 12, 1981). -2-

# ( 6. GLP Standards to comply with This test was conducted in compliance with Good Laboratory Practice Standards for industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests stipulated in article 4 ofthe Order Prescribing Those Items ofthe Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances (hereafter referred to as "GLP standards")" (March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles ofGoodLaboratoryPractice" (May 12, 1981). 7. Testperiod (1) Start oftest (2) Initiation ofexposure (3) Termination ofexposure (4) End oftest 8. Personnel Study director Test operator Stafffor fish care Personto conduct offish acute toxicitytest Person in charge ofstorage oftest substance andraw 

### data 9. Preparation offinal report Date Signature



## - 3- November 14, 1997 November 27, 1997 January 22, 1998 February 6, 1998 Yasuko Matsunobu YukaKida Yasuro Kawashima Yasuro Kawashima Kuniichi Kajiwara Tadashi Ohki February 6, 1998 Signed in original Yasuko Matsunobu



# rrr 1 ::1 I

# ( l 12. Performance ofacute toxicity test 12.1 Testmethod The test was performed in accordance with Japanese Industrial Standard (TIS K 0102-1993 section 71.), "Testing methods for industrial waste water, Acute toxicity test with fish". 12.2 Test fish (1) Species Orange-red killifish (Oryzias latipes) (2) Supplier Nakashima fish farm (Address: DaimyojinNagasu-cho Tamana-gun, Kumamoto 869-0100, Japan) (3) Conditions for fish care The fish were visually examined upon receiving and those demonstrating any abnormalities were removed. The fish were reared for 19 days in a flow through system following an external disinfection. (4) Conditions for acclimatization After rearing, the fish were transferred to an acclimatizing aquarium and acclimatized there after the second external disinfection. The fish those demonstrating any abnormality were removed and reared for 34 days in a flow through·system at the temperature of25 ± 2 °C. The fish were checked for health conditions and reared another 53 days after the external disinfection. (5) Weight average 0.17 g (6) Length 

### average 2.7cm (7) Certification Fish of the same lot No. TF0-970820 which passed the HgC12-test"2 

### were subjected to the test. *2 Kenji Tabata, Journal ofWater andWaste

## .1.1, 1297-1303 (1972)

-6-

# ( 12.3 Dilution water for test (1) Origin Ground water from the premises ofKurume Research Laboratories. (2) Water quality assessment The dilution water for test was taken out on August 1, 1997, and it was analyzed and measured (once every six months in this laboratory). The results are shown inReference 1. It was confirmed that the dilution water met the ministerial ordinance ofthe Ministry of Health and Welfare (December 21, 1992), water quality criteria for fisheries (Shadanhozin Nihon**Suis~igen** 

### Hogokyokai, March 1983), and OECD Guidelines for Testing ofChemicals 

## 210, Fish, Early-life Stage Toxicity Test (July 17, 1992). 12.4 Test conditions (1) Test tank 

### Round glass vessel (2) Volume oftest water 4 L/level (3) Temperature oftest water (4) Concentration ofdissolved oxygen in test water At initial exposure 8.1 mg/L Attermination ofexposure 7.1 mg/L (5) pHoftestwater At initial exposure 7.7 At termination ofexposure 7.7 (6) Number offish 10 /level (7) Duration ofexposure 48 hrs. (8) Exposure method Semi static system (Renewal oftest water, at every 8- 16 hrs.) 

# ( f1 

# 1ro -7-

# ( 12.5 Preparation ofstock solution (1) Dispersant HC0-40 (Hydrogenated castor oil) (2) Preparation Test substance and HC0-40 (100 times amount ofthe test substance) were dissolved in tetrahydrofuran.**Ion-exchan~ed** 

### water ofthe about same quantity as the tetrahydrofuran was added. Aftertetrahydrofuran was evaporated, the mixture was diluted with ion-exchangedwatertoprepare 1000 mg/L stock solution. 12.6 Performance oftest (1) Place 214 LC50 room (2) Date November 17, 1997- November 19, 1997 12.7 Estimation of48-hourLC50 value The 48-hour LC50 value was estimated bythe Doudoroffmethod. 12.8 Result oftest 48-hourLC50 value 

## > 100 mg/L (see Fig.3)



## - 8-**r·~-~·. -~** **~-····** 

# ·- ---···-**-~~~ -~·~-** ( \ 

### 13. Performance ofbioaccumulation test 13.1 Test fish (1) Species Carp (Cyprinus carpio) (2) Supplier Sugishima fish farm (Address: 123-2 GunchikuIchibancho, Yatsushiro-shi, Kumamoto 866-0024, Japan) Date received September 17, 1997 (3) Conditions for fish care The fish were checked visually at the delivery and removed those demonstrating any abnormalities. The fish were reared for 1 day in a flow through system following an external disinfection. (4) Conditions for acclimatization Afterrearing, the fish were medicated to eliminate parasites andtransferred to an acclimatizing aquarium. After the second external disinfection, they were acclimatized. The fish those demonstrating any abnormalities during this period were removed and reared for 51 days in a flow through system at the temperature or'25 ± 2 °C. The fish were then transferred to test tanks and reared at the same temperature in the flow through system for another 16 days, following the external disinfection. (5) Weight, length and lipid content at initial exposure Weight average 23.6 g Length average 9.8 em Lipid content average 3.7 % 

### Measured values ofLotNo. TFC-970917 Date ofdetermination November 6, 1997 (6) Feeding Feed Pelleted feed for carp Manufacturer Nippon FormulaFeedMfg. Co., Ltd. Feeding amount and interval Amount corresponding to about 2%oftotal body weightwas fed twice a day inhalves. On 

### the day before fish sampling, no feed was given. 13.2 Dilution water for test Same as 12.3. 

### -9-

**~-·~-.~~**

# ( 13.3 Conditions oftest and circumstances (1) Supply oftest water Flowthrough system assembled at this laboratory was used. (2) Test tank 

### 100-L glass tank (3) Flowrate oftestwater 2 mL/min for stock solution and 400 mL/min for dilution water ; 579 liters/day for test waterwere supplied. (4) Stock solution bottle 25-L glass bottle (5) Temperature oftestwater (6) Concentrations ofdissolved oxygen intesttank Levell 6.4-7.6mg/L (see Fig.ll) Level2 7.3-7.9mg/L (see Fig.12) Control 6.6-7.8mg/L (see Fig.13) (7) Number offish (at initial exposure) Level 1 and 2 11 · Control (8) Duration ofexposure (9) Place 13.4 Preparation ofstock solutions (1) Dispersant Same as 12.5 (1). (2) Preparation ·Level 1 

### 5 8 weeks 213 Aquatron room

100 mg/L stock solution was prepared in the same way as 12.5 (2). • Level2 10 mg/L stock solution was prepared in the same way as 12.5 (2). ·Control 250 g ofHC0-40 was dissolved 

### in ion-exchanged water and the solution was filled up to 25 L. 

# f1 

# 13

- 10-

# ( 13.5 Testconcentrations Based onpreliminarytest results for 48-hourLC50 and analytical detection limits, test concentrations ofthe test substance were decided on as follows. The control was set as a blanktest. Levell Level2 0.5 mg/L (as thetest substance) 0.05 mg/L (as the test substance) 13.6 Observation, measurement, etc. (1) Observation oftestfish Condition oftest fish wasobservedvisually twice a day. (2) Flowrate oftestwater Flowrate ofstock solution and dilution water were measured with graduated cylinder and recorded once a day. · (3) Temperature oftestwater Temperature of test water was measured with alcohol thermometer and recorded once a day. (4) Concentration ofdissolved oxygen in test tank · Concentration ofdissolved oxygen in test tank was measured with dissolved oxygen probe andrecorded twice a week. (5) The others. 

### Intest period, excreta ofcarp, dirt on test

### tank, etc. were removed once about a day.



### - 11-

# (

/ \ 

### 13.7 Analysis oftest water and fish 13.7

### .1 Frequency ofanalysis The concentrations ofthe test substance in the test water for both level 1 and 2 were analyzed 16 times ; twice a week intervals (n=l). The concentrations in the test fish for both levels were analyzed 4 times ; in weeks 2, 4, 6 and 8 (n=2). The control fish were analyzed atthe initiation and the termination ofexposure (n=2). 13.7.2 Pretreatment for analysis (1) Test water Aliquot oftestwaterwas taken from eachtest

### tank, Levell 40mL Level 2 400

## mL and pretreated for high-performance liquid chromatography (HPLC) analysis as follows:


# I 

# I Water layer 

# I 

# I Test water 

# I**~water** 

### for recoverytest 360 

### mL (graduated cylinder) (onlyLevel 1)**~sodium** 

### chloride 120g 

### (evenbalance)**~Ethyl** 

### acetate 100 

### mL (graduated cylinder) • Shaking for 10 min 

# I Ethyl acetate layer • Filtration and dehydration (IPS filter paper) • Evaporation to dryness

(rotary evaporator, about 40 °C, nitrogen purge) • Fillingup to 2 

### mL (tetrahydrofuran,

volumetric flask) 

# I Sample forHPLC analysis 

### .I

- 12-

# ( (2) Test fish


# I Test fish were taken from each test tank and pretreated for HPLC analysis as

follows: 

# I Test fish 

# I • Measurement ofweight and body length • Chopping into pieces

**~Tetrahydrofuran** 

### 100 mL (graduated cylinder)

• Homogenization (polytron, ca. 1 min) • Washing (tetrahydrofuran 20 mL) • Centrifugation (4000 x 

### g, 5 min) 

# I Residue 

# I I Supernatant 

# I • Filtration (absorbent cotton) • Filling up to 150 mL (tetrahydrofuran, volumetric flask) • Taking out 5 mL (transterpipet) • Column chromatography*3 

# I Supernatant 

# I • Filling upto 5 mL (tetrahydrofuran, volumetric flask) 

# I Sample for HPLC analysis 

# I *3 Conditions ofcolumn chromatography Sep-Pak Silicagel (Conditioning: Tetrahydrofuran 10 

### mL) Loading Whole volume ofthe solution was loaded. Test substance was eluted with the loaded solution.



## - 13-

# ( 13.7.3 Quantitative analysis for test substance The samples for HPLC analysis in pretreatment were analyzed by HPLC under the following analytical conditions. The concentration ofthe test substance in the sample for HPLC analysis was determined based on a comparison ofthe peak height on the chromatogram ofthe sample solution with that of a standard solution (see Tables-4, 5, Fig.6 andTables-7, 8, 9, Figs.8, 9, 10). (1)Analytical conditions Instrument 

### Pump Detector Column Eluent Flowrate Measurementwave length Sample size Detector output (2) Preparation ofstandard solution High-performance liquid chromatograph Shimadzu Corporation type LC-6A Shimadzu Corporation type 

### SPD-6A Asahipak GS-31OH 

### 25 em x 

### 7.6 mmi.D. stainless steel Tetrahydrofuran (containing 10 mmol/L sodiumperchlorate monohydrate) l.OmL/min 260nm (see Fig.14) 100 f.!L 0.8 

### V/AU The standard solution to determine the concentration ofthe test substance in the sample solutions wasprepared as follows. 100 mg of the substance supplied by the sponsor was dissolved in tetrahydrofuran to obtain 1000 mg/L solution of the test solution. 10 mg/L standard solution was then prepared from this solution by dilution with tetrahydrofuran. (3) Calibration curve 5.0, 10 and 20 mg/L standard solutions were prepared by the same method as described in (2). These solutions were analyzed according to·the analytical conditions described in (1). A calibration curve was drawn based ontherelation between the peakheight onthe chromatograms andtherespective concentrations. In consideration ofthe noise level, the lowest detectable peak height ofthe test substance 

### was regarded 

### as 300 

## J..lV, which corresponded to 0.89 mg/L ofthe test substance (see Fig.4).

- 14-

! iI \



### / 

### \.

( 

### 13.7.4 Recovery and blanktest (1) Method A specified amount ofthe test substance was added to test water and fish homogenate for the recovery test, followed by pretreatment described in 13.7.2. The blank test was also conducted in the same manner without the test substance. All the recovery and blanktests were carried out in duplicate. (2) Results ofrecovery test In the blank test, the chromatogram ofHPLC had no peak interfering with determination ofthe test substance concentration. The duplicate recovery rates and the average ofthem in the pretreatment are shown below (see Tables-3, 6 and Figs.5, 7). The average recovery rate was used as correction factors for the determination ofthe test substance concentrations in the analytical samples. Foranalysis oftestwater (20 p.g test substance added) 86.4 %, 84.9% average 85.6% Foranalysis oftestfish (1500 p.g test substance added) 77.7 %, 75.1% average 76.4%



### - 15-i 

### \ 13.7.5 Calculation of test substance concentration m sample and minimum limit of determination (1) Calculation oftest substance concentration in testwater The equation in Tables-4, 5 was used to obtain the concentrations, and they were rounded offto 

### 3 figures. (2) Determination limit oftest substance intestwater This determination limit*4 

### was calculated based on that in 13.7.3 (3) as follows. Levell Level2 0.052 mg/L 0.0052 mg/L (3) Calculation oftest substance concentration in test fish The equations in Tables-7, 8, 9 wereused to obtain the concentrations. But the measured concentrations oftest substance in.testfish were less than minimum determination limit oftest substance. (4) Determination limit oftest substance in test fish Based on the determination limit 13.7.3 (3), the determination limit*4 

### ofthe test substance in test fish was calculated as follows, assuming fish weight of30 g. 5.8 p.g/g *4 Minimum determination limit oftest substance (mg/L orp.g/g) 

## = A B 100 X 

### where CxE D 

### A:Minimum determination limit oftest substance onthe calibration curve (mg/L) B :Recoveryrate (%) C: Samplingvolume oftestwater (mL) orweight offish (g) D: Final volume ofsample solution (mL) E: Ratio ofthe portion, used for analysis to whole volume Results were rounded offto 2 figures.



### - 16-

# ( 13.8 Calculation ofbioconcentration factors (BCFs) The measured concentrations of test substance in test fish were less than minimum determination limit oftest substance. From the minimum limit ofdetection ofthe test substance in 13.7.5 (4), BCFs could be obtained for cases ofa BCF exceeding the followings : level 1 12 level2 129 13.9 Treatment ofnumerical values Values were rounded offin accordance with JIS Z 8202-1985 reference 3 rule B. 14. Results

14.1 Concentrations oftest substance in test water The measured concentrations of the test substance in test water are shown in Table-1. Each average concentration ofthe test substance in test water was maintained at more than 90 

### % ofeach the nominated concentration. Table- I 

### Measured concentrations oftest substance in test water (average value attimes from start ofexposure) (Unit: mg/L) 2 weeks 4weeks 6weeks 8 weeks Table Fig. Levell 0.473 0.460 0.465 0.468 4 6 Level2 0.0486 0.0457 0.0451 0.0452 5

- 17-I \

i '\_ 

### 14.2 Bioconcentration factors BCFs are shown in Table-2. BCFs in Table-2 plotted against duration ofexposure are shown in Fig.1 and 2. BCFs ofthe test substance were less than 12 atLevel1 and less than 129 at Level2. Table-2 BCFs 2 weeks 4weeks 6weeks 8 weeks Table Fig.

<12 <12 <t2 <t2 Levell 

### 7 8 <t2 <t2 <t2 <t2 <t29 <t29 <t29 <129 Level2 

## 8 9 <t29 <129 <t29 <129 14.3 Results oftest fish observation No abnormality in behavior or appearance was noted. 15. Factors possibly affecting accuracy No adverse effects onthe reliability ofthis test were noted. 16. Storage oftest substance, raw data, etc.

16.1 Test substance About 5 g ofthe test substance is sealed in a store vessel and stored, for theperiod designated byarticle 32 ofthe GLP standards, in a storage room 

### inthis laboratory. 16.2 Raw data and materials Raw data used for the preparation ofthe final report such as notebooks and results ofmeasurements and observations, protocol, a record ofthe request ofthe test from the sponsor, a record ofinformation about the test substance presented by the sponsor, etc. are stored with the final report, for the period designated by article 32 of the GLP standards, in a storageroom 

### in this.laboratory.



### - 18-I \



## ( 17. Remarks 17.1 Instruments, apparatuses, special apparatuses andreagents, etc. forthe test (1) Instruments for fish care Micro quantitative pump for supplying stock solution : Tokyo Rika Kikai Co., Ltd. type GMW Instrument for measuring concentration ofdissolved oxygen : IIJIMA ELECTRONICS CORPORATION type F-102 (2) Instruments, apparatuses, special apparatuses andreagents Instruments and apparatuses

Highperformance liquid chromatograph : Electronic analytical balance : see page 14 Sartorius type 1702MP8 Sartorius type 1404MP8 MetlerToledo type 

### PB602 Infrared spectrophotometer : Shimadzu Corporation type FTIR-8200PC Ultraviolet and visible spectrophotometer : Rotary evaporator : Mechanical shaker : Homogenizer (polytron): Centrifuge : Special apparatuses

Sep-Pak Silicagel: Reagents Shimadzu Corporation type UV-2200A Tokyo Rika Kikai Co., Ltd. type N-1 Iuchiseieido Co., Ltd. type MS-1 Kinematica Kubota Syouji Co., Ltd type 6900 Nihon Millipore Ltd. Tetrahydrofuran (HPLC) : Kanto Chemical Co., Inc. Ethyl acetate (reagent grade): Kanto Chemical Co., Inc. Sodiumperchlorate monohydrate (reagent grade) : Wako Pure Chemical Industries, Ltd. Sodium chloride (reagent grade): MANAC Incorporated HC0-40 : Nikko Chemicals Co., Ltd.

- 19- 

## ·1--r I IT

# (

\ 

### 18. Contents oftables and figures Contents oftables Table-I Table-2 Table-3 Table-4 Table-5 Table-6 Table-7 Table-8 Table-9 Reference 1 Measured concentrations oftest substance in test water (see text) BCFs (see text) Calculationtable for recovery andblanktest (analysis oftestwater) Calculationtable for analysis oftestwater (Levell) Calculationtable for analysis oftestwater (Level2) Calculationtable for recovery andblanktest (analysis oftest fish) Calculationtable for analysis oftest fish (Level 1) Calculationtable for analysis oftest fish (Level2) Calculationtable for analysis oftest fish (Control) Analytical results ofdilution water

# /~ 

# (OJ -20-

## (

( ' 

### Contents offigures Fig. 1 Correlation between exposure period and bioconcentration factor (Levell) Fig. 2 Correlation between exposure period and bioconcentration factor (Level2) Fig. 

### 3 Concentration-mortality curve Fig. 4 Analytical chart and calibration curve ofHPLC analysis (analysis oftest water) Fig. 

### 5 Fig. 6 Fig. 7 Fig. 

### 8 Fig. 9 

### Fig.lO . Fig.ll Fig.l2 Fig.l3 Fig.14 Fig.l5-l Fig.I

### 5-2 Reference 2 Recovery and blanktest (analysis oftest water) Analysis oftestwater Recovery and blanktest (analysis oftest fish) Analysis oftestfish (Level 1) 

### Analysis oftest fish (Level2) Analysis oftest fish (Control) Concentration ofdissolved oxygen (Level 1) 

### Concentration ofdissolved oxygen (Level 2) Concentration ofdissolved oxygen (Control) UV

### spectrum oftest substance 

## IR spectrum of test substance measured before the initiation of exposure to fish 

## IR spectrum of test substance measured after the termination of exposure to fish 

## IR spectrum suppliedbythe sponsor
-21 -

### Table-3 Calculation table for recovery and blank test (Analysis oftest water) Sample description A B 

## c Standard 10mg!L 3457 Recovery a 2986 1 2 Recovery b 2935 1 2 Standard lOmg!L 3477 Blank a n.d. l 2 Blank b n.d. 1 2 ( a. b : indh-idual sample ) A : Peak height (flV) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis C : Final volume (mL) D :Amount ofblank in test water (J.tg) E : Amount oftest substance recovered (J!g) E= P x ( A(t) I A(std) ) I B x C - D

... F: Recovery rate(%) F=E/Q><lOO P : Concentration oftest substance in standard solution (mg/L) Q: Amount oftest substance added (20!lg) See Fig. 5 D



# -
-

-

-

January 26. I998 Name Test No 43112 E F 17.3 86.-1-% 17.0 "84.9% Average 85.6%



# -

-



# f7 

# I(15

**--r~**

### : \ 

### Table-4-1 Calculation table for analysis oftest water (Levell) Sample description A 

### I Standard lOmg!L 3227 Test water at 2nd day 2450 0.443 Standard 10mg!L 3156 Test \Vater at 7th day 2834 0.524 Standard lOmg!L 3180 Test water at 8th day 2568 0.471 Standard 10mg!L 3033 Test water at 13th day 2358 0.454 Standard 1 Omg!L 3160 Test water at 16th day 2346 0.433 Standard lOmg!L 3098 Test water at 20th day 2264 0.427 Standard 1OmgiL 

### 3559 Test water at 24th day 2815 0.462 Standard 1 Omg!L 3577 Test water at26th day 2837 0.463 A : Peak height (J.lV) 

### A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis l 

### C : Final volume 2mL F : Recovery rate 85.6% H: Volume oftest ·water taken out 40mL I: Concentration oftest substance in test water (mg!L) I 

## =P x ( 

### A(t) I 

### A(std) ) I 

### B x 

### C I 

### F x 

### 100 I 

### H J: Average concentration oftest substance in test water (mg!L)

J 

## = (I(I)+ ... + I(n)) In



### n : Number oftest water analysis I (l) : 

### First analysis oflest water I (n) : Last analysis oftest water P : Concentration ofstandard solution lOmg!L See Fig. 6 January 21. 1998 Test No 43ll2 

### J
0.484 0.473
0.459



# - 0.460

Name



## ~ 

# ·?pkcv



# ~1 

# .( 0

# b

### : \

( \"'--

- 

### Table-4-2 Calculation table for analysis oftest water (Levell) Sample description A 

### I Standard IOmg!L 3669 Test water at 29th day 2745 0.437 Standard 1OmgiL 3566 Test ·water at 33rd day 2734 0.448 Standard IOmg/L 2897 Test \Vater at 39th day 2700 0.544 Standard lOmg/L 2840 Test water at41st day 2323 0.478 Standard lOmgiL 3178 Test water at 44th day 2587 0.475 Standard IOmg!L 3229 Test water at48th day 2492 0.451 Standard IOmg!L 3300 Test water at 51st day 3014 0.533 Standard lOmgiL 3220 Test water at 55th day 2495 0.452 A : Peak height (J.LV) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis 1 C : Final volume 2mL F : Recovery rate 85.6% H: Volume oftest water taken out 40mL I: Concentration oftesi substance in test water (mg/L) I 

## =P x ( A(t) 

### IA(std))

### IB x C 

### IF x IOO/ H J: Average concentration oftest substance in test water (mg/L)

J 

## = ( I( I) 

### + ... + l(n) ) 

### In

n : Number oftest water analysis I (l): First analysis oftest water I (n) :Last analysis oftest water P : Concentration ofstandard solution lOrngiL See Fig. 6 January 27, 1998 Test No 43II2 

### J
0.456 0.465
0.465



# . 0.468

Name 

# J6

**~/.** 

# f1

## ( \

( \ 

### Table-5-1 Calculation table for analysis oftest water (Level2) Sample description 

### A I Standard 1 Omg!L 3227 Test water at 2nd day 2635 0.0477 Standard lOmg!L 3156 Test water at 7th day 2369 0.0438 Standard lOmg!L 3180 Test water at 8th day 2685 0.0493 Standard 10mg!L 3033 Test ·water at 13th day 2778 0.0535 Standard lOmg!L 3160 Test water at 16th day 2674 0.0494 Standard 1 Omg!L 3098 Test water at 20th day 2189 0.0413 Standard 1OmgiL 3559 Test water at 24th day 245-l 0.0403 Standard lOmg!L 3577 Test water at 26th day 2464 0.0402 A : Peak height (JLV) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis 1 C : Final volume 2rnL F : Recovery rate 85.6% H: Volume oftest water taken out 400mL I : Concentration oftest substance in test water (mg/L)

I 

## =P x ( A(t) 

### I A(std) )

### I B x C 

### IF x 100 

### I H 

### J : Average concentration oftest substance in test water (mg/L)

J 

## = ( I( l) 

### + ... + I(n) ) I n

n : Number oftest water analysis I (I) : First analysis oftest v.:ater I (n) : Last analysis oftest \Vater P : Concentration ofstandard solution lOmg/L See Fig. 6 January 2L 1998 TestNo 43112 J
0.0458 0.0486
0.0475



# - 0.0457



### ,, . ('j _/, 

### 12. - Name 

## X/.

**~k_** 

# (rfi( 

### \.



# (



# ( Table-5-2 Calculation table for analysis oftest water (Level2) Sample description A I Standard lOmg/L 3669 Test water at 29th day 2481 0.0395 Standard 1OmgiL 3566 Test water at 33rd day 2650 0.0434 Standard 1 Omg/L 2897 Test water at 39th day 2269 0.0457 Standard lOmg/L 2840 Test water at41st day 2276 0.0468 Standard 10mg/L 3178 Test water at 44th day 2112 0.0388 Standard lOmg/L 3229 Test \Vater at 48th day 2447 0.0442 Standard 1OmgiL 3300 Test water at 51st day 2711 0.0480 Standard 1OmgiL 3220 Test water at 55th day 2876 0.0521 A : Peak height (/lV) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis l C : Final volume 2mL F : Recovery rate 85.6% H: Volume oftest water taken out 400mL I :Concentration oftest substance in test water (mg/L) I 

## =p X ( A(t) 

### I A(std)) 

### I B X 

# c IF X 100 

### I H 1 : Average concentration oftest substance in test water (mg/L)



### J 

## = ( I( l) 

### + ... + l(n) ) 

### I n

n: Number oftest water analysis I (l) : First analysis oftest water I (n) : Last analysis oftest water P : Concentration ofstandard solution lOrngiL See Fig. 6 January 27, 1998 Test No 43112 

### J
0.0448
0.0451
0.0446



## - 0.0452

Name 

# JG

**~?1/** 

# Pt (1J9

### Table-6 Calculation table for recovery and blank test (Analysis oftest fish) Sample description A B 

# c Standard 1OmgiL 3310 Recovery a 2571 5/150 

### 5 Recoveryb 2485 5/150 

### 5 Standard 10mg!L 3404 Blank a 

### n.d. 5/150 5 Blank b n.d. 5/150 

### 5 ( a, b : indhidual sample ) A : Peak height (!1V) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis C : Final volume (mL) D : Amount ofblank in test fish (!lg) E : Amount oftest substance recovered (!lg) E= P x ( A(t) I A(std) ) I 

### B x C • 

### D F: Recovery rate(%) F=E/Qx 100 P : Concentration oftest substance in standard solution (mg/L) Q: Amount oftest substance added (1500J.tg) See Fig. 7 D



# -
-

.

.

January 26, 1998 Name Test No 43112 E F 1170 77.7% 1130 75.1% Average 76.4%



# -

-

-


# f1 //Di \ 

### Table-7 Calculation table for analysis oftest fish (Levell) Sample description A D G

Standard 10mg!L 3169 Test fish after 2 weeks a n.d. 1 25.3 Test fish after 2 weeks b n.d. 1 25.8 Standard 1OmgiL 

### 3619 Test fish after 4 weeks a n.d. 1 35.6 Test fish after4 weeks b n.d. 1 27.8 Standard 1OmgiL 

### 3235 Test fish after 6 weeks a n.d. 1 31.4 Test fish after 6 weeks b n.d. 1 32.5 Standard 1OmgiL 

### 3055 Test fish after 8 weeks a n.d. 1 37.9 Test fish after 8 weeks b n.d. 1 33.5 (a, b : individual sample )

A : Peak height (p.V) A(std) : Standard solution A(t) : Sample B : Ratio ofportion used for analysis 51150 C : Finai volume 5mL D : Dilution factor E :Average concentration ofblank in analysis ofcontrol OJlgfg 

### F : Recovery rate 76.4% G: Weight oftest fish (g) .. 

### K : Concentration of test substance in test fish (p.g/g) K 

## ={P x ( A(t) 

### IA(std))

### IB x D x C

### IG- E }

### IF x 100 H : Average concentration oftest substance in test water (mg!L) 

### J: BCF J 

## = KIH P : Concentration of test substance in standard solution 10mgiL 

### See Fig. 8 Test No. 43112 K H 

### J



# - 0.473 

# -
- 0.473 

# -
-· 0.460 

# -
- 0.460 

# -

- 0.465 

# -
- 0.465 

# -

- 0.468 

# -- 

# - 0.468 

# -

January 23, 1998 Name 

# JC; . 

# 1j«Ail/ 

# f1 (/(

# (

/ I 

### Table-8 Calculation table for analysis oftest fish (Level2) Sample description A D G

Standard 10mg!L 3182 Test fish after 2 weeks a n.d. 1 

### 27.5 Test fish after 2 weeks b n.d. 1 23.2 Standard 10mg!L 3600 Test fish after 4 weeks a n.d. 1 31.2 Test fish after 4 weeks b n.d. J, 

### 27.9 Standard 10mg!L 3220 Test fish after 6 weeks a n.d. 1 32.9 Test fish after 6 weeks b n.d. 1 34.1 Standard lOmg!L 3042 Test fish after 8 weeks a n.d. 1 

### 31.9 Test fish after 8 weeks b n.d. 1 

### 33.6 (a, b: individual sample)

A : Peak height (p.V) A(std) : Standard solution A(t) : Sample 8 : Ratio ofportion used for analysis 51150 C : Final volume SmL D :Dilution factor E : Average concentration ofblank in analysis ofcontrol Opg/g F : Recovery rate 76.4% G :Weightoftest fish (g) K : Concentration oftest substance in test fish (ug/g) K 

## ={P x ( 

### A(t) 

### IA(std))

### I 8 x 

### D x 

### C

### IG- E }

### IF x 

### 100 H :Average concentration of test substance in test water (mg!L) J: 8CF J 

## = Kl H P : Concentration of test substance in standard solution lOmg!L See Fig. 9 K



# -
-

-
-

-


# -

-
-

January 27, 1998 Name TestNo 4311? 

# - H 

### J 0.0486 

## - 0.0486 

# - 0.0457 

# - 0.0457 

# - 0.0451 

# - 0.0451 

## - 0.0452 

# - - 0.0452 

# -

-

# ( Ffable-9 Calculation table for analysis oftest fish (Control) Sample description A Standard lOmg!L 

### 3216 Before the initiation ofexposure a n.d. Before the initiation ofexposure b n.d. ' Standard lOmg!L 

### 3043 After the termination ofexposure a n.d. After the termination ofexposure b n.d. ( a, b : individual sample ) A : Peak height (Jl.V) A(std) : Standard solution A(t) : Sample 

### B : Ratio ofportion used for analysis 

### 51150 C : Final volume SmL 

### E : Amount ofblank in analysis ofcontrol (Jl.g) 

### E= P x ( A(t) 

### I A(std) ) 

### I B x C 

### G : Weight oftest fish (g) I : Concentration ofblank in test fish (uglg) l 

### = EIG 

### E



# -
-

-

-

P : Concentration oftest substance in standard solution 10mgiL See Fig. 10 January 23, 1998 Name Test No 4311? 

# - G I 

### 20.7 

# - 20.4 

# - 36.6 

# - 32.1 

# -



# F1 /13/ \

( I \ 

## Reference 1 Analytical results of dilution water Sampling date August 1. 

## 1997 Item Unit Measured value Standard value Detection limit Total hardness (Ca. 

## Mg) mg/L 121 < 

## 300* 1 

## Suspended solid mg/L < 

## 1. 0 < 

## 20* 3 

## 1 

## pH 

## - 7. 8 6. 5 - 8. 5* 2 

## Total organic carbon mg/L 2. 0 < 

## 2q 0. 1 

## Chemical oxygen demand mg/L < 

## 0. 5 < 

## 5* 2 

## 0.5 

## Residual chlorine mg/L < 

## 0. 01 < 

## 0. 02* 2 

## 0. 01 

## Ammonia nitrogen mg/L 0. 01 < 

## 1*2 0. 01 

## Total cyan mg/L < 

## 0. 01 

## n. d. • 2 

## 0. 01 

## Alkalinity mg/L 90.5 

## - Electric conductivity 11 

## s/ern 

## 360 - 

## Organic phosphorus mg/L < 

## 0. 1 

## n. d.· 2 

## 0.-1 

## Alkylmercury mg/L < 

## 0. 0005 

## n. d. • 2 

## 0.0005 

## Mercury mg/L < 

## 0.0005 < 

## 0. 0005. 2 0.0005 

## Cadmium mg/L < 

## 0. 005 < 

## 0. 01. 2 

## 0.005 

## Cr 6 • 

## mg/L < 

## 0. 02 < 

## 0. 05 * 2 

## 0.02 

## Lead mg/L < 

## 0.005 < 

# o. 1 *

2 

## 0.005 

## Arsenic mg/L 0. 002 <

# o. 05. 2 

## 0.002 

## [ron mg/L 0.06 < 

### t. 

# o· 2 0. 01 

## Copper mg/L < 

## 0.005 < 

## 0. 005. 2 0.005 

## *1 Ministerial ordinance of the Ministry of Public Welfare No. 69 (Revised December 21. 1992) 

## *2 Water quality criteria for fisheries (Shadanhozin Nihon Suisansigen Hogokyokai, March 1983) *3 OECD Guideline for Testing of Chemicals 210, 

## Fish, Early-life Stage Toxicity Test 

# fr 111 ·---r---·\

/ \ 

## ... 0 0 <5 

### Test No. 43112



### IO' Ll**--~Tnh~e~mwinnllimnJUJIDm•uc~awlc~u~la~tuiounulwimwwit~ouf~bwiowcxnwn~ce~nwt~ra~tuiounLfua~cutour** 

### ________**~-------** 

### 0 

## 2 

## 4 6 

## 8 Exposure period (weeks) Eight data at 2, 4, 6 

### and 8 

### weeks were lower than detection limit. 

### Correlation between exposure period and bioconcentration factor (Level 1)

The minimum calculation limit ofbioconcentration factor.



## 2 

## 4 6 8 Exposure period (weeks) Eight data at 2, 4, 6 

### and 8 

### weeks were lower than detection limit. 

### Fig. 2 Correlation between exposure period and bioconcentration factor (Level 2) February 3, 1998 ·Name 

# ((1 f15'I I \

I \ 

## 100 /"""'\



### ~ '--' 

### >. ......, __, ro 

### ......, 

## 50 r.... 0



### ~



## F 

## i g. 

## 3 

## T e s t 

## No. 4 3 1 1 2 

# 48hourLC50= >100

**•~**



### 10 100 

## C 

## o n c e n t 

## r a t 

## o n (mg/L) 

## Cumulative Mortality Concentratior. 

### Cumulative Mortality 

## (%) 

# C•IL> **r-------~--------~--------~** 

### 2 hours 24 hours 48 hours Control 

## 0 0



# 1 0 0 0 

## 0 Concentration-Mortality Curve 1000

Date : Nov.21,1997 

# Name:J(__J(_~ 

# p1 //k

### Solvent Operating date : Oct.

**2~** 1997 PKNO Time Area Height Cone. (min) (ttV·sec) (t.LV} (%) Total 0 0. 00 ( 

### \ ?eak position 

# lad- " 0. 0 2. 5 5.0 7. 5 10.0 Operator : Yuka Kida 

### Standard solution 5. 0 mg/L Operating date : Oct. 22, 1997 PKNO Time Area Height Cone. (min) (J.LV·sec) (J.LV) (%} I 3. 65 73576 1683 10<;>. 00 Total 73576 100.00 1



# ~ I 

# \?:

**~(** 

# : 0. 0 2. 5 5. 0 7. 5 10.0 Operator : Yuka Kida 

### Standard solution 10 mg/L Operating date : Oct.22.1997 PKNO Time Area Height Cone. (min) (ttV·sec) (J.LV) (%) 1 3. 65 145993 3266 100. 00 Total 145993 100.00 1



# ~ \ ( 0. 0 2. 5 5. 0 7. 5 10.0 Operator : Yuka Kida 

### Standard solution 20 mg/L Operating date : Oct. 22. 1997 1 PKNO Time Area Height Cone. (min) (J.LV·sec) (J.LV) (%) 1 3. 65 295293 6756 100. 00 Total 295293 100. 00 

# \I 0. 0 2. 5 5. 0 7. 5 10. 0 Operator : Yuka Kida 

### Fig. 4 - I 

### Analytical chart of HPLC analysis for calibration curve. Date : Oct. 22. 1997 Name : 

# (h //7 r( \



# (

/ 8000

4000

0 

### Conditions ofHPLC Instrument Sample Solvent Injection vol. Column

Size Eluent

Flow rate Detector Wave length Output Rec. range y

# =336 X r = 1.000 10 

### Concentration (mg!L)
Shimadzu LC-6A, SPD-6A V-05H Tetrahydrofuran lOO,uL Asahipak GS-310H 25cm x 7.6mm LD. 

# •

20

lOmmoi/L NaCI04 • H2

### 0 in tetrahydrofuran l.OmL!min. 

# uv 260nm O.SV/AU SmV Test No. 43112



### Fig. 4- 2 Calibration curve and conditions ofHPLC analysis for Y-05H ' January 28, 1998 Name 

# }0**·1f~~-** 

# t4**1~**( 

## \ Standard solution 10 mg/L Operating date : Oct. 29, 1997 PKNO Time Area Height Cone. (min) (t.tV·sec) (ttY) (%) 3. 65 144809 3457 100.00 Total 144809 100.00

0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Recovery test from test water a Operating date : Oct. 29. 1997 PKNO Time Area Height Cone. (min) (t.tV·sec) (ttY) (%) 1 3. 64 132185 2986 11. 94 2 6. 02 975148 17515 88.06 Total 1107334 100.00

0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Recovery test from test water b Op,erating date : Oct. 29. 1997 PKNO Time Area Height Cone. (min) (t.tV·sec) (t.tV) (%) 1 3. 64 132315 2935 13. 53 2 6. 01 845548 19035 86. 47 Total 977863 100. 00
0.0 2. 5 5. 0 7. 5 10. o(min) Operator Yuka Kida 

### Fig. 5 - I Recovery and blank test (analysis of test water) Date : Oct. 29, 1997 Name 

# Or 

# 111( \ 

### Standard solution 10 mg/L Operating date :

**Oc~ 2~**  1997 PKNO Time Area Height Cone. (min) (JJ.V·sec) (JJ.V) (%) 1 3. 64 144662 3477 100. 00 Total 144662 100. 00 I



# ~

**~** ( 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Blank test of test water a Operating date : Oct. 29, 1997 

# v PKNO Time Area Height Cone. (min) (JJ.V·sec) (JJ.V) (%) Total 0 0. 00 

# 'v PMi position 

# 1

**n.~** 0. 0 2.5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Blank test of test water b Operating date : Oct.29, 1997 

# v PKNO Time Area Height Cone. (min) (JJ.V·sec) (JJ.V) (%) Total 0 0. 00 

# v Peak position 

# 1M 0.0 2.5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 5-2 

### Recovery and blank test (analysis of test water) Date Oct. 29, 1997 Name

-T""lF"'__,...-lT,_I-----·--

## -r-- --

### Standard solution 10 mg/L Operating date : Nov. 28, 1997 PKNO Time Area Height Cone. (min) (/l.V·see) (/l.V) (%) 1 3. 65 141570 3227 100.00 Total 141570 100. 00 1



# ~ ,,-- 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 2nd day (Leve I I ) Operating date : Nov. 28, 1997 

### I PKNO Time Area Height Cone. (min) (/l.V·see) (/l.V) (%) 

# v 1 3. 65 117831 2450 10.40 2 6. 02 1015634 20080 89. 60 Total 1133466 100.00



# ~ **~** 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 2nd day (Leve I 

### 2 ) Operating date : Nov. 28. 1997 

# v PKNO Time Area Height Cone. (min) (/l.V·see) (/l.V) (%) 1 3. 64 114007 2635 8. 58 2 6. 03 1214822 21182 91. 42 Total 1328828 100.00 

# h)

**~** 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 6 - I Analysis of test water Date Nov. 28, 1997 Name

( 

# r?7



# I~ r

### Standard solution 10 mg/L Operating date : Dec. 3, 1997 PKNO Time Area Height Cone. (min) (tLV·sec) (tLV) (%) 3. 64 143004 3156 100.00 Total 143004 100. 00

0. 0 2. 5 5. 0 7. 5' 10. o!min) Operator : Yuka Kida 

### Test water at 7th day (Level I ) Operating date : Dec. 3, 1997 PKNO Time Area Height Cone. (min) ( tLV· sec) (tLV) (%) 1 3. 64 128030 2834 10. 26 2 6. 02 1120233 19358 89. 74 Total 1248263 100. 00
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 7th day (Level 2)

**Op~rating** date : Dec. 3, 1997 PKNO Time Area Height Cone. (min) (tLV·sec) (tLV) (%) 1 3. 64 108234 2369 7. 92 \ 

### ' 2 6.02 1258936 20156 92.08 Total 1367169 100. 00
0. 0 2. 5 5.0 7. 5 10. o!min) Operator Yuka Kida 

### Fig. 

### 6 - 2 Analysis of test water Date Dec. 3. 1997 Name 

# X; 

# 7:fvh&1r

( 

# ;

**·--,-T~**

### Standard solution 10 mg/L Operating date : Dec. 4, 1997 PKNO Time Area Height Cone. (min) (ttV·sec) (ttV) (%) I 3. 64 146744 3180 100.00 Total 146744 100. 00 I



# ~ - 

# f-- 0. 0 2.5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 8th day (Level l ) Operating date : Dec. 4, 1997 

# v PKNO Time Area Height Cone. (min) (tJ.V·sec) (tJ.V) (%) I 3. 63 120174 2568 10. 86 2 6. 01 986385 18524 89. 14 Total 1106559 100. 00 

# h)

**~** 11 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 8th day (Level 2 ) Operating date : Dec. 4, 1997 

# v PKNO Time Area Height Cone. (min) (tJ.V ·sec) (tLvr (%) I 3. 63 115034 2685 10. 21 2 6. 01 1011497 18085 89. 79 Total 1126531 100.00



# ~ L3 I 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 6-3 

### Analysis of lest water Date Dec. 4. 1997 Name 

# /)-)( 

### \



# ( 0. 0

0. 0

0.0 

### Fig. Standard solution 10 mg/L

2. 5 5. 0 

### Test water at 13th day (Level l )

2. 5 5. 0 

### Test water at 13th day (Level 2)

2. 5 5.0 

### 6 - 4 

### Analysis of 

### test water PKNO

7. 5 10. o(min) PKNO 1 2

7. 5 10. o(min) PKNO 1 2

7. 5 10. o(min) Date Jan. 27. 1998 Operating date : Dec. 9. 1997 Time Area Height Cone. (min) (p.V·sec) (p.V) (%) 3. 64 144074 3033 100. 00 Total 144074 100. 00
Operator : Yuka Kida Operating date : Dec. 9. 1997 Time Area Height Cone. (min) (p.V•sec) (p.V) (%) 3. 64 127481 2358 9. 47 6. 02 1218105 19960 90. 53 Total 1345586 100.00 Operator : Yuka Kida

**Op~rating** date : Dec. 9. 1997 Time Area Height Cone. (min) (p.V·sec) (p.V} (%) 3. 63 127429 2778 12. 60 6.02 883918 16906 87. 40 Total 1011347 100. 00 Operator Yuka Kida

Name 

# J0 

# 1fclttvi i" 

### Standard solution 10 mg/L PKNO I 1

# h~ 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Test water al 16th day CLeveI I ) \ PKNO 

# \1'1 I 2 

# w**~** ' 10. o(min) 0. 0 2. 5 5.0 7. 5 

### Tes l water at 16th day CLeve I 2 ) 

# v \ PKNO 1 2

# ~~**~** 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Fig. 6-5 

### Analysis of lest water Date Dec. 12, 1997**r··~~-·· ~~~···** **·~ ---·~** **~---• ··~** **~~-** Operating date : Dec. 12. 1997 Time Area Height Cone. (min)**(tLV·se~)** (tLV) (%) 3. 63 143749 3160 100. 00 Total 143749 100. 00
Operator : Yuka Kida Operating date : Dec. 12. 1997 Time Area Height Cone. (min) (tLV·sec) (tLV) (%) 3. 63 104912 2346 10. 55 6.00 889327 16125 89.45 Total 994238 100. 00 Operator : Yuka Kida Operating date : Dec. 12, 1997 Time Area Height Cone. (min) (tLV·sec) (tLV) (%) 3. 51 108453 2674 11. 71 6. 00 817464 14469 88. 29 Total 925918 100. 00 Operator : Yuka Kida

Name

# ;~1. 

# /)5
T

# (

i \

\ 0.0

0. 0

0.0 

### Fig. Standard solution tO mg/L

2. 5 5. 0 

### Test water at 20th day (Level l )

2. 5 5. 0 

### Test water at 20th day (Level 2 )

2. 5 5.0 

### 6 - 6 

### Analysis of test water PKNO

7. 5 10. o{min) PKNO I 2

7. 5 10. o(min) PKNO 1 2

7. 5 10. o(min) Date Dec. I6, 1997 Operating date : Dec. 16, 1997 Time Area (min) (IJ.V· sec) 3. 51 142180 Total 142180 Height {IJ.V)

3098
Operator : Yuka Kida Cone. (%) 100.00 100.00
Operating date : Dec. 16, I997 Time Area Height Cone. {min) {JJ.V·sec) {JJ.V) (%) 3. 61 I 15137 2264 6. 28 5. 96 1717224 55245 93. 72 Total 1832361 100.00 Operator : Yuka Kida Operating date : Dec. 16. 1997 Time Area Height Cone. (min) {JJ.V·sec) (JJ.V) {%) 3. 61 113389 2189 8. 19 5. 98 1271517 17968 91. 8I Total 1384906 100. 00 Operator Yuka Kida

Name 

# J6 

# 1fw&Ct./

,... -Tr

### Standard solution 10 mg/L PKNO

0. 0 2.5 5. 0 7.5 10. o(min) 

### Test water at 24th day (Level l ) PKNO 1 2

0. 0 2. 5 5. 0 7.5 10. o(min) 

### Test water at 24th day (Level 2 ) PKNO 1 2

0. 0 2..5 5. 0 7. 5 10. o(min) 

### Fig. 6 - 7 

### Analysis of test water Date Dec. 24. 1997

( **t~v.** -.wl I C.. Operating date : Dec. 24. 1997 Time Area (min) (.uV·sec) 3. 64 146007 Total 146007 Height Cone. (.uV) (%)

3559 100.00 100. 00 Operator : Yuka Kida Operating date : Dec. 24, 1997 Time Area Height Cone. (min) (.uV· sec) (.uV) (%) 3. 63 133499 2815 12.50 6. 03 934589 15719 87.50 Total 1068088 100. 00 Operator : Yuka Kida Operating date : Dec. 24. 1997 Time Area Height Cone. (min) (.uV·sec) (.uV) (%) 3. 62 112463 2454 5. 63 6. 01 1884706 51752 94.37 Total 1997170 100.00 Operator Yuka Kida

Name 

# 26.

**~tV** 

# /)7 I'i \

\ 0.0

0. 0

0. 0 

### Fig. Standard solution 10 mg/L

2.5 5. 0 

### Test water at 26th day (Level I )

2.5 5. 0 

### Test water at 26th day (Level 2)

2. 5 5. 0 

### 6 - 8 Analysis 0

### f 

### test water PKNO

7. 5 10. o(min) PKNO 1 2

7. 5 10. o(min) PKNO 1 2

7. 5 10. o(min) Date Dec.24.1997 Operating date : Dec. 24. 1997 Time Area Height Cone. (min) (p.V ·sec) (tLV) (%) 3. 64 150547 3577 100. 00 Total 150547 100.00
Operator : Yuka Kida Operating date : Dec. 24. 1997 Time Area Height Cone. (min) (p.V·sec) (p.V) (%) 3. 63 140173 2837 12.21 6. 02 1007894 15759 87. 79 Total 1148068 100.00 Operator : Yuka Kida Operating date : Dec. 24. 1997 Time Area Height Cone. (min) (p.V·sec) (p.V} (%) 3. 62 115472 2464 8. 97 6.02 1172136 18481 91.03 Total 1287608 100. 00 Operator Yuka Kida

Name 

# /6,

**~/**'''-"• ......... , 14 

### Standard solution 10 mg/L Operating date : Dec. 26, 1997 PKNO Time Area Height Cone. (min) (J.LV· sec) (J.LV) (%) 3. 64 154609 3669 100.00 Total 154609 100. 00 

# (
0.0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 29th day (Level I ) Operating date : Dec. 26. 1997 PKNO Time Area Height Cone. (min) (p.V·sec) (p.V) (%) I 3. 62 134928 2745 6.85 2 6. 01 1833528 53354 93. 15 Total 1968456 100.00
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 29th day (Level 2 ) Operating date : Dec. 26. 1997 PKNO Time Area Height Cone. (min) (J.LV·sec) (p.Vt (%) 

## ( I 3. 63 124289 2481 6. 19 2 6. 01 1883837 53549 93.81 Total 2008127 100. 00
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator Yuka Kida 

### Fig. 6 - 9 

### Analysis or test water Date Dec. 26. 1997 Name



# !'/.~

**I"·~···~~-····~---····-~~~ ~~----··**  

### ,- i 

## rr( \. 

### Standard solution 10 mg/L PKNO I I



# ~

**~** 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Test water at 33rd day (Level l ) 

# v PKNO I 2 1



# ~ 0. 0 2. 5 5. 0 7. 5 10. o{min) 

### Test water at 33rd day (Leve I 

### 2 ) PKNO 1 2 1 0.0 2.5 5. 0 7. 5 10. o{min) 

### Fig. 5 -10 Analysis of test water Date Dec. 29, 1997 Operating date :

**De~ 2~**  1997 Time Area Height Cone. (min) (ttV·sec) (ttV) (%) 3.64 153058 3566 100. 00 Total 153058 100. 00 Operator : Yuka Kida Operating date : Dec. 29, 1997 Time Area Height Cone. (min) (ttV·sec) {ttV) (%) 3. 63 125594 2734 6. 92 6. 01 1689912 49161 93. 08 Total 1815506 100. 00 Operator : Yuka Kida

**Op~rating** date : Dec. 29. 1997 Time Area Height Cone. (min) (ttV·sec) {tLV) (%) 3. 62 123274 2650 5. 59 6. 01 2082429 53542 94. 41 Total 2205703 100. 00 Operator : Yuka Kida

Name 

# J3ra

### Standard solution 10 mg/L Operating date : Jan. 4, 1998 PKNO Time Area Height Cone. (min) ( t.LV·sec) (uV) (%) I 3.62 156979 2897 100. 00 Total 156979 100. 00 1



# ~ J 

# v . 10. o(min) Operator : Yuka Kida 0. 0 2. 5 5. 0 7. 5 

### Test water at 39th day (Level 1 ) Operating date : Jan. 4. 1998 PKNO Time Area Height Cone. (min) (uV·sec) (uVl (%) I 3.60 147272 2700 6.60 2 5. 96 2085439 58955 93. 40 Total 2232711 100.00 I



# ~ 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test water at 39th day (LeveI 2 ) Operating date : Jan. 4. 1998 PKNO Time Area Height Cone. (min) (uV·sec) (uVJ (%) I 3.60 118367 2269 4. 68 2 5. 96 2408287 54860 95.32 Total 2526654 100.00 1



# ~ v 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 6 -11 Analysis 

# or test water Date Jan. 4, 1998 Name



# ~1 

# /)/

I 

### 'Ill.

## (

( \ 

### Standard solution 10 mg/L PKNO I I



# ~ 

# ~Q ' 

# c.. 

# =: 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Test water at 41st day (Level I ) 

# \ PKNO I 2 I



# ~ I 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Test water at 41st day (Level 2 )\] PKNO I 2 1



# ~ 0. 0 2.5 5. 0 7. 5 10. o(min) 

### Fig. 6-12 Analysis of test water Date Jan. 6. 1998 t'C.:;,\. HV .. 6tVII&. Operating date : Jan. 6. 1998 Time Area Height Cone. (min) (.uV·see) (,uV) (%) 3. 62 157951 2840 100.00 Total 15795.1 100.00 Operator : Yuka Kida Operating date : Jan. 6. 1998 Time Area Height Cone. (min) (.uV·see) (.uV) (%) 3. 61 131561 2323 7. 05 5. 96 1733559 52604 92. 95 Total 1865120 100. 00 Operator : Yuka Kida Operating date : Jan. 6. 1998 Time Area Height Cone. (min) (.uV·see) (.uV) (%) 3. 61 131096 2276 6. 10 5.96 2019023 54202 93. 90 Total 2150120 100. 00 Operator : Yuka Kida

Name



# !3~/ \ 

### Standard solution 10 mg/L
1



# ~ 0. 0 2. 5 5. 0 

### Test water at 44th day (Level l )



# ~ 0. 0 2. 5 5. 0 

### Test water at 44th day (Level 2 )
1
0. 0 2. 5 5. 0 

### Fig. 6 -13 Analysis of test water PKNO 1

7. 5 10. o(min) PKNO 1 2



# ~ 7. 5 10. o(min) PKNO 1 2



# ~ 7. 5 10. o(min) Date Jan. 9. 1998 Operating date : Jan. 9. 1998 Time Area Height Cone. (min) (t.LV·secl (t.LV) (%) 3. 63 130726 3178 100.00 Total 130726 100. 00
Operator : Yuka Kida Operating date : Jan. 9, 1998 Time Area Height Cone. (min) (t.LV·see) (t.LV) (%) 3. 63 133432 2587 7. 12 5. 97 1739760 50230 92. 88 Total 1873192 100.00 Operator : Yuka Kida Operating date : Jan. 9. 1998 Time Area Height Cone. (min) (t.LV·see) (t.LV) (%) 3. 62 120648 2112 5. 70 5. 97 1995270 55649 94.30 Total 2115917 100. 00 Operator : Yuka Kida

Name 

# /1}( \ 

### Standard solution 10 mg/L PKNO 1 1

# t?:~ : ;= 

# c- I 0. 0 2. 5 5.0 7. 5 10. o(min) 

### Test water at 48th day (Level I )

## ~ PKNO 1 2 1 1

# ~ 0. 0 2. 5 5. 0 7.5 10. o(min) 

### Test water at 48th day (LeveI 2 )

# \ PKNO 1 2

# ~**\~** 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Fig. 6 -14 Analysis of test water Date Jan. 13, 1998 Operating date : Jan. 13. 1998 Time Area Height Cone. (min) (tLV·see) (tJ.V) (%) 3. 62 146080 3229 100. 00 Total 146080 100. 00
Operator : Yuka Kida Operating date : Jan. 13. 1998 Time Area Height Cone. (min) (tJ.V·see) (tJ.V) (%) 3. 61 128316 2492 6. 71 5. 97 1782742 54044 93. 29 Total 1911058 100. 00 Operator : Yuka Kida Operating date : Jan. 13, 1998 Time Area Height Cone. (min) (tJ.V·see) (tJ.V) (%) 3. 61 115673 2447 5. 83 5.97 1868253 53133 94. 17 Total 1983927 100. 00 Operator : Yuka Kida

Name 

# /3Y

### Standard solution 10 mg/L Operating date : Jan. 16. 1998 PKNO Time Area Height Cone. (min) (tLV·see) (tLV) (%) 1 3. 62 144170 3300 100.00 Total 144170 100.00 1



# ~

**~** 0. 0 2. 5 5. 0 7. 5 10. 0(min) Operator : Yuka Kida 

### Test water at 51st day (Level l ) Operating date : Jan. 16. 1998 

### "'\) PKNO Time Area Height Cone. (min) (tLV·see) (tLV) (%) 1 3. 60 121842 3014 9. 78 2 5. 96 1124603 20346 90. 22



# ~ **~** Total 1246445 100.00



# - 0. 0 2. 5 5. 0 7. 5 10. o!min) Operator : Yuka Kida 

### Test water at 51st day (Level 2 ) Operating date : Jan. 16. 1 998 PKNO Time Area Height Cone. (min) (tLV·see) (.uV) (%) 1 3. 60 123895 2711 7. 85 2 5. 97 1454769 22387 92. 15



# ~ Total 1578663 100. 00
0. 0 2. 5 5.0 7. 5 10. o!min) Operator : Yuka Kida 

### Fig. 6 -15 Analysis of test water Date Jan. 16. 1998 Name


# ."

- 

# (35(

( 

### \.



# ( Standard solution 10 mg/L
1



# ~ __.}--- 0.0

**2~** 5 5.0 

### Test water at 55th day (Level l )

**~**
1 __)

. 0. 0 2. 5 5. 0 

### Test water at 55th day (Level 2 ) 

# v 1 ) 0. 0 2. 5 5.0 

### Fig. 6 -16 Analysis of test water PKNO A 1



# \ 7..5 10. o(min) PKNO 1 2



# ~ 7. 5 10. o(min) PKNO 1 2



# ~ 7. 5 10. o(min) Date Jan. 20. 1998 Operating date : Jan. 20, 1998 Time Area Height Cone. (min) (p.V ·sec) (.uV) (%) 3. 61 146036 3220 100.00 Total 146036 100.00
Operator : Yuka Kida Operating date : Jan. 20. 1998 Time Area Height Cone. {min) {p.V·sec) (.uV) (%) 3. 60 127197 2495 6. 41 5. 95 1857047 53123 93.59 Total 1984244 100.00 Operator : Yuka Kida

**Op~rating** date : Jan. 20. 1998 Time Area Height Cone. {min) {p.V· sec) (p.Vj (%) 3. 60 121077 2876 6. 05 5. 96 1879872 54306 93. 95 Total 2000950 100. 00 Operator : Yuka Kida

Name 

# /?6

### Standard solution 10 mg/L Operating date : Nov. 6. 1997 PKNO Time Area Height Cone. (min) (J.LV·see) (t.LV) (%) 3. 64 142834 3310 100. 00 Total 142834. 100. 00 ' I \ 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Recovery test from test fish a Operating date : Nov. 6, 1997 PKNO Time Area Height Cone. (min) (J.LV·sec) (t.LV) (%) 1 3. 64 100297 2571 4. 01 2 5. 35 2398622 106240 95. 99 Total 2498919 100.00
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Recovery lest from test fish b Operating date : Nov. 6, 1997 PKNO Time Area Height Cone. (min) (J.LV·see) (tLVt (%) 1 3. 64 100565 2485 5. 10 2 5. 35 1869761 74968 94.90 Total 1970326 100. 00
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator Yuka Kida 

### Fig. 7 - I 

### Recovery and blank test (analysis of test fish) Date : Nov. 6, 1997 Name

·.\.._ 

# f1

### Standard solution 10 mg/L Operating date : Nov. 6, 1997 PKNO Time Area Height Cone. (min) (t!V·see) (tLV) (%) 3. 64 142228 3404 100. 00 Total 142228 100. 00 ( \
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Blank test of test fish a Operating date : Nov. 6, 1997 PKNO Time Area Height Cone. (min) (tLV·see) (tLV) (%) Total 0 0. 00 

### Peak. position

# l~d0. 0 2.5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### B Iank test of test fish b**Op~rating** date : Nov. 6, 1997 PKNO Time Area Height Cone. (min) (tLV·sec) (tLV) (%) 

## ( Total 0 0. 00 

### Peak position
0. 0 2. 5 5. 0 7. 5 10. o(min) Operator Yuka Kida 

### Fig. 7-2 

### Recovery and blank test (analysis of test fish) Date : Nov. 6, 1997 Name

### (



## ( Standard solution 10 mg/L

0.0 2. 5 5. 0 7. 5 10.0 

### Test fish after 2 weeks (Level 1-a) Peak. position



# l~ 0. 0 2. 5 5. 0 7. 5 10.0 

### Test fish after 2 

### weeks (Level !-b) 

### Peak position



# ta~ 0.0 2. 5 5. 0 7. 5 10. 0 

### Fig. 8 - I Analysis of test fish (Level I ) Operating date : Dec. 11, 1997 PKNO Time Area (JLV ·sec) 141045 Height Cone. (min) 3. 62 Total 141045
Operator : Yuka Kida (JLV) {%) 3169 100. 00 100. 00

Operating date : Dec. 11. 1997 PKNO Time Area Height Cone. (min) (JLV·sec) (.uV) (%) Total 0 0. 00
Operator : Yuka Kida Operating date : Dec. 11. I997 PKNO Time Area Height Cone. (min) (tLV·sec) (.uV1 (%) Total 0 0. 00
Operator Yuka Kida Date : Dec. 11, 1997 Name 

# 13

# r

### Standard solution 10 mg/L Operating date : Dec. 25. 1997 PKNO Time Area Height Cone. (min) (.u.V·sec) (.u.V) (%) 1 3. 64 151972 3619 100. 00 1 Total 151972 100. 00



# ~ - ' 10. o(min) Operator : Yuka Kida 0. 0 2. 5 5. 0 7. 5 

### Test fish after 4 

### weeks CLeve I 

### 1-a) Operating date : Dec. 25. 1997 PKNO Time Area Height Cone. (min) (.u.V·sec) (.u.V) (%) Total 0 0. 00 

### Peak position 

# ln.d. 

# f'--J\
0. 0 2. 5 5.0 7. 5 10. o(min) Operator : Yuka Kida 

### Test fish after 4 

### weeks CLeve I 1-b) Operating date : Dec. 25. 1997 (



# ~ **~** PKNO Time Area Height Cone. (min) (.u.V·sec) (.u.V) (%) Total 0 0. 00 

### Peak position 

# 1

•.

**~** 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 8 - 2 Analysis of test fish CLevel I ) Date : Dec. 25. 1997 Name



# r0. 0

0. 0

0.0 

### Standard solution !0 mg/L

2. 5 5. 0 

### Test fish after 6 

### weeks (Level 1-a)
Peak 

### position 

# 1

n.d. 2. 5 5. 0 

### Test fish after 6 

### weeks (Level !-b)


### Peak position 7. 5

7. 5 nv. ""t\oJI IL. Operating date : Jan. 8. 1998 PKNO Time Area Height Cone. (min) {.uV·see) (t.tV) (%) 3. 62 136345 3235 100.00 Total 136345 100. 00
IO.o(min) Operator : Yuka Kida Operating date : Jan. 8. 1998 PKNO Time Area Height Cone. (min) (t.tV·see) (t.LV) (%) Total 0 0. 00
IO.o(min) Operator : Yuka Kida Operating date : Jan. 8. 1998 PKNO Time Area Height Cone. (min) (uV·see) (t.tVT (%) Total 0 0. 00

2. 5 5. 0 7. 5 10. o(min) Operator Yuka Kida 

### Fig. 8-3 Analysis of test fish (Level 1 ) Date : Jan. 8. 1998 Name 

# /f{(

### Standard solution \0 mg/L Operating date : Jan. 22, 1998 PKNO Time Area Height Cone. (min) (ttV·see) (ttV) {%) I 3. 66 147124 3055 100.00 I Total 147124 100.00



# ~ \_ 0. 0 2. 5 5. 0 7. 5 10. o!min) Operator : Yuka Kida 

### Test fish after 

### 8 weeks (Leve I 

### 1-a) Operating date : Jan. 22. 1998 PKNO Time Area Height Cone. (min) (ttV·secl (ttV) (%) Total 0 0.00 

### Peak position 

# l

•.

**~** I 0. 0 2. 5 5. 0 7. 5 10. o!mih) Operator : Yuka Kida 

### Test fish after 

### 8 weeks (Leve I 

### 1-b) OP.erating date : Jan. 22. 1998 PKNO Time Area Height Cone. (min) (f.LV•see) (t.LV) (%) 

# ( Total 0 0. 00 

### Peak position 

# 1 n.d. ) 

# I 0. 0 2. 5 5. 0 7. 5 10. o!min) Operator : Yuka Kida 

### Fig. 8-4 Analysis of test fish (Level I ) Date : Jan. 22, 1998 Name

. ' 1---"'1[ 

## ·-;-rr-

### Standard solution 10 mg/L Operating date : Dec. 11. 1997 PKNO Time Area Height Cone. (min) (t.LV·sec) (t.LV) (%) 1 3. 62 140595 3182 100. 00 Total 140595 100.00 1



# ~ \. \



# - 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test fish after 2 

### weeks (Leve I 

### 2-a) Operating date : Dec. 11, 1997



# ~ PKNO Time Area Height Cone. (min) (t.LV·sec) (t.LV) (%) Total 0 0.00 

### Peak uosition



# ~~ '



# 1~~ 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test fish after 2 

### weeks (Level 2-b) Operating date : Dec. 11, 1997 PKNO Time Area Height Cone. (min) (t.LV·secl (t.LV) (%) 

## ( Total 0 0.00 

### Peak position



# l~~



# ~ 0. 0 2. 5 5. 0. 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 9 - I · Analysis of test fish (Level 2) Date : Dec. 11. 1997 Name

### Standard solution 10 mg/L Operating date : Dec. 25, 1997 PKNO Time Area Height Cone. (min) (JLV ·sec) (JLV) (%) 1 3. 64 158534 3600 100. 00 Total 158534 100. 00 1



# ~ - (
0. 0 2. 5 5. 0 7.5 10. o(min) Operator : Yuka Kida 

### Test fish after 4 weeks CLeveI 2-a) Operating date : Dec. 25, 1997 PKNO Time Area Height Cone. (min) (JLV·sec) (JLV) (%) Total 0 0. 00 

### Peak position 

# 1

n.d.



# ~ 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Test fish after 4 weeks (LeveI 2-b) Operating date : Dec. 25. 1997 

# \ PKNO Time Area Height Cone. (min) (JLV ·sec) (tM (%) 

## ( Total 0 0.00 

### Peak position 

# 1

n.d.

**~** 0. 0 2. 5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

### Fig. 9 - 2 

### Analysis of test fish (Level 2) Date : Dec. 25. 1997 Name



### lT I.i \ 

### Standard solution 10 mg/L PKNO 1 1



# ~ **~** 

# :-

**~** 0. 0 2. 5 5.0 7.5 10. o(min) 

### Test fish after 6 

### weeks (Leve 1 

### 2-a) - PKNO 

### Peak. position 

# l

n.d. 

# f'--..---. 0. 0 2. 5 5. 0 7. 5 10. o(min) 

### Test fish after 6 

### weeks (Leve 1 

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10. o(min) Operator : Yuka Kida Operating date : Jan. 22. 1998 PKNO Time Area Height Cone. (min) (ttV·see) (ttV) (%) Total 0 0. 00

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**Ja~ 2~**  1998 Name



### ·-;-,- 

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### Standard solution 10 mg/L Operating date : Nov. 26, 1997 PKNO Time Area Height Cone. (min) (JLV· sec) (JLV) (%) 1 3. 63 145789 3216 100.00 Total 145789 100.00 1



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### Fig.I0-2 Analysis of test fish ( Control Date : Jan. 23. 1998 Operating date : Jan. 23. 1998 Time Area (min) (t.tV·sec) 3. 65 151819 Total 151819 Height (tLVl

3043
Operator : Yuka Kida Cone. (%) 100. 00 100. 00

Operating date : Jan. 23. 1998 Time Area Height Cone. (min) (uV·sec) (uVl (%) Total 0 0. 00
Operator : Yuka Kida Operating date : Jan. 23. 1998 Time Area Height Cone. (min) (t.LV ·sec) (t.LV} (%) Total 0 0. 00
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# Amendment and addition to Final Report

1. Title 2. Testnumber 3. Content 4. Reason Kurume Research Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan Bioaccumulationtest ofV-05H in carp 43112 

# CD 10.2 Chemical structure, etc. molecularweight (Page 4) 

# ® 10.2 Chemical structure, etc. polymerization degree (Page 4) 

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## m~MJ~?\fn*rncf:l:9=!:arr19~:t-&14

### Receipt No. T96-2575

**R~ort** 

### No. T-4743 STUDY CODE: K01-1861 

## FINAL REPORT BACTERIAL REVERSE MUTATION TEST OF 

### V-OSH

January 1997 Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute Japan 

# f1 /)C

# ( STATEMENT I, the undersigned, hereby declare that this report provides a correct English translation ofthe final report (Study Code K01-1861, issued on January 28, 1997). Chemicals Inspection & 

### Testing Institute, Japan (CITI) formally changed its name to Chemicals Evaluation and Research Institute, Japan (CERI) as ofNovember 1, 1999. The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to "NISSHINBO INDUSTRIES, INC. R & 

### D CENTER" and "1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan", respectively by Sponsor's request. CAS No. 157299-02-0 oftest substance was added by Sponsor's request. In the structural formula, n=180 was changed to n=2-800. Molecular weight of3,000 was changed to 30,000.



# J~ **!~,** 2002 Shozo Ogura HitaLaboratory Chemicals Evaluation and Research Institute, Japan Date

TC

# ( QUALITY ASSURANCE STATEMENT Hita Laboratory, Chemicals Evaluation and Research Institute, Japan Sponsor: NISSIDNBO INDUSTRIES, INC. R&D CENTER Title: Bacterial Reverse Mutation Test of V-OSH Study code: KO 1-1861
This report was audited by the Quality Assurance Section. I, the undersigned, hereby declare that this report reflects the original Japanese report.

Section Chief, Quality Assurance (Date) 

# J

**~vacr** 1g-, ..2. f:J()

## 2. (Signature) 

# ~ii!:Ei>:._

# • Keiji Shiraishi, B.S.

# (

( 

### \ Sponsor: Title: GLP STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Bacterial Reverse Mutation Test ofV-05H **_l(,.Ul-l~bl**



### Study Code: KOl-1861 I, the undersigned, hereby dec_lare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's Ministry of Labor, No.76, September 1, 1988), and "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 ofthe Planning and Coordination Bureau, Environment Agency (EA), No. 229 ofthe Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) & 

### No. 85 (1984) ofthe BasicJndustries Bureau, Ministry ofInternational Trade and Industry (MIT!), March 31, 1984; Notification No. 233 ofthe Planning and Coordination Bureau, EA, No. 38 ofthe Environmental Health Bureau, MHW & 

### No. 823 (1988) oftheBasic Industries Bureau, MITI, revised on November 18, 1988). Management: Signed in original January 28, 1997 Shigetaka Yamane, Ph.D.

### ( \ K.Ul-lH6l 

### QUALITYASSURANCE STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan Sponsor: NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Title: Bacterial Reverse Mutation Test ofV-05H Study Code: K01-1861 This study was audited and inspected by Quality Assurance Section ofHita Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & 

### Testing Institute, Japan. The dates audited and/or inspected and the dates reported these results to the study director and management are as follows. Dates of Dates ofReports to Dates ofReports to Inspections/ Audits Study Director Management November 26, 1996 November 29, 1996 December 3, 1996 December 3, 1996 December 3, 1996 December 3, 1996 January 27, 1997 January 28, 1997 January 28, 1997 January 28, 1997 January 28, 1997 January 28, 1997 I, the undersigned, hereby declare that this report provides an accurate description ofthe methods and procedures used in this study and that the reported results accurately reflect the raw data obtained. Section Chief, Quality Assurance: Signed in original January 28, 1997 Keiji Shiraishi, B.S. 

# f1 /to

# (

( 

### \. 

### TITLE Bacterial Reverse Mutation Test ofV-05H SPONSOR NISSHINBO INDUSTRIES, INC. R & 

### D CENTER 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan TESTING FACILITY **.li....Ul-l~bl** 

### Study Code: K01-1861 Test Substance Code: HR3392 Sponsor Code: N-680

Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877, Japan PURPOSE OF STUDY The mutagenic potential of the test substance is examined using Salmonella typhimurium 

### and Escherichia coli. 

### TESTING METHOD This study was conducted in accordance with "Standards for Toxicity Investigations" (Japan's Ministry ofLabor, No.77, September 1, 1988), and "Notification on Partial Revision ofTesting Methods Relating to the New Chemical Substances (Notification No. 700 ofthe Planning and Coordination Bureau, Environment Agency (EA), No. 1

039 ofthe Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare (MHW) & 

### No. 1014 (1986) ofthe Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986)". GLP COMPLIANCE This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's Ministry of Labor, No.76, September 1, 1988), and ••concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 ofthe Planning and Coordination Bureau, EA, No. 229 of the Pharmaceutical Affairs Bureau, MHW & 

### No. 85 (1984) of the Basic Industries Bureau, MIT!, March 31, 1984; Notification No. 233 of the Planning and 

# ?1 (£.(/ 

### \



# (

( \ 

### KOI-1861 Coordination Bureau, EA, No. 38 ofthe Environmental Health Bureau, MHW & 

### No. 823 (1988) ofthe Basic Industries Bureau, MITI, revised on November 18, 1988). PERIOD OF STUDY Commencement ofStudy: November 27, 1996. Commencement ofa Dose-Range-Finding Test: November 28, 1996 Completion ofObservation: Completion ofStudy: December 5, 1996 January 28, 1997 LOCATION AND PERIOD FOR RETENTION OF DATA Data and the remaining test substance will be retained in the archives and test substance storage room, respectively, of Hita Research Laboratories for I0 years following the date of the notification specified under Item 1 of Article 57-2 of Industrial Safety & 

### Health Law. Date of the notification will be communicated from the Sponsor to Hita Research Laboratories. After termination ofthe retention period, any measures taken will be done so with the approval ofthe Sponsor. AUTHOR AND PERSONNEL CONCERNED WITH STUDY

AUTHOR

Study Director: Signed in original Tsunehiko Inai, B.S. Mutagenicity Section Hita Research Laboratories PERSONNEL CONCERNED WITH STUDY

Study Staff: Shozo Ogura Person in charge ofStorage: Shizuka Kouda January28, 1997

### ( \ 

### K01-1861 TABLE OF CONTENTS 

### SUMMARY 1 MATERIALS AND METHODS I. TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 2 2. BACTERIAL STRAINS 3. MEDIUM AND S9 MIX 4. PRE-CULTURES OF TEST STRAINS 5. PREPARATION OF TEST SUBSTANCE AND POSITIVE 4 

### 5 

### 6 CONTROL SUBSTANCES ···················································· 6 6. METHODS 7 

### 7. OBSERVATION AND MEASUREMENT ···························· 8 8. JUDGEMENT CRITERIA OF TEST RESULTS ······················· 8 UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 8 RESULTS 9 

### CONCLUSION 9 

### REFERENCES 9 

### TEST RESULT TABLES AND DOSE-RESPONSE CURVES....... 1-6 

# (/1 

# /£)

# (

( \ 

### KOl-1861
SUMMARY The mutagenicity of V-05H was examined in 

### Salmonella typhimurium strains TAtOO, TA1535, TA98 and TA1537 and 

### Escherichia coli strain WP2 

### uvrA using a preincubation method with and without metabolic activation system (S9 mix). The results showed that the number ofthe revertant colonies was less than two times that ofnegative control in all ofthe test strains with and without S9 mix. The number ofrevertant colonies in the negative and positive controls were confirmed to be within a range ofthe historical data in our laboratory. Based on the above results, V-05H was considered to have no reverse mutagenic potential under the condition the present study.



# f1 /£i -1-

# f;/~5

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## I Partition coefficient: - Solubility: Soluble in oil Degree ofsolubility: Water: <5 w/v% (measured in our laboratory) DMSO: <5 w/v% (measured in our laboratory) Acetone: 

## > 10 w/v% (measured in our laboratory) Others: 8) Storage conditions Stored within a desiccator in a cold and dark place. 9) Precautions KOl-1861

Gloves, a mask, a head cap and a lab coat were worn. Hand-washing and gargling were carried out at the end ofhandling. 1.2 Positive Control Substances 1) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) Manufacturer: Lot No.: Properties: Purity: Grade: 2) Sodium azide (NaN3) Manufacturer: Lot No.: Properties: Purity: Wako Pure Chemical Industries, Ltd. LEN0571 Reddish-:-orange crystalline powder 99.5% Special grade Wako Pure Chemical Industries, Ltd. DLP2438 White crystal 99.4% Grade: Special grade 3) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine · 2HCI (ICR-191) Manufacturer: Polysciences, Inc. Lot No.: 412795 Properties: Yellow crystalline powder Purity: Grade: 4) 2-Aminoanthracene (2AA) Manufacturer: LotNo.: Properties: Purity: Grade: Wako Pure Chemical Industries, Ltd. DLR7869 Yellowish-green-brown powder 95.7% 

## -3-

i I( \ 

### 5) Storage conditions Stored in a cold and dark place. 6) Precautions Gloves, a mask, a head cap and a lab coat were worn. 2. BACTERIAL STRAINS 2.1 Kinds ofBacterial Strains K01-1861



### Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 were obtained from Dr. B. N. Ames, University ofCalifornia, on June 20, 1990. 

### Escherichia coli strain WP2 

### uvrA was obtained from Japan Bioassay Laboratoy on April6, 1995. S. 

### typhimurium strains TAlOO, 

### TA1535, and 

### E. coli strain WP2 

### uvrA were used for detection ofbase-pair substitution mutagens, and S. 

### typhimurium strains TA98 and T A1537 for the detection offrameshift mutagens. 2.2 Storage After 0.045 ml ofdimethyl sulfoxide for spectrophotometric grade (DMSO; Lot No.CF103, Purity >99%, DOJINDO Laboratories) was added to 0.5 ml of the bacterial suspension, the mixture was stored at -80oC in an ultra-deep freezer (MDF-291, SANYO Electric Co., Ltd.) until use. 2.3 Characteristics ofStrains and Date ofCharacterization 1) Characteristics ofstrains Mutations in amino acid Mutations in Mutation in R-factor Strains excision- membrane (pKM101) synthesis genes repair genes 

### Salmonella typhimurium TA1535 

### hisG46 l::::.uvrB rfa TA1537 

### hisC3076 l::::.uvrB rfa TA98 

### hisD3052 !::::.uvrB rfa 

## + TAIOO 

### hisG46 l::::.uvrB rfa 

## + Escherichia coli WP2 

### uvrA trp l::::.uvrA 

## + The amino acid requirement was determined as histidine for 

### S. typhimurium strains and tryptophan for E. 

### coli strain. The presence or absence ofR-factor was determined by ampicillin resistance, and mutations in membrane and DNA repair were determined by sensitivity to crystal violet, and UV, respectively. 

# (fl**1~7** -4-( \

**(~** \ 

### K01-1861 2) Date ofcharacterization Strains Date ofcharacterization Salmonella typhimurium 

### TA1535 July 18, 1996 TA1537 June 7, 1996 TA98 June 7, 1996 TA100 October 18, 1996 Escherichia coli 

### WP2 uvrA 

### April 18, 1996 3. MEDIUM AND S9 MIX 3.1 Medium

3.2 1) Minimal glucose agar plate (prepared in our laboratory)

2) 1) 

### An aliquot of 30 ml of medium consisting of the following components was poured into a plastic dish. Components Amount contained in one liter 20xVogel-Bonner Minimum medium E 40 w/v% Glucose 50 ml 50ml 15 g Agar (1) 

### Agar: Bacto-Agar (Lot No. 90800JA, Difco Laboratories) (2) Date ofmanufacture: Dose-range-finding test: November 8, 1996 Main test: November 25, 1996 Soft agar A solution containing 0.5 mM histidine and 0.5 mM biotin for Salmonella typhimurium 

### strains or 0.5 mM tryptophan for E. coli 

### strain was added to a soft agar solution containing 0.6 w/v% agar (Bacto-Agar, Lot No. 71892AJB, Difco Laboratories) and 0.5 w/v% NaCl at a ratio of 1 : 10. S9mix Rat liver S9 (Kikkoman Co., Ltd.) Induction method: Phenobarbital (30 mg/kg x 1 time, 60 mg/kg x 3 times) and 5,6-benzoflavone (80 mg/kg x 1time) were intraperitoneally administered to male 7weeks old SpragueDawley rats (body weight 194-232 g). Lot No.: RAA-352 (manufactured on September 27, 1996 and purchased on October 9, 1996) Storage conditions: Stored at -80 

## oC in an ultra-deep freezer (MDF-291, SANYO Electric Co., Ltd.). 

# (// IG



# ~ -5-' I 

### \ I , 1 

### 2) Cofactor for S9 mix (Oriental Yeast Co, Ltd.) Lot No.: 999602 Storage conditions: Stored at -20oC in a bio freezer (GS-2603, Nihon Freezer Co., Ltd.). 3) Composition ofS9 mix K01-1861

One milliliter ofS9 mix comprised 8 J.tmol MgCh, 33 J.tmol KCl, 5 J.tmol G-6- p, 4 J.tmol NADPH, 4 J.tmol NADH, 100 J.tmol ofsodium-phosphate buffer (pH 7.4) and 0.1 ml ofS9. 4. PRE-CULTURES OF TEST STRAINS After 20 J.tl ofeach working stock was inoculated to 10 ml ofNutrient Broth No. 2 (OXOID Ltd., Lot No. OI6 572I2) in L tubes (27 ml volume), it was incubated at 37 ±0.5°C for 8 hours by shaking at 50 times/minute in a Monod shaker (MONOSIN

### llA, Taitec Corporation). The number ofviable cells at the end ofthe incubation, which was calculated from the value of O.D. at 660 nm measured by spectrophotometry (NOVASPEC, LKB Japan) was shown as follows. TA100 TA1535 WP2 uvrA 

### TA98 TAI537 Dose-range-finding test 2.1 3.1 2.7 1.2 2.4 Main test 1.4 3.7 3.I 1.2 2.2 ( x 

### I09 

### viable cells/ml) 5. PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 5.1 Preparation ofthe Test Substance I) Preparation method The test substance was added to acetone (Lot No. DLP4532) and dissolved to make a I0 w/v% solution for irradiating with ultrasonic wave for 5 min, and it was diluted with the same solvent to give each required concentration. 2) Stability oftest substance solution Denaturation of the test substance solution in color and exothermic reaction was not observed until 2 hours after preparation. 3) Preparation time The test substance was prepared just before use and used within 0.5 hours. The test substance solutions were kept at room temperature until use. 

## -6-

> > 

### l ·- _,. '·-- ---. **--·---r:'--·~·** 

## ·- 11

# ( KOl-1861 5.2 Preparation ofPositive Control Substances 1) Preparation method NaN3 was dissolved in distilled water (Lot No. K6B74, Otsuka Pharmaceutical Factory Inc.). AF-2, ICR-191 and 2AA were dissolved in DMSO (Lot No. CF103). 2) Preparation time and storage condition The working solutions were prepared every 3 months and stored at -80oC m an ultra-deep freezer (MDF-291, SANYO Electric Co., Ltd.). 6. METHODS The test was carried out inS. typhimurium 

### TA1535, TA1537, TA98 and TAIOO as well as E. 

### coli 

### WP2 uvrA 

### using the pre-incubation method with and without a metabolic activation system. Three plates were used for the negative control group, and two plates for a dose of the test substance and for the positive control group. The test code number, presence or absence ofS9 mix and dose levels were noted on the plates. 6.1 Procedures After 0.05 ml ofthe test substance solution (0.1 ml ofthe positive control), 0.5 ml ofO.l M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 ml ofthe bacterial culture were added to a test tube, and the mixtures was shaken for 20 min at 37± 0.5°C. Two ml of the soft agar was then added to each tube, and the mixture poured onto a minimal glucose agar plate. After incubation at 37±0.5°C for 48 hours, the number ofrevertant colonies was counted. In the sterility test, 0.1 ml ofeach bacterial culture, the test substance solution, S9 mix and 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal glucose agar plate, which was incubated at 37±0SC for 48 hours. Acetone was used as a negative control, and the following positive controls were used corresponding to the tester strains. TA100 TA1535 WP2 uvrA 

### TA98 TA1537

AF-2 NaN3 AF-2 AF-2 ICR-191 S9 mix(-) 0.01 0.5 0.01 0.1 2AA 2AA 2AA 2AA 2AA S9 mix(+) 1 2 10 0.5 2 (f.!g/plate) 

# f? 

# /70 -7-

### K01-1861 6.2 Dose Selection 1) Dose-range-finding test The test was carried out at seven dose levels consisted ofthe highest dose of 5,000 f..lg/plate and 6 following doses of 1,000, 500, 100, 50, 10, and 

### 5 f..lg/plate. 2) Main test Based on the results of the dose-range-finding test, growth inhibition and increases in the revertant colonies were not observed. Therefore, a main test was carried out at 5,000 J..tg/plate as the highest dose and 4lowerdoses of2,500, 1,250, 625, and 313 f..tg/plate diluted with a geometric progression of2. 7. OBSERVATION AND MEASUREMENT 7.1 Observation Appearance ofrevertant colonies (size and number ofcolonies), deposition ofthe test substance, and the growth inhibition were examined with a stereo microscope. 7.2 Measurement The number ofrevertant colonies was counted with a manual counter or a colony analyzer (CA-9,

**Toyo~sokki** 

### Co., Ltd.). The number of revertant colonies obtained using a colony analyzer was corrected by a supplementary coefficient. Each plate was measuredthree times, and the average ofthese three measurements was adopted as the number of the revertant colonies on each plate. The mean value was calculated for each dose from the number of plates set up, and the decimal point rounded off. 8. JUDGEMENT CRITERIA OF TEST RESULTS The test substance was judged positive when the number of revertant colonies increased two times or more than that ofthe negative control with a dose-dependent manner, and also when reproducibility of the test results was determined. In all other cases, it wasjudgednegative. Statistical procedures were not applied.

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpected circumstances which might have affected the quality ofthe study and no deviation from protocol. 

# 01/71 -8-

## ( K01-1861 

## RESULTS The test results showed that the number ofrevertant colonies for all of the test strains was less than two times that in the negative control at any ofthe dose levels regardless ofthe presence or absence ofS9 mix. Opaque oily deposition ofthe test substance were observed at more than 1,000 Jlg/plate with and without S9 mix, however, these did not interfere with measurement of the revertant colonies. The present .study was confirmed to be valid because the positive control substances induced mutations to the tester strains, and the number of revertants in the positive controls and the negative control was within the range of background data in our laboratories. The sterility test demonstrated that the test system was free from bacterial contamination. Therefore, there were no fluctuations which affected the test results. 

## CONCLUSION In conclusion, V-05H was considered to have no reverse mutagenic potential under the present test conditions. 

## REFERENCES 1. Ministry ofLabor (1991) Guidebook on Mutagenicity Tests using Micro-organisms, New Edition (in Japanese) published by Japan Industrial Safety and Health Association, 2. Ministry ofHealth and Welfare (1992) Guideline for toxicity testing ofchemicals, revised edition, published by the Chemical Daily Co., Ltd. 3. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using Trp+ reversion in Escherichia coli. Mutation Res., 38: 3-32. 4. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113: 173-215. 

# vYJ 

# 17Y -9-

## '," K01-1861 

## Dose-range-finding test Test substance: V-05H With(+) or Test substance Number ofrevertants perplate without(-) dose Base-pair substitution type Frameshift type

S9mix (1-!g/plate) TA 100 TA 1535 WP2 uvrA 

### TA98 TA 1537 Negative 115 8 21 31 13 

### 102 (115) 12 (11) 29 (24) 25 (29) 13 

### (13) control 127 12 22 30 13 

### 5 143 (134) 14 (11) 33 (32) 30 (32) 7 (8) 124 7 31 34 9 10 143 (145) 13 (14) 27 (29) 36 (33) 13 

### (13) 146 14 31 29 13 

### 50 127 (131) 19 (18) 30 (32) 29 (30) 9 ( 9) -S9 mix 134 16 33 31 9 100 113 (128) 11 (12) 29 (28) 24 (24) 19 (19) 143 13 27 24 18 500 116 (129) 22 (18) 28 (26) 37 (33) 21 (20) 142 13 24 28 19 

# • 1000 130 (136) 17 (18) 27 (26) 28 (32) 16 (18) 14I 18 25 35 19 

# • 5000 I19 (122) I4 (I2) 27 (28) 37 (37) IS (I4) I25 10 29 37 I2 Negative I23 8 33 39 22

control 122 (122) 14 (11) 33 (34) 29 (37) I8 (19) 121 12 36 42 16 5 II9 (130) 6 ( 9) 26 (28) 29 (34) 19 (18) I40 I1 30 39 16 10 142 (147) 8 (10) 28 (31) 33 (34) 14 (16) 152 12 34 34 ' 18 50 I29 (130) I2 (II) 35 (34) 47 (39) 25 (26) +S9 mix 130 IO 32 30 27 100 138 (125) 11 (1 0) 32 (34) 39 (40) 25 (24) 1I2 9 35 40 23 500 122 (13I) 11 (10) 37 (34) 39 (39) 22 (21) 140 8 3I 38 19 

# • 1000 I32 (129) 9 ( 7) 29 (27) 42 (35) 22 (21) 126 5 24 28 20 

# • 5000 126 ( 135) 15 (13) 33 (33) 44 (43) 24 (23) I43 IO 33 41 22 Chemical AF-2 NaN3 AF-2 AF-2 ICR-I91 Positive control Dose O.OI 0.5 O.ot 0.1 I (Jlg/plate) -S9 mix Number of 505 303 (3I4) 111 (124) 55I (535) 2792 (2804) (521) revertants/plate 536 324 137 519 2815 Chemical 2AA 2AA 2AA 2AA 2AA Positive control Dose 1 2 IO 0.5 2 +S9 mix (!lg/plate) Number of 834 (833) 138 (144) 423 (448) 316 (307) 137 (145) revertants/plate 831 150 472 297 152 [Notes] Parenthesis shows the mean ofeach plate. A: Deposition oftest substance. • AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide • NaN3: Sodium azide • ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine·2HC1 · 2AA: 2-Aminoanthracene 

### -1- 

# Pi !73I \ 

### K01-1861 

## Main test Test substance: V-05H With(+) or Test substance Number ofrevertants per plate without(-) dose Base-pair substitution type

S9mix (Jlg/plate) TA IOO TA I535 Negative I38 13 131 (134) I6 (I5) control 133 I6 313 166 (153) 10 (11) 139 12 625 126 (130) 11 (15) -S9 mix 134 18 

# ... 1250 144 (140) 10 (13) 136 16 

# ... 2500 139 (137) 13 (14) 135 14 

# ... 5000 140 (138) 14 (I6) 135 18 Negative 140 11 129 (134) 14 (14) control 134 18 313 120 (133) 16 (16) 145 16 625 126 (130) I6 (13) +S9 mix 133 9 

# ... 1250 119 (128) 13 (12) 136 11 

# ... 2500 150 (142) 15 (13) 133 10 

# ... 5000 147 (137) 16 (17) 127 17 Chemical AF-2 NaN3 Positive control Dose 0.01 0.5 (J.l.g/plate) -S9 mix Number of 510 328 (326) revertants/plate 501 (506) 324 Chemical 2AA 2AA Positive control Dose 1 2 +S9 mix (Jlg/plate) Number of 770 (772) I57 (155) revertants/{>late 774 I53 [Notes] Parenthesis shows the mean ofeach plate. A.: 

### Deposition oftest substance. • AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide · NaN3 : 

### Sodium azide WP2 uvrA 24 20 (24) 27 25 (25) 25 26 (27) 27 30 (30) 29 22 (22) 22 19 (20) 21 21 28 (26) 30 27 (28) 28 26 (27) 28 22 (21) 19 23 (21) 19 30 (29) 27

AF-2 0.01 109 (Ill) 113 2AA

10 482 (537) 591 Frameshift type TA 98 TA I537 25 II 27 (23) IO (12) 18 14 34 (30) 10 (13) 25 16 23 (28) 16 (18) 32 19 25 (23) 15 (15) 21 15 25 (28) 16 (17) 30 17 32 (29) 15 (13) 25 11 30 25 37 (38) 15 (20) 46 19 38 (38) 22 (28) 37 33 39 (37) 22 (22) 34 22 36 (40) 21 (24) 44 27 39 (38) 34 (27) 37 19 43 (45) 27 (26) 47 24

AF-2 ICR-191

0.1 1 512 2671 527 (520) 2566 (2619) 2AA 2AA 0.5. 2 277 (273) 138 ·(142) 269 145
• ICR-I91: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine·2HCI · 2AA: 2-Aminoanthracene



### - 2-

## Dose-range-finding test K01-1861

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## 20 10 313 Fig. 7


## 40 30 20 10 625 1250 2500 Dose-response curve without S9 mix: Frameshift type 

## K01-1861 O:TA 98 D:TA 

### 1537



### 5000 

## (JLg/plate) O:TA 98 D:TA 

### 1537

**0~----,----------.----------~-----------.----------.** 

## 313 625 1250 2500 Fig. 8 Dose-response curve with S9 mix: Frameshift type -6- 

## 5000 (JLg/p 1ate) 

# f7

**/7~**

# (

( STUDY CODE: K06-0612 

## FINAL REPORT Receipt No. T97-2707 Report No. T-5194

CHROMOSOMAL ABERRATION TEST OF 

### V-OSH USING CULTURED MAMMALIAN CELLS

March 1998 Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute Japan

# (

/

**\~** 

### STATEMENT I, the undersigned, hereby declare that this report provides a correct English translation ofthe final report (Study Code K06-0612, issued onMarch 23, 1998). Chemicals Inspection & 

### Testing Institute,Japan (CITI) formally changed its name to Chemicals Evaluation and Research Institute,Japan (CERI) as on November 1, 1999. The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to "NISSHINBO INDUSTRIES, INC. R & 

### D CENTER" and "1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan", respectively by Sponsor's request. CAS No.157299-02-0 oftest substance was added by Sponsor's request. In the structural formula, n=2-800 was added. Molecular weight of3,000 was changed to 30,000. Wakamatsu Shinya, M.S. Hita Laboratory Chemicals Evaluation and Research Institute, Japan 

# b

**/~**

# / 2.0l72 

### Date 

# f1 (

**~()**

**~~-**

# (

( QUALITY ASSURANCE STATEMENT Hita Laboratory, Chemicals Evaluation and Research Institute, Japan Sponsor: NISSHINBO INDUSTRIES, INC. R&D CENTER Title: Chromosomal Aberration Test of V-OSH Using Cultured Mammalian Cells Study code: K06-0612
This report was audited by the Quality Assurance Section. I, the undersigned, hereby declare that this report reflects the original Japanese report.
(Date)



# J~ **l~, ..2..o~2.** 

**-;:;:)\** .......

# - 

# . ,/,

**~** 

# .

**~~--~** (Signature) Section Chief, Quality Assurance Keiji Shiraishi, B.S. 

# fr l1l

Tl

### Sponsor: Title: 

## GLPSTATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Chromosomal Aberration Test of V-05H Using Cultured Mammalian Cells Study Code: K06-0612 I, the undersigned, hereby declare that this study was conducted in compliance with "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and ofthe Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 ofthe Planning and Coordination Bureau, Environment Agency (EA), No. 229 of the Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare (MHW) & 

### No. 85 (1984) ofthe Basic Industries Bureau, Ministry ofInternational Trade and Industry (MITI), March 31, 1984; Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, MHW & 

### No. 823 (1988) ofthe Basic Industries Bureau, MITI, revised on November 18, 1988) and with "Organisation for Economic Cooperation and Development (OECD) Principles ofGood Laboratory Practice" (May 12, 1981). Management: Signed in original March 23, 1998 Yoshifumi Fujino, M.S.I \ 

### QUALITY ASSURANCE STATEMENT Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan Sponsor: NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Title: Chromosomal Aberration Test of V-05H Using Cultured Mammalian Cells Study Code: K06-0612 This study was audited and inspected by Quality Assurance Section of Hita Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & 

### Testing Institute, Japan. The dates audited and/or inspected and the dates reported these results to the study director and management are as follows. Dates of Dates ofReports to Dates ofReports to Inspections/ Audits Study Director Management December 11, 1997 December 12, 1997 December 12, 1997

January 12, 1998 January 12, 1998 January 12, 1998 January21, 1998 January 22, 1998 January 22, 1998

March 4, 1998 March 4, 1998 March 4, 1998 March 9, 1998 March 9, 1998 March 9, 1998 March 17, 1998 March 17,1998 March 17,1998 March 23, 1998 March 23, 1998 March 23, 1998 

## I, the undersigned, hereby declare that this report provides an accurate description ofthe methods and procedures used in this study and that the reported results accurately reflect the rawdata obtained. Section Chief, Quality Assurance: Signed in original March 23, 1998 Keiji Shiraishi, B.S.

# f1 (<6}

### Study Code: K06-0612. Test Substance Code: HR3539 Sponsor Code: N-680 TITLE Chromosomal Aberration Test ofV-05H Using Cultured Mammalian Cells SPONSOR NISSHINBO INDUSTRIES, INC. R & 

### D CENTER 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan TESTING FACILITY Rita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita, 877-0061, Japan PURPOSE OF STUDY The ability of the test substance to induce chromosome aberration is investigated using Chinese hamster lung fibroblast cells (CHL cells). TESTING METHOD This study was conducted in accordance with "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances (Notification No. 700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 ofthe Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare (MHW) & 

### No. 1014 (1986) ofthe Basic Industries Bureau, Ministry ofInternational Trade and Industry (MITI), December 5, 1986)". GLP COMPLIANCE This study was conducted in compliance with "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, EA, No. 229 ofthe Pharmaceutical Affairs Bureau, MHW & 

### No. 85 (1984) ofthe Basic Industries Bureau, MITI, March 31, 1984; Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, MHW & 

### No. 823 (1988) of the Basic Industries Bureau, MITI, revised on November 18, 1988) and with "Organisation for Economic Co-operation

# (

/ 

### \ 

### and Development Principles ofGood Laboratory Practice" (May 12, 1981). PERJOD OF STUDY Commencement ofStudy: Initiation ofPreliminary Test: Completion ofObservation: Completion ofStudy: December 11, 1997 December 17, 1997 March 13, 1998 March 23, 1998 LOCATION AND PERJOD FOR RETENTION OF RAW DATA AND SPECIMENS Data and specimens, and the test substance of this study will be retained in the archives and the test substance storage room, respectively, of Hita Reseach Laboratories for 10 years following the date of completion of study. After termination of the retention period, any measures taken will be done with the approval of the Sponsor. However, specimens that are liable to deteriorate markedly will be retained only for the period the quality of specimens permits evaluation, and the abandonment ofthem will be taken with approval ofthe sponsor. AUTHOR AND PERSONNEL CONCERNED WITH STUDY 

### AUTHOR Study Director: Signed in original Nahoko Yoshida, B.S. Hita Research Laboratories Mutagenicity Section March 23, 1998

# ( TABLE OF CONTENTS SUMMARY MATERIALS AND METHODS 1. 

### TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 2. CELLS AND MEDIUM 3. S9MIX 4. CELL CULTURE .................................................... 5. PREPARATION OF THE TEST SUBSTANCE 1 

### 2 4 4 

### 5 AND THE POSITIVE CONTROL SUBSTANCES ················ 5 6. PREPARATION OF SPECIMENS ················ 5 7. DETERMINATION OF DOSE LEVELS ················ 6 8. CHROMOSOMAL ABERRATION TEST ················ 6 9. OBSERVATIONAND SCORING ················ 7 10. JUDGEMENT CRITERIA OF RESULTS ················ 8 UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 8 TESTRESULTS 1. CELL GROWTH INHIBITION TEST AND CELL DIVISION INHIBITION TEST 2. CHROMOSOMAL ABERRATION TEST CONCLUSION REFERENCES TABLES AND FIGURES 8 

### 9 10 10 1-6



### T II \_



# ( SUMMARY The ability of V-05H to induce the chromosomal aberration was investigated using Chinese hamster lung fibroblast cells (CHL cells) without S9 mix (direct method) and with S9 mix (metabolic activation method). From the results ofthe cell growth inhibition test and the cell division inhibition test, chromosomal aberration test was carried out at 156, 313 and 625 f.!g/ml for the 24, 48 htreatment group by the direct method and the 18 h-recovery group without S9 mix, and at 1,250, 2,500 and 5,000 f.lg/ml for the 18 h-recovery group with S9 mix by the metabolic activation method. As the result, the test substance induced no chromosomal aberrations within the dose range, which included the doses producing cytotoxicity in the direct method and the metabolic activation method. On the other hand, Mitomycin C and Cyclophosphamide monohyderate obviously induced chromosomal aberrations inpositive control groups. Based on the above results, it is concluded that V-05H induce no chromosomal aberration with and without metabolic activation under the conditions of the present study.

-1-

### T.

# (

( 

### \ 

### Melting point: Boiling point:

.Vaporpressure: Partition coefficient: - Solubility: Soluble in oil Degree ofsolubility: Water: <50 mg/mL(measured in our laboratory) 8) Storage conditions DMSO: <50 mg/mL(measured in ourlaboratory) Acetone: 

## > 500 mg/mL(measured in our laboratory) Others: Stored in the sealed container at the cold and dark place. The hightemperature and humidity was avoided. 9) Precautions Goggles, gloves, a mask, a head cap and a lab coat were worn. Hand-washing and gargling were carried out at the end ofhandling. 1.2 Positive Control Substances 1) Mitomycin C (MMC) Manufacturer: Lot No.: Appearance: Composition: Kyowa Hakko Kogyo Co., Ltd. 158AGB royal purple crystals or crystalline powder It includes 2 mg (titer) of Japanese Pharmacopoeia (J.P.) MMC and 48 mg of J.P. sodium chloride in one vial 2) Cyclophosphamide monohydrate (CPA) Manufacturer: Lot No.: Appearance: Purity: 3) Storage conditions Wako Pure Chemical Industries, Ltd. PTL7433 and LEE7264 white crystalline powder 98.8% and 98.9% MMC and CPA were stored at room temperature and at a cold and dark place, respectively. 4) Precautions Gloves, a mask, a head cap and a lab coat were worn in handling.

- 3 - 

# fl

**t~r**

# ( 2. CELLS AND MEDIUM 2.1 Cell Line and Reason for Selection Chinese hamster lung fibroblasts (CHL cells, clone No. 11), which were supplied by the National Institute of Health Science (old name: National Institute of Hygienic Sciences) on September 28, 1988, were used. The modal number of chromosomes is 25, and the doubling time is about 15 hours. The passage number was 18 at receipt. Cells were used at 39 passage number in the chromosomal aberration test. CHL cells are widely used in in vitro 

### chromosomal aberration tests because they . have quite high sensitivity to chemical mutagens and a large amount of back ground data available. 2.2 Medium and Water Eagle's minimum essential medium (Lot No. 388706 and 398711, NISSUI PHARMACEUTICAL CO., LTD.) supplemented with newborn calf serum (Lot No. NBQ07, MITSUBISHI CHEMICAL CORPORATION) at a rate of 10 v/v% was used, which is referred to hereafter as 10% NCS/MEM. The medium was prepared using distilled water obtained with a glass still (WG220, YAMATO SCIENTIFIC CO., LTD.). 3. S9 MIX 3.1 Rat Liver S9 (Kikkoman Corporation) Induction method: Male Sprague-Dawley rats aged 7 weeks old (Body weight;

**RAA-371:205~244** 

### g, **RAA-372:186~225** 

### g) were Lot No.: intraperitoneally administrated phenobarbital (30 mg/kgx 1 time, 60 mg/kgx3 times) and 5,6-benzoflavone (80 mg/kgx1 time). RAA-371 (date manufactured: October 24, 1997; date purchased: November 12, 1997) and RAA-372 (date manufactured: October 31, 1997; date purchased: December 3, 1997) Storage condition: Stored at -80°C (Deep freezer, ULT-1285, REVCO CO., LTD.) 3.2 Composition ofS9 mix One milliliter ofS9 mix consists of4 J.tmol ofHEPES (pH 7.2), 

### 5 J.tmol ofMgCh, 33 J.tmol ofKCl, 5 J.tmol ofG-6-P, 4 J.tmol ofNADP and 0.3 ml ofS9. 3.3 S9 mix/MEM A halfmL ofS9 Mix was added to 2.5 mL of 10% NCS/MEM.

- 4-

# I 

# (



# ( 4. CELL CULTURE 4 

### Cells suspension of 1.5x104 

### or 0.5x10 cells/mL was seeded five milliliter of 10% NCS/MEM in a 60 mm diameter Petri dish (IWAKI GLASS CO., LTD.). Monolayer cells in logarithmic growth phase after cultivation for 2 or 3_days were applied to the tests. Cells were cultured in a C02 

### incubator, which was set at 37±0.5°C, almost 100% humidity and 5% C02 

### atmosphere_ 5. PREPARATION OF THE TEST SUBSTANCE AND THE POSITIVE CONTROL SUBSTANCES 5.1 

### 1) Test Substance Preparation Acetone (Lot No. TPN6909, Wako Pure Chemical Industries, Ltd.) was selected as a solvent, because degree ofsolubility ofthe test substance was less than 50 mg/ml in distilled water and DMSO and more than 500 mg/ml in acetone. The test substance was dissolved in acetone to make the original solution. The original solution was diluted with the same solvent to make the required concentrations. 2) Preparation time Test substance solution was freshly prepared just before use, and it was used within 2 hours. 5.2 Added Amount ofTest Substance Solution 5.3 Thetest substance solution was added to the medium to become 1%. Positive Control Substances 1) Preparation Positive control substances were dissolved in distilled water. 2) Preparation time Positive control substance solutions were freshly preparedjust before use, and they were used within 2 hours. 6. PREPARATION OF SPECIMENS 0.075 M KCl was added to the cells, which were removed from each dish with 2 ml of 0.25% trypsin and collected by centrifugation, and hypotonic treatment was carried out at 37°C for 15 min. Cell suspension fixed by Carnoy's solution (methanol: acetic acid=3:1) was dropped onto microscope slides and dried, and then stained with 2% Giemsa solution. One specimen and four specimens per dose (two specimens per dish) were made in the cell division inhibition test and the chromosomal aberration test, respectively.



### - 5-

**---~-=~~_..-.-..**.. -=..---

**-=--~--** 

## .... **--...---~-··"~'** 

## ..._..... ----,

### 7. DETERMINATION OF DOSE LEVELS The 50% growth inhibition concentration of the test substance was determined by the cell growth inhibition test. The highest dose, at which chromosome was observed, was determined by the cell division inhibition test. A concentration equivalent to 5 mg/ml was employed as the maximum dose when no cytotoxicity was observed. 7.1 Cell Growth Inhibition Test For the treatment by the direct method, the cells in logarithmic growth phase were treated with 5 ml ofthe 10% NCS/MEM including the test substance solution for 24 and 48 hours. For the treatment by the metabolic activation method; the cells were treated with 3 ml of 10%NCS/MEM (the treatment group without S9 mix) or S9 mix/MEM (the treatment group with S9 mix) including the test substance solution for 6 hours. The medium was removed after treatment. The cells were rinsed three times with 2 ml of Ca2

### +, Mg2

### +-free phosphate-buffered saline [PBS(-)], and subsequently the cells were cultured with 5 mL of 10% NCS/MEM for 18 hours on the group with and without S9 mix. Colcemid was added to the medium to give a final concentration of0.1 Jlg/ml 2 hours before the end ofincubation in either treatment method. After removal of the medium at the end of incubation, the cells were detached with 2 ml of0.25% trypsin and the number ofcells was counted using a Microcell Counter (F-500, Toa Medical Electronics Co., Ltd.) to obtain cell growth rate(%). The medium was changed 2 hours before the end of incubation because precipitation ofthe test substance prevented observing specimens. 7.2 Cell Division Inhibition Test Specimens ofcells were made which were used in the cell growth inhibition test and dividing cells were observed. Chromosome aberration was observed preliminarily. 8. CHROMOSOMAL ABERRATION TEST 8.1 Dose Levels Test substance: Three dose levels were made by a geometric progression of2, and limit was maximum dose from the result ofthe cell growth inhibition test and the cell division inhibition test.


## -6-

## ( '· 

### Positive control substances: The concentrations ofthe positive control substances were selected as follows according to our background data.
8.2 Method Direct method: MMC 0.05 )!g/ml Metabolic activation method: Without S9 mix: MMC 0.1 )!g/ml With S9 mix: CPA 10 )!g/ml Cultures and treatment ofcells were done and specimens were made by the same procedure as that in the cell growth inhibition test. A solvent-treated group was set in a negative control, and MMC or CPA-treated groups were set in a positive control. 8.3 Number ofDishes and Identification Two dishes were used for each dose. The study code number, treatment method and dose level were written on the dishes. 9. OBSERVATION AND SCORING The number ofcells with structural aberrations ofchromatid or chromosome (such as gaps, breaks or exchanges) and numerical aberrations (polyploid, endoreduplication) were examined by observing 100 metaphases for each dish. An aberration cell was defined had structural or numerical aberrations. Two hundreds ofmetaphases for each group were observed and incidence ofaberration cells were calculated. The incidence ofstructural aberration with or without gaps (larger than a width and less than twice width ofchromatid) was calculated. All specimens were coded and observed in a blind manner.



### - 7-

### 10. JUDGEMENT CRITERIA OF RESULTS Structural aberrations (including gaps) and numerical aberrations were judged as follows according to their incidences: negative (-) in less than 5%; falsely positive (±)in 5% or more but less than 10%; and positive(+-+++) in 10% or more. The positives were subdivided into (+) for an incidence in 10% or more but less than 20%; (++)in 20% or more but less than 50%; and(+++) in 50% or more. The test substance was judged to be positive for the induction of chromosomal aberrations was dose-related orreproducible.



### UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL There were no unexpected situations which might have affected the test results and no deviation from protocol. 

### TEST RESULTS The incidence of aberrant cells was not markedly different between two dishes. The incidence of cells with aberration did not exceed 5% in the negative control. The incidence of cells with structural aberrations excluding gap was 10% 

## oi more in the positive control. There were no fluctuations in the test conditions. The tests were therefore considered to be valid. 1. 

### CELL GROWTH INHIBITION TEST AND CELL DIVISION INHIBITION TEST (Table 1, and Fig. 1) The cell growth inhibition test and the cell division inhibition test were performed at doses shown in Tables 1. In the cell growth inhibition test, 50% cell growth inhibition concentrations of the test substance were about 630 J.lg/ml in 24 hours treatment group, and about 400 Jlglml 

### in 48 hours treatment group by the direct method, as 100% in the solvent control. However, the 50% cell growth inhibition concentration was not able to obtained even at 5,000 J.lg/ml with S9 mix because of the weak cytotoxicity ofthe test substance. 

### In the examination used acetone as a solvent, the cell growth rate was the lowest at 625 J.tg/ml, and increased above 1,250 J.lg/rnL because of the bad suspension of the test substance in the medium by the direct method or without S9 mix by the metabolic activation method. Therefore, dose determination test was performed to used 0.5% sodium carboxymethyl cellulose (CMC) solution as a solvent. However, cell growth inhibition and division inhibition were not observed all the doses, because ofthe bad suspension of the test substance in the medium. Based upon the above results, acetone was



### - 8-I 

### 1 

## ! 

# (

(

( \, 

### selected as the solvent. The results of the observation of specimens, increase remarkable the chromosomal aberrations were not observed at all doses by either the direct method or the metabolic activation method. The maximum dose was 625 J.lg/ml in the direct method and without S9 mix by the metabolic activation method, 5,000 J.lg/ml in S9 mix in the chromosomal aberration test. Based upon the above results, the following 3 dose levels were set in the chromosomal aberration test. 24 hours treatment by the direct method: 156, 313 and 625 Jlg/ml 48 hours treatment bythe direct method: 156, 313 and 625 Jlg/ml Without S9 mix by the metabolic activation method: 156, 313 and 625 J.lg/ml With S9 mix by the metabolic activation method: 1,250, 2,500 and 5,000 Jlg/ml 2. CHROMOSOMAL ABERRATION TEST 2.1 Direct Method (Table 2 and Fig. 2) 1) 24 hours treatment 2) The incidence ofcells with structural aberrations include gap was 0.5, 0.5 and 1.0% at 156, 313, 625 J.lg/ml, respectively. On the other hand, the incidence in the solvent control was 2.5%, which was within the normal range, and the positive with MMC induced the structural aberration at 57.0%. The incidence ofnumerical aberration was less than 5% in all treatment groups. 48 hours treatment The incidence ofcells with structural aberrations include gap was 1.0, 0.5 and 1.0% at 156, 313, 625 J.lg/ml, respectively. On the other hand, the incidence in the solvent control was 1.0%, which was within the normal range, and the positive with MMC induced the structural aberration at 55.0%. The incidence ofnumerical aberration was less than 5% in all treatment groups.



### - 9- 

# (/1 

# /1>1. 

### 2.2 Metabolic Activation Method (Table 3 and Fig. 3) 1) Without S9 mix The incidence ofcells with structural aberrations include gap was 1.0, 1.0 and 2.0% at 156, 313, 625

**~g/ml,** 

### respectively. On the other hand, the incidence in the solvent control was 0.5%, which was within the normal range, and the positive with MMC induced the structural aberration at 36.5%. The incidence ofnumerical aberration was less than 5% in all treatment groups. 2) With S9 mix The incidence ofcells with structural aberrations include gap was 0.5, 1.0 and 1.0% at 1,250, 2,500, 5,000

**~g/ml,** 

### respectively. On the other hand, the incidence in the solvent control was 0.5%, which was within the normal range, and the positive with CPA induced the structural aberration at 88.0%. The incidence ofnumerical aberration was less than 5% in all treatment groups. CONCLUSION In the chromosomal aberration test using CHL cells, V

### -05H induced no structural aberration or numerical aberration within the dose range including the cytotoxicity by either the direct method orthe metabolic activation method. It is concluded that V

### -05H induce no chromosomal aberration in the presence or absence ofmetabolic activation under the present test conditions. REFERENCES 1. 

### Ministry ofHealth and Welfare (1992) Guideline for toxicity testing of chemicals, revised edition. published by the Chemical Daily Co., Ltd. 2. Ishidate, M.Jr. (1979) Chromosomal aberration test in vitro for chemical mutagens in our environment (in Japanese). Tissue Culture, 5:115-122. 3. Ishidate, M.Jr. (ed.) (1987) Databook ofchromosomal aberration test in vitro. L.I.C. Inc., Tokyo. 4. Japan Environmental Mutagenicity Society/Mammalian Mutagenicity Study Group (ed.) (1988) Atlas ofchromosomal aberration induced with chemical substance (in Japanese). Asakura Publishing Co., Tokyo. 5. Yoshida, M. (1982) Shape of chromosome and chromosomal aberration (in Japanese). Metabolism, 19:427-438.

- 10 -

- T Lo0<• • -- **=--·---···-~** 

### -......-- --<> ........**,.-.---....,..,-~,** .......... ----·---· ' 0i 

### \. 

### 6. Yoshida, M. (1983) Chromosome analysis in cultured cells (in Japanese). Cell Technology, 2:1352-1360.

- 11 -_j I l l I \ 1 I l J I j **~** -...... **~** Table I Results ofthe dose determination test ofV-OSH Concentration of the test substance(l!g!ml)

24 hours treatment Direct method 48 hours treatment without S9 mix Metabolic activation method with S9 mix ---- -- ---- --- - *I

Mean value for two dishes.
*2 Judgement Growth rate 'I(%) Index amount of mitotic metaphase' 2 Chromosomal numerical aberrations •J (%) structural Concentration of the test substance(J!g/ml) Growth rate 'I (%) Index amount ofmitotic metaphase' 2 Chromosomal numerical aberrations' 3

(%) structural Concentration of the test substance(J!g/ml) Growth rate 'I (%) Index amount of mitotic metaphase ' 2 Chromosomal numerical aberrations ' 1

(%) structural Concentration of the

**t~st** substance(J-Lg/ml) Growth rate ' 1 (%} Index amount of mitotic metaphase ' 2 Chromosomal numerical aberrations ' 1

(%) structural

++ : abundant, + : a few • ± : rare • - : none

*3

50 cells/dose were observed. *Precipitation of the test substance was observed in culture medium. 0 313 100 85.3 ++ + 0 0 4 2 0 313 100 65.3 ++ + 2 0 0 0 0 313 100 91.1 ++ ++ 2 0 2 0 0 313 100 80.4 ++ ++ 0 0 0 0

- I K06-0612

* * * * *

625 1,250 2,500 5,000 50.2 72.1 83.3 90.8

+ + ++ ++

0 2 0 2

4 2 4 0

* * * * *

625 1,250 2,500 5,000 16.1 3D.4 54.0 82.9

+ + ++ ++

2 2 0 0

0 4 0 0 * * * * *

625 1,250 2,500 5,000 74.5 81.3 85.7 91.1

++ ++ ++ ++

0 4 0 0 I

2 2" 0 0 * * * * *

625 1,250 2,500 5,000 65.1 72.3 69.0 59.6

++ ++ ++ ++

0 0 2 0

0 0 0 0 I• I l _J j I

l l N

I l j

I
-l l 1 l -1 **~** .........._

l **~**

- \ l I \ **,--~--~\**

Name of test substance: Y-05H Table 2 Results of chromosomal aberration test (Without metabolic activation) K06-06

I I Treatment Concent- Number of cells umber and the percenta1esl%l ·of cells shmdn2 structural ch omosomal aberrations

Treatment

time(h) ration Observed Number of Judge- Gap Chromatid-type Chromosome-t }'pe

Others Total Judge

**(~g/ml)** IDolypJoids ment g ctb cte csb cse -g tg

100 0 0 3 n ·o 0 0 3 3

24 0 lllO 0 0 __l I 0 0 0 2 2

Solvent

200 0( 0. 0 Of 0. 0 ....ti 2.0 II IU 0( 0 .{) 0( 0.0 Oi il.O 5_( 2. 5 5( 2 5

(Acetone)

tOO 0 0 I 0 0 0 0 1 I

48 0 100 t' 0 0 0 I 0 0 1

200 l ( 0. 5 0( 0 0 I( 0,5 0 0.0 t( 0.5 0( 0.0 0{ 0. 0 2 ( I. 0 1( -,. 0

100 0 0 0 I -0

0 0 0

156 * Too

0 - 0 0 0 0 0 I

200 0( 0.0 Of 0.0 OJ 0 0 I 0.5 01 0. 0 0( 0.0 01 0 0 ll 0. 5 If 0. 5

100 0 Q _Q 0 0 0 0 0 0

24 JIJ * 100 0

- 0 I 0 0 0 0 1 l

-filo01 0.0 ot o.o· I ( 0. 5 ol o.o O( 0.0 0( 0.0 0( 0 0 I ( 0. 5 1 ( 0.

**~** l

100 0 0 1 c 0 0 0· I l

625 * 100 0 - 0 __Q 0 0 0 _l 1

Test 200

or o· o 0( 0.0 t ( 0 5 ( 0.5 0( 0.0 0( 0. 0 0( 0.0 2 ( I 0 2 ( 1. 0

Substance 100 I 0 0 0 0 0 0 0 0

156 * 100 2 - 0 0 0 I 0 2 2

2oo 31 1.5 01 0. 0 01 0 0 t I 0 5 O( 0.0 I ( 0. 5 01 0 0 2J 1.0 2 f I. 0

100 2 0 0 0 0 0 0 0 0

48 Jl:l* tOO 0 - . 0

Jl. 0 n I

. 0 I I

20o 2 f 1.0 0( 0.0 0( 0.0

. 0( 0.0 O( 0.0 l ( 0. 5 0( 0.0 ll 0. 5 t ( 0 '5)

100 t o. 0 0 0 0 0 0 0

625 ' 100 2 - _1 0 0 0 0 0 0 2

200 31 .1.5 Zl 1.0 nt JL_O_ O( 0.0 O( 0. 0 0( 0._0_ OJ 0 0 OJ 0.0 2 f I. 0

100 0 t 13 40 0 0 0 50 50

24 0.05 100 0 - I a 53 0 I 0 63 64

Positive

loo

Of 0 .o 2( 1.0 3U JB ._i 93 ( 4 6 5 O( 0.0 I ( 0. 5 O! 0 .Q l _1131 56.5 _114( 57.0

control 100

I 0 18 56 0 1 0 59 59

(HHC)

48 0.05 tOO 0 - I 26 34 0 I 0 50 51

200 t ( 0.5 l ( 0 5 44( 22.0 01 4' .0 0( 0.0 2( I. 0 0( 0 0 109{ 54.5 I tO! 55.0

-g: total number and the percentage of cells except those which has only gaps tg: total number and the percentage of cells including those which has only gaps

ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange Others: fragmentation etc.(except pulverization)

H/>IC: Hitomycin C

t : Precipitation of the test substance. ment - - - - - - ttl HI**/~·-.,** w

I i I

I -l l \ **~**
l **~·**

I **~**
I : I I **~~** ..

/---

Name of test substance: V-05H Table 3 **Result~** of chromosomal aberration**te~t** (With metabolic activation) K06-061 ··-- --.

I Concent- Number of cells Number and the oercentaeesl%1 or eel s showinr scructural chromosomal aberrations

Treatment S9 mix ration Observed Number of Judge- Gap Chromatid-type Chromosome-type

Others TotalJudge(!Jg/ml) jpolyploids ment g ctb cte csb cse -g tg

. ment

100 I 0 I 0 0 0 0 I I

- 0 100 0 0 0 0 0 0 0 0 0

Solvent 200 II 0. 5 0 ( _0_. Q_ H _0._5_ Ol o o ol o o

. 01 0.0 01 0 0 l ( 0. 5 I ( 0. 5

(Acetone) 100 0 I 2 1 0 (} 0 3 4

t 0 100 I 0 1 0

. 0 0

. 0 I I

200 II 0. 5 It 0 5 31 u I l 0. 5

. 01 0. 0 01 0 0 01 0.0 41 2.0 5 ( 2. 5

100 0 0 2 0 -0

0 0 2 2

156 * 100 I - 0 0 0 0 0 0 0 0

200 I ( 0. 5 O( 0.0 2 ( I 0 01 O.o Ol 0 0 ot o:O 6T o o 21 l.o 21 1.0

100 0 0 0 I 0 0 0 I I

- 313 * 100 0

- 0 I 0 0 0 0 I I

200 01 0 0 Ot 0 0 H 0. 5 l ( 0 5 01 (} .11 (}( 0 '0 0{ 0. 0 2 ( I. 0 21 I. 0)

100 0 0 2 0 0 0 0 2 2

625 * 100 0 - 0 I I 0 0 0. 2 2

res l 200

01 0 0 01

. 0.0 3l 1,5_ If II. 5 OT -oll or ·-o.o Ol 0. 0 4 ( 2. 0 4 ( 2. 0

Substance

100 0 '

0 I 0 0 0 0 I I

I, 250 * 100 l . 0 0 0 0 0 0 0 0

200 I ( 0. 5 0( 0.0 II 0. 5 ot o:OC OT olf 6{ 0.0 0( 0.0 l ( 0. 5 11 0. 5

100 0 0 l 0 0 0 0 I l

t 2' 500 j 100 0 - 0 1 0 0 0 0 I l

200 01 0.0 0( 0.0 2( 1.0 01 0. 0 Ol 0 0 Ol 0.0 01 0.0 2 I I. 0 2( 1.0

10 0 0

. _0_ 0 0 0 0 0 0

5. 000 * 10 I - 0

**·~** 0 0 0 0 2 2

201 II 0. 5 _Ot (LO H J.O 0( 0. 0 0( 0 0 0( 0 0 0( 0. 0 2 ( l 0 21 l 0

Positive· 10 0 J Jl 31\ . I 0 0 36 37

**cont~ll** - 0 .I 10 0 - 0 6 32 ·o 0 0 36 36

IHHC 20 0( 0 0 I l 0 5 IS( 9.0 62T11:0 11 o:s ot o o 01 0.0 721 36 0 731 36.5

Positive 100 0 l 62 79 0 0 0

. 89 89

**cont~\1** t 10 100 0

. - 0 28 81 0 0 0

. 87 87

I CPA 200 or o.o If L5 90f 45.0 I R?.{ Rl 0 · Of 0 0 01 o-n -or o o l7Bl BB. 0 178( 88 0

Amount of S9(5%), Treatment time of the test substance(6h), Recoven• time of cells after treatment of the test substanc·e(l8h)

-g: total number and the percentage of cells except those which has only gaps +g: total number and the percentage of cells including those which has only gaps

ctb: chromatid break cte: chromatid exchange csb: chromosome break cse: chromosome exchange Others: fragmentation etc.(except pulverization)

**~~C:** Mitomycin C CPA: Cyclophosphamide, monohydrate

• :

**Precipitati~h** of the test substance. - - - - - - tt ttt ll **~** I l **~** i -- j I ' I i ..p.. I **~ ~**  **~ ~**  ,.-_ **~** Q '--" Q) ...... **~** I- **~** ...... **~** 0 I0 100 50 100 ,.-_ **~** Q '--" Q) ...... **~** I- 50 **~** ...... **~** 0 I0 -----"

10 100 1,000 10,000 Concentration of the test substance ( J.l g/ml)

24 hours treatment by the direct method

10 100 1,000 10,00 Concentration of the test substance ( J.l g/ml)
48 hours treatment by the direct method .- **~** " '-' Q) ...... 1'1 I- .J:l ...... **~** 0 ).. 0 .- **~** Q '--" **~** ...... 100 50 100 e 5o -E **~** 0 ).. 0 **/~\** 1\06-0612 **~**

10 100 1,000 10,000 Concentration of the test substance ( J.l g/ml) Without S9 mix by the metabolic activation method **~**

10 100 1,000 10,000 Concentration of the test substance ( J.l g/ml) Wit}l S9 mix by the metabolic activation method

Fig. 1 Results of the dose determination test ofV-05H

# (_

**~** 

### "' 5 "' <... ·<... (1) .= 

### "' 

### "' E 0 en 0 E 0 <...

**~** .c:

**~** :>: 

### "' <ll <.> ._ 0 <1> '-' c:; (1) 

### """ '-' 

## ..::



# - en c:; 0

**~** 

### "' '- '- (1) ..0 

### "" "' E 0 

### "" 0 E 0 "- 

## -= '-' 

## -=

**~** :>: 

### "' <1.1 <.> 

# .... 0 <1.1 '-' c:; <ll 

### """ "-' c:; 

# - 30 o 

### numerical aberrations • structural aberrations (rg) 20 10



# 0~ 

# " 313" 

### 0 156 

### ConcentratLon(IJ.g/

### ml) 24 

### hours treatment by direct method 

### JO 0 

### numedeal aberrations • structural aberrations (rg) 20 

### tO 0\



### 0 

### 156 313 Concentration(IJ.g/ml) 

### 48 h.ours treatment by direct method Fig.2 Results of chromosomal aberration test of V-05H

- 5- 

### KOS-0612·

625

625! / 

# ( ..., "' <= 0 _, 

### "' 

# ... ... QJ .0 

### "' "' e 0 

### "' 0 E 0 "- <:; 

### 5 :;:

**~** 

# ... ""'

**~** 0 QJ 

## ""' <= <I> 

### ""' 

## ""' c::

/ 

# \

**~** 

## "' c: 0 _, 

### "' '- '- QJ .0 "' 

### "" E 0 

### "' 0 El 0 

# ... .<:: 

### ""' 5 ;: 

### "' 

# - QJ 

### ""' ._ 0 QJ <.l c:: QJ '"0 <.> c:: 

# -
/ 

### 30 

### 20 10



### JO 20 10 

### o numerical aberrations • structural .aberrations (tg)



### 156 313 Concent.ration( u.g/ml) Without S9 mix by metabo!Lc activation method 

### o numerical aberrations • s.tructural aberrations 

### (+g)

1,250 2,500 Concentration(u.g/ml) With S9 mix by metabolic activation method 

### 6Z5



### 5,000 Fig.3 Results of chromosomal aberration test of V-05H

- 6-



# ~1 

# ;Jo

# 3 KOS-0612

### STUDY CODE: Bll-0443 

# FINAL REPORT ReceiptNo. D97-1763 Report No. D-5317

TWENTY-EIGHT-DAY REPEATED-DOSE ORAL TOXICITY STUDY OF V-OSH 

### IN RATS
May1998 Hita Research Laboratories Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute Japan

# c

( Bll-0443 

### STATEMENT 

### I, the undersigned, hereby declare that this report provides a correct English translation ofthe final report (Study Code B11-0443, issued on May 29, 1998). Chemicals Inspection & 

### Testing Institute, Japan (CITI) formally changed its name to Chemicals Evaluation and Research Institute, Japan (CERI) as ofNovember 1, 1999. The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to "NISSHINBO INDUSTRIES, INC. R & 

### D CENTER" and "1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan", respectively by Sponsor's request. CAS No.l57299-02-0 oftest substance was added by Sponsor's request. In the structural formula, n=2-800.was added. Molecular weight of3,000 was changed to 30,000. Madoka Tada, B.S. Junior Laboratory Animal Technician Hita Laboratory Chemicals Evaluation and Research Institute, Japan 

### q~ /J' ' .,J-I)b.,L Q 

### Date

# (



# (



# ( QUALITY ASSURANCE STATEMENT Hita Laboratory, Chemicals Evaluation and Research Institute, Japan Sponsor: NISSHINBO INDUSTRIES, INC. R&D CENTER Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of V-OSH in Rats Study code: 

### Bll-0443
This report was audited by the Quality Assurance Section. I, the undersigned, hereby declare that this report reflects the original Japanese report.

Section Chief, Quality Assurance (Date)



## J~ t2", 2.c:>o2.. (Signature)



# ~ Keiji Shiraishi, B.S.t' 

# ( Sponsor: GLP STATEMENT 

### Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Bll-0443



### Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV-05H in Rats Study Code: B11-0443 I, the undersigned, hereby declare that this study was conducted in compliance with "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and ofthe Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 ofthe Planning and Coordination Bureau, Environment Agency (EA), No. 229 of the Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare (MHW) & 

### No. 85 (1984) ofthe Basic Industries Bureau, Ministry ofInternational Trade and Industry (MITI), March 31, 1984; Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, MHW & 

### No. 823 (1988) ofthe Basic Industries Bureau, MITI, revised on November 18, 1988) and with "Organisation for Economic Cooperation and Development (OECD) Principles ofGood Laboratory Practice" (May 12, 1981). Management: Signed in original May 29, 1998 Yoshifumi Fujino, M.S.

# (

( I I I 

### Bll-0443 

### QUALITY ASSURANCE STATEMENT Hita Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & 

### Testing Institute, Japan Sponsor: NISSHINBO INDUSTRIES, INC. R & 

### D CENTER Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV-05H in Rats Study Code: B11-0443 This study was audited and inspected by Quality Assurance Section ofHita Laboratory, Chemicals Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported ofthese results to the study director and management are as follows. Dates ofInspections Dates ofReports to Dates ofReports to and Audits Study Director Management October 28, 1997 October 31, 1997 October 31, 1997 January 21, 1998 January 22, 1998 January 22, 1998 January 27, 1998 January 27, 1998 January 27, 1998 February 2, 1998 February 3, 1998 February 3, 1998 February 3, 1998 February 3, 1998 February 3, 1998 February 18, 1998 February 18, 1998 February 20, 1998

March 3, 1998 March 4, 1998 March 4, 1998 March 13, 1998 March 13, 1998. March 13, 1998 March 23, 1998 March 24,. 1998 March 24, 1998 April21, 1998 April21, 1998 April21, 1998 April23, 1998 April 23, 1998 April23, 1998 April 28, 1998 April 28, 1998 April 28, 1998 May26, 1998 May 26, 1998 May 26, 1998 May28, 1998 May 29, 1998 May 29, 1998 May 29, 1998 May29, 1998 May 29, 1998 I, the undersigned, hereby declare that this report provides an accurate description ofthe methods and procedures used in this study and that the reported results accurately reflect the raw data obtained. Section Chief, Quality Assurance: Signed in original May 29, 1998 Keiji Shiraishi, B.S.

## (

I 

### \ Bll-0443 

### Study Code: B11-0443 Test Substance Code: HR3539 Sponsor Code: N-680 TITLE Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV-05H in Rats SPONSOR NISSHINNBO INDUSTRIES, INC. R & 

### D CENTER 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan TESTING FACILITY Hita Research Laboratories, Chemical Biotesting Center Chemicals Inspection & 

### Testing Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan PURPOSE OF STUDY The purpose of this study is to define the type, severity and reversibility of toxicological signs and to determine the no-observed-effect-level (NOEL) ofthe test substance by observing the functional and morphological changes in animals receiving repeated doses for 28 days. TESTING METHOD This study was conducted in accordance with "28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "Notification on Partial Revision ofTesting Methods Relating to the New Chemical Substances (Notification No. 700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry ofHealth and Welfare (MHW) & 

### No. 1014 (1986) of the Basic Industries Bureau, Ministry ofInternational Trade and Industry (MITI), December 5, 1986)", and with "Section 407, Repeated Dose Oral ToxicityRodent: 28-day or 14-day Study" prescribed in "OECD Guidelines for Testing of Chemicals (May 12, 1981)".I \

i 

## \ Bll-0443 GLP COMPLIANCE This study was conducted in compliance with "Concerning Testing Facilities Stipulated in Article 4 ofthe Order Prescribing the Items ofthe Test Related to the New Chemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, EA, No. 229 ofthe Pharmaceutical Affairs Bureau, MHW & 

### No. 85 (1984) ofthe Basic Industries Bureau, MITI, March 31, 1984; Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, MHW & 

### No. 823 (1988) of the Basic Industries Bureau, MITI, revised on November 18, 1988) and with ..OECD Principles of Good Laboratory Practice" (May 12, 1981). PERIOD OF STUDY Commencement ofStudy: Animal Receipt: Start ofDosing: Terminal Autopsy ofthe Dosing Period: Start ofthe Recovery Period: Terminal Autopsy ofthe Recovery Period: Completion ofStudy: October 31, 1997 January 27, 1998 February 3, 1998 March 3, 1998 March 3, 1998 March 17,1998 May 29, 1998 LOCATION AND PERIOD FOR RETENTION OF RAW DATA AND SPECIMENS Data and specimens, and each remaining lot ofthe test substance will be retained in the archives and test substance storage room, respectively, of Hita Research Laboratories for 10 years following the date of completion of study. After termination of the retention period, any measures taken will be done so with the approval of the Sponsor. Samples and specimens that are liable to deteriorate markedly will be retained only for as long as the quality ofthe preparation permits evaluation with the Sponsor's consent.

# ( I ,i ' i 

### AUTHOR AND PERSONNEL CONCERNED WITH STUDY AUTHOR Bll-0443 

### Study Director: Signed in original May 29, I998 Takaaki Umano, M.S., D.V.M.

General Toxicology Section HitaResearch Laboratories PERSONNEL CONCERNED WITH STUDY Study Staff: Takako Muroi Junior Laboratory Animal Technician Person in charge ofPathologic Examination: Kanji Yamasaki, Ph. D., D.V.M. Person in charge ofClinical Chemistry: Keiji Shiraishi, B.S. Person in charge ofChemical Analysis: Takeharu Wada, M.S. Person in charge ofDivided Work: Teruhiko Kanabayashi, Ph.D. Toxicological pathologist, Diplomate J.S.T.P. Bio pathology Institute, Ltd.

# (

( 

### Bll-0443


### TABLE OF CONTENTS SUMMARY 

### ...................................................... 1 MATERIALS AND METHODS 1. TEST SUBSTANCE 

### ······················································ 2 2. ANIMALS ······················································ 3 3. HOUSING CONDITIONS ...................................................... 3 4. 

### GROUPING ······················································ 4 

### 5. PREPARATION OFTEST SUBSTANCE ································· 4 6. DOSING 

### ······················································ 5 7. OBSERVATION AND MEASUREMENT 

### ................................ 5 8. STATISTICAL ANALYSIS ...................................................... 9 

### UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 9 RESULTS 1. CLINICAL SIGNS ...................................................... 10 2. BODY WEIGHTS ...................................................... 10 3. FOOD CONSUMPTION ...................................................... 10 4. HEMATOLOGICAL EXAMINATIONS ................................ 10 ································ 11 5. BLOOD CHEMICAL EXAMINATIONS 6. URINALYSIS ······················································ 11 7. ORGAN WEIGHTS ······················································ 11 8. AUTOPSY ······················································ 11 9. HISTOPATHOLOGICAL EXAMINATIONS ································ 12 DISCUSSION ······················································ 13

### FIGURES 1. Mean body weights 2. Mean food consumption TABLES 1. Clinical signs 2. Mean body weights 3. Mean food consumption 4. Hematology 5. Blood chemistry 6. Urinalysis 7. Absolute organ weights 8. Relative organ weights 9. Gross pathological findings 10.Histopathological findings PHOTOS 1, 2 ADDENDA 1. Clinical signs 2. Body weights in individual rats Bll-0443


### 3



### ······················································ 2



## ...................................................... 5

...................................................... 7 ······················································ 9



### ...................................................... 11 

### ······················································ 12

...................................................... 13



## ...................................................... 14 ······················································ 15

................................ 1



### ................................ 5 3. Food consumption in individual rats ................................ 10 

### 4. Hematology in individual rats 

### ................................ 13 5. Blood chemistry in individual rats ································ 17 6. Urinalysis in individual rats 7. Absolute organ weights in individual rats 

### ································ 21

................................ 23 8. Relative organ weights in individual rats 9. Pathological findings in individual rats APPENDIX 

### ································ 25 ································ 27 PHYSICOCHEMICAL REPORT

# ( Bll-0443 

### SUMMARY 

### A 28-day repeated-dose toxicity study oforally administered V-05H followed by a 14- day recovery period was performed in groups of6 male and 6 female Crj: CD (SD) rats at 5 weeks of age. Three dose levels were selected with the highest dose of 1 ,000 mg/kg/day, in addition to 200 and 40 mg/kg/day. Recovery groups were prepared for the 1,000 mg/kg and vehicle control groups. In this study, there were no death according to the test substance administration. No abnormalities were noted in the clinical signs, body weight, food consumption during the dosing period and in the hematological examination, blood chemical examination, urinalysis, autopsy and histopathological examination at the end ofthe dosing period. In the organ weight, increased relative kidney weights were noted in the female of 1 

### ,000 mg/kg group at termination ofdosing period. In the recovery study, no abnormalities were noted according to the treatment. Based on these results, NOEL ofV-05H for rat was considered to be 200 mg/kg/day in this study.

- 2-/ I \



### ( \_



### ( 

### \ 

### 1.8 Stability: Melting Point: Boiling Point: Vapor Pressure: Partition Coefficient: Solubility: Degree ofSolubility: Storage Conditions Bll-0443 

### The structure changes by additional reaction in the presence of active hydride (carboxylic acid, alcohol, amine and others). The stability test was conducted in our laboratories, and confirmed that the test substance was stable during the dosing period. Soluble in oil Water: DMSO: Acetone: Others: Stored in the sealed container at the cold and dark place. The high-temperature and humidity was avoided. 1.9 Precautions Goggles, glove, mask, head cap and lab coat were worn. Washing hands and gargle at the end ofhandling. 2. ANIMALS Cij: CD (SD) rats (SPF) were obtained from Charles River Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan). The animals were quarantined and acclimatized, and healthy animals with favorable weight gains were allocated to groups to ensure homogeneity of mean body weight using body weight-stratified randomization. The animals were 5 weeks old, and weighed 128.8-142.8 g for males and 111.1-125.7 g for females at the start ofdosing. The animals were identified by ear-tagging. 3. HOUSING CONDITIONS The barrier-system animal room was maintained at a temperature of23±2°C and a relative humidity of 55±10% with 10-15 air changes per hour and artificial light for 12 hours (between 7:00 and 19:00). The animals were housed individually in a hanging stainless steel cage with wire-mesh floor (165 W x 

### 300 D x 

### 150 H mm, TOKIWA KAGAKU KIKAI CO., LTD.). The trays were changed twice a weekly, cages once weekly and racks once four weeks. The racks and cages were

- 3-( 1\ 

### Bll-0443 

### identified by individual cards. The animals had free access to MF pelleted diet (Oriental Yeast Co., Ltd.) and water (chlorinated) from the Hita City supply via automatic watering system with sipper tubes. The diet and housing materials were autoclaved at 121

# oc for 30 min prior to use. Analysis ofcontaminants in both the diet and drinking water confirmed that they would not affect the test system. No environmental deviations, which might affect the study results, were noted during the quarantine, acclimation or study periods. 4. GROUPING The grouping was as follows: Dose Volume Concent- No. ofAnimals (Animal No.} GrouE

**(m&kg/da~)** 

### (ml/kg) ration(%} Males Females Vehicle control 0 10 0 6( I 

### - 6 ) 6 ( 37 - 42 ) Vehicle control (recovery) 0 10 0 6 ( 7 - 12 ) 6 ( 43 - 48 ) Low dose 40 10 0.4 6 ( 13 - 18 ) 6 ( 49 - 54 ) Intermediate dose 200 10 2.0 6 ( 19 - 24 ) 6 ( 55 - 60 ) High dose 1,000 10 10.0 6 ( 25 - 30 ) 6 ( 61 - 66 ) High dose (recovery) 1,000 10 10.0 6 ( 31 - 36 ) 6 { 67 - 72 ) 

### Reason for dosage selection: A 14-day preliminary repeated-dose oral toxicity study was carried out at 3 doses of 50, 250 and 1,000 mglkg/day. As a result, no death occurred in all groups. Abnormalities were noted in organ weights in female ofthe 1,000 mg/kg group. Therefore high dose level at 1,000 mg/kg/day and two lower doses at 200 and 40 mg/kg/day were set in the main study. Recovery groups were prepared for the 1,000 mg/kg and vehicle control groups. 5. PREPARATION OF TEST SUBSTANCE 5.1 Preparation The test substance was weighed accurately and ground with dry ice to a fine powder in an agate mortar. The powder was mixed with gum arabic (5.0 w/v% of final cone., Lot No. WTP0132, Wako Pure Chemical Co., Ltd.) and purified water (Lot No. A060, Takasugi Pharmaceutical Co., Ltd.) to make 10.0 and 2.0 w/v% once weekly. The lowest concentration of 0.4 w/v% was diluted from 10.0 w/v% prior to use.

- 4-



# ~11

# ( 5.2 

## 6. Bll-0443 

### Stability and Homogeneity Tests The stability and homogeneity of the test substance in above preparations were confirmed by our laboratories. DOSING Treatment by oral gavage was carried out daily using a Nelaton catheter (TERUMO CORPORATION) and a syringe (TERUMO CORPORATION) in the morning for 28 days, and the subsequent 14 days were used as a recovery period. 7. OBSERVATION AND MEASUREMENT The day ofthe start ofdosing was defined as day 1, and the day before as day -1. The week ofthe start ofdosing period was defined as week 1. Also, the next day ofthe final dosing was defined as recovery day 1, and the week ofthe start ofthe recovery period as recovery week 1. 7.1 Clinical Signs All animals were observed at least once daily. 7.2 Body Weights All animals were weighed as follows: Before Dosing: day -1 (at the time ofgrouping) During the Dosing Period: day 1 (at the start ofdosing), 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28 During the Recovery Period: day 1 (at the start ofthe recovery period), 3, 5, 8, 10, 12 and 14 In addition, immediately before autopsy, body weight was measured for calculation ofrelative organ weight. 7.3 Food Consumption Food consumption was measured as follows: Before Dosing: Once During the Dosing and Recovery Periods: Twice weekly

- 5-(

/ \ 

### B11-0443 7.4 Hematological Examinations All survivals were fasted overnight (16-20 hours) at the termination ofdosing and recovery periods, and blood samples were collected from abdominal aorta under ether anesthesia. Sodium citrate was used as an anticoagulant for examinations ofprothrombin time and activated partial thromboplastin time, and EDTA-2K for another items. Parameters Method 4 3 

### 1) Red Blood Cell Count (RBC) (xJO 

### /mm) System for detecting change in electrical resistance 2) White Blood Cell Count (WBC) 2 3 

### (xiO 

### /mm) System for detecting change in electrical resistance 3) Hemoglobin Cone. (Hb) (g/dl) Oxyhemoglobin method 4) 

### Hematocrit Value (Ht) (%) 

### System for detecting pulse 5) 

### Mean Corpuscular Volume (MCV) (Jlm3) __!!!__X 

### 103 

### RBC 6) 

### Mean Corpuscular Hemoglobin (MCH) (pg) Hb X 

### 103 

### RBC 7) 

### Mean Corpuscular Hemoglobin Cone. (MCHC) (%) 

### Hb X 102 

### Ht 4 

### 3 8) 

### Platelet Count (Platelet) (xiO 

### /mm) System for detecting change in electrical resistance 9) 

### Reticulocytes Count (Reticulo) (o/oo) 

### New methylene blue staining 10) Prothrombin Time (PT) (sec) Magnetic sensor system 11) 

### Activated Partial Thromboplastin Time (APTT)(sec) Magnetic sensor system 12) Differentiation ofLeukocytes (%) 

### Wright staining Stab Neutrophils (N-Band) Segmented Neutrophils (N-Seg) Eosinophils (Eosino) Basophils (Baso) Lymphocytes (Lymph) Monocytes(Mono) I) - 8) Microcell counter M-2000, Toa Medical Electronics 9), 12) MICROX HEG-120A, OMRON 1 0), 11) KC-1 OA, Amelung



### - 6-

### B11-0443 

### 7.5 Blood Chemical Examinations 

# ( Sera were separated from the blood samples collected at the same time of the hematological examinations and examined for following items. Parameters Method 1) GOT (IU/1) UV method (Method based on JSCC) 2) GPT (IU/1) UV method (Method based on JSCC) 3) Alkaline Phosphatase (ALP) (IU/1) p-Nitrophenyl phosphate method 4) Cholinesterase (ChE) (lUll) Butyrylthiocholine iodide method 5) y-GTP (IU/1) L-y-Glutamyl-p-nitroanilide method 6) Total Cholesterol (T-Cho) {mg/dl) COD·DAOS method 7) Triglyceride (TG) (mg/dl) GPO·DAOS method 8) Glucose (mg/dl) Glucokinase-G-6-PDH method 9) Total Protein (T-Protein) (g/dl) Biuret method 10) Albumin (g/dl) Bromocresol green method 11) AIG 

### Ratio Albumin T-Protein - Albumin 12) Blood Urea Nitrogen (BUN) (mg/dl) Urease indophenol method 13) Creatinine (mg/dl) Jaffe's method I 

### 14) Total Bilirubin (T-Bil) (mg/dl) Azobilirubin method \' 

### 15) Calcium (Ca) (mg/dl) OCPC method 16) Inorganic Phosphorus (IP) {mg/dl) Fiske-Subbarow method 17) Sodium (Na) (mEq/1) Crown-Ether membrane electrode method 18) Potassium (K) (mEq/1) Crown-Ether membrane electrode method 19) Chloride (Cl) (mEq/1) Coulometric titration method

1)-10), 12)-16) 7150 Automatic Analyzer, Hitachi 17)- 19) PVA- 

### a III, A & T 

### 7.6 Urinalysis Sixteen-hour urine samples were collected in individual metabolic cages, from all animals at the termination of the dosing (day 

### 28) and recovery (day 

### 14) periods and examined for volume, color and additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen using a test paper 

### (NMultistix®, Bayer·Sankyo ).



### - 7-( \



# (

( 7.7 Autopsy Animals were subjected to a detailed gross necropsy. 7.8 Organ Weight Following organs were measured in wet weight: Brain, Liver, Kidneys, Spleen, Adrenals, Testes, Ovaries 7.9 Histopathological Examinations 1) The following organs and tissues were preserved in 10% formalin: Bll-0443



### Brain (Cerebrum, Cerebellum), Hypothesis, Eyeball, Thyroid glands (with Parathyroid glands), Heart, Lung, Liver, Kidneys, Spleen, Adrenal glands, Stomach, Intestine (Duodenum to Rectum), Testes, Ovaries, Uurinary bladder, Bone marrow (Femur), Gross lesions. 2) Light microscopic examinations were performed for the following organs and tissues after embedding in paraffin, sectioning and hematoxylin and eosin staining. The histological preparation was conducted by Bio Pathology Institute, Ltd. according to the assignment protocol. Terminal Autopsy ofthe Dosing Period Vehicle control and 1 ,000 mg/kg groups: Liver, Kidneys, Spleen, Heart, Stomach, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Adrenal glands, Testes. 200 mg/kg/day group: Kidneys (females only), Stomach (females only) Terminal Autopsy ofthe Recovery Period Vehicle control and 1,000 mg/kg/day groups: Kidneys (females only), Stomach (females only), Testes Gross Lesions Terminal Autopsy ofthe Dosing Period 40 mg/kg group (Male, No. 15): Cerebrum Terminal Autopsy ofthe Recovery Period 1,000 mg/kg group (Male, No. 33): Kidneys Vehicle control group (Female, No. 43): Skin

- 8-i 

### \.

' 

# \ 8. Bll-0443 

### STATISTICAL ANALYSIS 

### Data regarding body weights, food consumption, hematological examination, blood chemical examination, urine volume and organ weight were analyzed using Bartlett's test for homogeneity ofvariance. Ifthe variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. When there was a significant difference inthis analysis, the difference between the vehicle control group and each ofthe treatment group was analyzed by Dunnett's test. Ifthe variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test was used. When there was a significant difference in this test, the difference between the vehicle control group and each ofthe treatment group was analyzed by nonparametric Dunnett's test. UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 

### There were no unexpected situations which might have affected the test results and deviations from protocol.

- 9-' ( \



### ( 

## RESULTS I. CLINICAL SIGNS (TABLE 1, ADDENDUM 1) 1.1 During Dosing Period No abnormalities were noted in all groups. 1.2 During Recovery Period Male: No abnormalities were noted in all groups. Female: Loss ofhair (1/6) was noted in the vehicle control group. 2. BODY WEIGHTS (FIG.I, TABLE 2, ADDENDUM 2) 2.1 During Dosing Period No abnormalities were noted in all groups. 2.2 During Recovery Period 

### 3. 3.1 3.2 No abnormalities were noted in all groups. FOOD CONSUMPTION (FIG.2, TABLE 3, ADDENDUM 3) During Dosing Period No abnormalities were noted in all groups. During Recovery Period ' 

### No abnormalities were noted in all groups. 4. HEMATOLOGICAL EXAMINATIONS (TABLE 4, ADDENDUM 4) 4.1 At Termination ofDosing Period No abnormalities were noted in all groups. 4.2 At Termination ofRecovery Period Male: No abnormalities were noted in all groups. Bll-0443



### Female: Decreased reticulocytes count and increased eosinophils ratio were noted in the 1,000 mg/kg group.

- 10 -



## ....---=---r--=-----r •---- -( \ '

( \. i 

### Bll-0443 

### 5. BLOOD CHEMICAL EXAMINATIONS (TABLE 5, ADDENDUM 5) 5.1 At Termination ofDosing Period Male: Decreased y-GTP activity was noted in the 40 mg/kg group. Decreased y-GTP activity and total protein level were noted in the 200 mg/kg group. Decreased total protein level was noted in the 1,000 mg/kg group. Female: Decreased GOT activity was noted in the 40 mg/kg group. 5.2 At Termination ofRecovery Period Male: Decreased albumin levels was noted in the 1,000 mg/kg group. Female: No abnormalities were noted in all groups. 6. URINALYSIS (TABLE 6, ADDENDUM 6) 6.1 At Termination ofDosing Period No abnormalities were noted in all groups. 6.2 At Termination ofRecovery Period No abnormalities were noted in all groups. 7. ORGAN WEIGHTS (TABLES 7,8, ADDENDA 7,8) 7.1 At Termination ofDosing Period Male: No abnormalities were noted in all groups. Female: Increased relative kidney weights were noted in the 1,000 mg/kg group. 7.2 At Termination ofRecovery Period No abnormalities were noted in all groups. 8. AUTOPSY (TABLE 9, ADDENDUM 9) 8.1 At Termination ofDosing Period Male: Blackish region of the mucosa in the glandular stomach (1/6) was observed in the vehicle control group. Partial defect ofthe cortex in the cerebrum (1/6) was observed in the 40 mg/kg group. Female: Pelvic dilatation ofthe kidney (1/6) and blackish region ofthe mucosa in the glandular stomach (1/6) were observed in the 1,000 mg/kg group. 8.2 At Termination ofRecovery Period Male: Apparent spottY pattern ofthe surface in the kidney (1/6) and small testis (1/6) were observed in the 1,000 mg/kg grouup. Female: Loss ofhair in the skin (1/6) was observed in the vehicle control group. -II-

# (

( Bll-0443 

### 9. HISTOPATHOLOGICAL EXAMINATIONS (TABLE 10, ADDENDUM 9) 9.1 At Termination ofDosing Period Male: Microgranuloma in the liver (+, 2/6), basophilic tubules (+, 116) and fibrosis(+, 116) in the kidney, and necrosis ofthe mucosa in the glandular stomach (+, 116) were observed in the vehicle control group. Partial defect of the cortex in the cerebrum (+, 111) was observed in the 40 mg/kg group. Microgranuloma in the liver(+, 1/6) was observed in the 1,000 mg/kg group. Female: Microgranuloma in the liver(+, 1/6) was observed in the vehicle control group. Basophilic tubules (+, 116), cyst formation (+, 116) and fibrosis (+, 1/6) in the kidney were observed in the 200 mg/kg group. Pelvic dilatation of the kidney (+, 1/6) and necrosis of the mucosa in the glandular stomach(+, 1/6) were observed in the 1,000 mg/kg group. 9.2 At Termination ofRecovery Period Male: Increased eosinophilic bodies in the kidney(++, 111) and focal atrophy of the seminiferous tubules in the testis(++, 1/6) were observed in the 1,000 mg/kg group. Female: Atrophy ofhair follicles in the skin(+, Ill) was observed in the vehicle control group. Cyst formation in the kidney(+, 116) was observed in the 1,000 mg/kg group.

- 12-/ 

### \ Bll-0443 

### DISCUSSION The oral toxicity ofV-05H was examined in male and female SD rats given a daily dose of40, 200 and 1,000 mg/kg/day for 28 days followed by the 14-day recovery period. In the present study, no deaths were observed throughout the study period. No abnormalities were noted in the clinical signs, body weights, food consumption during the dosing period or hematological examinations and urinalyses at termination ofdosing period, either. In the blood chemical examinations, decreased total protein levels were observed in male ofthe 200 and 1,000 mg/kg groups at termination ofdosing period. This change was considered to be of no toxicological significance, because this was within the historical control data in our laboratories and it had no other related findings. In organ weight, increased relative kidney weights was observed in female ofthe 1 ,000 mg/kg group at termination of dosing period. This finding was considered to be accidental one, since this was slight change and it had no related findings; absolute weight; blood chemical parameters, autopsy, and histopathological findings. But the elevation is suspectable change ofthe treatment. In autopsy, pelvic dilatation of the kidney, and blackish region of the mucosa in the glandular stomach were observed in female ofthe 1,000 mg/kg group at termination of dosing period. In histopathological examination, pelvic dilatation of the kidney, and necrosis of the mucosa in the glandular stomach were observed in female ofthe 1,000 mg/kg group at termination of dosing period. These changes were not considered related to administration of the test substance. Because they were limited to one each animal, and frequently found in normal rats. In the recovery study, the effect of the test substance noted in the dosing period was disappeared. At termination recovery period decreased albumin levels, apparent spotty pattern ofthe surface and increased eosinophilic bodies in the kidney, focal atrophy of

- 13 -( 

### Bll-0443 

### the seminiferous tubules in the testis were observed in male ofthe 1,000 mg/kg group, and increased eosinophils ratio and cyst formation in the kidney were observed in female of the 1,000 mg/kg group. These findings were considered to be no toxicological significance, since similar changes were not observed at the end of the dosing period; no related findings; histopathological changes of them might be spontaneous. The findings of testis were not considered related to administration of the test substance, since these changes were unilaterally and limited in one animal. The other statistically significant differences were not considered related to administration ofthe test substance, since there were no dose-relationship and with no related findings. Based on these results, the main effect ofV-05H was increased relative kidney weights in female ofthe 1,000 mg/kg group. But the effect was very weak. Accordingly, the NOEL ofV-05H for rats was considered to be 200 mg/kg/day in this study. Historical control data Parameter (unit)

Total Protein (g/dl) Mean±2S.D. At termination ofdosing period Male 5.9 ± 

### 0.2 n= 

### 186



### - 14- FemaleI _j I I **~** ! -1 l I l I I /-·---""' **~** y **~;** (g) 450 400 350 300 250 zoo 150 100 **~,** () : Vehicle control 0 : 40 mg/kg/day
£::. : 200 mg/kg/day
<> : 1,000 mg/kg/day **~·.** Bll-0443 Administration Recovery

-1 1 3 5 8 10 12 15 17 19 22 24 26 28 1
Figure 1 28-day repeated-dose oral toxicity study in rats
Mean

body weights : Male 3 5 8 10 12 14 (days)**~** ' l I N I
1 I I **~**

l i **~ ~**  **~** ' **~**

l -I i -I l (g) 250 200 150 100 () : Vehicle control

0 : 40 mg/kg/day
tJ. : 200 mg/kg/day
<> : 1,000 mg/kg/day Bll-0443 Administration Recovery

-1 1 3 5 8 10 12 15 17 19 22 24 26 28 1 3 5 8 10 12 14 (days)

Figure 1 -Continued Mean body weights : Female; **~** I -1 j I **~** I l ! i • w / **~** y **~** 0 40 30 >. o:! 20 "0 - ..., o:! .... - bO 10 0 **/--~----** .... Administration

1 4 8 11 15 18 22 Figure 2 28-day repeated-dose oral toxicity study in rats
Mean

food consumption : Male 25 28 4 0 : Vehicle control 0 : 40 mg/kg/day
t:. : 200 mg/kg/day
<> : 1,000 mg/kg/day Recovery 8 11 **/~** Bll-0443 14 (days)**~** I i **~** t 1 **~** ! I r i I l l i I **/--~ ~**  **~ ~**  .- 40 30 >. 201 <d "0 - ...., <d .... - 'b.() **~** 10 0 /--""'

,/ Bll-0443

__.-A Ill &:::,..__ **~** 0 : Vehicle control D :

40 mg/kg/day 6. : ZOO mg/kg/day

0 : 1,000 mg/kg/day Administration Recovery

1 4 8 11 15 18 22 25 28 4 8 11 14 (days)

Figure Z -Continued Mean food consumption : FemaleBll-0443 Table 28-day repeated-dose oral toxicity study in rats Clinical signs Sex Signs Administration Period Recovery Period 

## \ mg/kg/day 

### vc vc 40 200 1,000 1,000 

### vc 1,000 (R) (R) Male. ta ta ta ta ta ta ta ta 6' 6 6 6 6 6 6 6 No-abnormalities detected 6 6 6 6 6 6 6 6 Female ta ta ta ta ta ta ta ta 6' 6 6 6 6 6 6 6 No abnormalities detected 6 6 6 6 6 6 5 6 ------------ Loss ofhair 

## *Num'ler ofanimals examined. VC, Vehicle control;(R),Recovery. ta,terminal autopsy.



### - 1 -// /---, ,/--"'

Table 2 28-day repeated-dose oral toxicity study in rats Bll-0443

Mean

body weights(g) Sex Exp.group Number of Administration (mg/kg/day) animals -1 1 3 5 8 10 12 (days)

Vehicle 12 127.7 134.5 153.3 171.2 196.8 215.7 232.0

control ± 3.1 ± 2.8 ± 4.1 ± 5.2 ± 5.8 ± 7.0 ± 7.6

40 6 127.9 134.8 150.9 167.7 191.1 208.0 222.6

Hale ± 3.0 ± 3.0 ± 3.7 ± 4.7 ± 7.1 ± 8.0 ± 8.5

200 6 127.9 135.2 151.4 166.3 190.1 206.6 221.6

±

3.3 ± 4.3 ± 6.8 ± 8.3 ± 11.5 ± 13.6 ± 20.1

1,000 12 127.8 135.3 150.7 168.8 193.3 211.8 227.7

±

3.1 ± 3.0 ± 3.7 ± 4.1 ± 5.1 ± 7.7 ± 9.5

Vehicle 12 113.1 118.9 129.7 140.3 151.5 161.3 166.5

control ± 2.8 ± 2.7 ± 3.5 ± 4.4 ± 6.8 ± 8.6 ± 10.1

40 6 113.2 118.2 128.9 140.7 150.4 161.2 163.9

Female ± 2.9 ± 2.9 ± 4.6 ± 2.4 ± 2.8 ± 5.2 ± 5.0

200 6 113.2 118.7 131.0 139.7 155.4 161.6 170.3

±

2.9 ± 4.5 ± 6.1 ± 8.2 ± 12.6 ± 11.6 ± 14. 1

1,000 12 113.5 117.7 129.4 138.7 150.7 160.5 166.6

**~**

± 2.5 ± 3.2 ± 4.5 ± 6.4 ± 7.7 ± 9.2 ± 10.4

Hean ±S.D. t-v

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol.

! l l I **~**

1 l y **~ ~**  **~**

I I l l l I**/-~ ,~-~~-**  /-----

Table 2 -Continued Bll-0443

Mean body weights(g) Sex Exp.group Number of Administration (mg/kg/day) animals 15 17 19 22 24 26 28 (days)

Vehicle 12 257.2 277.3 291.7 315.5 331.5 341.3 352.9

control ± 10.4 ± 10.5 ± 12.0 ± 14.8 ± 15.6 ± 15.8 ± 17.2

40 6 244.0 260.8 274.0 295.3 307.6 318.4 327.4

Hale ± 10.1 ± 10.4 ± 12.1 ± 14.8 ± 14.2 ± 16.1 ± 16.1

200 6 245.8 263.4 276.4 296.0 310.0 320.0 331.1

±

20.3 ± 22.5 ± 26.4 ± 28.0 ± 29.5 ± 32.2 ± 31.3

1,000 12 252.3 268.9 282.7 304.1 318.3 328.1 339.9

±

11.5 ± 12.8 ± 15.2 ± 16.5 ± 19.1 ± 20.9 ± 20.9

Vehicle 12 172.8 183.0 187.0 197.3 202.0 207.8 210.8

control ± 10.1 ± 10.8 ± 12.3 ± 11.9 ± 14.5 ± 12.6 ± 15.7

40 6 173.5 179.1 187.2 199.0 201.0 206.1 208.8

Female ± 6.1 ± 8.4 ± 9.3 ± 7.1 ± 10.6 ± 11.1 ± 11.3

200 6 178.0 187.3 193.4 202.0 211.3 213.2 218.1

±

12.7 ± 10.4 ± 12.7 ± 13.7 ± 15.8 ± 15.5 ± 19.4

1,000 12 172.9 181.6 187.3 196.4 202.0 205.8 211.3

----1

± 12.5 ± 13.2 ± 14.0 ± 18.6 ± 16.7 ± 17.3 ± 17.7

I l w

i ' Hean ±S.D. * :

Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol.

i l ! j l **~**
I **~ ~** 

I **~**

I 1 **~** l I,_,--,, **/~**

Table 2 -Continued Bll-0443

Mean

body weights(g) Sex Exp.group Number of Recovery (mg/kg/day) animals 1 3 5 8 10 12 14 (days)

Vehicle 6 362.1 372.6 376.3 397.4 409.0 413.1 423.0

Male control ± 14.4 ± 15.9 ± 16. 4. ± 17.8 ± 18.9 ± 20.6 ± 21.6

1,000 6 348.8 360.7 367.4 385.4 396.5 401.0 415.5

±

20.3 ± 21.7 ± 25.4 ± 27.7 ± 28. 1 ± 29.3 ± 30.2

Vehicle 6 216.4 220;9 224.2 229.7 237.5 238.8 246.2

Female control ± 17.2 ± 17.4 ± 17.1 ± 21.5 ± 21.2 ± 19.7 ± 22.1

1,000 6 211.6 214.9 219.7 225.2 230.1 232.0 235.2

±

17.4 ± 17.9 ± 18.6 ± 19.3 ± 22.5 ± 20.0 ± 21.3

Mean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol.

**~** ' l ...,. ! **~** l j l **~ ~**  **~** ._.)

i V\ I . **-~** l I l I---, CJl l **~ ~**  **~ ~**  **~** <f'

l ; **-~** 1 I /-- Table 3 28-day repeated-dose oral toxicity study in rats
Mean

food consumption(g/rat/day)
Sex Exp.group Number of (mg/kg/day) animals 1 4 Vehicle 12 16.2 20.2 control

± 1.3 ± 1.2

40 6 16.3 19.7
Hale

± 1.4 ± 1.0 200 6 15.5 19.6

±

1.0 ± 1.3 1,000 12 15.7 20.2

±

1.0 ± 1.0 Vehicle 12 13.9 16.3 control

± 1.3 ± 0.9

40 6 14.5 16.5 Female

± 1.0 ± 0.7 200 6 13.6 16.5
±

2.2 ± 1.9 1,000 12 12.3 16.2

±

1.7 ± 1.1
Mean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. 8 - 23.1 1. 3. 22.1 ± 1.5 ± 21.6 1.8 22.8 1.2 17.4 ± 1.4 ± 17.4 ± 1.9 17.9 ± 2.0 ± 17.1 ± 1.1 ± Bll-0443 Administration 11 15 18 22 25 28 (days)
25.1 26.1 27.4 28.0 26.1 26.7

1.5 ± 1.8 ± 1.6 ± 1.9 ± 1.6 ± 1.9
23.8 24.3 24.9 25.5 24.8 24.9

1.7 ± 1.8 ± 1.7 ± 1.3 ± 1.6 ± 2.0
23.3 25.0 25.4 26.1 25.9 25.8

2.9 ± 3.1 ± 3.4 ± 2.9 ± 2.9 ± 2.7
25.2 26.1 26.5 27.1 27.0 26.9

2.0 ± 1.8 ± 1.8 ± 2.2 ± 2.1 ± 1.9
17.7 17.7 16.6 17.9 18.1 18.2

1.7 ± 1.8 ± 1.5 ± 1.2 ± 2.2 ± 1.8
17.9 17.6 15.5 17.8 18.6 18.3

1.9 ± 1.2 ± 1.8 ± 1.4 ± 2.0 ± 1.3
18.1 18.0 16.8 18.5 19.2 19.1

1.7 ± 1.3 ± 1.5 ± 1.5 ± 2.9 ± 2.6
17.8 17.7 16.7 18.1 18.5 18.8

1.1 ± 1.2 ± 1.1 ± 1.2 ± 0.9 ± 1.2_j **~** l I 1 l i **~** I ! l -i l 1 I (J) **~** ---..:> **~** :...::J Table 3 -Continued Mean

food consumption(g/rat/day)
Sex Exp.group Number of

( mg/kg/ day) animals 4 8 Vehicle 6 26.3 27.0

Hale control ± 1.3 ± 1.2 1,000 6 25.7 27.1

±

2.9 ± 2.5 Vehicle 6 18.5 19.2

Female control ± 2.4 ± 2.6 1,000 6 17.9 18.5

±

1.2 ± 1.3

Mean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. , .. **.-----.~"** Bll-0443 Recovery 11 14 (days) 27.8 27.5

±

1.5 ± 2.0 27.3 27.9

±

1.7 ± 2.2 19.7 20.4

±

2.2 ± 3.2 18.1 18.8

±

1.8 ± 1.7---: ,, ' l **~ ~**  ! l i -I I ' '

l ! I --J **~ ~**  **~** "" **~** ( Table 4 28-day repeated-dose oral toxicity study in rats Hematology Sex Hale Female Mean ±S.D. Exp,group (mg/kg/day) Vehicle control 40 200 1,000 Recovery __ _ Vehicle control 1,000 Vehicle control 40 200 1,000 Number of animals 6 6 6 6 6 6 6 6 6 6 RBC WBC Hb (x10 4 /mm 3 ) (x10 2 /mm 3 ) (g/dl) 741 112 15.3

±

41 ± 16 ± 0.6 765 95 15.3

±

30 ± 22 ± 0.5 754 100 15.2

±

21 ± 25 ± 0_4 762 100 15.4

±

3L

_____ LB__ ± 0.4 Ht (%) 44.1 ± 1. 7 44.7 ± 1.4 44.4 ± 1. 2 45.2 HCV (.um3) 59.6 ± 1. 6 58.4 ± 1. 8 58.9 ± 1. 0 59.4 __ LU

______ L2. 3 HCH (pg) 20.7 ± 0.6 19.9 ± 0.7 20.2 ± 0.2 20.2 HCHC (%) 34.8 ± 0.6 34.1 ± 0.3 34.3 ± 0.6 34.0
± 0&

__ **~** 0.3

825 93 15.8 45.9 55.7 19.1 34.3

±

23 ± 11 ± 0

. 4 ± 1. 3 ± 2 . 5 ± 0

. 7 ± 0 • 7

816 107 15.7 45.6 55.9 19.2

**34~4**

± 36 ± 25 ± 0.3 ± 1.0 ± 1.7 ± 0.6 ± 0.5

761 79 14.9 42.8 56.3 19.7 34.9

±

37 ± 26 ± 0. 3 ± 1. 5 ± 1. 8 ± 0. 8 ± 0. 6

763 75 15.3 44.0 57.7 20.0 34.7

±

27 ± 22 ± 0.5 ± 1.3 ± 1.4 ± 0.5 ± 0.3

769 79 15.4 43.7 56.8 20.0 35.2

±

33 ± 22 ± 0. 4 ± 1. 3 ± 0. 9 ± 0. 5 ± 0. 6

772 90 15.5 44.1 57.2 20.1 35.2

±

40 ± 29 ± 0.6 ± 1.1 ± 1.8 ± 0.5 ± 0.7 i\ecove"fY-------------------------------------------------------
Vehicle 6 789 75 15.4 43.6 55.3 19.5 35.3

control ± 24 ± 15 ± 0. 6 ± 1. 7 ± 0. 9 ± 0. 2 ± 0. 5
1,000 6 813 77 15.6 44.9 55.2 19.2 34.7

±

18 ± 25 ± 0.2 ± 0.7 ± 1.0 ± 0.4 ± 0.3

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. **~~-----,** Bll-0443 Platelet Reticulo P T APTT (sec) (xl0 4 /mm 3 ) (%,) (sec) 126.1 ± 14.6 124.6 ±_ 14.8 118.5 ± 12.8 117.6 ,± 4. 9 29 ±

9 25 ± 4 26 ± 5 30 ± 5 13.4 24.4 ±0.9

±3.2 13.4 25.3 ±

0.9 ± 2.5 13.6 27.5 ±1.3

±2.5 13.6 25.9
± o ._9 ±**_L~-----** 120.8 24 13.6 28.6

±

17.6 ± 7 ± 1.3 ± 2.2 116.6 21 13.5 28.6

±

7.3 ± 4 ± 2.2 ± 3.3 111.3 22 11.6 21.4

±

18. 2 ± 4 ± 0. 9 ± 3. 1 126.7 17 11.0 24.6

±

17.9 ± 5 ± 0.7 ± 5.3 121.9 26 11.3 24.2

±

10.5 ±10 ± 1.2 ± 3.3 124.1 22 11.5 24.2

±

10. 4 _ _!:_!.9

______ Lt·_L

___ **Ll~-~------** 113.7 ± 6.9 106.8 ± 10.7 26 ±

5 20* ± 3 12.4 ±

0.6 12.4 ± 0.4 21.7 ±

2.8 21.8 ± 1. 5**~** l I

' ::0

' I

i j I j **~** j l **~** ......_s.., **~ ~** 

l **~**

l **~**

**~** J l • I I _ ... **~----....__ /-~** 

Table 4 -Continued Hematology Bll-0443

Sex Male Female Mean ±S.D. Exp.group (mg/kg/day) Vehicle control 40 200 1,000 Recovery Vehicle control 1,000 Vehicle control 40 200 1,000 Number of animals 6 6 6 6 6 6 6 6 6 6 Differentiation of leukocyte (96)

-- N-Band N-Seg Eosino Baso LYJllph Mono

0.0 12.6 0.7 0.0 86.3 0.4
±0.0

± 4.0 ±0.8 ±0.0 ± 4.3 ±0.8

0.2 8.6 0.4 0.0 90.7 0.2

±0.3

± 1. 7 ±0.5 ±0.0 ± 1. 9 ±0.3

0.2 11.3 0.8 0.0 87.6 0.1
±0.3

± 3.3 ±0.4 ±0.0 ± 3.1 ±0.2

0.3 10.2 0.7 o.o 88.8 0.1

±o_.

3 ± 5.4 tQ.:..L_

±o.o

___ ± 6.2

____ ±o__,_?

_____

0.0 5.4 0.9 0.0 93.4 0.3
±0.0

± 1.8 ±1.1 ±0.0 ± 2.0 ±0.3

0.0 6.0 0.6 0.0 93.2 0.3
±0.0

± 2.6 ±0. 5 ±0.0 ± 3.0 ±0.3

0.3 9.2 1.3 0.0 89.3 0.0
±0.3

± 2.5 ±1.0 ±0.0 ± 2.6 ±0.0

0.2 8.6 0,8 0.0 90.4 0.1
±0.3

± 3.7 ±0.7 ±0.0 ± 4.1 ±0.2

0.2 10.9 1.1 0.0 87.8 0.1

±0.3

± 7.6 ±1.0 ±0.0 ± 7.6 ±0.2

0.1 9.1 1.1 0.0 89.4 0.3
±0.2

± 3.5 ±0.5 ±0.0 ± 3.4 ±0.4 Re-c-overy--------------------------------------------------------------
Vehicle 6 0.0 6.1 0.3 0.0 93.4 0.2

control ±0.0 ± 4.2 ±0.3 ±o.o ± 4.2 ±0.3
1,000 6 0.0 6.8 1.1* 0.0 92.0 0.2
±0.0

± 2.2 ±0.8 ±0.0 ± 2.1 ±0.3

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol.! **~** j I I **~** ' l I <0 **~ ~**  **~ '"~**  0 /- Table 5 28-day repeated-dose oral toxicity study in rats Blood

chemistry Exp.group Number of GOT GPT

Sex (mg/kg/day) animals (IU/1) (I U/1) Vehicle 6 81 20 control

±12 ± 1
40 6 76 20 ±17

± 3 200 6 74 20

Hale ±11 ± 3 1,000 6 81 21 ± 9 ± 2 Recovery Vehicle 6 67 19 control

± 8 ± 2 1,000 6 66 24 ± 7 ± 8 Vehicle 6 75 17 control

± 5 ± 2
40 6 62* 16 ± 6 ± 2 200 6 71 18

Female ±12 ± 5 1,000 6 78 17 **/~ ~·**  .. Bll-0443

ALP ChE y-{ITP T-Cho TG Glucose T-protein Albumin A/G ratio

(IU/1) (IU/1) (IU/1) (mg/dl) (mg/dl) (mg/dl) (g/dl) (g/dl)

363 57 0.7 73 110 116.0 6.0 2.9 0.93

±104 ± 7 ±0.2 ± 8 ± 32 ± 8.4 ±0. 2 ±0.2 ±0.11

383 61 0.4* 69 96 106.9 5.8 2.8 0.92

±

90 ± 23 ±0 .1 ± 8 ± 55 ± 10.6 ±0 .1 ±o .1 ±0. 05

350 53 0.4* 63 124 124.5 5.7* 2.8 0.95

±

97 ± 18 ±0 .1 ± 9 ± 24 ± 15.2 ±0.1 ±0.1 ±0.06

440

59 0.6 64 114 105.2 5.7** 2.8 0.95 **±_~**__

± 17

___ ±O.L

___ ±_s

___ ± 42_ ± 4.4 ±0.1 ±0 .1

_____ J.9...:..9_l

_____

328 54 0.6 64 81 138.8 6.0 2.8 0.86

±

42 ± 22 ±0.1 ±12 ± 24 ± 22.8 ±0.1 ±0 .1 ±0.02

274 51 0.7 54 so 152.5 5.9 2. 7* 0.83

±

67 ± 7 ±0. 2 ± 5 ± 20 ± 13.0 ±0. 2 ±0.1 ±0. 04

249 338 0.7 55 39 108.7 6.0 3.0 0.98

±

69 ±102 ±o .1 ± 7 ± 9 ± 9.5 ±0.4 ±0.2 ±0.07

253

414 0.7 54 37 125.2 6.1 3.1 1. 02

±

61 ± 82 ±0.3 ± 9 ± 10 ± 8.8 ±0.3 ±0. 2 ±0.07

232 359 0.9 64 38 119.3 6.0 3.0 0.98

±

50 ±137 ±0.1 ±12 ± 6 ± 15.2 ±0. 2 ±0.1 ±0 .10

216

325 0.9 66 34 125.0 6.1 3.0 0.99

± 8 ± 2 ± 23 ± 73 ±0.3 ± 8 ± 5 ± 11.1 ±0.3 ±0.1 ±0.06 Re-covery------------------------------------------------------------------------------------------------------------

Vehicle 6 70 19 181

control ± 5 ± 3 ± 42 1,000 6 66 17 197 ± 2 ± 4 ±

47

Mean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. 385 0.9 ±144

±0.3 417 0.9 ±191

±0.2 69 43 140.8 6.5 3.2 0.96

±15 ± 14 ± 28.9 ±0. 3 ±0 .1 ±0.04

72 38 133.7 6.4 3.0 0.90

± 8 ± 6 ± 23.0 ±0 .2 ±0. 2 iO .07----1 0 **~** l l l l **~** <::::::> **~**
I **~**
I **~**

l

**~**

I ' -l ! **~~,**

Table 5 -Continued Bll-0443

Blood chemistry Exp,group Number of BUN Creatinine T-Bil Ca IP Na K Cl

Sex

(mg/kg/day) animals (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mEq/l) (mEq/1) (mEq/l)

Vehicle 6 13.9 0.37 0.27 10.2 7.7 144 4.2 107.1

control ± 1.8 ±0.11 ±0.05 ± 0.2 ±0.4 ± 1 ±0.2 ± 1.5

40 6 12.0 0.32 0.28 10.6 7.7 144 4.3 106.4

±

2.1 ±0.05 ±0.04 ± 0.2 ±0.4 ± 1 ±0.3 ± 1.6

200 6 13.7 0.38 0.26 10.1 7.9 143 4.4 106.5

Hale ± 1. 5 ±0.05 ±0.05 ± 0.3 ±0.3 ± 1 ±0.3 ± 1.0

1,000

6 13.1 0.38 0.25 10.2 7.4 144 4.4 107.1

±

1.0 ±0.03 _±0. O,?__

___ i_O. 4 ±0.5

__ _j_

__ l_

__ ____tQ.,L

____ L_Q_._?_

__

Recovery

Vehicle 6 12.4 0.40 0.23 9.8 7.4 144 4.4 105.5

control ± 1.1 ±0.11 ±0.07 ± 0.2 ±0.3 ± 1 ±0.4 ± 1.1

1,000

6 12.6 0.39 0.20 9.6 7.7 144 4.3 105.5

±

1.0 ±0.04 ±0.02 ± 0.3 ±0.7 ± 2 ±0.4 ± 1.3

Vehicle

6 15.1 0.47 0.20 10.5 7.6 142 4.2 107.8

control ± 0.8 ±0.04 ±0.02 ± 0.5 ±1. 2 ± 1 ±0.3 ± 0.5

40 6 13.6 0.46 0.21 10.6 7.4 141 4.3 108.1

±

0.8 ±0.03 ±0.02 ± 0.3 ±0.6 ± 1 ±0.1 ± 0.5

200 6 14.3 0.44 0.21 10.5 7.5 142 4.3 108.0

Female ± 1. 6 ±0.05 ±0.04 ± 0.3 ±0.7 ± 1 ±0.3 ± 1.1

1,000

6 14.4 0.51 0.18 10.8 7.6 142 4.2 108.0
Rec-overT

±

**--~~_7_**_______ i<?

.. ! _ _o_1

____ _lg_,_Q_?

________ j

__ g_,_L

_____ **tQ_,_~**_________ t

.... L ___ ±__g_,_3

_______ L_!_._I__

___

Vehicle

6 14.9 0.45 control

± 2.2 ±0.06 1,000 6 14.3 0.40
±

1. 7 ±0.05
Hean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. 0.21 9.7 ±0.02

± 0.2 0.24 9.6 ±0.05

± 0.3 6.8 142 4.2 106.8 ±0.6

± 1 ±0.3 ± 0.4
6.8 142 4.1 107.2 ±0.3

± 1 ±0.3 ± 1.8 **~**----lI1-llilI-iIiJ·\Table628-dayrepeated-doseoraltoxicitystudyinratsUrinalysisSexMaleFemaleExp.group(mg/kg/day)Vehiclecontrol402001,000RecoveryVehiclecontrol1,000Vehiclecontrol402001,000RecoveryVehiclecontrol1,000a)Mean±S;D.Numberofanimals666666666666Volume•>(ml)25±1124±1218±618±1021±812±612±58±414±415±1310±711±4ColorSYY54 254 252 4554 23 32 43 3*SignificantlydifferentfromvehiclecontrolatP<0.05.**SignificantlydifferentfromvehiclecontrolatP<O.Ol.SYSlightlyyellowYYellowPH6.06.57.0033033024033024042z32 400420420244_ProteinKetonesBilirubin± +++- ± +05006060420060604200606023060604200606050056050600603z6006060060062 3060063 2060060506006OccultBlood±6060605606056060606060Glucose666666666666/.----......_Bll-0443Urobi(EU/dl)0.1666666666666---1 >-' N
I l -1 l l 1 l I **~ ~**  **~**

! l -c.. v

l ; I **··~** l • i I Table 7 Sex Male Female 28-day repeated-dose oral toxicity study in rats Bll-0443

Absolute

organ weights Adrenal Exp.group Number of Spleen Liver Kidney Brain Testis gland Ovary Body weight

(mg/kg/day) animals (g) (g) (g) (g) (g) (mg) (mg) (g)

Vehicle 6 0.60 9.58 2.26 1. 95 2.95 53.9 - 320.6

control ± 0.09 ± 1.10 ± 0.19 ± 0.08 ± 0.19 ± 3.4 - ± 18.6

40 6 0.56 8.86 2.31 1. 90 2.84 49.3 - 304.4

0.07 ± 0.54 ± 0.14 ± 0.04 ± 0.10 ± 7.0 - ± 15.6

200 6 0.59 8.89 2.22 1.85 2.77 47.4 - 307.6

± 0.11 ± 1.45 ± 0.23 ± 0. 06 ± 0.21 ± 8.0 - ± 30.8

1,000 6 0.59 8.48 2.33 1. 94 2.87 46.8 - 306.1

±

0.11 ± 0.89 ± 0.18 ± 0.09 ± 0.14 Recovery ---------------------------------------------- 5.7 **-----=------~____!1.:_7.._**___

Vehicle 6 0.66 11.13 2.51 2.00 3.20 51.5 - 396.1

control ± 0.07 ± 1. 02 ± 0.13 ± 0.09 ± 0.16 ± 4.0 - ± 19.1

1,000

6 0.67 11.12 2.65 1. 99 3.09 55.9 - 386.9

±

0.15 ± 0.96 ± 0.21 ± 0. 06 ± 0.54 ± 10.6 - ± 28.5

Vehicle 6 0.48 5.77 1. 53 1.81 60.4 99'. 0 199.5

control ± 0.08 ± 0.32 ± 0.11 ± 0.10 ± 3.8 ± 28.6 ± 14.6

40 6 0.41 5.90 1. 61 1. 79 65.9 95.8 195.6

0.04 ± 0.32 ± 0.12 ± 0. 09 ± 9.3 ± 12.2 ± 11.6

200

6 0.43 5.96 1.60 1.82

- 61.2 93.4 202.9

0.06 ± 0.90 ± 0.23 ± 0.11

- ± 8.9 ± 14.7 ± 15.5

1,000 6 0.41 5.93 1.64 1.85

- 66.9 96.7 194.1

±

0.06 ± 0.48 ± 0.14 ± 0.06

- ± 9.5 ± 22.9 ± 17.1 Recovery------------------------------------------------------------------------------------------------

Vehicle 6 0.45 6.37 1.60 1.87

- 63.7 90.8 225.2

control ± 0.04 ± 0.70 ± 0.08 ± 0.05

- ± 8.8 ± 8.9 ± 19.4

1,000 6 0.46 ±

0.06 6.07 ±

0.55 1. 71 ±

0.14 1.81 0.08 66.3 9.2 89.1 18.7 218.6 20.4

Mean ±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol.

**~~**_J ...... w **~** 1 ; _, ! l l -1S -.so .,...:> **~** .....c: ) I **-~** l , I l ,./- '"', Table 8 Sex Hale Female /- 28-day repeated-dose oral toxicity study in rats Relative

organ weights Bll-0443
Exp.group Number of (mg/kg/day)

animals Vehicle control 40 200 1,000 6 6 6 6 Spleen (g/lOOg) Liver (g/lOOg) 0.19 2.98 ±0.02

±0.22 0.18 2.91 ±0.02

±0.15 0.19 2.88 ±0.02

±0.25 0.19 2.77 ±0.03

±0.15 Kidney (g/lOOg) Brain (g/lOOg) Testis (g/lOOg) Adrenal gland (mg/lOOg)

Ovary Body weight

(mg/lOOg) (g)
0.71 0.61 0.92 16.8

- 320.6

±0.05 ±0.03 ±0.05 ± 0.8 - ± 18.6
0.76 0.63 0.93 16.3

- 304.4

±0.06 ±0.02 ±0.06 ± 2.5 - ± 15.6
0.72 0.61 0.91 15.7

- 307.6

±0.03 ±0.05 ±0.11 ± 4.0 - ± 30.8
0.76 0.63 0.94 15.3

- 306.1 ii"fico'Ver:-y----------------------- **_j_~_Q..iL_**_____ i_Q_._9_1

____ ±o :.9L..

_ **_l__l~**___ - ___ L..l7...:...7

__ _

vehicle

6 0.17 2.81 0.63 0.51 0.81 13.0

- 396.1

control ±0.02 ±0.15 ±0.04 ±0.02 ±0.06 ± 1.2 - ± 19.1

1,000

6 0.18 2.88 0.69 0.52 0.80 14.4

- 386.9

Vehicle control 40 200 1,000 6 6 6 6 ±0.03 ±0.12 ±0.05 ±0.03 ±0.13 ± 2.3 - ± 28.5

0.24

2.90 0.77 0.91

- 30.5 49.2 199.5

±o. 04 ±o. 07 ±o. 04 ±o. 07 - ± 3. 6 ± 11. 5 ± 14. 6

0.21

3.02 0.82 0.92

- 33.8 49.1 195.6

±o.01 ±0.13 ±0.02 ±0.06 - ± 5.7 ± 7.0 ± 11.6

0.21

2.93 0.79 0.90

- 30.3 46.0 202.9

±0.03 ±0.26 ±0.06 ±0.02

- ± 4.9 ± 6.7 ± 15.5

0 . 21 3 . 0 6 0

. 8 5** 0

. 9 6 - 3 4 . 4 4 9 . 5 19 4 . 1

±0.02 ±0.06 ±0.04 ±0.07

- ± 3.3 ± 9.0 ± 17.1 iiecoveii-------------------------------------------------------------------------------

Vehicle 6 0.20 2.83 0.72 0.84

- 28.5 40.4 225.2

control ±0.03 ±0.16 ±0.05 ±0.05 - ± 5.2 ± 3.3 ± 19.4

1,000

6 0.21 2.78 0.78 0.83

- 30.4 40.7 218.6

±0.01 ±0.18 ±0.07 ±0.07

- ± 3.8 ± 7.3 ± 20.4

Hean

±S.D.

* : Significantly different from vehicle control at P<0.05.

** : Significantly different from vehicle control at P<O.Ol. **~- ~**  l -l l 1 I I I i -1 l l l --, Table 9 28-day repeated-dose oral toxicity study in rats Gross

pathological findings Findings Vehicle control Vehicle control ,/-- Male 40 200 1,000 1,000 Vehicle control Vehicle control Female 40 **-~---** Bll-0
200 1,000 1,000
(Recovery) __ (Recovery) (Recovery) (Recovery) (mgtkg/d

ta*

ta ta ta ta ta ta ta ta ta ta ta

6**

6 6 6 6 6 6 6 6 6 6 6

Kidney Apparent spotty pattern of surface 0 0 0 0 0 1 0 0 0 0 0 0 .........................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

Pelvic dilatation 0 0 0 0 0 0 0 0 0 0 I 0

Glandular

stomach Blackish region of mucosa 1 0 0 0 0 0 0 0 0 0 0

Testis Small 0 0 0 0 0 Cerebrum Partial defect of cortex 0 0 0 0 0 0 0 0 0 0 0

Skin ..,. Loss of hair 0 0 0 0 0 0 0 0 0 0 0

*

ta,tern1inal autopsy. **Number of animals examined. ::)? **~** -C. """i -1 l **~** l I :.n **~** '3i **~** //_. ___ Table 10 28-day repeated-dose oral toxicity study in rats Histopathological findings Findings Vehicle control Vehicle control .. ....--------... Male 40 200 1,000 1,000 Vehicle control Vehicle control Female 40 **,~'** Bll-0 200 1,000 1,000
(Recovery) (_Recovery) {Recovery) (Recovery) (mg/kg/c

ta* ta ta ta ta ta ta ta ta ta ta ta

Grade 6** 6 6 6 6 6 6 6 6 6 6 6

Liver .. :t:!:<:>

.. **~.~-~<:>~.~l.\~~~-~** .. **~~~~-~~~~•••••••ou•ouoouoououuouo~/~.,~~•~•••uuuuu•u•=••••••••••••••••••ou•u••••=•••uuu•u•••••••uu•u•••=u••U''''"'''"''''''"'''''?.!.~••••••Um''"'"''''''''''"'='"''''"'''"'''"'''"''"'''''"'"''''''?./~''''''''''''''''"'"'''"''"'='''"''''''''''"''''''''''''** '''"'"''"'"'''''"'"''''"''"''''
Microgranuloma

+ 2/6 l/6 l/6 016

Spleen No abnormalities detected 6/6 6/6 6/6 6/6

Kidney **.,;t:!:<:>.,~.~-~.?.~.~-l.i~~~~.,~~~~-~~~~** . ., ... .,.,.,.,.,.,.,.,.,., . **.,.,?./~_,** . .,., ..... .,.,., .. .,.,.,.,::::., .. .,.,.,., .... .,.,.,.,.,.,.,::::.,.,.,,., • .,., .. .,.,.,., . .,.,:::::.,.,.,,.,.,., ..**.,.,.,.,.,.,~!.~** . .,.,., .. .,., . .,., ..... .,.9.!.l.,.,.,.,.,

.. .,., . .,., . **.,.,.,.,.,.,,.?.(,~.,.,.,.,** .. ., ..... ., ...... ?.!?.

. ., ...... .,., ... ., .. ., .. .,.,::::.,

.... ., .. ., .... ., .. .,., ... **~!?.** .. ., ...... ., .... ., ........ **?./~** ..... .,.,., ... ., .......... ?.!?..,

... ., ............... ., .. ., ..... ..

. **1.3..~-~<:>P!Y.F~** .. **~?.1:!.!~~** .......... ., .. ., ..... ., .. . ., .. ., .. .,.,., . ., .. **.,.,l/~** .. ., ........ ., .. ., ... ., .. .,.:::::

..... ., . ., ..... .,., .... ., .. .,::::

.. ., .... ., .. ., ... ., .......... :::::

.. ., ........... ., .. ., ...... Q!.?.

.................... ., .... 2!..1

... ., .... .,., ..... ., ................ **2t.~** .......... .,., .. ., ....... 9.!.?.

.......... ., ......... .,., . .,::::

......................... .,.!.!.?.

..... .,., .. ., ... ., .. ., .... 9/?.

........ ., . ., ............. 2!?.

. ., . .,.,., ....... .,., .... ., .... ., ..

... **~.Y..~~.,f.?.~.~~~<:>~** .... ., .. .,,.., .. ., ..... ., ..... ., .. ::

.. ., ..... ., ...... ., .. .,,9/?.

... .,.,., ................... :::::.,

... ., .. ., .. .,.,., .. .,., . .,:::::.,

....... .,., ....... ., ... ., .. ::::

........... ., ............... Q!..?.

.. ., ... ., ......... ., ... .,.2!..!

. .,.,.,.,., ........... ., ... ., .... **.,.9/~_,** .... ., ... ., .. ., ..... .,.9.!?.

.. .,., ... .,.,,. .... .,., ..... ::::.,

.. .,, .............. ., .... .U?.

... .,., .......... .,.,., .. .9.'.?.

....... ., .. .,., ........ .,)!?..,

.... ., ......... ., . ., .... ., ..... ..

**.!.)~r.<:>~.\.~_,** ........... .,., ............. ., ...... ., ........... ::

.... ., ..... ., .. ., .. ., .. .Y?..,

.......... ., . ., ... ., .. ., .. ::::

............ .,., .............. ::::

............................. :::::

.......... .,.,.,,.., .... .,9.!.?..,

.. ., ......... ., ... .,.,.,9.!.I

.......... .,., .. ., .... ., ........ ., .. **9.(.~** ... ., .. ., ........... ., .. .2!.?.

........... ., ............... ::::

.. ., . .,.,.,,..,,.., .. ., .. ...l.!.?.

............... .,.,.,., .. 9/?.

.. ., ..... .,,.,.,.,., . .,.,.9.!.?..,.,

. ., .. .,., ..... .,.,.,.,., ..... ..

Increased

eosinophilic bodies ++ 016 - - - 0/6 111 0/6 0/6 - 0/6 0/6 016 ........................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

Pelvic dilatation + 0/6 - - - 0/6 Oil 016 016 016 J/6 016

Heart No abnormalities detected 6/6 - - - 6/6 - 6/6 6/6
Forestomach No abnormalities detected 6/6 - - - 6/6 - 6/6 6/6 6/6 6/6 6/6

Glandular stomach

.. :t:!:<:>

.. **~.\?.~<:>~~-~-~~i.~~** .. **~~~~-~-~~~** ............................... **?./~** ............................ :::::

............................. ::::

............................. :::::

........................... ?.!.?.

............................ ::::

..................................... **~t.~** ......................... §!.§

............................ ::::

........................... ?.!.?.

.......................... ?./.?.

.......................... ?.!?.

.................................. .

Necrosis of mucosa + 1/6 - - - 0/6 - 0/6 0/6 - 016 1/6 0/6

* ta, terminal autopsy. **Number of animals autopsied.

* * * Ntunber of animals affected I Ntunber of animals examined. -,Not examined. +,slight;++, moderate.-l l -1 l l ...... O"l **~ ~**  -=1 **-~··** Table 10- Continued Histopathological fmdings Findings Grade Duodenum No abnonnalities detected Jejunum No abnonnalities detected ileum No abnonnalities detected Cecum No abnonnalities detected Colon No abnonnalities detected Rectum No abnonnalities detected Testis Vehicle control ta 6 6/6 6/6 6/6 6/6 6/6 6/6 Vehicle control 40 Male 200 I,OOO 1,000 (R.ecoveryl_ . ___ _ _ __ (Recovery) ta ta ta ta ta 6 6 6 6 6 6/6 6/6 6/6 6/6 6/6 6/6

No abnonnalities detected 6/6 6/6

- - 6/6 5/6 Vehicle control ta 6 6/6 6/6 6/6 6/6 6/6 6/6 Vehicle control 40 Female 200 Bll-C 1,000 1,000 (Recovery) ______ _ _ (Recovery) (mgikg/c ta ta ta ta ta 6 6 6 6 6 6/6 6/6 6/6 6/6 6/6 6/6 ..........................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

Focal atrophy of seminiferous tubules ++ 0/6 0/6 016 116
Adrenal No abnonnalities detected 6/6 6/6 6/6 6/6

Cerebrum Partial defect of cortex + Ill
Skin Atrophy of hair follicles + Ill

# (

Photo. 1

Photo. 2
( I \ .l:Hl-U44..;

Testis ofa male rat given vehicle orallyfor 28 days (recovery group). Normal. ' No. 7 animal. HE. x90.

Testis of a male rat given test substance at a dose level of 1,000 mg/kglday orally for 28 days (recovery group). Focal atrophy ofseminiferous tubules. No. 31 animal. HE. x90.



### - 1-**---~~~---**/ \

\ Addendum I 28-day repeated-dose oral toxicity study in rats Clinical signs Vehicle control Administration Signs Sex 2 3 1,2,3,4,' 1,2,3,4, 1,2,3,4, 5,6,7,8, 5,6,7,8, 5,6,7,8, 9,10,1 1, 9,10,1 I, 9,10,1 I, No abnormalities detected Male 12 12 12 37,38,39, 37,38,39, 37,38,39, 40,41,42, 40,41,42, 40,41,42, 43,44,45, 43,44,45, 43,44,45, Female 46 47 48 46 47 48 46 47 48 Loss ofhair (ventral neck) Male Female.

- 1 - BJ 1-0443 Recovery 4 2 (week) 1,2,3,4, 5,6,7,8, 7,8,9,10, 7,8,9,10, 9,10,11, 11,12 I 1,12 12 37,38,39, 40,41,42, 43,44,45, 44,45,46, 43,44,45, 46,47,48 47,48

. 46 47 48 43

# (

\ Addendum 1 -Continued Clinical signs 40 mg/kg/day Signs No abnormalities detected



### * Animal number. Bll-0443
Administration Recovery Sex 1 2 3 4 1 2 (week) Male 13,14,15, 13,14,15, 13,14,15, 13,14,15,


### ...................J.§,J.?.,J.?.......

## !..?.!!..?.!!..?.....J?.!!..?.!!..?.......**~.?.!n~~---·····························································** 49,50,51, 49,50,51, 49,50,51, 49,50,51, Female 52,53,54 52,53,54 52,53,54 52,53,54



### - 2 -( 

### \



# ( Addendum 1 -Continued Clinical signs 200 mg/kg/day Signs No abnormalities detected

* Animal number. Bll-0443
Administration Recovery Sex 2 3 4 1 2 (week) M I 19 20,21 • 19,20,21, 19,20,21, 19,20,21,

a e t ' 

### ..........••...•....&.'?.,&.:?.,&.4.......

## ?..?.!t2.!?.:±......**?.?:!?.~.~?.±**

## .....**.?.?.~?.~.'.?.~** 

## ................................................................. 55,56,57, 55,56,57, 55,56,57, 55,56,57, Female 58,59,60 58,59,60 58,59,60 58,59,60



### - 3 -Bll-0443 Addendum I -Continued Clinical signs 1,000 mg/kg/day 

# ( ,- Administration Recovery Signs Sex 2 3 4 I 2 (week) 25,26,27,

# . 25,26,27, 25,26,27, 25,26,27, No abnormalities detected Male 28,29,30, 28,29,30, 28,29,30, 28,29,30, 31,32,33, 31,32,33, 31,32,33, 31,32,33, 31,32,33, 31 ,32,33, 34,35,36 34,35,36 34,12 36 34,35,36 34,35,36 34,35,36 61,62,63, 61,62,63, 61,62,63, 61,62,63, Female 64,65,66, 64,65,66, 64,65,66, 64,65,66, 67,68,69, 67,68,69, 67,68,69, 67,68,69, 67,68,69, 67,68,69, 70,71,72 70,71,72 70 71 72 70 71 72 70 71 72 70 71 72



### - 4 -Addendum 2 28-day repeated-dose oral toxicity study in rats Bll-0443

Body weights(g) Sex Exp.group Administration (mg/kg/day) Animal No. -1 1 3 5 8 10 12 (days)

1 130.7 134.0 158.2 175.7 203.1 220.2 238.0

2 128.5 133.8 152.3 176.3 199.4 220.3 237.0

3 126.5 135.7 155.6 172.6 199.5 218.6 235.6

4 132.1 136.5 156.3 172.6 196.2 214.3 234.6

5 123.7 130.8 147.6 162.5 183.9 199.0 215.0

Vehicle 6 124.3 132.2 148.7 168.3 196.1 214.3 228.1

control 7 125.7 136.6 155.4 175.5 199.3 220.4 236.9

8 127.0 136.0 154.2 172.3 201.5 217.7 237.2

9 130.7 134.7 151.0 166.8 193.1 213.3 228.1

10 128.2 136.3 151.0 170.7 191.5 213.2 225.2

11 131.3 138.6 160.8 178.6 204.7 227.2 242.3

12 123.4 128.8 149.0 163.0 193.6 209.4 226.5

13

124.2 130.5 147.2 161.6 182.4 198.2 214.6

14 130.3 136.9 151.8 168.9 196.9 213.3 224.7

40 15 126.6 133.6 149.2 167.6 192.2 210.1 225.4

16 125.2 132.7 147.8 164.6 185.7 202.6 214.7

17 131.8 136

.. 7 157.3 175.6 201.1 220.0 237.0

Hale 18 129.3 138.4 152.2 168.0 188.1 203.5 218.9

19

126.1 135.6 150.9 168.3 198.2 216.0 234.3

20 128.3 132.3 147.1 162.9 190.0 206.4 216.9

200 21 133.5 142

.. 8 163.1 178.1 203.2 219.8 243.0

C.l1

22 129.3 136.6 154.9 170.4 192.4 210.9 227.2

23 126.0 132.7 148.7 165.1 186.6 205.3 223.4

24 124.1 131.0 143.7 153.2 169.9 181.2 184.9

25 132.1 141.9 155.9 171.9 197.7 218.0 233.3

l

26 126.3 133.7 147.8 164.5 184.9 200.2 210.4

27 130.6 135.7 152.7 168.7 192.1 211.2 225.4

**~**

28 124.0 131.8 147.4 162.8 187.8 204.1 219.5

29 128.3 135.2 149.8 166.9 190.1 205.1 218.5

1,000 30 125.4 132.9 145.4 167.4 193.4 210.0 226.9

31 130.8 135.8 149.0 168.2 191.6 207,0 221.9

1 32 123.2 132.5 146.9 165.5 190.4 208.8 225.8

33 128.7 138.8 154.9 172.9 198.4 220.0 233.7

34 125.4 133.6 151.4 168.7 192.9 213.0 234.7

I 35 131.9 138.0 156.7 178.1 203.8 227.2 244.7

l

36 126.7 133.1 150.9 170.0 196.2 217.0 237.2

l **~ ~**  **~**

i I **~** 'vJ

1 l I I i**/~~**

Addendum 2 -Continued Bll-0443

Body

weights(g) Sex Exp.group Administration (mg/kg/day) Animal No. 15 17 19 22 24 26 28 (days)

1 263.6 282.5 297.6 321.1 337.0 344.7 357.1

2

256.8 279.0 288.0 307.7 322.5 329.4 337.5

3

264.7 286.5 297.9 332.2 346.6 354.1 369.1

4

260.5 278.9 301.4 323.3 345.2 356.5 371.1

5

234.1 253.1 264.5 285.5 301.8 311.4 321.0

Vehicle

6 251.5 269.8 285.5 302.2 322.1 326.0 337.2

control

7 257.2 277.8 292.7 317.0 328.6 342.0 354.1

8

263.1 280.7 297.6 317.4 334.0 346.9 359.8

9

253.8 274.6 287.4 310.7 321.9 333.9 341.6

10

254.4 278.3 290.0 313.1 329.6 339.9 352.9

11

277.2 296.6 314.0 344.7 363.6 372.4 383.6

12

249.8 269.5 283.6 311.2 325.6 338.6 349.8

13

239.7 255.1 269.8 292.3 304.8 318.2 326.7

14

247.1 264.9 279.1 305.7 315.2 322.1 333.4

40

15 245.1 265.6 277.5 297.9 314.7 327.2 333.8

16

233.7 250.0 261.5 280.4 294.1 307.0 314.0

17

261.8 277.4 293.7 316.9 327.2 340.9 350.9

Hale

18 236.7 251.8 262.5 278.6 289.7 294.7 305.5

_j

19 259.7 277.1 294.6 315.2 331.4 339.7 355.6

20

245.2 260.9 271.0 291.2 306.4 316.4 322.9

200

21 263.7 284.6 301.1 322.9 336.3 352.4 360.3

22

250.4 269.7 280.2 298.9 310.2 320.5 330,6

O'l 23 248.7 267.2 284.1 304.3 320.8 331.0 342.9

24

206.8 220.6 227.1 243.7 254.7 259.8 274.1

25

263.7 282.2 298.5 322.7 339.3 354.7 368.4

26

232.4 246.7 256.2 278.9 288.9 296.6 313.5

27

247.0 261.7 275.2 295.1 305.9 320.2 329.7

28 248.4 262.8 274.6 296.7 307.1 314.3 327.7

29 240.5 258.1 267.2 285.3 301.6 308.7 317.2

1,000 30 250.8 268.2 283.8 302.7 315.5 329.2 334.7

31 245.1 260.6 273.4 293.0 307.4 312.4 326.9

32 248.8 265.7 279.6 304.9 314.5 325.6 334.0

33 251.1 266.1 280.1 298.2 313.1 318.6 333.7

34 265.5 284.2 299.7 324.3 345.3 356.6 372.5

l 35 273.0 291.3 308.5 333.2 351.9 362.9 374.1

1 I

36 261.3 279.4 295.1 313.8 329.4 337.6 346.1

**~ ~**  **~ ~.**  -c..
I l 1 j I**~'**

Addendum 2 -Continued Bll-0443

Body weights(g) Sex Exp.group Administration (mg/kg/day) Animal No. -1 1 3 5 8 10 12 (days)

37 116.2 121.4 133.1 145.1 157.7 165.1 173.0

38 111.2 116.5 127.9 135.3 144.1 157.6 153.6

39 110.0 117.3 126.1 138.5 148.9 157.5 165.5

40 115.0 118.1 129.5 143.6 156.1 168.3 172.4

41 113.6 120.2 132.5 144.3 158.9 170.1 176.0

Vehicle 42 112.9 119.3 124.6 136.5 142.5 148.9 156.4

control 43 114.9 118.5 133.9 137.8 147.5 155.3 154.3

44 110.6 115.6 127.5 137.7 154.7 163.2 174.0

45 113.6 121.4 132.9 144.1 158.9 174.2 177.3

46 107 .,8 114.5 124.3 134.0 143.9 152.2 157.4

47 113.7 119.6 130.5 138.8 145.3 151.8 157.4

48 117.8 123.8 133.3 147.6 160.0 171.7 180.2

49

113.6 120.9 128.7 141.1 149.2 156.6 159.1

50 114.5 li9.1 134.6 143.4 153.8 167.1 168.7

40 51 115.3 118.5 130.5 138.0 146.8 154.7 157.0

52 108.2 113.1 121.0 139.2 153.9 166.5 168.7

53 111.7 116.9 127.2 139.0 149.2 158.9 163.2

Female 54 115.9 120.9 131.5 143.7 149.6 163.5 166.4

**~**

55 109.1 114.3 126.5 130.8 145.3 147.7 161.0

56 112.1 116.3 130.7 139.6 154.8 160.0 169.9

200 57 115.0 122.9 137.4 148.3 168.4 175.3 183.7

--.]

58 111.9 116.5 127.2 138.3 150.5 161.8 168.2

59 117.5 125.7 139.5 150.1 172.5 174.5 188.3

60 113.8 116.6 124.6 131.3 141.1 150.3 150.4

61

112.3 117.7 131.0 139.2 152.7 160.2 170.5

j

62 116.8 121.9 136.6 149.5 159.2 171.2 177.6

63 115.5 117.4 129.9 142.3 155.0 164.7 170.5

i 64 114.2 119.9 134.7 144.5 165.0 173.1 182.3

---1

65 110.5 116.6 126.9 138.6 144.7 156.8 159.6

1,000 66 111.4 111.1 120.6 126.2 138.8 147.8 149.4

I 67 114.7 119.1 126.8 131.4 140.2 143.8 152.4

' **~**

68 110.8 113.5 125.7 134.0 148.4 159.8 167.3

69 115.4 122.2 135.0 143.6 156.5 169.8 178.2

70 112.7 116.6 128.9 137.7 151.8 166.9 171.0

71 117.2 119.2 129.9 142.2 150.5 159.5 160.5

l 72 110.2 117.4 126.6 135.0 146.0 152.6 159.5

-I **~ ~·**  . '·:

-"" **·~** '-1\

I , i I I • 1 ; I**~-**

Addendum 2 -Continued

Bll-0443

Body weights(g) Sex Exp,group Administration (mg/kg/day) Animal No. 15 17 19 22 24 26 28 (days)

37 179.9 186.6 196.3 198.2 214.1 208.1 218.6

38 169.5 177.7 181.9. 194.2 189.0 203.3 197.4

39 164.8 182.5 177.3 197.6 201.5 207.9 212.7

40 181.7 195.9 198.5 212.1 216.5 227.1 228.5

41 177.8 192.9 200.1 207.1 216.1 214.0 228.9

Vehicle 42 156.5 168.3 168.4 178.5 185.3 189.7 190.4

control 43 165.2 173.1 175.7 184.4 183.5 194.9 191.2

44 175.5 184.6 191.0 198.3 208.6 214.6 214.1

45 190.5 197.9 207.4 217.2 219.1 227.8 231.2

46 159.5 169.1 173.4 182.3 186.3 194.9 196.9

47 171.3 173.1 180.7 192.2 188.2 195.6 195.6

48 181.6 194.8 192.9 206.0 216.3 216.0 224.5

49 172.3 183.8 187.9 197.5 204.9 199.6 213.9

50 177.7 184.7 187.7 202.6 197.6 209.5 204.2

40 51 163.7 163.1 173.9 187.6 186.0 188.2 195.6

52 181.5 184.6 202.1 207.2 218.3 220.9 228.5

53 171.0 176.5 182.1 195.0 201.1 207.0 206.2

Female 54 174.5 182.0 189.4 203.8 198.0 211.3 204.6

55 163.3 178.0 178.1 191.7 195.6 199.5 201.3

-1 56 177.3 190.0 194.4 200.6 210.5 218.2 211.4

200 57 191.9 194.1 206.0 219.4 224.1 228.9 234.0

ex:> 58 176.7 186.3 188.5 197.5 207.5 208.8 211.2

59 193.4 201.8 210.1 217.3 234.9 230.8 249.3

60 165.2 173.7 183.1 185.4 195.0 192.7 201.1

61 174.6 180.3 185.4 188.3 199.6 194.6 210.3

62 173.6 197.6 191.7 212.2 215.4 219.6 223.3

1 63 184.3 192.5 199.3 218.4 213.2 214.5 225.1

• 64 193.1 191.1 211.0 222.1 228.6 231.4 239.2

**~** 65 169.1 178.5 183.9 192.5 202.1 203.5 210.1

1,000 66 150.8 157.5 165.0 165.8 174.3 176.7 181.8

67 157.1 161.1 166.6 168,0 178.2 178.7 187.9

68 168.9 181.6 188.1 197.0 203.1 211:7 212.4

69 188.3 197.0 202.6 213.1 220.1 224.8 230.3

70 lSi. 8 191.9 197.2 206.8 209.0 216.5 217.9

l 71 168.0 174.4 180.2 185.0 184.8 194.3 189.4

72 H)5. 3 176.0 176.6 187.4 195.9 203.3 207.8
**--------~**

i **~**

l i **~,** I

'-1\ **~**

l l l **~** l 1 I**----~,** __...------.._

**_,/---~** Addendum 2 -Continued

Bll-0443

Body weights(g) Sex Exp.group Recovery (mg/kg/day) Animal No. 1 3 5 8 10 12 14 (days)

7 359.2 369.1 369.3 387.2 398.2 397.9 404.4

8 359.9 372.5 373.8 393.0 404.3 409.7 418.9

Vehicle 9 348.6 354.3 360.7 382.9 391.0 395.7 403.9

control 10 357.9 368.5 373.9 395.2 409.7 413.4 425.8

11 390.3 402.3 408.2 432.6 445.1 452.7 463.0

Male 12 356.4 368.7 371.6 393.5 405.7 409.1 422.1

31 331.1 341.7 342.3 353.0 366.5 368.6 380.2

32 335.4 345.4 351.0 370.9 377.0 382.1 398.9

1,000 33 333.7 347.5 353.0 369.6 380.8 385.0 399.0

34 371.3 384.0 395.3 410.3 421.8 430.9 448.0

35 377.3 392.3 403.1 426.7 438.7 442.3 456.6

36 343.8 353.4 359.5 381.9 394.1 396.9 410.0

43 201.0 207.4 208.9 208.5 217.9 220.6 224.0

44 220.3 229.0 232.7 232.6 243.3 241.6 258.2

Vehicle 45 242.7 247.9 248.7 259.1 269.5 266.9 275.0

control 46 201.3 203.2 207.7 214.4 219.8 219.8 224.3

47 204.4 208.5 211.4 212.4 221.4 227.3 232.4

Female 48 228.9 229.3 235.5 250.9 252.9 256.8 263.5

j

67 191.4 189.0 196.7 202.2 198.1 209.3 208.4

68 219.0 219.8 229.3 235.3 236.9 244.8 241.1

I 1,000 69 232.4 235.4 239.7 245.7 255.1 250.7 259.8

£) 70 223.6 229.6 231.8 237.6 246.4 241.1 249.8

71 189.9 198.8 196.4 200.1 207.0 203.9 209.6

72 213.1 217.0 224.2 230.1 236.9 242.2 242.4

l " l l l -- i **~ ~**  **~ ~** 

• V)

I l ......:::r

l l l iAddendwn 3 28-day repeated-dose oral toxicity study in rats

Bll-0443

Food consumption(g/rat/day)
Sex Exp,group Administration (mg/kg/day)

Animal No. 1 4 8 11 15 .18 22 25 28 (days)

1

16.7 21.0 24.6 26.1 27.0 27.7 29.3 27.1 27.1

2 15.7 21.1 24.3 26.4 26.0 26.5 26.8 24.1 24.9

3 15.9 21.2 24.5 26.4 28.2 29.2 30.4 27.2 27.7

4 15.2 20.5 23.1 24.8 27.9 28.3 29.4 28.4 29.8

5 14.5 18.6 20.6 21.9 22.2 23.3 24.4 23.3 23.4

Vehicle 6 16.6 18.5 22.2 23.5 25.6 27.1 28.0 25.5 25.8

control 7 17.9 21.8 23.1 26.1 24.9 26.6 26.2 25.2 25.9

8 19.1 21.2 24.3 26.2 27.9 27.9 27.7 26.1 28.7

9 16.4 18.4 22.4 24.3 23.9 26.3 25.7 25.1 24.7

10 15.8 19.6 22.3 24.4 26.1 28.8 28.8 26.1 25.3

11 16.0 20.9 24.1 27.0 27.2 29.1 30.8 28.8 27.7

12 14.3 19.5 21.9 24.4 26.0 27.5 28.9 25.9 28.8

13 15.8 18.7 20.9 22.8 23.5 24.3 25.4 24.1 24.6

14 17.3 20.4 23.4 23.2 23.4 24.9 25.3 23.6 25.0

40 15 16.9 19.4 22.3 25.0 25.1 26.2 25.9 26.3 24.4

16 13.8 18.5 19.7 21.6 21.9 22.0 23.7 23.1 22.6

17 16.6 20.7 23.6 26.5 27.0 26.9 27.6 27.2 28.7

Hale 18 17.5 20.7 22.4 23.6 25.1 25

.. 0 25.3 24.5 24.2

19 15.3 19.4 22.1 24.8 26. 1. 25.2 27.5 27.0 26.0

---1

20 14.0 18.8 21.2 22.2 25.1 25.0 25.4 26.4 26.5

I 200

21 17.0 21.3 23.9 26.7 27.6 28.8 29.2 29.3 28.7

I-' 22 15.7 20.5 22.6 24.7 26.4 27.6 26.1 25.0 26.1

0 23 15.5 20.0 21.4 23.2 26.1 26.4 27.6 27.0 26.8

24 15.7 17.6 18.5 18.3 18.9 19.1 20.9 20.7 20.7

25 15.9 21.0 22.3 24.7 26.9 27.0 29.3 27.4 29.3

26 13.6 18.8 20.5 22.5 23.0 23.5 23.4 24.1 25.2

27 15.5 20.6 22.8 24.1 25.3 25.9 25.4 26.1 26.0

28 15.4 18.9 21.7 24.2 25.4 25.8 26.5 25.4 25.7

29 17.7 21.4 23.2 24.6 25.5 26.7 26.9 27.1 26.7

1,000 30 14.1 19.3 22.2 23.3 25.2 25.2 26.0 25.3 26.3

31 16.1 19.7 22.1 23.4 24.3 25.1 25.3 26.0 25.7

32 15.8 19.6 23.0 25.5 26.0 27.6 27.5 26.7 25.7

33 16.3 20.5 23.9 25.7 25.9 24.6 25.6 25.9 25.9

34 15.9 21.0 23.0 28.2 29.7 29.2 30.7 30.8 31.0

35 16.3 22.1 25.4 29.1 28.4 29.5 30.5 31.0 29.5

36 15.8 19.9 23.1 26.8 27.1 27.4 27.6 27.7 26.0

-I S> %) -1 **~**/-- **--~,** Addendum 3 -Continued

Bll-0443

Food consumption(g/rat/day)
Sex Exp.group Administration (mg/kg/day)

Animal No. 1 4 8 11 15 18 22 25 28 (days)

37

12.1 16.1 17.2 17.8 17.7 15.2 17.7 19.5 17.9

38 12.6 14.8 15. 9. 17.2 17.1 16.0 17.5 16.4 16.6

39 13.9 16.4 17.1 17.2 18.0 17.7 18.2 18.4 18.3

40 13.9 16.2 17.2 17.3 17.5 17.0 17.5 18.0 17.6

41 14.1 l6.3 17.5 18.8 18.1 17.8 18.7 20.0 19.8

Vehicle 42 12.5 15.4 16.1 15.8 15.8 16.2 16.9 15.7 16.4

control 43 13.1 16.1 16.2 16.3 15.5 14.5 15.6 15.5 16.2

44 12.7 15.5 18.9 18.4 18.3 16.3 19.1 19.0 19.8

45 16.1 17.7 19.9 21.2 21.3 18.9 19.8 21.7 21.5

46 14.3 16.4 16.6 17.8 15.9 15.0 16.2 16.6 17.2

47 15.2 16.9 16.4 15.0 16.9 15.7 17.9 15.3 16. 7.

48 15.7 17.9 20.0 19.4 20.6 19.0 19.2 20.7 20.6

49 13.0 15.8 15.5 16.2 16.6 15.7 16.4 18.5 17.3

50 14.2 17.2 17.7 18.6 17.5 15.1 17.4 17.4 18.2

40 51 14.1 16.2 16.1 15.8 16.2 13.0 16.4 15.7 17.3

52 15.2 15.8 19.4 19.6 19.1 17.3 19.6 21.7 20.7

53 14.4 16.4 15.9 16.6 17.0 14.1 17.4 19.0 18.0

Female 54 15.8 17.3 19.8 20.4 19.0 17.6 19.4 19.4 18.5

55 14.3 15.2 16.4 16.0 16.9 16.0 17.7 17.6 17.7

--:

56 11.9 16.2 17.2 17.6 17.7 15.5 18.3 18.1 17.1

200 57 15.0 18.8 20.7 20.6 19.4 18.9 20.0 20.2 22.0

,_. 58 10.2 14.9 16.2 17.3 16.5 15.2 16.9 16.7 16.8

- 59 16.3 18.8 20.2 19.9 19 .a 18.3 20.7 24.6 22.9

60 13.7 15.0 16.6 17.4 17.6 16.9 17.4 18.2 18.1

61 12.1 16.0 17.4 17.5 18.0 16.0 17.8 17.8 18.4

l 62 14.0 17.3 17.6 19.4 18.3 17.8 19.3 19.1 19.4

63 10.8 15.9 17.8 18.5 17.9 16.8 17.3 19.4 18.2

64 13.8 17.8 18.8 18.4 19.6 16.6 20.3 18.6 21.5

**-~** 65 13.8 16.8 16.5 17.1 16.8 16.2 17.9 18.4 19.7

1,000 66 9.7 15.8 17.2 17.2 17.7 16.6 17.9 18.2 18.9

I 67 12.1 15.2 14.3 15.4 15.4 14.0 15.7 17.0 17.4

**~** 68 9.6 14.4 16.3 17.3 17.2 16.4 17.9 19.0 17.9

69 14.4 17.7 18.3 19.1 19.5 17.9 19.8 20.2 19.7

- 70 11.3 16.5 17.4 18.2 18.6 17.2 18.3 17.5 18.7

l 71 12.4 15.1 17.1 17.8 16.6 17.9 17.2 18.9 17.4

I 72 13.5 15.4 16.7 18.1 16.8 16.6 17.9 18.2 18.6

:y **~** J\ **~**-l " ' l I I **~** I I 1 I ['..J 5> **~ ~**  **~** /------, Addendum 3 Sex Hale Female -Continued Food

consumption(g/rat/day) Exp.group (rog/kg/day) Animal No. 4 7 25.4

.
8 27.0 Vehicle

9 25.5 control

10 24.6 11

27.9 12

27. 3

.. 31 22.2 32

25.2 1,000

33 24.4 34

27.3 35

30.5 36

24.7 43

16.5 44

20.5 Vehicle

45 :20.1 control

46 16.4 47

16.3 48

2L3 67

16.2 68

18.1 1,000

69 18.1 70

t8.4 71

16.8 72

19.6 -------- ,/-----......_,
·---- -·- ---- **----~--·-·-** Bll-0443 Recovery
8 11 14 (days) 25.6 26.6 24.4 26.9

27.3 27.1 26.1

26.6 27.1 26.9

27.6 28.7 28.9

30.7 30.5 27.6

28.2 27.3 23.4 . 25.5 24.4 26.9

26.7 27.7 26.5

26.3 26.7 27.4

28.1 29.0 31.2

30.3 30.4 27.0

27.1 29.4 16.3

17.6 16.919.3

20.3 21.5 21.8

22.2 21.9 16.9

17.5 17.3 18.1

18.2 19.6 22.8

22.1 25.4 16.8

15.3 18.6 18.1

17.7 20.0 19.5

19.3 19.4 18.3

18.5 17.3 17.8

17.4 16.5 20.3

20.6 20.9,/--, Addendum 4 28-day repeated-dose oral toxicity study in rats Bll-0443

Hematology Exp.group RBC WBC Hb Ht HCV HCH HCHC Platelet Reticula p T APTT

Sex

(mg/kg/day) Animal No. (xl0 4 /mm3 ) (xl0 2 /mm 3 ) (g/dl) (%) ( J.tm3 ) (pg) (%) (xl0 4 /mm 3 ) (%,) (sec) (sec)

1 769 128 15.7 44.0 57.2 20.4 35.7 108.7 44 12.5 24.1

2 768 109 16.2 46.3 60.3 21.1 35.0 107.2 29 13.2 24.7

3 748 130 15.2 44.1 59.0 20.3 34.5 139.1 17 13.2 27.4

4 686 116 14.7 42.2 61.5 21.4 34.8 140.1 35 13.8 24.2

5 694 91 14.7 42.2 60.8 21.2 34.8 130.3 25 12.8 18.6

Vehicle 6 779 95 15.5 45.8 58.8 19.9 33.8 131.1 25 15.1 27.5

control iie-c-ove-ii-----------------------------------c------------------------------------------------------------------------------------------------------------------------------

7 832 98 16.3 46.3 55.6 19.6 35.2 137.4 30 13.5 31.6

8 793 85 15.6 44.8 56.5 19.7 34.8 96.5 31 15.6 25.9

9 860 91 15.2 43.9 51.0 17.7 34.6 127.3 25 12.4 27.3

10 834 77 15.8 46.8 56.1 18.9 33.8 140.7 27 13.5 29.4

11 810 94 15.8 47.3 58.4 19.5 33.4 105.3 13 14.4 27.2

12 819 110 15.8 46.5 56.8 19.3 34.0 117.3 18 12.1 30.4

13 727 134 14.4 42.7 58.7 19.8 33.7 145.9 28 13.0 23.3

14 810 80 15.8 46.5 57.4 19.5 34.0 132.6 26 13.4 27.3

40 15 778 71 14.8 43.6 56.0 19.0 33.9 121.6 20 14.4 27.9

16 781 97 15.4 45.1 57.7 19.7 34.1 120.7 29 14.3 21.8

17 746 91 15.4 45.0 60.3 20.6 34.2 125.5 22 12.3 27.0

**~** Hale 18 750 99 15.7 45.4 60.5 20.9 34.6 101.1 22 12.7 24.7

19 738 131 14.8 42.8 58.0 20.1 34.6 100.0 27 14.0 29.5

20 769 101 15.6 45.5 59.2 20.3 34.3 126.2 29 12.8 25.3

..... 200 21 724 127 14.7 43.7 60.4 20.3 33.6 123.3 28 16.1 28.2

w 22 761 72 15.3 45.5 59.8 20.1 33.6 126.4 24 13.5 31.1

23 753 81 15.4 43.6 57.9 20.5 35.3 130.6 32 12.6 24.9

24 780 86 15.5 45.4 58.2 19.9 34.1 104.7 17 12.6 25.7

1

25 711 139 14.7 42.8 60.2 20.7 34.3 120.4 33 13.0 23.8

26 804 93 15.8 46.4 57.7 19.7 34.1 117.4 24 15.0 26.7

I 27 793 74 15.5 45.4 57.3 19.5 34.1 110.9 23 14.5 27.6

**~** 28 783 92 15.1 44.9 57.3 19.3 33.6 123.8 31 12.9 25.0

29 738 102 15.5 45.4 61.5 21.0 34.1 113.0 37 13.2 26.0

1,000 30 745 98 15.7 46.5 62.4 21.1 33.8 120.0 29 13.2 26.0 iiecoveri-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------

31 8'26 143 15.6 45.8 55.4 18.9 34.1 104.4 19 12.4 24.9

32 843 89 16.2 46.0 54.6 19.2 35.2 121.9 18 12.9 29.1

33 861 98 15.9 47.2 54.8 18.5 33.7 115.9 21 13.2 29.1

J 34 757 96 15.4 44.9 59.3 20.3 34.3 113.1 20 17.8 34.4

i

35 807 134 15.5 45.2 56.0 19.2 34.3 124.8 28 12.7 28.1

J 36 803 82 15.5 44.5 55.4 19.3 34.8 119.4 22 11.9 26.0

**~ ~**  **~** -...../
____, ...... ....

l **~** I **~** •

1 l **~** :i<
I **~** J **~**

! **~)i** Addendum 4 -Continued Hematology Sex Hale Exp,group (rog/kg/day) Vehicle control 40 200 1,000 ------·---- . **---·-~-----** --------· -------·-------- Bll-0443 Differentiation of leukocyte (%) Animal

No. N-Band N-Seg Eosino Baso Lymph Mono

1 0.0 8.5 0.0 0.0 89.5 2.0

2 0.0 17.0 0.5 0.0 82.5 0.0

3 0.0 9.5 0.0 0.0 90.5 0.0

4 0.0 17.0 2.0 0.0 80.5 0.5

5 0.0 9.0 1.0 0.0 90.0 0.0

6 0.0 14.5 0.5 0.0 85.0 0.0 R.ec-ovii-ii-------------------------------------------------------------------------------------------------------

7 0.0 5.5 1.0 0.0 93.0 0.5

8 0.0 6.5 1.0 0.0 92.0 0.5

9 0.0 8.5 0.0 0.0 91.0 0.5

10 0.0 4.0 3.0 0.0 93.0 0.0

11 0.0 4.0 0.0 0.0 96.0 0.0

12 0.0 4.0 0.5 0.0 95.5 0.0

13 0.0 10.5 0.0 0.0 89.5 0.0

14 0.5 9.5 0.0 0.0 90.0 0.0

15 0.0 8.5 0.0 0.0 91.0 0.5

16 0.0 6.5 1.0 0.0 92.5 0.0

17 0.0 6.5 0.5 0.0 93.0 0.0

18 0.5 10.0 1.0 0.0 88.0 0.5

19 0.5 15.5 0.5 o.o 83.5 0.0

20 0.0 6.5 1.0 0.0 92.5 0.0

21 0.0 12.0 1.0 0.0 86.5 0.5

22 0.0 11.0 0.5 0.0 88.5 0.0

23 0.0 14.0 0.5 0.0 85.5 0.0

24 0.5 9.0 1.5 0.0 89.0 0.0

25 0.0 5.0 0.0 0.0 95.0 0.0

26 0.0 9.5 1.5 0.0 89.0 0.0

27 0.5 8.0 0.5 0.0 91.0 0.0

28 0.5 6.5 0.0 0.0 93.0 0.0

29 0.5 20.0 2.0 0.0 77.5 0.0

30 0.0 12.0 0.0 0.0 87.5 0.5 R.e-coviiri--------------------------------------------------------------------------------------------------------

31 0.0 6.0 0.5 0.0 93.0 0.5

32 0.0 5.5 1.0 0.0 93.5 0.0

33 0.0 6.0 0.0 0.0 94.0 0.0

34 0.0 2.5 0.0 0.0 97.5 0.0

35 0.0 5.5 1.0 0.0 93.0 0.5

36 o.o 10.5 1.0 0.0 88.0 0.5**~** ' l **~ ~**  i - CJ1 **~ ~**  **~ ~**  **~ ,/·~**  Addendum 4 -Continued Sex Female

Hematology Exp.group (mg/kg/day) Vehicle control 40 200 1,000 **.~** Bll-0443

RBC WBC Hb Ht MCV HCH MCHC Platelet Reticula P T

. APTT

Animal

No. (xl0 4 /mm 3 ) (xl0 2 /mm 3 ) (g/dl) (%) (,um 3 ) (pg) (%) (xl0 4 /mm 3 ) (%,) (sec) (sec)

37

748 53 15.0 43.6 58.3 20.1 34.4 121.1 21 12.0 17.2

38 763 127 14.7 41.8 54.8 19.3 35.2 79.2 25 13.0 18.3

39 774 70 15.1 42.9 55.4 19.5 35.2 101.9 18 11.8 24.9

40 825 62 15.3 45.1 54.7 18.5 33.9 118.9 21 11.6 24.3

41 735 77 14.5 40.8 55.5 19.7 35.5 130.3 27 10.4 21.6

42 718 86 14.9 42.3 58.9 20.8 35.2 116.4 17 10.8 22.1

**Recover:Y---------------------------------------------------~----------------------------------------------------------------------------------------------------------------------**

43

792 77 15.5 43.4 54.8 19.6 35.7 105.6 24 12.6 20.9

44 767 98 14.8 41.6 54.2 19.3 35.6 122.6 30 12.2 26.0

45 774 60 14.9 42.2 54.5 19.3 35.3 115.7 20 11.9 18.6

46 815 83 16.1 45.1 55.3 19.8 35.7 119.8 34 11.6 23.5

47 764 59 15.0 43.1 56.4 19.6 34.8 107.0 25 13.2 21.7

48 820 74 15.9 46.2 56.3 19.4 34.4 111.4 24 12.6 19.2

49 746 40 14.7 43.0 57.6 19.7 34.2 129.1 12 11.4 28.6

50 765 106 15.8 45.3 59.2 20.7 34.9 114.8 23 11.4 22.0

51 749 62 15.2 43.9 58.6 20.3 34.6 158.1 19 11.8 31.0

52 735 76 14.6 42.0 57.1 19.9 34.8 129.5 20 11.0 28.1

53 810 82 15.6 44.8 55.3 19.3 34.8 123.1 12 10.0 18.5

54 773 86 15.6 45.1 58.3 20.2 34.6 105.4 13 10.1 19.6

55 759 62 15.7 43.6 57.4 20.7 36.0 118.9 34 13.1 22.7

56 769 54 15.4 43.2 56.2 20.0 35.6 117.1 12 12.6 21.5

57 746 109 14.9 43.2 57.9 20.0 34.5 128.8 27 10.6 30.6

58 827 62 16.0 45.7 55.3 19.3 35.0 114.3 15 10.8 24.3

59 734 87 14.9 41.8 56.9 20.3 35.6 112.4 37 10.6 23.2

60 780 97 15.4 44.5 57.1 19.7 34.6 140.0 32 10.1 22.6

61 821 70 16.2 45.3 55.2 19.7 35.8, 138.3 12 12.9 22.2

62 727 90 14.8 42.8 58.9 20.4 34.6 121.9 15 13.0 21.5

63 751 71 14.8 43.5 57.9 19.7 34.0 134.3 37 11.6 28.8

64 766 143 15.9 45.0 58.7 20.8 35.3 115.4 33 11.1 28.1

65 821 99 16.1 44.9 54.7 19.6 35.9 111.9 17 10.0 20.7

66 744 65 15.2 43.0 57.8 20.4 35.3 122.8 20 10.6 23.7 Re-cove-r:Y------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

67 807 79 15.4 43.9 54.4 19.1 35.1 99.3 16 12.9 20.9

68 811 89 15.8 45.5 56.1 19.5 34.7 98.1 18 12.0 21.4

69 840 93 15.7 45.3 53.9 18.7 34.7 107.3 20 12.4 24.5

70 807 48 15.5 44.4 55.0 19.2 34.9 125.6 23 12.1 20.6

71 825 46 15.5 45.5 55.2 18.8 34.1 111.8 18 12.7 20.7

72 787 107 15.4 44.5 56.5 19.6 34.6 98.9 25 12.0 22.8-1 ' 0 l ......

' O"l

I i • l **~ ~**  l 1 **~ ~**  ($"""-., **~ /-~,**  Addendum 4 -Continued Hematology Exp.group Bll-0443 Differentiation of leukocyte ( %)

Sex

(mg/kg/day) Animal No. N-Band N-Seg Eosino Baso Lymph Mono

Female Vehicle control 40 zoo 1,000 37 0.5 13.0 1.5 0.0 85.0 0.0

38

0.5 9.5 1.0 0.0 89.0 0.0

39

0.0 8.0 0.0 0.0 92.0 0.0

40

0.0 5. 5 3.0 0.0 91.5 0.0

41

0.5 9.0 0.5 0.0 90.0 0.0

42

0.5 10.0 1.5 0.0 88.0 0.0 iiecaveir"Y------------------------------------------------------------------------------------------------------

43 0.0 5.0 0.0 0.0 95.0 0.0

44

0.0 14.0 0.5 0.0 85.5 0.0

45

0.0 6.0 0.5 0.0 93.5 0.0

46

o.o 6.0 0.5 0.0 93.0 0.5

47

o.o 2.0 0.5 0.0 97.5 0.0

48

0.0 3.5 0.0 0.0 96.0 0.5

49

0.5 10.5 0.0 0.0 89.0 0.0

50

0.0 7.0 0.5 0.0 92.5 0.0

51

0.5 8.5 0.5 0.0 90.5 0.0

52

0.0 15.0 2.0 0.0 83.0 0.0

53

o.o 5.0 1.0 0.0 94.0 0.0

54

0.0 5.5 0.5 0.0 93.5 0.5

55

0.0 4.5 0.5 0.0 95.0 0.0

56

0.5 23.0 1.0 0.0 75.5 0.0

57

0.0 8.5 2.0 0.0 89.5 0.0

58

0.0 7.0 0.5 0.0 92.5 0.0

59

0.5 17.5 0.0 0.0 81.5 0.5

60

0.0 5.0 2.5 0.0 92.5 0.0

61

0.0 11.0 0.5 0.0 88.0 0.5

62

0.0 15.0 1.5 0.0 83.5 0.0

63

0.0 7.0 1.5 0.0 91.5 0.0

64 o.o 9.5 1.0 0.0 89.0 0.5

65 0.5 6.5 1.5 0.0 91.5 0.0

66

o.o 5.5 0.5 0.0 93.0 1.0 iiec-over_y

__________________________________________________________________________________________________________

67 0.0 5.5 1.0 0.0 93.5 0.0

68 0.0 4.0 1.0 0.0 95.0 0.0

69

ci.o 10.0 1.0 0.0 89.0 0.0

70

0.0 6.0 2.5 0.0 91.5 0.0

71

0.0 6.5 1.0 0.0 92.0 0.5

72

0.0 8.5 0.0 0.0 91.0 0.5Addendum 5 28-day repeated-dose oral toxicity study in rats Bll-0443

Blood chemistry Exp.group GOT GPT ALP ChE y-GTP T-Cho TG Glucose T-protein Albumin A/G ratio

Sex (mg/kg/day) Animal No. (IU/1) (IU/1) (IU/1) (IU/1) (IU/1) (mg/dl) (mg/dl) (mg/dl) (g/dl) (g/dl)

1 84 20 283 57 0.4 79 116 114.8 6.1 3.2 1. 10

2 70 20 523 62 0.5 62 99 120.5 6.2 3.1 1. 00

3 77 21 294 60 0.8 85 157 122.8 6.2 2.9 0.88

4 85 22 256 67 0.9 73 125 125.1 5.8 2.7 0.87

5 101 20 386 51 0.8 70 59 103.5 5.9 2.8 0.90

Vehicle 6 71 19 436 47 0.5 67 102 109.0 5.8 2.6 0.81

control Recovery----------------------------------------------------------------------------------------------------------------------------------------------------------------------------

7 79 21 284 41 0.7 65 56 109.7 6.1 2.8 0.85

8 62 17 382 49 0.6 80 77 128.5 5.8 2.7 0.87

9 65 20 374 31 0.6 56 78 160.5 6.0 2.8 0,88

10 60 20 293 46 0.5 47 74 148.0 6.0 2.7 0.82

11 75 20 334 92 0.5 62 127 165.9 6.0 2.8 0.88

12 63 17 303 64 0.8 72 75 119.9 5.8 2.7 0.87

13 57 18 376 106 0.3 83 154 116.2 5.9 2.7 0.84

14 73 22 445 56 0.5 60 58 97.9 5.9 2.9 0.97

40 15 65 19 341 40 0.3 71 153 111.7

. 5. 7 2.7 0.90

16 77 16 284 51 0.6 67 128 119.9 5.8 2.8 0,93

17 107 20 528 49 0.4 64 35 93.4 6.0 2.8 0.88

Hale 18 75 23 321 61 0.2 68 47 102.2 5.7 2.8 0.97

_, 19

72 17 395 47 0.3 69 122 117.2 5.7 2.7 0.90

20 60 20 247 83 0.6 76 106 112.5 5.9 2.9 0.97

..... 200 21 68 17 397 42 0.2 59 144 147.1 5.7 2.8 0.97

-.) 22 84 21 250 67 0.4 50 95 140.3 5.8 2.8 0.93

23 71 23 313 41 0.4 57 159 118.3 5.8 2.7 0.87

24 91 24 496 40 0.5 64 118 111.8 5.5 2.8 1. 04

I

25 78 21 527 45 0.6 51 167 107.3 5.7 2.7 0.90

26 69 19 454 59 0.5 63 58 99.9 5.8 2.8 0.93

27 87 23 496 84 0.7 72 124 100.2 5.9 2.7 0.84

l 28

91 20 328 37 0.6 73 122 107.4 5,6 2.8 1.00

l

29 88 24 484 68 0.4 64 71 110.9 5.5 2.8 1. 04

1 1,000 30 72 21 350 61 0.9 61 141 105.4 5.7 2.8 0.97 Recove-ry-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------

- 31 65 25 193 44 0.6 59 65 171.0 5.9 2.6 0.79

32 56 23 356 45 0.7 51 83 159.5 6.0 2.8 0.88

33 74 40 288 49 0.6 54 82 143.1 6.1 2.7 0.79

l 34 60 19 318 48 0.5 62 110 159.8 5.8 2.6 0.81

i 35 67 18 193 62 1.0 52 88 136.2 5. 7 2.6 0.84

I 36 72 21 293 55 0.9 48 51 145.5 5.6 2.6 0.87

I **~ ~**  • I **~ ~** 

I \.f\

1 I I**/~,** Addendum 5 Sex
--j Hale

' j **~**

' co

I l I

' j l J - l i **~ ~** 

I **~** _J **~**

I ···--------- ----- __ I -Continued Blood chemistry Bll-0443
Exp.group (mg/kg/day) Vehicle control 40 200 1,000 BUN Animal No. (mg/dl)
1 15.0
2

14.5
3

12.4
4

14.9
5

15.7 Creatinine (mg/dl) 0.35 0.23 0.49 0.46 0.43 T-Bil (mg/dl) 0.21 0.31 0.26 0.27 0.23 Ca (mg/dl) 10.5 10.3 10.2 10.0 10.1 IP (mg/dl) 7.4 7.9 7.5 7.4 8.3 Na (mEq/1) 143 146 145 144 144 K (mEq/1) 4.0 3.9 4.0 4.3 4.5 Cl (mEq/1) 107.3 105.4 108.5 107.1 108.9

6 11.0 0.23 0.34 10.3 7.7 143 4.4 105.2 iiEicoverY:--------------------------------------------------------------------------------------------------------------------------------------

7 10.6 0.29 0.27 9.4 7.0 143 5.0 105.3

8 11.6 0.47 0.20 9.8 7.2 145 4.1 106.5

9 12.4 0.48 0.17 10.0 7.8 144 4.1 105.2

10 12.7 0.42 0.20 9.8 7.3 145 4.2' 106,4

11 13.9 0.48 0.17 9.7 7.4 143 4.2 105.8

12 13.0 0.23 0.36 9.9 7.8 143 4.5 103.5

13 12.5 0.32 0.27 10.4 7.1 144 3.9 106.4

14 12.3 0

0 36, 0.24 10.8 7.5 145 4.3 108.3

15 8.7 0.33 0.27 10.4 7.7 144 4.4 107.3

16 11.9 0.37 0.25 10.6 7.9 143 4.1 104.1

17 15.1 0.22 0.36 10.7 8.4 143 4.9 104.8

18 11.3 0.31 0.28 10.6 7.6 143 4.4 107.2

19 14.3 0.40 0.26 9.8 8.1 142 4.2 105.4

20 15.8 0.36 0.33 10.2 7.5 144 4.4 106.3

21 12.9 0.36 0.25 10.4 8.3 144 4.3 105.8

22 13.0 0.38 0.19 9.9 7.6 143 4.1 107.7

23 11.4 0.31 0.27 10.6 8.0 142 4.9 106.2

24 14.5 0.47 0.23 9.8 7.6 143 4.2 107.8

25 13.3 0.39 0.27 10.1 7.9 142 4.3 107.3

26 11.5 0.35 0.23 10.8 6.8 143 4.3 108.0

27 13.2 0.36 0.25 10.0 7.0 144 4.3 106.9

28 14.3 0.43 0.23 9.9 8.0 144 4.6 106.6

29 13.9 0.37 0.28 9.9 7.3 144 4.3 107.7

30 12.2 0.35 0.23 10.4 7.6 144 4.4 106.2 Rec-overY:-----------------------------------------------------------------------------------------------------------------------------------------

31 11.7 0.46 0.17 10.2 8.9 146 4.7 103.8

32 13.7 0.39 0,20 9.6 7.6 143 3.8 104.2

33 11.2 0.35 0.21 9.6 7.2 145 4.0 105.8

34 12.6 0.38 0.19 9.5 7.1 142 4. 1 107.5

35 13.7 0.38 0.19 9.4 7.3 142 4.9 105.7

36 12.7 0.38 0.23 9.4 7.8 143 4.2 105.7 **'~**----1 ' I

l ._.

' <.0

I. I

**~** .I ' 1 ' 1 -l **~** ; **~** i I 1 l **~ ~**  l **~** l **~** '-J

1 ' l 1 /-'-..._ **,----~**

Addendum 5 -Continued Blood

chemistry Bll-0443

Sex Female Exp.group (mg/kg/day) GOT Animal No. (IU/1) GPT (IU/1) ALP (IU/1) ChE (IU/1) y-GTP (IU/1) T-Cho (mg/dl) TG (mg/dl) Glucose (mg/dl) T-protein (g/dl) Albumin (g/dl) A/G ratio

37 69 15 169 287 0.7 64 45 119.2 6.3 3.0 0.91

38 77 18 235 349 0.7 44 29 97.7 6.0 2.9 0.94

39 73 20 281 365 0.6 55 36 105.4 5.9 3.1 1.11

40 84 16 357 192 0.6 56 55 109.4 5.6 2.8 1.00

41 73 14 266 330 0.9 50 36 120.1 5.6 2.7 0.93

Vehicle 42 74 18 183 504 0.9 62 35 100.1 6.6 3.3 1.00

control iiecoveri----------------------------------------------------------------------------------------------------------------------------------------------------------------------

43 65 17 226 247 0.9 64 39 112.6 6.2 3.0 0.94

44 69 20 201 336 1.2 73 31 124.8 6.5 3.2 0.97

45 72 19 169 315 0.7 96 70 135.4 6.7 3.2 0.91

46 77 16 168 486 1.0 60 37 141.9 6.8 3.3 0.94

47 65 16 110 630 0.6 61 35 133.9 5.9 3.0 1.03

48 73 24 211 297 1.2 57 47 195.9 6.6 3.2 0.94

49 56 17 265 428 0.3 37 30 125.4 5.8 2.9 1.00

50 64 16 347 397 0.8 57 52 113.6 5.9 3.1 1.11

40 51 71 15 273 286 1.0 56 38 135.1 6.5 3.3 1.03

52 56 13 264 423 0.5 60. 43 115.8 5.8 2.9 1.00

53 59 15 183 544 0.9 52 28 131.6 6.2 3.2 1.07

54 67 18 186 408 0.9 63 29 129.9 6.1 2.9 0.91

55 61 16 207 244 1.0 55 29 103.6 5.7 2.8 0.97

56 65 23 267 349 0.8 74 34 111.3 6.2 2.9 0.88

200 57 62 12 195 296 0.9 74 42 141.5 6.2 3.1 1.00

58 68 15 209 624 1.0 78 39 113.1 6.0 3.2 1.14

59 94 23 196 363 0.8 54 44 135.2 6.2 2.9 0.88

60 75 16 318 276 1.0 50 41 111.3 5.9 3.0 1.03

61 82 16 216 245 0.5 53 40 119.1 5.8 3.0 1.07

62 70 19 217 420 0.6 64 32 116.4 6.3 3.0 0.91

63 72 16 230 404 0.9 6! 29 115.9 6.2 3.0 0.94

64 83 16 210 277 0.8 71 36 145.6 6.2 3.1 1.00

65 89 15 245 276 1.2 66 27 125.8 5.7 2.9 1.04

1,000 66 72 18 175 329 1.3 77 38 126.9 6.4 3.2 1.00 iie-cover"Y------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

67 64 24 182 791 0.8 73 43 110.9 6.3 3.0 0.91

68 67 11 155 **33~** 1.0 77 40 133.9 6.4 2.8 0.78 69 64 18 290 303 1.3 78 33 132.0 6.4 3.0 0.88

70 68 15 194 274 0.7 59 30 126.7 6.5 3.1 0.91

71 66 18 181 433 0.6 80 46 121.3 6.8 3.3 0.94

72 65 14 181 362 0.9. 67 36 177.5 6.1 3.0 0.97/.-
___, : I

; N 0 I

I i . **~ ~**  **~** l **~** :5?

! **~** I **~ ~**  I I Addendum 5 -Continued Blood

chemistry Bll-0443

Sex Female Exp.group (mg/kg/day) Vehicle control 40 200 1,000 BUN Animal No. (mg/dl) Creatinine (mg/dl) T-Bil (mg/dl) Ca (mg/dl) IP (mg/dl) Na (mEq/1) K (mEq/1) Cl (mEq/1)

37 15.1 0.46 0.23 10.4 6.5 143 4.5 108.4

38 15.4 0.48 0.23 10.2 7.0 142 3.9 107.7

39 16.4 0.48 0.19 10.2 6.9 141 4.0 108.3

40 13.8 0.47 0.20 10.2 7.5 140 4.5 107.6

41 15,0 0.39 0.19 10.4 8.2 141 4.5 107.5

42 14.8 0.51 0.17 11.5 9.7 143 4.0 107.1 Recover::y-------------------------------------------------------------------------------------------------------------------------------

43 13.5 0.42 0.22 9.4 6.5 142 4.3 107.1

44 13.4 0.48 0.22 9.7 6.4 143 4.2 107.5

45 15.1 0.46 0.21 9.7 6.1 143 3.9 106.8

46 13.2 0.44 0.19 9.9 6.9 141 3.9 106.3

47 15.0 0.35 0.24 9.7 7.6 142 4.6 106.7

48 19.1 0.53 0.17 10.0 7.5 142 4.3 106.4

49 13.2 0.41 0.21 10.1 7.4 141 4.5 107.4

50 14.2 0.45 0.24 10.5 7.7 142 4.4 108.8

51 14.2 0.49 0.23 10.4 6.2 141 4.1 108.6

52 14.2 0.46 0.22 10.7 7.8 142 4.4 108.1

53 13.6 0.43 0.18 11.0 7.3 141 4.3 107.8

54 12.3 0.49 0.19 10.6 7.7 140 4.3 107.8

55 15.1 0.38 0.23 10.3 7.0 141 4.5 108.4

56 13.8 0.43 0.22 10.4 6.5 141 4.0 107.9

57 12.8 0.42 0.21 10.5 7.6 142 4.2 106.0

58 16.9 0.51 0.20 10.4 7.7 142 4.2 109.3

59 14.6 0.47 0.13 10.6 7.9 142 4.0 108.5

60 12.8 0.40 0.25 11.0 8.4 141 4.6 108.1

61 11.8 0.39 0.22 10.4 7.4 142 4.3 108.7

62 16.3 0.51 0.18 11.2 7.6 141 4.1 108.5

63 16.0 o:54 0.20 10.9 6.7 143 4.1 110.1

64 14.0 0.60 0.17 11.0 7.9 143 3.9 105.5

65 13.6 0.51 0.16 10.7 8.3 140 4.6 106.3

66 14.8 0.51 0.17 10.4 7.5 142 3.9 108.7 Rec-av-er::y-----------------------------------------------------------------------------------------------------------------------------------------

67 14.0 0.36 0.22 9.8 7.0 141 3.7 107.1

68 14.1 0.46 0.20 9.7 6.5 142 3.9 106.3

69 14.0 0.36 0.31 9.5 6.9 141 4.5 105.4

70 16.1 0.39 0.27 9.3 6.4 142 4.4 108.9

71 15.9 0.38 0.24 10.1 6.7 143 4.0 110.0

72 11.5 0.46 0.19 9.4 7.0 141 3.9 105.7 **~··.**_j I

' l **~ ~**  ! l **~** I i l I

i I N y )...:> **~ ~**  **--~--~ ---~**  **-~-**

Addendum 6 28-day repeated-dose oral toxicity study in rats

Urinalysis 811-0443

Sex Male Exp.group (mg/kg/day) Vehicle control 40 200 1,000
SY : Slightly yellow

Y : Yellow Animal No. VolUJDe (ml) Color pH Protein Ketones Bilirubin Occult Blood Glucose Urobi I inogen (EU/dl)

1 24 SY 6.5 ± ± - - - 0.1

2 7 y 6.5 + ± - - - 0.1

3 35 SY 6. 5 ± ± - - - 0. 1

4 21 SY 7.0 ± ± - - - 0.1

5 39 SY 7. 0 ± ± - - - 0.1

6 23 SY 7. 0 ± ± - - - 0. 1 -!lecciver'Y·---·----·-·---·--·-------·----------------------·--·-·----···---------------·----····--··----·-------------------------------------------------------

7 14 SY 6.5 ± ± - - - 0.1

8 21 SY 6.5 ± ± - - - 0.1

9 10 y 7.0 + ± - - - 0.1

10 31 SY '7.0 ± ± - - - 0.1

11 18 SY 7.0 + ± - - - 0.1

12 29 SY 7. 0 ± ± - - - 0.1

13 35 SY 7.0 ± ± - - - 0.1

14 28 SY 6. 5 ± ± - - - 0. 1

15 34 SY 7. 0 ± ± - - - 0.1

16 8 y 6.5 + ± - - - 0.1

17 25 SY 7. 0 ± ± - - - 0.1

18 11 y 6.5 + ± - - - 0.1

19 20 SY 7.0 ± ± - - - 0.1

20 25 SY 7. 0 ± ± - - - 0 .1

21 14 SY 6.5 + ± - - - 0.1

22 21 SY 7.0. ± ± - - - 0.1

23 20 SY 7.0 ± ± - - - 0.1

24 8 y 6.5 + ± - - - 0.1

25 33 SY 7. 0 ± ± - ± - 0. 1

26 26 SY 7. 0 ± ± - - - 0.1

27 16 SY 6.5 + ± - - - 0.1

28 7 y 6. 5 t+ ± - - - 0.1

29 9 y 6.5 + ± - - - 0.1

30 14 SY 7. 0 + ± - - - 0. 1

·ife.ca'Ver'Y _____________________ .......................................................................................................................................................................................... .

31 14 SY 6.5 + ± - - - 0.1

32 22 SY 7. 0 ± ± - - - 0. 1

33 9 y 6. 5 + ± - - - 0.1

34 12 y 6.5 + + - - - 0.1

35 9 y 7.0 + ± - - - 0.1

36 5 y 6.5 + ± - - - 0.1__, 1 l l **~** j **-~** N N **~ ~**  )-) ...._j **~** Addendum 6 Sex Female -Continued Urinalysis Exp.group (mg/kg/day) Vehicle control 40 zoo 1,000
SY : sllgntly yellow Y : Yellow **-~~** Bll-0443

Animal No. Volume (rnl) Color pH Protein Ketones Bilirubin Occult Blood Glucose Urobilinogen (EU/dl)

37 10 SY 6.0 ± - - - - 0.1

38

4 y 6.0 + - - - - 0.1

39

14 SY 6.5 ± - - - - 0.1

40

12 SY 6.5 ± - - - - 0.1

41

11 SY 6.5 ± - - - - 0.1

42

19 SY 7.0 ± - - ± - 0.1 -fecovei:;;·-··-------------------------------------------------------------------------------------------------------------------------------------------------------

43 24 SY 7. 0 - - - - - 0 .1

44

6 y 6. 5 + - - - - 0.1

45

12 SY 7.0 ± - - - - 0.1

46

4 y 6. 5 + - - - - 0 .1

47

10 y 7.0 ± - - - - 0.1

48

5 y 7.0 ± - - - - 0.1

49 15 SY 6.5 ± - - - - 0.1

50

5 y 6.0 + - - - - 0.1

51

3 y 6.0 ++ - - - - 0.1

52

10 y 6.5 ± - - - - 0.1

53

5 y 6. 5 + - - - - 0.1

54

10 y 6.5 ± - - - - 0.1

55

13 SY 6. 5 ± - - - - 0. 1

56

16 SY 6.5 ± - - - - 0.1

57

10 y 7.0 ± - - - - 0.1

58

ZO SY 7. 0 ± - - - - 0 .1

59

13 SY 6.5 ± - - - - 0.1

60

9 y 6. 5 ± - - - - 0 .1

61

39 SY 7. 0 - - - - - 0.1

62

18 SY 6. 5 ± - - - - 0. 1

63

12 SY 7. 0 ± - - - - 0.1

64

7 y 6.5 + - - - - 0.1

65

6 y 6.5 + - - - - 0.1

66

5 y 6.5 + - - - - 0.1 'lfecover"Y······----······-·-·····----------------------·-------------------------------------------------------------------------------------------·----------·--------------------------------------------

67 1 Z SY 6. 5 ± - - - - 0. 1

68

6 y 6.5 ± - - - - 0.1

69

15 SY 6.5 ± - - - - 0.1

70

10 y 7. 0 ± - - - - 0.1

71

15 SY 6.5 ± - - - - 0.1

72

5 y 6.0 + - - - - 0.1-i -I **~** 9 **~:')** w -1 **~** iv -1 **~** I l j I Addendum 7 28-day repeated-dose oral toxicity study in rats Absolute

organ weights Bll-0443

Sex Male Exp. group Spleen Liver (g) Kidney (g) Brain (g) Testis (g) Adrenal gland (mg) Ovary

(mg) Body weight (g)

(mg/kg/day) Animal No. (g) Vehicle control 40 200 1,000 1 0.66 10.16 2.27 2.09 3,03 53.6 - 328.5

2

0.55 8.63 2.46 1.93 2.63 56.5 - 311.4

3

0.70 10.41 2.29 1.97 3.05 58.0 - 337.3

4

0.68 10.52 2.44 1.98 3.17 55.5 - 342.4

5

0.54 7.83 1.94 1.89 2.95 50.2 - 294.1

6

0.49 9.93 2.17 1.86 2.87 49.7 - 309.7 iiec-overi-----------------------------------------------------------------------------------------------------------

7 0.66 9.99 2.70 1.97 3.33 49.7 - 381.0

8

0.70 11.51 2.46 1.90 2.95 46.0 - 396.8

9

0.54 10.44 2.33 1.97 3.09 55.5 - 380.2

10

0.65 10.52 2.42 1.94 3.30 56.6 - 396.7

11

0.76 12.78 2.61 2.16 3.13 51.6 - 432.1

12

0.62 11.55 2.51 2.04 3.37 49.4 - 389.8

13

0.63 9.12 2.08 1.92 2.92 53.3 - 304.6

14

0.47 8.39 2.32 1.96 2.81 40.9 - 308.6

15

0.49 9.06 2.20 1.88 2.76 41.4 - 311.9

16

0.57 9.34 2.40 1.90 2.99 58.3 - 295.2

17

0.65 9.24 2.47 1.92 2.83 53.0 - 326.1

18

0.53 7.99 2.37 1.83 2.71 49.0 - 280.2

19

0.79 9.50 2.48 1.86 2.72 35.3 - 334.5

20

0.54 9.35 2.14 1.86 3.11 46.7 - 298.5

21

0.63 10.35 2.48 1.91 2.55 42.0 - 335.6

22

0.56 8.60 2.04 1.83 2.69 56.5 - 306.5

23

0.55 9.37 2.23 1.88 2.92 48.8 - 317.9

24

0.48 6.16 1.93 1.74 2.61 55.0 - 252.3

25

0,78 10.00 2.45 2.01 2.86 48.4 - 334.4

26

0.50 7.29 2.19 1.83 2.93 47.4 - 283.9

27

0.51 8.60 2.27 1.95 3.05 45.8 - 302.6

28

0.49 8.58 2.64 1.81 2.81 41.9 - 303.9

29

0.64 8.00 2.21 2.03 2.90 40.6 - 294.5

30

0.61 8.40 2.21 1.98 2.64 56.5 - 317.4 iiecover-i------------------------------------------------------------------------------------------------------------------------------

31 0.63 10.09 2.33 1.90 2.14 46.8 - 354.6

32

0.51 11.01 2.64 1.94 3.18 44.6 - 373.7

33

0.54 10.51 2.88 2.02 3.60 62.6 - 368.7

34

0.81 12.70 2.81 2.06 3.53 50.8 - 414.3

35

0.88 11.77 2.75 2.02 3.24 72.5 - 428.5

36

0.63 10.63 2.49 1.97 2.87 58.0 - 381.4 ,/----..._,_---' l i

-I **~** 1 : l **~** N ..;::. **~ ~**  **~** ...j **~** ---------···· ------------·--------- ---·-------- -- ----·--

Addendum 7 -Continued Bll-0443

Sex Female

Absolute organ weights Exp.group Spleen Liver (g) Kidney (g) Brain (g) Testis (g) Adrenal gland (mg) Ovary

(mg) Body weight (g)

(mg/kg/day) Animal No. (g) Vehicle control 40 200 1,000 37 0.40 5.88 1.47 1.71

- 60.0 83.0 203.4

38 0.43 5.45 1.50 1.74

- 64.7 93.4 187.0

39 0.54 5.67 1.55 1.75

- 53.8 99.4 201.3

40 0.39 6.07 1.54 1.86

- 62.5 102.9 212.9

41 0.60 6.17 1.72 1.98

- 59.2 150.3 214.7

42 0.49 5.38 1.41 1.82

- 62.4 64.9 177.5 ii"Eic-over"Y-------------------------------------------------------------------------------------------------------------------------

43 0.47 5.52 1.61 1.83

- 53.9 96.4 206.4

44 0.51 6.31 1.63 1.92

- 55.0 91.8 232.9

45 0.47 7.22 1.73 1.94

- 64.6 99.5 254.5

46 0.44 6.19 1.55 1.83

- 78.1 77.8 206.2

47 0.43 5.81 1.59 1.82

- 65.9 82.2 214.2

48 0.39 7.19 1.51 1.89

- 64.8 97.2 236.7

49

0.45 5.80 1.61 1.73

- 68.7 79.4 197.2

50 0.43 5.51 1.56 1.71

- 50.7 85.1 194.1

51 0.38 5.70 1.46 1.82

- 77.4 106.5 177.7

52 0.46 6.36 1.81 1.93

- 72.5 106.5 213.4

53 0.38 5.82 1.57 1.85

- 63.2 91.1 191.6

54 0.38 6.20 1.65 1.72

- 62.9 106.2 199.6

55

0.44 5.45 1.39 1.72

- 65.6 81.9 190.2

56 0.40 5.68 1.61 1.84

- 65.3 99.0 201.2

57 0.50 7.36 1.86 1.95

- 67.6 108.1 219.1

58 0.34 5.12 1.42 1.78

- 43.4 69.2 198.6

59 0.45 6.78 1.91 1.93

- 61.5 101.6 223.7

60 0.47 5.35 1.42 1.69

- 63.6 100.3 184.8

61

0.42 5.71 1.62 1.86

- 50.8 71.1 182.9

62 0.44 6.29 1.86 1.87

- 72.5 124;4 211.3

63 0.48 6.09 1.66 1.89

- 72.1 123.1 198.5

64 0.44 6.57 1.69 1.87

- 76.8 99.4 213.1

65 0.35 5.64 1.57 1.89

- 68.2 84.5 190.1

66 0.34 5.25 1.43 1.73

- 60.8 77.9 168.6 iiec-over-:Y-------------------------------------------------------------------------------------------------------------------------------

67 0.38 5.46 1.61 1.77

- 63.4 79.7 194.0

68 0.50 6.71 1.96 1.92

- 72.8 122.3 225.7

69 0.55 6.23 1.71 1.73

- 55.6 88.9 239.3

70 0.45 5.86 1.59 1.84

- 72.5 77.6 232.1

71 0.41 5.48 1.60 1.71

- 56.2 69.7 192.0

72 0.47 6.65 1.77 1.87

- 77.1 96.5 228.7 **/~**__J N CJl

l I

' l -1 **~** • 1 i **~**
I • **~ ~** 

i **~**

I _J i ' i I 1 l Addendum 8 28-day repeated-dose oral toxicity study in rats Relative

organ weights Bll-0443

Sex Hale Exp.group Spleen (mg/kg/day)

Animal No. (g/lOOg) Liver (g/lOOg) Kidney (g/lOOg) Brain (g/lOOg) Testis (g/lOOg) Adrenal gland

Ovary Body weight

(mg/lOOg) (mg/lOOg) (g)

1 0.20 3.09 0.69 0.64 0.92 16.3 - 328.5

2 0.18 2.77 0.79 0.62 0.84 18.1 - 311.4

3 0.21 3.09 0.68 0.58 0.90 17.2 - 337.3

4 0.20 3.07 0.71 0.58 0.93 16.2 - 342.4

5 0.18 2.66 0.66 0.64 1.00 17.1 - 294.1

Vehicle 6 0.16 3.21 0.70 0.60 0.93 16.0 - 309.7

control Recover'Y--------------------------------------------------------------------------------------------------------------------------

7 0.17 2.62 0.71 0.52 0.87 13.0 - 381.0

8 0.18 2.90 0.62 0.48 0.74 11.6 - 396.8

9 0.14 2.75 0.61 0.52 0.81 14.6 - 380.2

10 0.16 2.65 0.61 0.49 0.83 14.3 - 396.7

11 0.18 2.96 0.60 0.50 0.72 11.9 - 432.1

12 0.16 2.96 0.64 0.52 0.86 12.7 - 389.8

13

0.21 2.99 0.68 0.63 0.96 17.5 - 304.6

14 0.15 2.72 0.75 0.64 0.91 13.3 - 308.6

40 15 0.16 2.90 0.71 0.60 0.88 13.3 - 311.9

16 0.19 3.16 0.81 0.64 1.01 19.7 - 295.2

17 0.20 2.83 0.76 0.59 0.87 16.3 - 326.1

18 0.19 2.85 0.85 0.65 0.97 17.5 - 280.2

19

0.24 2.84 0.74 0.56 0.81 10.6 - 334.5

20 0.18 3.13 0.72 0.62 1.04 15.6 - 298.5

200 21 0.19 3.08 0.74 0.57 0.76 12.5 - 335.6

22 0.18 2.81 0.67 0.60 0.88 18.4 - 306.5

23 0.17 2.95 0.70 0.59 0.92 15.4 - 317.9

24 0.19 2.44 0.76 0.69 1.03 21.8 - 252.3

25

0.23 2.99 0.73 0.60 0.86 14.5 - 334.4

26 0.18 2.57 0.77 0.64 1.03 16.7 - 283.9

27 0.17 2.84 0.75 0.64 1.01 15.1 - 302.6

28 0.16 2.82 0.87 0.60 0.92 13.8 - 303.9

29 0.22 2.72 0.75 0.69 0.98 13.8 - 294.5

1,000 30 0.19 2.65 0.70 0.62 0.83 17.8 - 317.4 iiec-ove-r'Y-------------------------------------------------------------------------------------------------------------------------------

31 0.18 2.85 0.66 0.54 0.60 13.2 - 354.6

32 0.14 2.95 0.71 0.52 0.85 11.9 - 373.7

33 0.15 2.85 0.78 0.55 0.98 17.0 - 368.7

34 0.20 3.07 0.68 0.50 0.85 12.3 - 414.3

35 0.21 2.75 0.64 0.47 0.76 16.9 - 428.5

36 0.17 2.79 0.65 0.52 0.75 15.2 - 381.4I l I ----! I

I N 0>

J I

I i **~ ~**  l n -I **~** 1---' ._,_j

l I -c:

! 1 l I I l ' l I **/--~** Addendum 8 -Continued Bll-0443

Sex Female

Relative organ weights Adrenal Exp.group Spleen Liver (g/lOOg) KidneY (g/lOOg) Brain (g/lOOg) Testis (g/lOOg) gland Ovary Body weight

(mg/kg/day) Animal No. (g/lOOg) (mg/lOOg) (mg/lOOg) (g)

Vehicle control 40 200 1,000 37 0.20 2.89 0.72 0.84 - 29.5 40.8 203.4

38

0.23 2.91 0.80 0.93 - 34.6 49.9 187.0

39

0.27 2.82 0.77 0.87 - 26.7 49.4 201.3

40

0.18 2.85 0.72 0.87 - 29.4 48.3 212.9

41

0.28 2.87 0.80 0.92 - 27.6 70.0 214.7

42

0.28 3.03 0.79 1.03 - 35.2 36.6 177.5 Recover'Y-------------------------------------------------------------------------------------------------------------------------

43 0.23 2.67 0.78 0.89 - 26.1 46.7 206.4

44

0.22 2.71 0.70 0.82 - 23.6 39.4 232.9

45

0.18 2.84 0.68 0.76 - 25.4 39.1 254. 5.

46

0.21 3.00 0.75 0.89 - 37.9 37.7 206.2

47

0.20 2.71 0.74 0.85 - 30.8 38.4 214.2

48

0.16 3.04 0.64 0.80 - 27.4 41.1 236.7

49

0.23 2.94 0.82 0.88 - 34.8 40.3 197.2

50

0.22 2.84 0.80 0.88 - 26.1 43.8 194.1

51

0.21 3.21 0.82 1. 02 - 43.6 59.9 177.7

52

0.22 2.98 0.85 0.90 - 34.0 49.9 213.4

53

0.20 3.04 0.82 0.97 - 33.0 47.5 191.6

54

0.19 3.11 0.83 0.86 - 31.5 53.2 199.6

55

0.23 2.87 0.73 0.90 - 34.5 43.1 190.2

56

0.20 2.82 0.80 0.91 - 32.5 49.2 201.2

57

0.23 3.36 0.85 0.89 - 30.9 49.3 219.1

58

0.17 2.58 0.72 0.90 - 21.9 34.8 198.6

59

0.20 3.03 0.85 0.86 - 27.5 45.4 223.7

60

0.25 2.90 0.77 0.91 - 34.4 54.3 184.8

61

0.23 3.12 0.89 1.02 - 27.8 38.9 182.9

62

0.21 2.98 0.88 0.88 - 34.3 58.9 211.3

63

0.24 3.07 0.84 0.95 - 36.3 62.0 198.5

64

0.21 3.08 0.79 0.88 - 36.0 46.6 213.1

65

0.18 2.97 0.83 0.99 - 35.9 44.5 190.1

66

0.20 3.11 0.85 1.03 - 36.1 46.2 168.6 Rec-overY:-------------------------------------------------------------------------------------------------------------------------------

67 0.20 2.81 0.83 0.91 - 32.7 41.1 194.0

68

0.22 2.97 0.87 0.85 - 32.3 54.2 225.7

69

0.23 2.60 0.71 0.72 - 23.2 37.2 239.3

70

0.19 2.52 0.69 0.79 - 31.2 33.4 232.1

71

0.21 2.85 0.83 0.89 - 29.3 36.3 192.0

72

0.21 2.91 0.77 0.82 - 33.7 42.2 228.7 ,----------

# (

( Addendum 9 Sex 28-day repeated-dose oral toxicity study in rats Pathological findings Bll-0443 Exp.group Animal Fate Gross findings (mg/kglday) No. Histopathological findings 1 

### ta Glandular stomach Glandular stomach Blackish region ofmucosa Necrosis of mucosa+


## ..................................._ ..........................G...**E~8.!.?..~~?.**

### ..**~P.?..~!>..'l..**

## ..................................................................................................................................................... 2 ta NAD Liver 

### ·····-··---·····-····-········-·..·······················--·-··-····-··············-········..

## ·················-·························-·············~·!.~~?..W..~?.~~~.~~...



## ~

........................................... 3 ta NAD Liver 

### .................................................................................................................................................... -....................**~.!.~!..?..~~?.~!..9..~-~-~······························-·····-·-·** 4 ta NAD NAD ·········-····-················-·--············-·····-·········-···················-························

### ..·········

### ..·-·······················-··················-··--···················-·············-········-················-······-·········· 

### 5 

### ta NAD NAD .............................................................................................................................................................................................................................................................................. 

### Vehicle control 6 

### ta NAD Kidney Basophilic tubules 

### + Fibrosis+ Recovery group 7 ta NAD NAD 

### 8 ta NAD NAD 9 

### ta NAD NAD Male 10 ta NAD NAD 11 ta 

### NAD NAD 12 . ta 

### NAD NAD 13 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

### 14 ta NAD Not examined 

### 15 ta Cerebrum Cerebrum 

### 40 Partial defect ofcortex Partial defect ofcortex+ 

### 16 ta NAD Not examined 17 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

### 18 ta NAD Not examined 

### 19 ta NAD Not examined 20 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

### 200 21 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

### 22 ta NAD Not examined 23 ta NAD Not examined 

### .......................................

# -.........-........................................................................................................................................................................................................................ 

### 24 ta NAD Not examined ta, terminal autopsy. NAD, no abnormalities detected. +,slight; ++, moderate.



### - 27 - 

# P1 )-75

I 1'

### Addendum 9 Sex

Male

( \ .l:Hl-U44J



### - Continued

Pathological findings Exp.group Animal Fate Gross findings Histopathological findings (mglkg/day) No.

1,000 

### 25 

### ta NAD Liver 

## ······-··································································································································································~·i·~~~W..~!:?:~.l.~.I?.~...

### :!:........................._ ..._

........... 26 

### ta NAD NAD ··················-···-·········-··-·····--··-···········-·········-

### ..-··-·······

### ..··················-······················-·

### ..·····--·················--·······························-···-··-

### ..········-···-·-··············-···-····-···· 

### 27 

### ta NAD NAD ............................................................................................................................................................................................................................................................................... 

### 28 ta 

### 29 ta 30 

### ta Recovery group 31 

### ta NAD NAD NAD Testis

Small (right) NAD NAD NAD Testis

Focal atrophy of seminiferous tubules++ 32 

### ta NAD NAD ...............................·-···--··-············

### ..··---··----·-·--··

### ..-·..--

### ..

## --.................................

## _ .....

# -..............................................

## _ ...,,..._ ......................

## _ .....

## -.......

# -............. 

### 33 

### ta Kidney Kidney Apparent spotty pattern of Increased eosinophilic



## -···-··-········-··-··-·····-······················-··~·~E!.~.~~..

## Q?.i!~.t.~~.~!2...

# ....................**-···········-··--··········~.?..9.~~**..2:.±.······················-·-··········-·····-···-··-·-·-······ 34 

### ta NAD NAD
.......,_................................................................................

## ,_...........................................................................................................................................

### -................................. 

### 35 

### ta NAD NAD 36 

### ta NAD NAD

- 28 -

### ( \

/ 

### Bll-0443 Addendum 9 - Continued Pathological findings Sex

Female Exp.group (mglkg/day) Animal No. 37 

### Fate ta NAD Gross findings Histopathological fmdings NAD 38 

### ta NAD NAD ·····················-······-·································-························-·······························································································-································································-··············· 

### 39 ta NAD NAD 40 ta NAD NAD .............................................................................................................................................................................................................................................................................. 

### 41 ta NAD NAD 42 ta NAD Liver



## ··---·-------·--·--·····-··---------------·-······-·······-···········---····--·-·····--··-···········-·················-·····-················--------~!-~~?.~~~~-!.?..~~-::!:.__________: __________________________ Vehicle control Recovery group 43 ta Skin Skin



## ······--------·-············--····-·--······-----------!:.?.~~--~-f..~~i~

# ..**{~~!Y~.~~Q**________________________________

## ~!!.~P.-~Y...**?..f..?..~~~--f.?.!!~~-!-~-~---:!::······-·····-·-·** 44 

### ta NAD NAD 45 ta NAD NAD ··-···-·-····-·-········-·--·--···-··················-·-·-········-······--······-·-···

### ..········-·····-······-··········

### ..·····-···-···-·---········.................................................._.,,

### ..,

## ____..................... 

### 46 ta NAD NAD 47 ta NAD NAD ..........................................................................................,_,...........................

# _ ....................................................

## _ ....................................... ,_,,,.............,_, _______________

### .. 48 

### ta NAD NAD 

### 49 ta NAD Not examined 

### 50 ta NAD Not examined ····················-·····-············-·······································································-·································--·--

### ..··-··--...................................................................................................... 

### 40 51 ta NAD Not examined 52 ta NAD Not examined 53 ta NAD Not examined 54 ta NAD Not examined 

### 55 ta NAD Kidney



## ··········--········--··--···········-·················--··········--··---·--------------······--···--····------·-······--···············--···--················--··----··gy~!..

## f.?.~.~~i?.~

# ..::!: ...................................._ .......... 56 

### ta NAD NAD 57 ta NAD NAD .............................................................................................................................................................................................................................................................................. 

### 200 58 ta NAD Kidney Basophilic tubules + Fibrosis+ 

### 59 ta NAD NAD 

### 60 ta NAD NAD

- 29 -

# (_ Bl1-0443 Addendum 9 - Continued Pathological findings Sex Histopathological fmdings Exp.group Animal (mglkg/day) No. Fate Gross findings 61 

### ta Kidney Kidney



### .....

## ·-·-······-··-············-·····-·····-···········}~~~Y.~~--~~~-~!~!.!~~..

## J~!.!~!.~~-~!!.

# .....................- ....**~~-~Y.!.~**..

## ~~~-~-~£!.~~---~·····-·········-···-····-········-·- 62 ta NAD NAD 63 ta NAD NAD ···········-··-··-··-·-····--·

### ..···-···········--··-

### ..······-······--·--·-·······--···········-········-································-·······--·······-····-········--·······--···········-···-·····-········--···-···--···· 

### 64 ta NAD NAD 65 

### ta Glandular stomach Glandular stomach Blackish region ofmucosa Necrosis ofmucosa 

### +

## ······-··--·--·····--·······-···-···--·······-····(9_::?..~..

## ~...

## ~>...- 

# ..·····-··-·······---··-········-·············-··--··-·····-···-·-····-·-···-·······-··--···-·----·--··-··-··-·-··-· Female 1,000 66 

### ta NAD NAD Recovery group 67 ta NAD NAD ....,_.................................

# _ .........

# __.,_......,

### _____,,,

### ................................................._,,.......................;

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### Bll-0443 

# ( APPENDIX

PHYSICOCHEMICAL REPORT

Person in charge ofChemical Analysis: Signed in original May 29, 1998 Takeharu Wada, M.S.

**-I~**

# (

( Bll-0443 

### Sponsor: NISSHINBO INDUSTORIES, INC. R & 

### D CENTER Testing Facilities: Hita Research Laboratories, Chemical Biotesting Center, Chemicals Inspection & 

### Testing Institute, Japan Test substance: V-OSH (Lot No. 70616) Tests: 1) Stability Test ofTest Substance 2) Stability and Uniformity Tests ofTest Substance in Formulation Date ofTest: 1) December 24, 1997 and March 3, 1998 2) January 22, 26, 29, 1998 Methods: 1) Stability Test ofTest Substance The test sample was taken (n=l) before and after the dosing period, and analyzed with infra-red spectrophotometer (IR-470, Shimadzu). 2) Stability and Uniformity Tests ofTest Substance in Formulation Formulations were prepared before dosing and following tests were performed. The formulations were stored in the cold and dark place during the test period. (1) Stability Test Middle layers of the formulation were taken (n=3) just after preparation, at interval, and 7 days after. After pre-treatment, the test substance in the formulations was quantitatively analyzed by the high performance liquid chromatography (HPLC). (2) Uniformity Test Upper, middle and lower layer of formulations were taken (n=3) after preparation. After pre-treatment, the test substance in formations was analyzed by HPLC to estimate the uniformity oftest substance. Stability test and uniformity test were performed at the same time, and the mean value (n=3) of results of the uniformity test was served as the data of the stability testjust after preparation.

-2-

### Results: 

## ( Bll-0443 

### 1) Stability Test ofTest Substance (Fig. 1, 2, 3) IR spectrum of the test substance was identical with IR spectrum provided from the Sponsor. There were no differences of IR spectrum pattern between before and after dosing periods. Therefore, the test substance was stable during the test period. 2) Stability and Uniformity Tests of Test Substance in Formulations (Table 1, 2) The test substance in formulations was stable (within 

### ±10%) for 

### 7 days. The uniformity ofthe test substance among the three layers was virtually good with less than 5% relative standard deviation (R.S.D.%).



### -3-

**~~--·-~-**( \, 

### Bll-0443 1. Pre-treatment ofFormulation 1.1 10 w/v% formulation Accurate 1 ml offormulation was added to tetrahydrofuran (THF) to make 50 ml, and treated in an ultrasonic bath for 2 minuites. Accurate 0.5 ml ofthis solution was added to THF to make 50 ml and served for HPLC. The dilution rate ofthe sample was 5,000. 1.2 2 w/v% formulation Accurate 1 ml offormulation was added to THF to make 25 ml. Accurate 0.5 ml ofthis solution was added to THF to make 20 ml, and served for HPLC. The dilution rate ofthe sample was 1,000. 2. Preparation ofStandard Solution Approximately 0.5 g (0.5500 g) ofthe test substance was accurately weighted, and dissolved in THF to make 100 ml (undiluted standard solution; 5,500 J.tg/ml). Accurate 1 ml ofthis solution was added to THF to make 25 ml. Accurate 2 ml of this solution was added to THF to make 20 ml (standard solution; 22.0 J.tg/ml). 3. Analytical Conditions Instrument: High performance liquid chromatography (Model L-6200, Hitachi Ltd.) Column: Shodex Asahipak GF-310 HQ 8.0 mm l.D. X 

### 300 mm T t 4o

# oc

empera ure: Eluent: 10 mM sodium perchlorate tetrahydrofuran solution Flow rate: 1.0 mllmin Detector: Wave length: Recorder: Injected amount: UV-VIS detector (Model L-4250, Hitachi Ltd.) 260nm HPLC system manager (Model D-7000, Hitachi Ltd.) 100 J.tl Under the analytical conditions mentioned above, the. waking curve was a straight lines which passed through the origin of the coordinates, and obtained linearity with a correlation coefficient of0.999 (Fig. 4). The typical chromatogram is shown in Fig. 5.



### -4-

# (

( Bll-0443 

### 4. Calculation The total peak area of the test substance in each HPLC sample was measured to calculate concentration ofthe test substance in each dosing solution (C: w/v%) using the following equation:

-C= CsxAxD Asx 10,000 where: Cs: Concentration ofthe test substance in the standard solution (J.tg/ml) As: Total peak area ofthe test substance in the standard solution A: Total peak area ofthe test substance in each HPLC sample D: Dilution rate in each HPLC sample



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4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 BOO 500 700 600

Fig.3 IR spectrum provided by the sponsor

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### 10 20 30 40 50 Concentration (!lg/ml) 

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### 11.0 34,495 22.0 71,176 55.0 178,935

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### 8 9 10 

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### Table 1 Chemical stability ofthe test substance in dose formulations I I 

### \ Time point of Nominal Found cone. Dilution Actual cone. Mean cone. R.P. measurement conc.(w/v%)

**(~g/ml)** 

### rate (w/v%) (w/v%) (%) Immediately 10.0 9.61 after preparation 2.0 1.99 19.407 5,000 9.70 10.0 18.855 5,000 9.43 9.63 100 4 days after 19.534 5,000 9.77 preparation 19.939 1,000 1.99 2.0 19.055 1,000 1.91 1.94 97.3 19.089 1,000 1.91 19.782 5,000 9.89 10.0 19.473 5,000 9.74 9.88 103 7 days after 20.074 5,000 10.0 preparation 20.391 1,000 2.04 2.0 20.267 1,000 2.03 2.04 102 20.376 1,000 2.04 R.P.(%): Rate to the concentration measured immediately after preparation Table 2 ( 

### Physical stability ofthe test substance in dose formulations 

### \ Nominal .Layer of Found cone. Dilution Actual cone. Mean cone. R.S.D. conc.(w/v%) measurement

**(~g/ml)** 

### rate (w/v%). (w/v%) (%) 18.845 5,000 9.42 top 18.523 5,000 9.26 9.41 19.118 5,000 9.56 19.496 5,000 9.75 10.0 middle 19.409 5,000 9.70 9.74 9.61 2.4 19.559 5,000 9.78 19.497 5,000 9.75 bottom 19.294 5,000 9.65 9.67 19.190 5,000 9.60 20.191 1,000 2.02 top 20.059 1,000 2.01 2.01 20.002 1,000 2.00 20.507 1,000 2.05 2.0 middle 20.416 1,000 2.04 2.05 1.99 3.3 20.601 1,000 2.06 18.359 1,000 1.84 bottom 19.845 1,000 1.98 1.92 19.459 1,000 1.95 R.S.D.(%) :Relative standard deviation / 

# \ -11-



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### Accepted number Study number P03-2314 82314



# STUDY ·REPORT - I

Measurement ofmolecular weight distribution ofV-05H

September 9, 2003 Kurume Laboratory Chemicals Evaluation and

**~esearch** 

### Institute, Japan 

# f1

**~1D**( 

### \ Sponsor Title Studynumber 

# STATEMENT

NISSHINBO INDUS1RIES, INC. 82314

Kurume Laboratory Chemicals Evaluation and Research Institute, Japan
Measurement ofmolecularweight distribution ofV-05H 82314 I, the undersigned, hereby declare that this report provides a correct English translation ofthe Study Report (Study No.82314, issued on September 9, 2003).

Date StudyDirector Takanori Matsumoto

# ( 

# SUMMARY Title Measurement ofmolecular weight distribution ofV-05H Test conditions (1) Test method GPCmethod 2000mg!L 82314

(2) Concentration oftestitem (3)Datatreatment system Waters Corporation MILLENNIUM 201O(GPCN) Results The molecular weight ofthe test item was calculated from the calibration curve ofthe polystyrene standards. The average content ofthe component ofthe molecular weight less than 1000 for the test item was 5.53 

### %. Results ofmolecular weight distribution oftest item Number average molecularweight (Mn) 

### 4.}2 X 

### 103 

### Weight average molecular weight (Mw) 1.65 X 

### 104 

### Degree ofdispersion (Mw/Mn) 4.01 Average content ofcomponent ofmolecular weight less than 1 000 (%)· 

### 5.53

# (

> 

### e 

# (

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# .. ,g 

### "' :;:, 3: 

### "' Molecular weight distribution bypolystyrene standards Figure ofmolecularweight distribution Number average molecularweight 

### (Mn) Weight average molecularweight (Mw) Z average molecularweight ( 

### Mz ) Degree ofdispersion ( Mw

### IMn) See figures 4.12 X 

### 103 

### 1.65 X 

### 104 

### 3.39 X 

### 104 

### 4.01 Average content of component of molecular weight less than 1000 (%) 

### 5.53

40.00 30.00 20.00 10.00 0.00 -10.00 -20.00 

### (Thevalues 

### inthe table are the average ofduplicate measurement.) 

## GPO chromatogram**.~,** '' 

### i \ ; I , I I ' 

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...........................................................................______, .............L--............ .

0.00 5.00 10.00 15.00' 20.00 25.00 30.00 35.00 40.00 0.80 0.60 0.40 0.20 0.00 min 

### Sample Test item-1 Vial 6 Date 2003/07/16 15:24:12



### . gl _ ....... -1 00 00**~** 

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## -o.oo ' 4.o'o**~.oo 2~-**  5.00 4.50 3.50**LDg~Mw**
.............. Molecularweight distribution

.............. Total area 

### 82314

### 82314 

## ( 

# CONTENTS page Title 1 Sponsor 1 Test facility 

### 1 Objective 1 Dates 1 

### Personnel · 1 

# ( Approval ofstudy report 1 1. 

### Testitem 2 2. Performance oftest 3 3. Results 4 4. Remarks 4

### Figures 82314 

### Fig. 1 Chromatogram ofGPC analysis and calibration curve for standard polystyrene Fig. 2 Chromatogram and results ofGPC analysis for test item -1 Fig. 3 Chromatogram and results ofGPC analysis for test item -2 Fig. 4 Chromatogram ofGPC analysis for solventblank

# (

(

( 

### 82314 Title Measurement ofthe molecularweight distribution ofV-05H Sponsor 

## NISSHINBO INDUSTRIES, INC. 1-2-3, Onodai, Midori-Ku, Chiba, 267-0056, Japan Testfacility ObjectiveKurume Laboratory ChemicalsEvaluation and Research Institute, Japan 19-14 Chuomachi, Kurume, Fukuoka 830-0023, Japan This study was performed to evaluate the molecular weight and the molecular weight distribution ofV-05H. Dates Studyinitiation date July 15,2003 Study completion date September 9, 2003 PersonnelStudyDirector Takanori Matsumoto Studypersonnel Tamaki Hata Approval ofstudy report StudyDirector Date September 9, 2003 . Signature Signed in original Takanori Matsumoto 1

## ( "'--

# (

( 82314 2. Performance oftest

2.1 Test method The molecular weight distribution of the test item was measured by using high-peiformance liquid chromatography (gel permeation chromatography). The measurement was conducted in duplicate. The number average molecular weight and the weight average molecular weight were calculated from the calibration curve ofthe standard items ofmolecular weight. 2.2 Preparation oftest item solution 20 mg ofthe test item was dissolved and filled up to 10 mL with the eluent; The mixture was allowed to stand at 25±1 

# oc for about 24 hours to prepare 2000 mg/L solution ofthe test item. Then the solution was filtrated with a membrane filter (pore size 0.45 11m) andthe filtrate was analyzed. 2.3 Preparation ofstandard solution The polystyrene (PS) standards were used 

## as the standard items of molecular weight. The PS standards, whose average molecular weights are described

### bello~, were dissolved in the eluent to prepare the standard solution. The concentration of each PS standard in the solution was 1000 mg/L. Then the standard solution was filtrated with a membrane filter (pore size 0.45. 11m) and the filtrate was analyzed. PS Standard (TSK Standard) F-700 F-128 F-40 F-10 F-4 F-1 A-2500 A-500 2.4 Measurement conditions Instrument GPC system Datatreatment system Column Columntemperature Eluent Flowrate Sample size Average molecular weight 6770000 1260000

355000 J02000

43900

9500 '2800 526 High-performance liquid chromatograph Showa Denco K.K. GPC SYSTEM 21 Waters Corporation MILLENNIUM 2010(GPCN) Shodex GPC KF-806L x 

### 2 30 em x 

### 8.0 mm I.D. stainless steel Shodex GPC KF-800D x 

### 1 10 em x 

### 8.0 mmI.D. stainless steel 40°C Tetrahydrofuran 0.8mL/min 100 JlL 3/ \

( \ 

### 3. Results Results ofmolecularweight distribution oftest item No.1 Number average molecularweight 

### (Mn) 4.13 X 

### 103 

### Weight average molecularweight (Mw) 1.65 X 

### 104 

### Degree ofdispersion ( Mw

## I

## Mn ) 4.00 Content of component of molecular 5.50 weight less than 1000 (%) 4.Remarks Instruments and apparatuses High-performance liquid chromatograph : seepage 3 Datatreatment system : seepage 3 No.2 4.10 X 

### 103 

### 1.65 X 

### 104 

### 4.02 5.55

Electronic analytical balance: MetlerToledo type AT201 82314 Average 4.12 X 

### 103 

### 1.65 X 

### 104 

### 4.01 5.53

Special apparatus Membrane filter : ADV ANTEC DISMIC-25HP pore size 0.45 J..lffi 

### Reagent Tetnihydrofuran: Wako Pure Chemical Industries, Ltd.

4( 

## Study No. 

## 82314 Date 

## 2003/07/17 Page 1 

## I 1

> E 

### GPO chromatogram 30.00-.----------,--------- 0 :=--;-o"o--------------, 0


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### Sample PS STD Vial 5 Date 2003/07/16 11:29:58

I 16.00 

## .. ,__.

### GPO calibration curve

I 18.00 I 20.00 Elution volume I 22.00 

### GPO calibration table Molecular weight Retention time(min.) Calculated M.W. 

### 6670000 19.079 6649582 1260000 21.356 1323919

355000 23.017 320041 102000 24.313 98435

43900 25.074 48828

9500 26.841 9948 2800 28.476 2588

526 30.907 536 I 24.00



## Chromatogram of GPC analysis and calibration curve for standard polystyrene. I ' 40.00

I 26.00

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## Study No. 82314 Date 2003/07/17 40.00 30.00 20.00 10.00 

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### Sample Test item-1 Vial 6 Date 2003/07/16 15:24:12 

## Results 

# •Ret. time(time) Mn Mw Mw1Mn r 

### Mz Mz/Mw <MW1000(%) 1 26.380 4127 16498 3.998004 33889 2.054170 5.499 0.80- 

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.............. Molecularweight distribution
.............. Totalarea 2.50


## Fig. 2-1 Chromatowam and results of GPO analysis for test item -1.

# (

( Study No. 82314 Date 2003/07/17 GPC slice table 

## Page 2 

## I 2 lp: 

# . arne: ea 

## M 14924216522N P k1 

# r. Retention time(min.) Peak area M.W. Log M.W.



# -~" dwt/d(logM)



### 1 23.627 19 184513 5.266026 0.002 0.000531 2 23.904 171

**14~261** 

### 5.156129 0.026 0.004699 3 24.181 837 111098 5.045704 0.164 0.022898 4 24.459 2722 86089 .f.934949 0.680 0.074346 5 24.736 6550 66690 4.824058 2.063 0.178826 6 

### 25.o13 12017 51669 4.713226 4.904 0.328553 7 25.290 17152 40054 4.602650 9.395 0.470476 8 25.567 . 21513 31083 4.492525 15.288 0.593036 9 25.844 25930 241.57 4.383046 22.516 . 0.719654 10 26.121 29078 .·18811. 4.274409 30.923 0.814061 11 26.399 29997 14683 4.166810 39.926 0.848687 12 26.676 . 29085 11493 4.060443 48.896 0.833247 13 26.953 26894 .9026 3.955505 57.375 0.781769 14 27230 23800 7115 3.852191 65.038 0.703450 15 27.507 20151 5632 3.750697 71.665 0.606968 -. 

### 16 27.784 16369 4479 3.651218 77.155 0.503640 

### 17 28.061 12945 3581 3.553949 81.543 0.407858 18 28.339 10274 2878 3.459087 85.013 .0.332348 19 28.616 8223 2327 3.366827 87.783 0.273925 20 28.893 6483 1894 3.277363 89.985 0.223070 21 29.170 5077 1552 3.190893 91.713 . 0.181038 22 29.447 4044 1281 3.107611 93.075 0.149996 23 29.724 3333 1066 3.027712 94.179 0.129162 24 30.001 2825 894 2.951394 95.088 0.114852 25 30279 2488 757 2.878850 95.892 0.106731 26 30.556 2298 646 2.810276 96.625 0.104573 27 30.833 2223 557 2.745869 97.290 0.108123 28 31.110 1403 485 2.685823 97.862 0.073514 29 31.387 1731 427 2.630334 98.268 0.098592 30 31.664 3072 380 2.579598 99.068 0.192574 31 31.941 1286 342 2.533810 99.734 0.090007 32 32.219 286 311 2.493166 99.931 0.022808 33 32.496 90 287 2.457861 99.977 0.008382 34 32.773 33 268 2.428091 99.995 0.003703 35 33.050 0 254 2.404051 100.000 0.000000 

## Fig. 2-2 Chromatogram and results of GPC analysis for test item -1.

# ~-.

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## Study No. 82314 Date 2003/07/17 40.00

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### Sample Test item-2 Vial 7 Date 2003/07/16 16:09:10 

## Results 

# • Ret. time(time) 

### Mn Mw Mw/Mn Mz Mz/Mw <MW1 000(%) 1 26.384 4100 16472 4.017202 33914 2.058943 5.548

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--- Total area I 2.50 

## Fig. 3-1 Chromatogram and results of GPO analysis for test item -2.' ! \



## ( Study No. 

## 82314 Date 

## 2003/07/17 GPC slice table Page 2 

## I 2



# . . Mp· 14875 818326 

## Name: Peak1 

# • Retention.time(min.) Peak area M.W. Log M.W.



# -~" dwt/d(logM)



### 1 23.581 15 192432 5.284277 0.003 0.000398 2 23.862 130 148927 5.172973 0.021 0.003482 3 24.143 703 115106 5.061097 0.132 0.018804 4 . 24.424 2438 88890 4.948852 0.579 0.065097 5 24.705 6143 68619 4.836443 1.843 0.163954 6 24.986 11711 52975 4.724073 4.542 0.312922 7 25.267 17124 40921 4.611945 8.943 0.458985 8 25.548 21639 31642 4.500264 14.802 0.582858 9 25.829 26211 24504 4.389233 22.023 0.710821 10 26.110 29589 19013• 4.279056 30.474 0.809412 11 26.390 30634 14789 4.169936 39.571 0.846950 12 26.671 29736 11537 4.062078· 48.656 0.832564 13 26.952 27499 9030 3.955684 57.247 0.781355 14 27233 24306 7095 3.850959 65.006 0.702413 15 27.514 20534 5599. 3.748107 71.707 0.604917 16 27.795 16622 4439 3.647331 77.241 0.500373 17 28.076 13104 3539 3.548834 81.651 0.404123 18 28.357 10381 2837 3.452821 85.129 0.328900 19 28.638 8285 2288 3.359495 87.900 0.270455 20 28.919 6508 1858 3.269061 90.095 0.219603 21 29.200 5082 1520 3.181720 91.811 0.177861 22 29.481 4054 1252 3.097679 93.164 0.147767 23 29.762 3337 1040 3.017139 94.262 0.127204 24 30.043 2876 872 2.940305 95.171 0.115177 25 30.324 2527 737 2.867380 95.972 0.106930 26 30.605 2282 629 2.798569 96.703 0.102691 27 30.886 2253 542 2.734074 97.378 0.108546 28 31.167 1287 472 2.674101 97.907 0.066970 29 31.448 2189 416 2.618851 98.366 0.124334 30 31.729 2901 370 2.568529 99242 0.182103 31 32.010 982 334 2.523340 99.794 0.069261 32 32290 216 304 2.483485 99.940 0.017431 33 32.571 83 281 2.449170 99.978 0.007904 34 32.852 32 263 2.420598 99.995 0.003702 35 33.133 3 250 2.397972 100.000 0.000518 

## Fig. 3-2 Chromatogram and results of GPC analysis for test item -2.
il
**··-~~---**

## Study No. 82314 Date 2003/07/17 Page 1 

## I 1

> E 

### GPO chromatogram 40.00.,-----------,.------------'-----------, 30.00 20.00 10.00 0.00 -10.00 -20.00 I 0.00 

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### ··············· Sample BL THFVial 8 Date 2003/07/16 16:54:12 I I I I I '. 40.00 

## Fig.4 Chromatogram of GPO analysis for solvent blank.( '\__ 

### Accepted number P03-2315 Study number 82315 

# STUDY REPORT - IT

Study on "SafetyEvaluationFlowScheme for Polymer" ofV-OSH

September 9, 2003 Kurume Laboratory Chemicals Evaluation and Research Institute, Japan

# (

( Sponsor Title Studynumber 

# STATEMENT

NISSHINBO INDUSTRIES, INC. Kunnne Laboratory Chemicals Evaluation and Research Institute;- Japan
Study on "SafetyEvaluationFlow Schemefor Polymer" ofV-05H 82315 823l5

I, the undersigned, hereby declare that this report provides a correct English translation ofthe StudyReport (StudyNo.82315, issued on September 9, 2003).

Date 

# Octohw 23 r ) 

## 003 StudyDirector Takanori Matsumoto 

# P1 

# Jo 7j J I I l **~** l ] **~** I **/~' ~',**  **.~** 82315
Suqunruy of study results of flow scheme for polymer

I. Number average molecular weight (by polystyrene standard) Mn=4.J2 X 10 3
2. Stability test **~ ~**  'rJ **~** Test solutions pH 1.2 (2000mg/L) pH4 (2000mg/L) pH7 (2000mg/L) pH9 (2000mg/L) Before 403.8 404.5 411.1 405.4 402.7 407.5 405.3 400.9 Weight change Weight **(m~:)** Rate of weight Average After b..W change (%) (%) 404.1 -0.3 -0.1

0.0

404.2 0.3 0.1 409.3 1.8 0.4

0.3

405.1' 0.3 0.1 401.6 1.1 0.3

0.4

405.5 2.0 0.5 404.5 0.8 0.2

oA

398.7 2.2 0.5

\ TOCchange Concentration ofTOC (m C/L) After Control b.TOC Average
6.1 0.7 5.4

6.0

7.2 6.5 /

!/ I/ v
8.8 8.2 '

0.6 8.7

9.8 9.2 6.3 0.9 5.4

6.9

9.3 8.4 Results of molecular weight distribution Views by IR spectrum Average molecular weight (by polystyrene standard) Mnxi0 3 Mwxi0 4 Mw/Mn 4.31 1.83 4.24

- No difference was observed in the 4.22 1.80 4.26

IR spectra before and after the test. 4.37

1.85 4.24

.

. 4.32 1.83 4.24

I Before 4.38 1.83 4.18

b. W = weight before test - weight after test Rate of weight change AW = weight before test X 100 (%)

# (

( \



# ( 82315 3. Molecular weight distribution by polystyrene standards

> E



# %**~ ~**  3: 

### .., 40.00 30.00 20.00 10.00 0.00 -10.00 -20.00 

### Figure ofmolecular weight distribution Numberaverage molecularweight 

### (Mn) Weight average molecularweight (Mw) Z average molecularweight ( 

### Mz) Degree ofdispersion ( Mw

### I Mn) 

### Average content of component of molecular weightless 

## than 1000 (%) See figures 4.12 X 

### 103 

### 1.65 X 

### 104 

### 3.39 X 

### 10 4 

### '4.01 5.53 (The values 

### inthetable are the average ofduplicate measurement.) 

## GPC chromatogram 0 

## J, 

# 8 

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### 20~00**25~00 35~00** 

### 40~00
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### ~.oo I 5.00 4.50 I 4.00 3.50 3.00 Log Mw Molecularweight distribution Total area I 2.50


# ----·**·-----.~-~**

### 82315 

# CONTENTS ( page Title 

### 1 Sponsor 

### 1 Test facility 

### 1 Objective 

### 1 Test method 

### 1 Dates 

### 2 

# ( Personnel 

### 2 Approval ofstudyreport 

### 2 1. Test item 

### 3 2. Stabilitytest 4 

### 3. Results 

### 9 4. Summary 

### 12 5. Remarks 

### 12



# ---

**-~---~-,**

### 82315 FigliresFig. 1 IR spectrum oftest item before stability test Fig. 2 IR spectrum oftest item (pH 1.2) after stabilitytest Fig.3 IRspectrum oftest item (pH4) after stability test Fig;4 IR spectrum oftestitem (pH 7) after stability test Fig. 5 IR spectrum oftest item (pH 9) after stability test Fig. 6 · GPC chromatogram ofpolystyrene standards and calibration curve Fig. 7 GPC

### chromatogram andresults oftest itembefore stabilitytest Fig. 8 GPC chromatogram andresults oftest item after stabilitytest (pH 1.2) Fig. 9 GPG

### chromatogram andresults oftest item after stabilitytest (pH4) Fig. 10 GPC chromatogram andresults oftest item after stabilitytest (pH 7) ( 

### Fig. 11 GPC chromatogram and results oftest item after stabilitytest (pH 9) 

## \ Fig. 12 GPC chromatogram ofsolventblank

# ~f 

# }!(

# ( '



# (

( 82315 Title Study on "Safety Evaluation Flow Scheme for Polymer" ofV-05H Sponsor · NISSHINBO INDUSTRIES, INC. 1-2-3, Onodai, Midori-Ku, Ch;iba, 267-0056, Japan Test facility Kurume Laboratory Chemicals Evaluation and Research Institute, Japan 19-14 Chuo-machi,Kurume-shi, Fukuoka 830-0023, Japan Objective This study was performed to evaluate the information on safety evaluation for polymer. Test method This study was conducted according to "Stability Test and Solubility Test 

### in Water . and Organic Solvents" based on "Safety Evaluation Flow Scheme for Polymer (The Report Compiled byPolymer Commission,November, 1985)".


# P1 )IV

# (

(

( \ 

### Dates Studyinitiation date Study completion date Personnel Study Director Studypersonnel Approval ofstudyreport StudyDirector 82315 

### July 28, 2003 September 9, 2003 Tak:anori Matsmnoto 

## Tamak:i Hata Date September9, 2003 Signature Signed 

### inoriginal. Tak:anori Matsmnoto

2 

# rfJ( '3/3

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----,-=--

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### 82315 2. Stability test

2.1 Test method
2.2

2.3 About 400 mg ofthe test item was accurately weighed and 200 mL ofthe buffer solution described 

### in 2.3 was added to obtain test solutions. After the test**sol~tions** 

### were stirred 

### in a constant temperature bath at the test temperature (40 ± 

### 2 °C) for two weeks (24 hours for the test solution ofpH 1.2), theywere equilibrated at 25 ± 2 °C, and thenanalyzed forthe stabilitytest ofthe test item. Testconditions (1) Testconcentration 2000 mg!L (400 mg 

### I 200mL) (2) Testtemperature 40±2°C (3) Testperiod 2 weeks (24 hours forthetest solution ofpH 1.2.) ( 4) Equilibrium temperature 25 ±2

# oc (5) Equilibriumtime 24 hours (6) Light**Room~light ~**  

### (7) Numberoftest repetition 2 Preparation ofbuffer solution

··Abuffer solution ofpH 1.2 16.1 mL of2.0 mol/L hydrochloric acid and 12.5mL of2.0 mol/L potassium chloride solutionwere mixed and filled up to

### 500 mLwithpurifiedwater. • A buffer solution ofpH 4 2.0 mL of 0.1 mol/L sodium hydroxide solution and 100 mL of 0.25 mol/L potassium hydrogen phthalate solution were mixed and filled up to 500 mL with purified water. · A buffer solution ofpH 7 14.8 

### mL of 1.0 mol/L sodium hydroxide solution and 50.0 

### mL of 0.5 mol/L potassium phosphate solution were mixed and filled up to 500 

## mL with purified water. · A buffer solution ofpH 9 10.7 

### mL of 1.0 mol/L sodium hydroxide solution and 50.0 

### mL of 0.5 mol/L potassium chloride and 0.5 mol/L boric acid solution were mixed and filled up to 500 mLwithpurified water. 4 

## tf}( 

# 715"( \

/ 

### ( 

### 82315 2.4 Analysis oftest solution The test solutions were pretreated for each analysis according to the following flow scheme. Flow scheme (pH1.2) Test solution • Decantation Supernatant Residue. ·Washing(purified water) • Drying(underreducedpressure, 40 °C) Sample for TOC analysis Sample for gravimetric analysis Sample for IRanalysis • Weighing 20 mg (electronic analytical balance)

**~Tetrahydrofuran** 

### 10 mL

(measuringpipet) • Standing (25 °C, about 24hours) • Filtration (DISMIC-25HP) Sample for HPLC analysis

5

# ( Flow scheme (pH4, pH7 and pH9) ·Takingout

(komagome pipet) 82315

Supernatant Residue • Taking out 10 mL

(measuringpipet) • Centrifugation

(1000 X 

### g, 10

### min) • Washing (purified water) • Drying (underreducedpressUre, 40 °C) Sample for gravimetric analysis Sample for TOC analysis (Exceptfor the test

soluion ofpH4) Sample for IR analysis • Weighing 20mg (electronic analytical balance)**~Tetrahydrofuran** 

### 10

## mL (measuringpipet) • Standing (25 °C, about24 hours) • Filtration (DISMIC-25i-IP) Sample for HPLC analysis

6 

### !){ 

# 117

# (

( 82315 (1) Analysis oftotal organic carbon (TOC) The samples for TOC analysis were analyzed under the following conditions. The test solutions of pH 4 were not analyzed because these solutions contained potassium hydrogen phthalate. Analytical conditions Instrument Temperature offurnace Flowrate Injection volume ·sensitivity (2) Gravimetric analysis Total organic carbon analyzer Shimadzu Corporation type TOC-5000 680°C 150mL/min 

### 33 J.tL Range 5
· The ·samples for··gravimetric analysis were weighed after the constant weight were obtained. 

### (3) Measurement ofinfrared absorption spectrum (IR) The samples for IRanalysis were measured under the following conditions. Measurement conditions Instrument Sample preparationmethod

7 

### Fouriertransform infrared spectrophotometer Shimadzu Corporation type FTIR-8200PC Neat

### \

( \



# ( 82315 (4) Measurement ofmolecular weight distribution The samples for HPLC analysis were applied to measurement ofthe molecular

. weight distribution under the following conditions. The molecular weight

disutribution: ofthe test item was also mesured. Measurement conditoions

Instrument _GPC system High.,-performance liquid chromatograph ShowaDenco K.K. GPC SYSTEM21 Datatreatment system Waters Corporation MILLENIUM 2010 (GPCN) Column Shodex GPC KF-806L x 

### 2 Coltnniltemperature Eluent Flowrate Sample size 30 em x 

### 8.0 mmJ.D. stainless steel Shodex GPC KF-800D x 1 10 em x 8.0 mmJ.D. stainless steel 

### 40°C Tetrahydro:furan 0.8rnUmin 

### 100 J!L ·Standard items for measurement ofmolecularweight Polystyrene standards Average molecular weight 6770000, 1260000,3§5000, 102000,43900,9500,2800,526

8( \ 

### 82315 3. Results 

### (1) Analytical results ofTOC The concentrations ofTOC in the test solutions are as follows. *3 *2 

### Concentration ofTOC after test Theoretical Solubilization Test (mgC/L) concentration rate of solution ofTOC component (mgC/L) carbon(%) After Control ATOC Average Average test pH 1.2 6.1 0.7 5.4 6.0 1574.8 0.3 

## OA 7.2 6.5 1577.6 0.4 pH7 8.8 0.6 8.2 8.7 1570.5 0.5 0.6**9~8** 

### 9.2 1589.3 0.6 pH9 6.3 0.9 5.4 6.9 1580.7 0.3 0.4 9.3 8.4 1563.5 0.5 *2 Theoretical concentration ofTOC: The theoretical concentration ofTOC means the concentration ofTOC in the
- 

### case that the test iteni is completely dissolved in the test solution, and was calculated by the following equation. Theoretical concentration ofTOC (mgC/L)

_ Weight oftest itembefore test (mg) [ Weight ofcarbon in segment 

# J

- Volume oftest solution (L) 

### x Morecular weight ofsegment intest itemj [ Weight ofcarbon in segment .

# J where M 1 .gh.t f 1 . 

### t t .t - was calculated from the orecu arwet o segmen m es 1

### em structural formula described in 1.2, and the results was 0.78. *3 Solubilizationrate ofcomponent carbon : · Percentage of the measured concentration of TOC in the test solution to the theoretical concentration ofTOC, expressedbythe following formula.
• • • · 0 _ 

### Concentration ofTOC Solubthzationrate ofcomponent carbon 

### (Yo) - Theoretical concentration ofTOC x 100
9I \ 

### 82315 (2) Weight change and rate ofweight change (%) 

### in gravimetric analysis Test Weight (mg) Weight change (%)*5 

### solution Beforetest Aftertest 

### A.w*4 Average pH 1.2 403.8 404.1 -0.3 -0:1 0.0 404.5 404.2 0.3 0.1 pH4 411.1 409.3 1.8 0.4 0.3 405.4 405.1 0,3 0.1 pH7 402.7 401.6 1.1 0.3 0.4 407.5 405.5 2.0 0.5 pH9 405.3 404.5 0.8 0.2 0.4 400.9 398.7 2.2 0.5 *4 f).

### W =weightbeforetest -· weight aftertest 

## *5 Rate ofweight change(%)= W 'gh f . 

### bee. · x 100 e1 t o test Item 10retest

10

I I I i I I I( 

### 82315 (3) Results ofmeasurement ofinfrared spectra (IR) (see Figs. 1 - 6) No difference was observed between the IR spectra ofthe test item before the test and those after the test. The characteristic fundamental vibrations ofthe test item are asfollows. Wave number (cm- 1) 

### 3000-2800 Around2270 Around2140 1600-1400 Reversion C - H Stretching vibration - N 

## =C = 

## 0 Stretching vibration ·- N = 

### C 

## =N- Stretching vibration C = 

### C Ring vibration (4) Results ofmeasurement ofmolecular weight distribution (see Figs. 7- 11) The number average molecular weight· and the weight avarage**molecul~** 

### weight ofthe test itembefore and afterthe test ate as follows. Results ofmeasurement ofmolecularweight distribution Average molecular weight · bypolystyrene standards Sample Numberaverage Weight average Degree ofdispersion molecular weight molecular weight

- 

### (Mw) (Mw/Mn) (Mn) 

### Before 4.38 X 

### 103 

### 1.83 X 

### 104 

### 4.18 pH 1.2 4.31 X 

### 103 

### 1.83 X 

### 104 

### 4.24

pH4 4.22 X 

### 103 

### 1.80 X 

### 104 

### 4.26 pH7 4.37 X 

### 103 

### 1.85 X 

### 104 

### 4.24 pH9 4.32 X 

### 103 

### 1.83 X 

### 104 

### 4.24

11\



## (



# ( 82315 4. Summary The weight change ofthe test item before and after the test was less than 2.2 mg (the rate ofweight change was less than 0.5 

### %) and the concentration ofthe dissolved organic carbo.n was less than 9.2 mgCIL (solubilization rate of component organic carbon was less than 0.6 

### %). In the measurement ofinfrared absorption spectra and molecularweight ofthe test item, no difference was observed between those measured before the test andthose measured afterthe test. 5.Remarks Instruments and apparatuses High-performance liquid chromatograph : see page 8 Fouriertransform infrared : see page 7 ·Total organic carbon analyzer : see page 

### 7 Constant-temperature incubator :Thomas Scientific Instrument Co., Ltd.· typeAT-24R Electronic analytical balance : Datatreatment system : Special apparatus Membrane filter : Reagents Purifiedwater: Tetrahydrofuran: Hydrochloric acid : Potassium chloride : Sodiumhydroxide : Potassiumhydrogenphthalate : Potassiumphosphate : Boric acid: Metler type AT201 see page 8 Advantec INC. DISMIC-25HP pore size 0.45Jlm Takasugi Seiyaku Co., Ltd. Wako Pure Chemical Industries, Ltd. Kanto C)J.emical Co., Inc. Wako Pure Chemical Industries, Ltd. Kanto Chemical Co., Inc. Wako Pure Chemical Industries, Ltd. Wako Pure Chemical Industries, Ltd. WakoPure Chemical Industries, Ltd.

12

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( 100.0 --;-,---------.----------------- 

# " 1 I i "i

20.0 ····· ....... -- ----- 0.0 +-,--r-,-.---+-.---.---.-.-t----,,--,---r-,--+---r-.--.--..-i--,-.,.-.,--,-+---l

4000.0 3000.0 2000.0 1500.0 1000.0 500.0 1/cm 

## Fig. 1 

# IR spectrum of test item before stability test. Study No. : Method Date Name 82315 Neat September 1, 2003



# ·rAM

### fl fC 

### 1. H A 1 A

# (

\ 100:0 **-:---~------,---------------** 

### j %T



# ~ 

# t.J .."'· 

# ::

**~~-~** I ! 40.0 --······· ··-·-···-····-···-·····-·····-·-·-! ···----·--·--------·----- .- ----·-······--·-····-·- ····--·-· 

# I --·-··· ·····--··..····---······ --·-···1 

## I i 

# I 20.0 - - .... ........ ----- 

# -·r --·----·- ···- ... -·-·-· ..... ---- ......... l· 

# i

.......;...... 

## ! I 0.0 **--+--.--i---.--.--+-.-.--.----.--!----J-.~-.-+--,---r'--r-.---+----.--.--,---,---+---l**

4000.0 3000.0 2000.0 1500.0 1000.0 

# Fig.2 

# IR spectrum of test item (pH 1.2) after stabiIi

# ty test. Study No. : Method.· Date Name 82315 Neat September 1, 2003 "TAHA!<=-L HAI.A 500.0 1/cm( 100.0**-,-----:-----~--r-------------"------,** 

### %T



## I 20.0 ········· -- - ---- .---- -

# --l ---- ---- --- --- -- --·-- -··-- -- -- :--- 1 ' 

## i ··--· ··-····· ... ! I 0.0 -j--,----,---.---,---j-,--,--,-,-----;!---J----.---.---.-+1 

### ---.--.--.----,---+---.----.--.--.----+---l 4000.0 3000.0 2000.0 1500.0 1000.0 500.0 1/cm 

## Fig.3 

# IR spectrum of test item (pH 4) after stabiIity test. Study No. : Method · Date Name 82315 Neat September 1, 2003 \AMA!<:1- HATA 

# f1 ))b T·-.---1-,**..-~~·---· ·~~** I \ 100.0 ,,------------,-----------.,..-------. 

# l %T 

# l 80.0

# J

40.0 t 

# I l i I 

## ········-IJY_·····



# ·-·--------·-··-·-·-·t ...··-············-·· -·-·---············-········- ! ! I . . t-·· - ---- ...



### ! 

### : l

' 0.0 -j--,--,--,--,--i---,--,--,--,--j-.,-.,-.,-,-+1 ---,---,-,--;r,-+---,--,--,--,--;--j 4000.0 3000.0 2000.0 1500.0 1000.0 

## Fig.4 

# IR spectrum of test item (pH 7) after stab iIity test. Study No. : 82315 Method · Neat Date September Name TAt-1 A

### ~ 

# r 

# 1' 2003 HATA 

### soo:o 1/cm

# ( 100.0

**~** %T 

# l 80.0



# ~'v~ (\ i / .


# ~~ 1'( 

# .... **¥tJtwtvJ:~v** 60.0

--.-,ll...........--.- 40.0 _ ................____ - -·---- - i ! i 

## .........,____, ____ ....... --- -..... ·"'t--... i '.i f i 

# I 20.0 ---- -- ________

# l ..--..... ---- ------------------- --- ---- 

# r· -------- 0.0 --l------.--,---,--.--+--.----.--.--,------cl----r--r--r----r--+--.-.---,--'--r--+----,---,--,---,--+--1

4000.0 3000.0 2000.0 1500.0 1000.0 

# Fig.5 

# IR spectrum of test item (pH 9) after stab i 

# Ii

# ty test. Study No. : Method Date Name 82315 Neat September 1, 2003 IAMA!c-1 HATA



### -...

**----T~- ~**  

### ----·----

**·~·** 

### i I I I 500.0 1/cm

## Study No. 82315 Date 2003/08/27 Page 1 

## I 1 GPC chromatogram -so.oo,.J_______ ___,_________________ ......._,...------, 4o.ooJ 0 00 

## o 8889o 

## g**g:g~o** . 0 . co ' <?**~"""** 

## :g g > ::.:::**~** .1 

## ! A I 0) . E . co I ,, ''"**~** C\1 co 10.00 .**~ ~**  I: it:: !i I C\1 ·**i~** : \ J \: \ :. •1\ LO !I :\ I: i Y' ! I P 1 I \ ! \ ; i I l \ I \ ; i 

### ,.1', 30.00 20.00

0.00 I '- .f ·-{ V i \. ; '} \/ "".**-························n·-----··---------------------..l~-----~------l---~----X**.....**~**........:............____"\ 

### r·, ,.--···---------------···· \I \ ., I -10.00 ,; t' 1\ l : 1\} 

## \I -20.00 

# v**~** 

# .. 0 -' I I I I ! I I I I I I I I I I I I I I I I I I I I I I I I I I I·' ' I I I I I I I I 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 8.00 7.00 

# -- 6.00 5.00 4.00 3.00 Min. 

### Sample PS STD Vial1 Date 2003/08/26 11:51:05 

### GPC calibration curve

' ' 

### .

# --s-~.· : ..**·~** 

# ....**~** .··-. t...**"'-.....~,** . 

# . .. ;-....__............ 

### ,.,

## ~~---------~ 

# ...,

# ----**2.00+--,--.---r-r-...----r---r--r ---o-T-.---,I,--.,--,--,---,---,.-,---~....--,---r---r--f** **14~0 16~0** **18~0 20~0** **22~0 24~0**  .**26~0** Elution volume 

### GPC calibration table 

# • Molecular weight Retention time(min.)**Calculat~d** M.W.



### 1 6770000 19.174 6726918 2 1260000 21.463 1335798 3 355000 23.153 319889 4 102000 24.469 98087 5 .43900 25.243 48520 6 9500 27.024 9953 

### 7 2800 28.669 2605 8 526 31.118 535 

## Fig.6 Chromatogram of GPC analysis and calibration curve for standard polystyrene.

I '' I \



## ( Study No. 82315 Date 2003/08/27 50.00 40.00 30.00 20.00 > E 10.00 

### GPO chromatogram
co 0 

### ,_ co .,.... 

# ,+\ ! \ 

# I \ I I 

## i \ I ' i \ 

### i \ I \ 

## Page 1 

## I 2

0.00 

# . ! \___J\**·································································································-zsL·~·-··········--·······7S"\** (\ .**~-------················** 

# . . v\ {\ t -10.00 .-20.00 0.00 

# •

1 0.80 0.60 5.00 10.00 15.00 20.00 25.00 Min.

### 30~00 

## \IV \i 11 

### i/ .\1**~**

### 35~00 

### Sample Test item Via19 Date 2003/08/26 18:01:35 

### Results 40.00 

### Ret. time(min.) Mn Mw Mw/Mn Mz 

### Mz/Mw 

### 26.430 4383 18311 4.177234 37216 2:032453
-80.00 51 -

## ~ 1 

# /W'"' ____..**--·-···-~**

# _ ....---------·---·-··--**~1**

## oo.oo r- / 

### jg \. ----

### ~**~I ~**  

# \ //·-·· N I**~ ~** **~** ;:Et 

# I' , //<0 ....r I I \ C'? M :/, )(**~ ~I**  

### .,/\\!: II / /.. \ ;::E 

## .,.... I ' . -60.00**~** -40.00 + / ,,

## ~/ / 

# ' y // \ I .!/

# \"--·-~ : / I

# I ./__../ '·-

. ... - _(\, -20.00 I ' I .. / --------- I \ 0.40 0.20

•, 

# /// \.)\ . \ 0.00 ..........-.,c::.,...--··-·// '-·- -D.OO 5.00 4.50 4.00 3.50. Log Mw Molecularweight distribution

--------- Total area 3.00 2.50 

## Fig. 7-1 Chromatogram and results of GPC analysis for test item before stability test. 

# J

## Study No. 82315 Page 2 

## I 2 Date 2003/08/27 GPC slice table ( Mp: 16706.362227 

## Name: Peak1

( \ 

# - Retention time(min.) Peak area M,W. log M.W.

# -~" dwt/d(logM) 

### 1 23.549 2 224996 5.352174 0.000 0.000066 2 23.831 60 174627 5.242112 0.008 0.001671 3 24.114 427 135328 5.131387 0.072 0.011800 4 24.396 1718 104759 5.020191 0.379 0.047345 5 24.679 4738 81044 4.908721 1.342 0.130379 '6 24.961 9942 62686 4.797170 3.592 0.273622

7 25.243 15927 48499 4.685732 7.631 0.439178 8 25.526 20570 37549 4.574603 13.310 0.569274 9 25.808 24589 29106 4.463975 20.271 0.684204 10 26.090 28207 22597 4.354045 28.436 0.790586 11 26.373 29833 17579 4.245006 37.416 0.843775 12 26.655 29304 13710 4.137053 46.530 0.837946 13 26.938 27317 10725 4.030379 55.227 0.791261 14 27.220 24391 8417 3.925180 63.153 0.717165 ' 

### 15 27.502 20878 6632 3.821650 70.077 0.624413 16 27.785 17131 5248 3.719984 75.876 0.522334 17 28.067 13585 ·. 4172 3.620375 80.545 0.423284 18 28.350**1066~** 

### 3334 3.523018 84.219 0.340556 19 28.632 8488 2680 3.428107 87.123 0.278279 20 28.914 6739 2167 3.335838 89.436 0.227607 21 29.197 5295 1764 3.246403 91.260 0.184795 22 29.479 4200 1445 3.159999 92.697 0.151972 23 29.761 3428 1193 3.076819 93.852 0.129084 24 30.044 2826 993 2.997057 94.807 0.111218 25 30.326 2545 834 2.920908 95.634 0.105160 26 30.609 2312 706 2.848566 96.365 0.100834 27 30.891 2163 60,3 2.780226 97.039 0.100172 28 31.173 1780 520 2.716083 97.687 0.088172 29 31.456 1300 453 2.656330 98.095 0.069397 30 31.738 3005 399 2.601162 98.757 0.174643 31 32.020 1909 355 2.550773 99.591 0.122239 32 32.303 478 320 2.505358 99.909 0.034224 33 32.585 100 292 2.465111 99.977 0.008143 34 32.868 37 269 2.430227 99.995 0.003569 35 33.150 0 252 2.400900 100.000 0.000000 

## Fig. 7-2 Chromatogram and results of GPO analysis for test item before stability test.' 

### I 

### '··



# (

( Study No. 82315 Date 2003/08/27 Page 1 

## I 2

> 

## GPO chromatogram**50.00.------------~----------------~--.** 40.00 30.00 20.00 10 '<t 10 <0 

## ,t, I' f ·\ ; \ I \ I \ ! \ E 10.00 

## I \ ! \



# % ..9 ';:! '- 

# ... l: 

## ., I \ I \ . I '\__ 0.00 --·······················----····················•··:··-··-···-·-·r;·)·:······--·

## -~~~~\. ....1\'"l ;"o r·-··-················· , ___

\**'--~** \ 1 \ I -10.00 -20.00 0.00 

# •

1 0.80 0.60 0.40 0.20 5.00 10.00 15.00 20.00 25.00 30.00 Min. 'J .I 

## i\ ! : i\} 1! I: 

### \i 

## ii •I 

### l! 35.00 

### Sample pH1.2 Vial 10Date 2003/08/26 18:46:35 

### Resu:lts 40.00 

### Ret. time(mln.) Mn Mw Mw/Mn Mz Mz/Mw 26.441 4307 18276 4.243566 37437 2.048449**~** 

# ..... /1r'"" g ........-··_....... -1

## oo.oo
.

### ~ I

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## ......----/ \__ -o.oo 0.00 ' I ' 5.00 I I I I I I I I I I I I I I I I I I .4.50 4.00 . 3.50 3.00 Log Mw

Molecularweight distribution
.........,..... Total area I I ,- 2.50 

## Fig. 8-1 Chromatogram of GPC analysis and results for residue after stability test ( pH 1.2 ).( \ 

## Study No. 82315 Date 2003/08/27 GPO slice table 

## Page 2/ 2 Mp: 16544.618685Name: Peak1 

# 18 Retention time(min.) Peak area M.W. Log M.W.

## -~~ dwt/d(logM)



### 1 23.549 5 224996 5.352174 0.001 0.000155 2 23.831 64 174627 5.242112 0.010 0.001831 3 24.114 439 135328 5.131387 0.078 0.012400 4 24.396 1732 104759 5.020191 0.396 0.048774 5 24.679 4690 81044 4.908721 1.378 0.131852 6 24.961 9733 62·686 4.797170 3.637 0.273700 7 25.243 . 15520 48499 4.685732 7.664 0.437257 8 25.526 19982 37549 4.574603 13.310 0.565022 9 25.808 23821 29106 4.463975 20.207 0.677257 10 26.090 27355 22597 4.354045 28.291 0.783368 11 26.373 29013 17579 4.245006 37.202 0.838396 12 26.655 28555 13710 4.137053 46.268 0.8342'49 13 26.938 26683 10725 4.030379 54.938 0.789707 14 27.220 23872 8417 3.925180 62.856 0.717132 15 27.502 20478 6632 3.821650 69.788 0.625754 16 27.785 16834 5248 3.719984 75.604 0.524443 

### 17 28.067 13361 4172 3.620375 80.295 0.425340 1.8 28.350 10481 3334 3.523018 83.985 0.341818 19 28.632 8335 2680 3.428107 86.898 0.279219 .· 20 28.914 6624 2167 3.335838 89.219 0.228565 21 29.197 5201 

### 1764 3.246403 91.052 0.185450 22 29.479 4127 1445 3.159999 92.494 0.152586 23 29.761 3404 1193 3.076819 93.660 0.130967 24 30.044 2792 993 2.997057 94.629 0.112278 25 30.326 2520 834 2.920908 95.460 0.106388 26 30.609 2285 706 2.848566 96.198 0.101834 27 30.891 2147 603 ·2.780226 96.882 0.101585 28 31.173 2066 520 2.716083 97.581 0.104533 29 31.456 1370 453 2.656330 98.071 0.074713 30 31.738 2837 399 2.601162 98.712 0.168506 31 32.020 1971 355 2.550773 99.557 0.128927 32 32.303 521 320 2.505358 99.900 0.038119 33 32.585 109 292 2.465111 99.976 0.009082 34 32.868 37 269 2.430227 99.995 0.003623 35 33.150 0 252 2.400900 100.000 0.000000 

## Fig. 8-2 Chromatogram of GPO analysis and results for residue after stability test ( pH 1.2 ).> E

## Study No. 82315 Date 2003/08/27 50.00 40.00 30.00 20.00 10.00 

## GPC chromatogram

It) It) <? <0

**~** 

# J, I \ i \ 

### ! \ I \ 

# I \ ! \ 

### f \ : \ 

## Page 1 

## I 2

0.00 J \ 

### ··---------···········--·-································:................_._______i.'S):............. . · . ....._..J\ w.'. (\ r-················· \t I ("I -10.00 -20.00' 0.00



# • 1 0.80 0.60 0.40 0.20- 5.00 v \ 

# !V I! ii L ij

**10~0 15~0**  **20~0 25~0**  **30~0 35~0**  Min. 

### Sample pH4 Via,111 Date 2003/08/26 19:31:35 

## Results 40.00 

### Ret. time(min.) Mn Mw Mw/Mn Mz Mz/Mw 26.455 4224 17986 4.258285 37013 2.057809

**~·**

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### f I \



### ~~ , I \ 

### I I ' .1 

# I / \ ..."' :-80.00 

### H>o.oo

**~** 

# i .s 40.00 II' // ..'- ,i ,, ..... i·, -20.00

............ ! f \ / ..... 't--···-"""\ 

### j \ I ./ I \.i \ . .// _/__./ I \ ..

0.00- .......

**,.--~e:._**........-·- 

# ! ''--o.oo 5.00 4.50 4.00 3.50 Log Mw·

Molecularweight distribution

----------- Total area 3.00 2.50 

## Fig. 9-1 Chromatogram of GPC analysis and results for residue after stability test ( pH4 ) .

## Study No. 82315 Page 2 

## I 2 Date 2003/08/27 GPO slice table 

## C Mp: 16354.943394

## Name: Peak1

**~** Retention time(min.) Peak area M.W. log M.W.



# -~" dwt/d(logM)



### 1 23.550 

## ·o 224804 5.351804 0.001 0.000012 2 23.833 63 174328 5.241367 0.009 0.001870 3 24.117 400 134978 5.130264 0.075 0.011890 4 24.400 1585 104397 5.018689 0.382 0.046991 5 24.683 4338 80694 4.906840 1.337 0.128422 6 24.967 9061 62361· 4.794913 3.553 0.268297 7 25.250 14500 48206 4.683104 7.521 0.430182 8 25.533 . 18744 37292 4.571610 13.104 0.558157 9 25.817 22459 . 28882 4.460627 19.960 0.672466 10 26.100 25830 22405 4.350352 28.021 0.779044 11 26,383 27400 17417 4.240980 36.911 0.834008 12 £6.667 26994 13574 4.132709 45.961 0.830801 13 26.950 25251 .1 0610 4.025735 54.625 0.787361 14 27.233 22618 8322 3.920253 62.547 0.715986 15 27.517 19421 6553 3.816461 69.489 0.625469 16 27.800 15978 5183 3.714554 75.31.9 0.524708 17 28.083 12698 4118 3.614730 80.026 0.426211 1.8 28.367 10008 3290 3.517184 83.740 0.344181 19 28.650 7993 ' 2643 3.422113 86.685 0.282428 20 28.933 6364 2137 3.329714 89.040 0.231715 21 29.217 5012 1739 3.240182 90.902 0.188639 22 29.500 3989 1425 3.153714 92.372 0.155735- 23 29.783 3288 1176 3.070506 93.561 0.133662 24 30.067 2718 979 2.990756 94.550 0.115536 25 30.350 2474 822 2.914658 95.413 0.110488 26 30.633 2290 696 2.842411 96.193 0.107977 27 30.917 2133 595 2.774209 96.909 0.106883 28 31.200 1744 513 2.710249 97.595 0.093535 29 31.483 1366 447 2.650728 98.043 ' 

### 0.079075 30 31.767 2931 394 2.595843 98.767 0.184976 31 32.050 1721 351 2.545789 99.607 0.119879 32 32.333 418 317 2.500762 99.913 0.032655 33 32.617 83 289 2.460960 99.978 0.007449 34 32.900 33 267 2.426578 99.995 0.003416 35 33.183 2 250 2.397814 100.000 0.000219 

## Fig. 9-2 Chromatogram of GPC analysis and results for residue after stability test ( pH4 ).

"""t I

# (

> E



### 'i 

# ..**~** ....... .... 

# ,. . 

### .., 

## Study No. 82315

Date 2003/08/27 Page 1 

## I 2 

## GPO chromatogram 50.00 40.00 30.00**~** 

### /1\ ! \ 20.00 ! \ 

### ! \ f \ 10.00 

## f \ ! \ 

### I \ : \. 0.00 

### / "'--·,..1\ ••••••••••·-·-••••-•••••-••·-·---·-·--··•--•-••-o•mo-oom•ooooooom•.l<.•-'••-••-""""mmoou I' ' .r\ J'_....................**'--~** W \It .,., ! -10.00 -20.00 0.00 

# •

1 5.00 10.00 15.00 20.00 25.00 30.00 Min. 

### \I\ fl.! 

# v·: j

# v I: l/ 

## \i :I 

### v 35.00 40.00 

### Sample pH7 Vial 12 Date 2003/08/26 20:16:30 

## Results Ret. time(min.) Mn Mw Mw/Mn Mz Mz/Mw 26.428 4367 18535 4.244455 37847 2.041903 

### g .--·-······-1 00.00 

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## o:8o**~** 

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## ·t; -80.00

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## -~F ':ii./ . \ 6h 

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### \ 

# ! / \ -40.00 

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# / 

# "' 

# I / , I / ""· r, 

## -2o.oo 

## I ___.../ ................... /\ / 1 / ..**t-·---·~-/"'\/** 

### \ // ' - 

## o.oo ···········-··.,<........---· "· 

## -o.oo 5.00 4.50 4.00 3.50 Log Mw

Molecularweight distribution ··············· Total area 3.00 2.50 

## Fig. 1**0~1** 

## Chromatogram of GPC analysis and res1,.1lts for residue .after stability test ( pH7 ) .

# (

(

## ~-



# ( Study No. 82315 Date 2003/08/27 GPO slice table 

## Page 2 

## I 2 Mp: 16739.626799 

## Name· Peak1 

# . 

# •• Retention time(min.) Peak area M.W. Log M.W.

## -~% dwt/d(logM)



### 1 23.549 1 224996 5.352174 0.001 0.000020 2 23.831 72 174627 5.242112 0.009 0.002002 3 24.114 480 135328 5.131387 0.082 0.013249 4 24.396 1868 104759 5.020191 0.418 0.051361 5 24.679 5006 81044 4.908721 1.445 0.137408 6 24.961 10280 62686 4.797170 3.785 0.282229 7 25.243 16250 48499 4.685732 7.916 0.446994 8 25.526 20783 37549 4.574603 13.664 0.573753 9 25.808 24670 29106 4.463975 20.651 0.684765 10 26.090 28215 22597 4.354045 28.807 0.788864 11 26.373 29796 17579 4.245006 37.760 0.840639 12 '26.655 29226 13710 4.137053 46.833**0~833643** 

### 13 26.938 27220 10725 4.030379 55.482 0.786504 14 27.220 24289 8417 3.925180 63.357 0.712406 15 27.502 20786 6632 3.821650 70.234 0.620135
.. 

### 16 27.785 17049 5248 3.719984 75.992 0.518562 17 28.067 13504 4172 3.620375 80.626 '0.419721 18 28.350 10587 3334 3.523018 84.266 0.337092 19 28.632 8412 2680 3.428107 87.137 0.275109 20 28.914 6678 2167 3.335838 89.424 0.224987 21 29:197 5245 1764 3.246403 91.227 0.182584 22. 29.479 4160 1445 3.159999 92.647 0.150152 23 29.761 3439 1193 3.076819 93.795 0.129213 24 30.044 2814 993 2.997057 94.751 0.110496 25 30.326 2536 834 2.920908 95.568 0.104546

.. 

### 26 30.609 2310 706 2.848566 96.295 0.100518 27 30.891 2147 603 2.780226 96.966 0.099193 28 31.173 1915 520 2.716083 97.628 0.094628 29 31.456 1297 453 2.656330 98.063 0.069091 30 31.738 2932 399 2.601162 98.698 0.169999 31 32.020 2034 355 2.550773 99.548 0.129936 32 32.303 540 320 2.505358 99.896 0.038602 33 32.585 111 292 2.465111 99.974 0.009035 34 32.868 42 269 2.430227 99.994 0.004046 35 33.150 0 252 2.400900 100.000 0.000000 

## Fig. 10-2 Chromatogram of GPC analysis and results for residue after stability test ( pH7 ). 

# fJ'i 

# 317

# (

( 

### \. 

# I ! 

## Study No. 82315 Date 2003/08/27 Page 

## 1 

## I 2

> E 50.00 40.00 30.00 20.00 10.00 0.00 

## GPO chromatogram "' 

# - co co 

# - A, j \ 

### ! \. 1 \ I I I \ i \ j \ I ' 

## ! \ i \.,_ I -..,-'\.**···································································-,-----·---------·zs~······--··--. -··········'-~\**  /\ n r-··-----·············· \} l 1\ !



### \ i ') -10.00 -20.00 

# \! 'I \; if v I I I I I I I I I I I I 1. I I I I I I I I I I I I I I I I I I I I I I I I I I 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 

# • 1 0.80- 0.60- 0.40 0.20 Min.



### -- Sample pH9 Vial 13 Date 2003/08/26 21:01:32 

### Results Ret. time(minJ Mn Mw Mw/Mn Mz Mz/Mw 26.437 4322 18329 4.240298 37474 2.044569

I 5.00 4.50 

## g 

# ----------!- ....-····-**~/··-** 

### ]

. 4.00 3.50 Log Mw Molecularweight distribution ·············· Total area 

# ..

.

### ~

3.00**~---·······** .

I 2.50 100.00 80.00 f--60.00

40.00 20.00**~** 

# i = 

# ...

Fig. 11-1 Chromatogram of GPC 

## analysis and results for residue after stability test ( pH9 ).( 

### \ ' \ 

## Study No. 82315 Date 2003/08/27 Page 2 

## I 2 GPC slice table

- Retention time(min.) Peak area M.W. log M.W.



# -~" dwt/d(logM) 

### Mp: 16611.60547QName: Peak1 1 23.550 3 224804 5.351804 0.001 0.000094 2 23.833 62 174328 5.241367 0.008 0.001812 3 24.117 441 134978 5.130264 0.079 0.012728 4 24.400 1732 104397 5.018689 0.405 0.049859 5 24.683 4681 80694 4.906840 1.410 0.134556 6 24.967 9688 1 

### 62361 4.794913 3.719 0.278539 7 25.250 15392 48206 4.683104 7.823 0.443394 8 25.533 19739 37292 4.571610 13.553 0.570701 9 25.817 23494 28882 4.460627 20.535 0.683015 10 26,100 26908 22405 4.350352 28.703 0.788010 11 26.383 28426 17417 4.240980 37.676 0.840135 12 26.667 27881 13574 4.132709 46.770 0.833206 13 26.950 25969 10610 4.025735 55.440 0.786231 14 27.233 23166 8322 3.920253 63.333 0.712035 15 27.517 19817 6553 3.816461 70.224 0.619688 16 27.800 16238 5183 3.714554 . 75.988 0.517762 17 28;083 12846 4118 3.614730 80.621 . 0.418660 18 28.367 10069 3290 3.517184 84:260 0.336227 19 28.650 8015 2643 3.422113 87.132 0.274988 20 28.933 6357 2137 3.329714 89.420 0.224747 21 29.217 4986 1739 3.240182 91.222 0.182222 22 29.500 3955 .1425 3.153714. 92.641 0.149937 23 29.783 . 3276 1176 3.070506 93.788 0.129284 24 30.067 2676 979 ·2.990756 94.741 0.110427 25 30.350 2408 822 2.914658 95.561 0.104399 26 30.633 2213 696 2.842411 96.292 0.101345 27 30.917 2097 595 2.774209 96.970 0.102063 28 31200 1812 513 2.710249 97.642 0.094349 29 31.483 1352 447 2.650728 98.089 0.075983 30 31.767 2953 394 2.595843 98.787 0.180949 31 32.050 1754 351 2.545789 99.618 0.118630 32 32.333 419 317 2.500762 99.918 0.031788 33 32.617 83 289 2.460960 99.979 0.007158 34 32.900 26 267 2.426578 99.995 0.002626 35 33.183 0 250 2.397814 100.000 0.000000 

## Fig. 

## 11-2 Chromatogram of GPC analysis and results for residue after stability test ( pH9 ).
I, 

### ,T

# I

> E 

# (

___ j 

## Study 

## No. 82315 Date 

## 2003/08/27

50.00 40.00 30.00 20.00 10.00 

## PageL/ 1 GPO chromatogram

0.00 

## ---············································--------··············--------------,f\ 

# r····

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## v l 

## {'I ' j \; 

### I! -10.00 -20.00 I I I I I I I I I I I I I I I I I . I· I I I I I I I I 0.00 5.00

**10~0 15~0**  **20~0 25~0**  **30~0** Min. 

### i/ I• 

### q

**~** I I I I I I 35.00 40.00



### -- Sample BL THF Vial14 Date 2003/08/26 21:46:30 

## Fig.12 Chromatogram ofGPC analysis for solvent blank.