U .S. ENVIRONMENTAL PROTECTIONAGENCY



## PREMANUFACTURE NOTICE Date of receipt 

# , :PA

Enterthe total number ofpages in the Premanufacture Notice

. Form Approved O

.M .B. No. 2070-0012. Approv$Wpires 10-31-96 

### AGENCY USE ONLY

391 GEER &_ INSTRUCTIONS 

## Company Sanitized



# RNN 

### TS0-1 0 4 A You must provide all information requested in this formto the extent that it is known to orreasonably ascertainable by you. Make reasonable estimates if youdo not have actual data. Before you complete this form, youshould read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available fromthe Toxic Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603) . Ifa user fee has been remitted for this notice (40CFR700 .45), indicate in the boxes above the TS-user fee identification number you have generated . Remember, your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQAccounting Operations Branch (PM-226), P.O. 360399M, Pittsburgh, PA 15251-6399, Arm . TSCA User fee. Part I -GENERAL INFORMATION You must provide the currently correct Chemical Abstracts (CA) Name ofthe new chemical substance, even ifyou claimthe identity as confidential . You may authorize another person to submit chemical identity information for you, but your submission will not be complete and the reviewwill not begin until EPA receives this information. A letter in support ofyour submission should reference your TS user fee identification number . You must submit an original and two copies ofthis notice including all test data. Ifyou claimed any infomiation as confidential, a single sanitized copy must also be submitted. Part II-HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Ifthere are several manufacture, processing, or use operations to be described in Part II, sections A and Bof this notice, reproduce the sections as needed. Part III - LIST OF ATTACHMENTS Attach additional sheets if there is not enough space to answer a question fully. Label each continuation sheet withthe corresponding section heading. In Part III, list these attachments, any test data orother data and any optional information included in the notice. OPTIONAL INFORMATION You may include any information that you want EPA to consider in evaluating the new substance. On page 11 of this form, space has been provided foryouto described pollution prevention and recycling information you may have regarding the new substance. So-called "binding" boxes are included throughout this formfor you to indicate your willingness to be bound to certain statements you make in this section, such as use, production volume, protective equipment . . . This option is intended to reduce delays that routinely accompany the development ofconsent orders or Significant New Use Rules. Except in the case ofexemption applications (such as TMEA, LVE, LOREX) where certain infortnation provided in such notification is binding on the submitter when the Agency approves the exemption application, checking a binding box in this notice does not by itselfprohibit the submitter fromlater deviating fromthe information (except chemical identity) reported in the form . CONFIDENTIALITY CLAIMS You may claimany information in this notice as confidential . To assert a claimon the form, mark (X) the confidential box next to the information that youclaimas confidential . To assert a claimin an attachment, circle or bracket the information you claimas confidential . If vou claiminformation in the notices as confidential, You nmst also provide a sanitized version ofthe notice, (including attachments)

. For additional instructions on claiming information as confidential, read the Instructions Manual . ® Mark (x) ifany information in this notice is claimed as confidential . TEST DATAANDOTHERDATA Youare required to submit all test data in your possession or control and to provide a description ofall otherdata known to or reasonably ascertainable by you, ifthese data are related to the health and environmental effects on the manufacture, processing, distribution in commerce, use, or disposal ofthe new chemical substance

. Standard literature citations may be submitted for data in the open scientific literature. Complete test data (written in English) not summaries ofdata, must be submitted ifthey do not appear in the open literature. You should clearly identify whether test data is on the substance or on an analog. Also, the chemical composition ofthe tested material should be characterized . Following are examples of test data and other data. Data should be submitted according to the requirements of§720.50 ofthe Premanufacture Notification Rule (40CFRPart 720) .

Test Data (Check Below any included in this notice) Environmental fate data ® Yes " Other data ® Yes " Health effects data ® Yes Risk assessments " Environmentaleffects data ® Yes Structure/activity relationships PhysicaVChemical Properties` ® Yes Testdata not in the possession orcontrol of the submitter ' A physical and chemical properties worksheet is located on the last page ofthis form . TYPE OF NOTICE (Check Only One) ® 

# a PMN (Premanufacture Notice)

INTERMEDIATE PMN (submitted in sequence with final product PMN) SNUN (Significant NewUse Notice) tv r~ . c~atTMEA (Test Marketing Exemption Application) ; rl LVE (Low

Volume Exemption) @ 40 CFR 723.50(c)(I)~

N LJ r`-' i

t ' LOREX (Low

Release/LowExposure Exemption) L 40 CFR 723:59(W) LVE Modification IS THIS A CONSOLIDATED PMN? ® J LOREX Modification , Yes C,IT

N # ofchemicals ' (Prenotice Communication # required, enter # on page 3) EPAFORM

7710-25 (Rev. 5-95) Replaces previous editions of EPA Form7710-25. +Public reporting burden for this collection ofinformation is estimated to average 110 hours per response, including time for reviewiFg fistructions,

searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding the burden estimate or any other aspect ofthis collection ofinformation, including suggestions for reducing this burden, to Chief, Information Policy Branch, PM-223, U .S. Environmental Protection Agency, 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: I I. The company named in Part I, sectionA, subsection 1 a ofthis notice formintends to manufacture or import for a commercial purpose, other than in smallquantities solely for research and development, the substance identified in Part I, ', Section B

. 2. All information provided in this notice is complete andtruthful as of the date ofsubmission. 3. 1 am

submitting with this notice all test data in my possession or control and a description of all otherdata known to or reasonably ascertainable by me as required by §720.50 of the Premanufacture Notification Rule. Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNIJN, checkthe following user fee certification statementthat applies: ®The Company named in Part I, SectionAhas remitted the fee of $2500 specified in 40 CFR700 .45(b), or F-1 TheCompany named in Part I, Section Ahas remitted the fee of$1000 for an Intermediate PMN(defined @ 40 CFR 700 .43) in accordance with 40 CFR700 .45(b), or 0The Company named in Part I SectionAis asmall business concern under 40 CFR700 .43 and has remitted a fee of $100 in accordance with 40 CFR700.45(b) . Ifyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR723.50(c)(1) or a Low release and low exposure exemption (LoRex) application in accordance with 40 CFR723.50(c)(2), check the following certification statements: 0The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under theterms of40 CFR723.50 . 0Themanufacturer is familiar with the terms of this sectionand will comply with those terms; and F-1 The new

chemical substance for which thenotice is submitted meets all applicable exemption conditions . 0 If this application is foran LVE in accordance with 40 CFR723.50(c)(1), the manufacturer intends to commence manufacture ofthe exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day review period. The accuracy ofthe statements you make in this notice should reflectyour best prediction of the anticipated facts regarding the chemical substance described herein. Any knowing and willful misinter pretation is subject to criminal p enalty pursuant to 18 USC 1001 . Confidential

_ Signature and title ofAuthorized Official (Original Signature Required) Date , Signature ofagent -(ifapplicable) Date X

EPAFORM

7710-25 (Rev. 5-95) Page 2

~'" ~ `Part I -- GENERAL INFORMATION

Section A -- SUBMITTER IDENTIFICATION -~ Confidential I a. Person Name ofauthorized official Position Submitting Notice (in U.S.) Company

Mailing address (number and street) City, State, ZIPCode b. Agent (if Name of authorized official Position applicable) Company

Mailing address (number and street) City, State, ZIPCode Telephone Area Code Number

c. Ifyou are submitting this notice as part ofajoint submission, mark (X) this box. Joint Submitter (if Name of authorized official Position applicable) Company

Mailing address (number and street) City, State, ZIPCode Telephone Area Code Number 2. Technical Name ofauthorized official Position Contact (in X U.S.) Company

Mailing address (number and street) City, State, ZIPCode Telephone Area Code Number 3. Ifyou have had a prenotice communication (PC) concerning this notice and EPA assigned a PC Number to the notice, enter the number . Mark (X) if none 4. Ifyou previously submitted an exemption application for the chemical substance covered by this notice, enter the exemption number assigned by Mark (X) -~ ® EPA . Ifyou previously submitted a PMNfor this substance enter the if none PMN number assigned by EPA (i .e. withdrawn or incompletej---- " 5. If you have submitted a notice of Bona fide intent to manufacture or import for the chemical substance covered by this notice, enter the notice number Mark (X) -IN ® assigned by EPA

. --t . if none

1. ~ Manufacture 2. ® Import 6. Type ofNotice - Mark (X) Only Only 3. ~ Both Binding Option Q Binding Option Mark (X) Mark Qthe "Confidential" box next to an subsection you claimas confidential

EPA FORM

7710-25 (Rev. 5-95) Page 3p. 4 Part I -- GENERAL INFORMATION-- Continued Section B-- CHEMICAL IDENTITY INFORMATION : You must provide a currently correct Chemical Abstracts (CA) name ofthe substance based on

the ninth Collective Index 9CI ofCA nomenclature rules and conventions . Mark (X the "Confidential" box nextto an itemyou claimas confidential Complete either item1 (Class 1 or 2 substances) or 2 (Polymers) as appropriate. Complete all other items. Ifanother person will submit chemical identity information for you (for either ItemI or 2), mark (X) the box at the right . ConfiIdenti the name, com

p any, and address of that p

erson in a continuation sheet. - 10 E] dential I . Class 1 or 2chemical substances (for definitions ofclass 1 and class 2 substances, see the Instructions Manual) a. Class ofsubstance - Mark (X) 1 0 Class 1 or 2 [] Class 2 b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCAInventory listings for similar substances. For Class 1 substances a CAIndex Name must be provided. For Class 2substances either a CAIndex Name or CAPreferred Name must be provided, which ever is appropriate based on CA9CI nomenclature rules and conventions) .

c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice: (check one) . ® Method I (CAS Inventory Expert Service - a copy of the Identification ~ Method 2 (Other Source) report obtained fromthe CAS Inventory Expert Services must be submitted as an attachmentto this notice) d. Molecular formula andCAS Registry Number (if a number already exists for the substance) X CAStt X e. For a class I substance, provide a complete and correct chemical structure diagram . For a class 2substance - (1) Listthe immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction orprocess. (3) Indicate the range of composition and the typical composition (where appropriate) . (4) Provide a correct representative or partial chemical structure diaeram . ac comnlete as can be known . ifone can he reasonahlv ascertained .

® Mark (X) this box if youattach a continuation sheet. EPA FORM

7710-25 (Rev. 5-95) Page 4p. 5 Part I -- GENERAL INFORMATION-- Continued Section B -- CHEMICAL IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) narne ofthe substance based on the ninth Collective Index (9C[) of CA nomenclature rules and conventions . Mark (X) the "Confidential" box nextto any itemyouclaim as confidential Complete either itemI (Class I or 2substances) or 2(Polymers) as appropriate . Complete all other items. Ifanother person will submit chemical identity information for you (for either ItemI or 2), mark (X) the box at the right. ConfiIdentify the name, company, and address ofthat person in a continuation sheet. 0 dential I . Class 1 or 2 chemical substances (for definitions ofclass I andclass 2 substances, see the Instructions Manual) a . Class of substance - Mark (X) I E] Class I or 2 [:] Class 2 b. Chemical name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances . For Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA Preferred Name must be provided, which ever is appropriate based on CA 9CI nomenclature rules and conventions) .

c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice: (check one) . ® Method 1 (CAS Inventory Expert Service - a copy ofthe Identification ~ Method 2 (Other Source) report obtained fromthe CAS Inventory Expert Services must be submitted as an attachment to this notice) d. Molecular formula and CAS Registry Number (ifa number already exists for the substance) X CAS# X e. For a class 1 substance, provide a complete and correct chemical structure diagram . For a class 2 substance - (1) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of the reaction or process. (3) Indicate the range ofcomposition and the typical composition (where appropriate) . (4) Provide a correct representative or partial chemical structure diaeram . as comnlete as can he known. ifonecan he reasonahlv ascertained

® Mark (X) this box if youattach a continuation sheet. EPA FORM

7710-25 (Rev. 5-95) Page 4 t,L, 

# 000005p. 6

03/0512004~ 09: 49 202-872-0745 PAGE 11/12 Part I -- GENERAL INFORMATION -- Continued Seciinn B - CHEMICAL 1 DENTiTY TNTORMATION - Continued 2. Polymer, ; (ror a definition of polymer, cce the Instracrinns Manual,) dential o Indicate the number-avcrAge wcight of the lowest molecular wcight composition oCrhc polymer you intend to manufacture . Indicate moximum

weight percent oClnvn, molecular weight spccics (not including residual monomer;, rr_ictants, or so)vents) below500and below1,000 absolute molecular wr .ight. of that composition . Describe the mcthods of measurement nr the bsgie tar your estimates: GPC O other [] . (Speciry)

) lo-Oct nuntl)cr uveragc molecular uleight: ti) maximumwcig}tt ^/o below 500 molecular weigiht : iii) mnximumwe~ght °,~ belnwl OD0 malecular wetght ® Mark X) lhts box if you attach aenritinuaticin sheet. b. You must make sepntrote confidentiality claims for monomer or other reactant identity, composition information, and residual information, Tvtark (X) the "CnnC+dcntial" box next to any itemyou claimas confidential (1) - Provide thc spccrtic chemical name and CAS Rcgistry Number (iia number cxists) ofcaelt monomer or other rcactaflt used i11 the mltllttfaclure of ',

the polymer I (2) - Mark (X) this coiurttn iCentry in column (1) is confidentisl . I (3) - Tndicate the tyj)ic,al weight percent oCeach monomer or other reactant in tlhe polymer, (4) . Marl< (X) the identity column ifyou want a monomer or othcr reacrant used at two weight percent or less to be listed as part af thc polymer i

tltscription nn ilhc TSCACherntcal Substancc lnventory. (5) - Mark (X) this column if entries in columns (3) and (4) arc canGdent9al . (6) - Indicate the maxi mum weight pcrcenl of cach monomer or otbcr rctctant that may he present as n residusl in the polymer as mttnttfacturod tpr commcrcial purposes, 7 Mark ( thi . columsl if on in column 6 is conPdcntial Monomer or other reactant and CAS l:cgistry Nurnbrr Confi- Typical Identity Confi- Maximum Core, - dential compoaition Mark (X) dentiat residual denual 1 2 3 (4' 5 fG 7' °`n 

### X nrq X ~ro X ^/n X X n/f1 X h n/v ~io

% c/n Mark (X) this b(ix ifyou atrach a continuation sheet, c Me=identify which method you used to developor obtatrt the specified chemical identity information reported in this notice (check one)

Method 1 (CAS Inventory Lxperr $,ice - a . copy of the identification report QMethod 2 (otlter source) )( obtained fromCAS Inventory Expett Service must be submittcd as as attaclhmcnt to tl)is notice' d. ThecuiTrntly correct Chcmtcal Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers. )( c, Provide ti correct representative or pari,ial chomical structure dtagram, as compietc as can 1)c known, if nne can !~c ably atcertimcd. I 

### ' , See page 5A

Mark (?C) ihis botc ifyou at.lach a continutation shrct. FPA FORM

771a25 (Rev. 5

.g5) Page 5Page 5A 

# 0000070.3I05/2004 99:49 202-872-0745 TSr, p. 8 PAGE 12/12 Part I -- GENERAL CNFORI1iATION -- Continued Section S - CHEMICAL IDENTITY INFORMATION - Continued 2, PntymMS (Fare dafnitton ofpolyn'cr, see tile Instructions Min ual .) ContidcntiAl a. Indicate the number-.2vcrage weight of the lowest molecular wcight compositinn of thc polymer you inicnd to mAnufacturc. Indrcatc maximum

weight percent of 1mv molecultlr weight spccics (nor. includrng rcsidusl mor+omcrs. rcactonts, or so)vcnts) ?lclow500 and bclow1,000absolute mnlccular weight of that cninpo.,ftion Describe tile methods ofmcGsurcmcnt or the bnsjc for your C9timStCC . CrPC ~ Other [ :] . (Specify) i) lowcat ntlmbcr avcrsgc molccu1arWeight : i ii) maximumweight % below500tttolecular weight : iii) maximum

wclghl "/o hclow1000 molecular weip-_lht Mark (X) this box if you attach a continuation ilicct, b. You must makeseparate conridcnli :tli .ry claims for mornomer or otlicr reactant identity, cornpqsition infortrsa.tinn, and rcsidua.l informatirm . Mark (X) thc "Coin fidcntra.l" box nest to any itcrn you claim es confidential (1) - Provide the specific chemical name and CAS Regtsiry Ntlmbcr (i f a number exist ;) ofeach monomer or other reaci2nt used in 11he manttfacture of

tic polymer . (2) - Mark (X) this column if entry in column (l) is confidctntial (3) - lndicatr_ tile tyitical weiglht percent of eacli monorner or other TcAaInt. In the polymer. (4) - AAsrk (X) the identity column if you wart a monomer or othcr reactant used at two %cight pertrcnt or 1cR!t to be listed as part ofVic polymer

description on the TSCAC?,rmical Substancc Inventory (il - Mark (X) tliis column if entrica in colamn: (3) and (4) are confidcntial, (G) - Indicate tile maximum wcight peircnt ofcach monomererr other roactant tlhat may lrc present ns a rcsidual in the polymer as manufactumd for commercial purposc

.a. r7Mark X this column if cnt mcolomn (,6 is cnnfidential . Monomer or other rcsctatnt and CAS Registry Number Confi- Typical ldantity CanC" Maximum ConCitlentia1 composition Mqtk(X) tlential rceidual dential 1 (2 (? 4 (5 (6 7 `%a x "/o X % X % X

"ln X °/v 0/y °lo

^~n "/a Q Mark (Y) thia box ifyou attaclt a continuation sheet. c. !'lease Identify which method you used to develop or obtain the .cpcciFed cthcmical tdcntity information reported in this noticc (chcck one). a Method t (CAS Inventory Expert Service - a copy ofthe tdcnti ficslttott report a Mctliod 2 (other sourcc) )(

obtained fromCAS Inventory Expert Service must be submitted as as attuchmcntto this notice) d T1ic currently carreet Chemical Abstracts (CA) nnr,te for the polyrnr .r that is consistent with TSC.4 Inventory listings for similar polymm . e. Provide a eorrctit represenrative or parttal chemical .vtruct, :re diagram, as complcte as can be known, if one can be reasonably wseertained,



## - See page 5C

® Mark (X) 1hSg box if youattach a continutation sheet. LPA FORM

7710-25 (Rev. 5-95) Page 5 BPage 5C 

# 000009p. 10 

### Part I -- GENERAL INFORMATION-- Continued Section B - CHEMICAL IDENTITY INFORMATION -Continued 3. Impurities (a) - Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose.

CAS Registry Number ifavailable. If there are unidentified impurities, enter "unidentified." (b) - Estimate the maximum weight %

ofeach i mpurity. If there are unidentified i mpurities, estimate their total wei g ht % . Provide the Impurity and CAS Registry Number

(a) Maximum percent (b) Confidential % X

- %

% %

% %

% Mark (X) this box ifyou attach a continuation sheet. 4 . Synonyms - Enter any chemical synonyms for the new chemical identified in subsection I or 2 . Confidential

Mark (X) this box ifyou attach a continuation sheet. X

5. Trade identification - List trade names for the new chemical substance identified in subsection 1 or 2 .

Mark (X) this box ifyouattach a continuation sheet. X

6 . Generic chemical name - Ifyou claimchemical identify as confidential, you must provide a generic name for your substance thatreveals the specific chemical identity of the new chemical substance to the maximum extent possible. Refer to the TSCA Chemical Substance Inventory, 1985 Edition, Appendix Bfor guidance on developing generic names.

~ Copolymer of phenol and aromatic hydrocarbon

Mark (X) this box if youattach a continuation sheet: 7. Byproducts - Describe any byproducts resulting fromthe manufacture, processing, use, or disposal of the new chemical substance. Provide the CAS Registry Number ifavailable. , Byproduct (1) CAS Registry Number (2) Confidential 

### X

Mark (X) this box ifyou attach a continuation sheet. EPA FORM

7710-25 (Rev. 5-95) Page 6 

# 000010p. 11 

### Part I -- GENERAL INFORMATION-- Continued Section C-- PRODUCTION, IMPORT, ANDUSE INFORMATION:

Mark (X) the "Confidential" box next to an item you claim

as confidential . 1 . Production volume -- Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum production volume for any consecutive 12-month period during the first three years ofproduction. Estimates should be on 100%new chemical substance basis. Fora Low Volume Exemption application, ifyouchoose to have your notice reviewed at a lower production volume than 10,000 k /yr, specify the volume and mark (x) in the bindingbox. If anted, youare boundto this volume Maximum

first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confi- Binding (100% new chemical substance basis) (100%new chemical substance basis) denthal 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. (1) -- Describe each intended category of use of the newchemical substance by function and application. . (2) -- Mark (X) this column if entry column (1) is confidential business information (CBI) . (3) -- Indicate your willingness to have the information provided in column (1) binding. (4) -- Estimate the percent oftotal production for thefirst threeyears devotedto each category of use. (5) -- Mark (X) this column if entry in column (4) is confidential business information (CBI) . (6) -- Estimate the percentofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites underyour control associated with each category of use . (7) -- Mark (X) this column if entry in column (6) is confidential business information (CBI) . (8) -- Indicate %

ofproduct volume expected for the listed "use" sectors. Mark more than one box if appropriate. Mark (X) to indicate your willingness to have the use type provided in (8) binding. (9) -- Mark (X)this column if entry(ies) in column (8) is (are) confidential business information (CBI) . Category of use (1) CBI Binding Produc- CBI % in CBI n/o ofsubstance expected per use CBI option tion % Form- (8) (by function and application i .e. a dispersive dye Mark Ixl ulation Site- Con-* Indus- Com- B inding for finishing p

ol y ester fibers (2) (3) (4) (5) (6 (7) limited sumer trial mercial O ption (9) X x X X o~ o~ X X o/ o/

o~ o~

o~ o~ o/ . % % o/

o~ o~ * If you have identified a "consumer" use, please provide on a continuation sheet a detailed description ofthe use(s) of this chemical substance in consumer products. In addition include estimates of the concentration of the new chemical substance as expected in consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product. []Mark (X

) this box ifyou attach a continuation sheet . b . Generic Ifyou claim anycategory of use description in subsection 2a as confidential, enter a generic description of that category. Read the use Instructions Manual for examples of generic use descriptions . description Binder Component

~ Mark (X) this box if you attach a continuation sheet. 3. Hazard Information - Include in the notice a copy ofreasonable facsimile of any hazard warning statement, label, material safety data sheet, or other Binding information which will be provided to any Person who is reasonably likely to be exposed to this substance regarding protective equipment or practices Option Ma~k1xl for the safe handing, transport, use, or disposal of the new substance. List in part III hazard information you include. ® Mark (X) this box if youattach hazard information. EPAFORM

7710-25 (Rev. 5-95) Page 7 000011p. 12 

### Part II-- HUMAN EXPOSURE ANDENVIRONMENTAL RELEASE Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X) the "Confidential" box nextto any itemyou claimas confidential Complete section Afor each type ofmanufacture, processing, or use operation involving the new chemical substance at industrial sites you control . Importers do not have to complete this section for operations outside the U.S. ; however, you may still have reporting requirements if thereare further industrial p rocessing or useo

perations after im

p ort. Youmust describe theseoperations. See instructions manual 1 . Operation description Confidential a. Identity -- Enter the identity ofthe site at which the operation will occur.

Name Not applicable because PMN substance will be imported Site address (number and street) City, County, State, ZIP code If the same operation will occur at more than one site, enter the number of sites. Identify the additional sites on a continuation sheet, and if any of the sites have significantly different production rates or operations, include all the information requested in this section for those sites as attachments. Mark (X) this box ifyou attach a continuation sheet. b. Type -- Mark (X) DManufacturing aProcessing E]Use c . Amount and Duration -- Com plete 1 or 2as appropriate Maximum

kglbatch (too°i new

chemical Hours/batch Batches/year substance) 1 . Batch Maximum kg/batch (tooi new

chemical Hours/batch Batches/year substance) 2. Continuous d. Process description El Mark (X) to indicate your willingness to have your process description binding. (1) Diagramthe major unit operation steps and chemical conversions. Include interimstorage and transport containers (specify- e.g. 5 gallon pails, 55 gallon drum, rail car, tank truck, etc.) . (2) Provide the identity , the approximate weight (by kglday or kg/batch on a 100%new chemical substance basis), and entry point ofall starting materials and feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily or per batch.) . (3) Identify by number the points of release, including small or intermittent releases, to the environment ofthe newchemical substance. Not applicable because PMN substance will be imported

~ Mark (X) this box if you attach a continuation sheet. EPA FORM

7710-25 (Rev. 5-95) Page 8 

# OQ0012

### Part II-- HUMAN EXPOSURE ANDENVIRONMENTAL RELEASE -- Continuedp. 13

Section A-- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER-- Continued 2 . Occupational Exposure -- You must make separate confidentiality claims for the description of worker activity, physical formof the newchemical substance, number ofworks exposed, and duration ofactivity. Mark (X) the "Confidential" box nextto any itemyou claim as confidential . (1) --Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling, cleaning, etc.) in which workers may be exposed to the substance. (2) --Mark (X) this column ifentry in column (1) is confidential business information (CBI) . (3) --Describe any protective equipment and engineering controls used to protect workers. (4) and (6) -- Indicate your willingness to have the information provided in column (3) or (5) binding. (5) --Indicate the physical form(s) of the newchemical substance (e.g., solid: crystal, granule, powder, or dust) and % newchemical substance (ifpart ofamixture) at the time ofexposure. (7) --Mark (X) this column ifentry in column (5) is confidential business information (CBI) . (8) --Estimate the maximumnumber 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) -- Estimate the maximum duration ofthe activity for any worker in hours per day and days per year . (12) -- Mark (X) this column ifentries in columns (10) and (11) are confidential business inform ation (CB1) . Worker activity CBI Protective Equipment/ Binding Physical forms(s) Binding CBI # of CBI Maximu m duration CBI (i .e., bag dumping, filling drums)

(1) (z) Engineering Controls (3) option Mark (x) (4) and %

new substance (5) Option

Mark (x) (6) (7) Worker

Expose (9) Hrs/day

(10) Dayslyr

(I1) d (8) Not applicable, PMN substance will be imoorted

~ Mark (X) this box ifyou attach a continuation sheet. 3. Environmental Release and Disposal -- You must make separate confidentiality claims for the release number and the amount ofthe new chemical substance released and other release and disposal information. Mark (X) the "Confidential" box next to each itemyou claim as confidential . (1) --Enter the number ofeach release point identified in the process description, part 11, section A, subsection l d(3) . (2) --Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology (in kg/day or kg/batch) . (3) --Mark (X) this column ifentries in columns (1) and (2) are confidential business information (CBI) . (4) --Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, POTW, or other (specify)) to which the new

substance will be released fromthat release point . (5) -- a. Describe control technology, if any, and control efficiency that will be used to limit the release ofthe newsubstance to the environment. For releases disposed ofon land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet, for each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day) . (6) --Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CBI) . (7) -Identify the destination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or NPDES numbers ofthe POTW (Publicl Owned Treatment Works). Mark (X) ifthe POTW name or NPDES# is confidential business information (CBI) . Release Amount ofnewsubstance CBI Mediaof Control technology and efficiency (you may wish to optionally attach efficiency data) Number released release (1) r (2a) (2b) (3 e.g. dackair (4) (5a Binding Mark (x) (Sb) (6) Not applicable because PMN substance will be imported

(7) Mark (X) the ~ POTW provide name(s) below

: CBI [] Navigable ~ Other -Specify provide NPDES# CBI destination(s) of releases to water . waterway

11 Mark (X) this box if you attach a continuation sheet. EPA FORM

7710-25 (Rev. 5-95) Page 9 

# 000013p. 14



### Part II-- HUMANEXPOSURE AND ENVIRONMENTAL RELEASE -- Continued Section B- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control . Importers do not have to complete this section for operations outside the U .S. ; however, you must report anyprocessing or use activities after import . See the Instructions Manual . Complete a separate section B

for each type ojprocessing, or use operation involving the new chemicalsubstance. If the same operation is performed at more than one site describe the typical operation common to these sites. Identif y additional sites on a continuation sheet . 1 . Operation Description -- To claiminformation in this section as confidential, circle or bracket the specific information that you claimas confidential . (1) -- Diagram

the major unit operation steps and chemical conversions, including interim storage and transport containers (specify - e.g. 5 gallon pails, 55 gallon drums, rail cars, tank trucks, etc) . On the diagram, identify by letter and briefly describe each worker activity. (2) -- Provide the identity, the approximate weight (by kg/day or kg/batch, on an 100%new chemical substancebasis), and entry point ofall feedstocks (including reactants, solvents and catalysts, etc) andall products, recycle streams, and wastes. Include cleaning chemicals (note frequency if not used daily or per batch) . (3) -- Identify bynumber the points ofrelease, including small or intermittent releases, to the environment of thenew chemical substance. (4) Please enter the #of sites (remember to identify the locations of these sites on a continuation sheet) : # ofsites

®Mark (X) this box ifyou attach a continuation sheet. 2. Worker Exposure/Environmental Release (1) --Fromthe diagram above, provide the letter for each worker activity. Complete 2-8 for each worker activity described. (2) --Estimate the number ofworkers exposed for all sitgs combined. (4) -- Estimate the typical duration ofexposure per worker in (a) hours per day and (b) days per year . (6) -- Describe physical formofexposure and %new chemical substance (if in mixture), and anyprotective equipmentand engineering controls, if any, used to protect workers. (7) -- Estimate the percent of the new substance as formulated when packaged or used as a final product . (9) -- From

the process diagramabove, enter the number of each release point. Complete 9-13 for each release point identified. (10) -- Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kg/batch) . (12) -- Describe media ofrelease i .e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, or other (specify) and control technology, if any, that will be used to limitthe release of thenew substance to the environment . (14) -- Identify byproducts which may result from the operation. (3) , (5), (8), (11 , (13) and (15) --Mark (X) this column ifan of the proceeding entries are confidential business information (CBI) . Letter # of CBI Duration CBI Protective Equip. / % in CBI Release Amount of CBl Media of Release CBI of Workers of Engineering Controls/ Form- Number New & Control Act- Exposed Exposure Physical Form and % ulation Substance Released Technology ivity new substance (1) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (l0a) (l0b) (11) (12) (13) X X X X



### L 14) -- Byproducts : (15) X ® Mark (X) this box if youattach a continuation sheet, EPA FORM

7710-25 (Rev. 5-95) Page 10 

# 00003 .4Page 10A 000015p. 16



## OPTIONAL POLLUTION PREVENTION INFORMATION To claim

information in this section as confidential circle or bracket the sp ecific information that y ou claimas confidential . 

### In this section you mayprovide information not reported elsewhere in this formregarding your efforts to reduce or minimize potential risks associatedwith activities surrounding manufacturing, processing, use and disposal of the PMNsubstance. 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 of chemical wastes by technological modification, process and procedure modification, product reformulation, raw materials substitution, and/or inventory control . Recycling refers to the reclamation of useful chemical components fromwastes 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 I~ of risks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure) . In addition, information on the relative cost or performance characteristics of the PMNsubstance to potential alternatives maybe provided. All information provided in this section will be taken into consideration during the reviewof this substance

. See Instructions Manual and Pollution Prevention Guidance manual for guidance and exam ples. Describe the expected net benefits, such as (1) an overall reduction in risk to humanhealth or the environment; (2) a reductionin the volume manufactured; (3) a reduction in the generation of waste materials through recycling, source reduction or other means; (4) a reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in the cost ofproduction and/or improved operation efficiency of the new chemical substance in comparison to existing chemical substances used in similar application; or (6) the extent to which the new chemical substance may be a substitute for an existing substance that poses a eater overall risk to human health or the environment.

0Mark (X) this box if you attach a continuation sheet. EPAFORM7710-25 (Rev. 5-95) Page 11 

# 000016p. 17 

## Part III -- LIST OF ATTACHMENTS Attach continuation sheets for sections of the formand test data and other data (including physical/chemical properties and structure/activity information), and optional information after this page. Clearly identify the attachment and the section ofthe form to which it relates, ifappropriate. Number consecutively the pages of the attachments. In the column below, enter the inclusive page numbers of each attachment. Mark (X) the "Confidential" box next to any attachment name you claim as confidential . Read, the Instructions Manual for guidance on how to claimany information in an attachment as confidential . You must include with the sanitized copy of the notice forma sanitized version of any attachment in which you claiminformation as confidential . Attachment name Attachment a e number(s) Confidential Material Safetv Data Sheet (MSDS) 14-16 Chemical Identity 4A,5A, 5B, 5C ' Off-site process description 10A Physical and Chemical Properties Worksheet 13 Infrared spectrum 17 NMRspectrum 18 GPC data 19-24 Mutagenicity Testing in Bacterial Mutation Assay 25-50 Chromosonal Aberration Test in Cultured Mammalian Cells 51-75 Twenty-Eight Day Repeated Dose Oral Toxicity in Rats 76-164 Biodegradation Study by Microorganisms 165-203 Bioconcentration Study in Carp 204-290 Evaluation of the Potential Occupational and Environmental Hazards Associated with the Proposed Uses of the PMN substance 291-382 PMNfee documentation 383 CASIES Report 384,384A CMAelectronic PMN documentation 385 In vivo Micronucleus Test 386-410

~Mark (X) this box ifyou attach a continuation she et . Enter the attachment name and number . EPA FORM7710-25 (Rev. 5-95) Page 12p. 18 PHYSICAL ANDCHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical and chemical properties data,please complete the followingworksheet for data you provide and include it in thenotice. Identify the property measured, the page ofthe notice on which the property appears, the value ofthe property, the units in which theproperty is measured (as necessary), and whether or not the property is claimed as confidential . The physical state ofthe neat substance should be provided. These measured properties should be for the neat (100%pure) chemical substance. Properties that are measured for mixtures or formulations should be so noted (%PMNsubstance in -) . You are notrequired to submit this worksheet ; however, EPA strongly recommends that you do so, as it will simplifyreview and ensure that confidential information isproperly protected. You should submit this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission oftest data. Property Mark Page Value Measured o Confdential (x) if number Estimate Mark (x) (a) provided (b) (c) (Mor E) tdl X 15 Phy sical state of neat substance X(s -(1) -( ) Vapor pressure Tem perature °C Torr Density/relative density /cm3 Solubility @ Temperature °C g/L Solvent Solubili ty in water Tem p erature °C X 15 Meltingtem

perature (softening point) 60-130 °C M Boiling /sublimation temerature torr p ressure oc

X 17,18 M Spectra : infrared, nmr Dissociation constant Particle size distribution Octanol/water p

artition coefficient Henry's Law

constant Volitalization ftomwater Volitalization from

soil H concentration Flammabili ty Ex lodability Adsorption / coefficient ' X 119-24 M ' Other - Specify: gpcdata

ATTACHMENT oO0018 PAGE 132004/01/28 11 :44:54



# Safety Data Sheet Date of issue : January 28, 2004 Product Identity No. Trade name : L 1 .ident iflcation of the product and of the company. 1 .1 Product name : I 1 .2 Company name : Address : Emergency phone 

### 2.Composition/information on :ingredients 

# 7



# 7

3.Hazards identification Emergency Overview

: Red to brown solid with no odor . May cause skin irritation. Avoid contact with skin. Avoid breathing substance. Avoid ingestion. Use respiratory and skin protection. 

### 4.First aid measures 4.1 Eye contact : Rinse eyes with running water for 15 minutes. Consult a doctor . 4.2 Skin contact : Wash promptly exposed areas with soap and water. 4.3 Inhalation of aerosol/vapour/dust : Remove victim to fresh air immediately. 4.4 Ingestion or accident : Get medical attention promptly.lf awallowed,seek medical advice immediately (treat symptometically) . 5.Fire fighting measures 5.1 Extinguishing media: Dry chemical,CO z, foam, water spray 5.2 Special advice in case of fire: Firefighters should wear self-contained breathing apparatus and eye protection in fighting significant fires in which this material is ivolved. 

# C 

# ]

6.Accidentai release measures 

# naa0z4 0000192004/01/28 11 :44:54 r ' ~ Pag2-Zp. 20



### After spillage Sweep or scoop up for disposal or salvage, avoiding dust condition. Flush trace residues from area with water . Do not allow residues to enter water ways. 7.Handling and storage 7.1 Handling To avoid contact with skin and eyes, protective goggles, gloves, aprons etc. . 

### 7.2 Storage Store in a cool, dry,weil-ventilated location. 8.Exposure controls and personal protection Personal protective equipment Respiratory : Dust respirator 

### Eye protection: Face shields Hand,skin and body protection : Impervious clothing, Chemical-resistanct gloves, apron and impervious boots 9.Physical and chemical properties * Form :Solid * Colour :Yellow * Softening point :60-r130°C * Hydroxyl group equivalent Wt . :195-250 * Appearance :Transparent yellow to orange flake 10.Stability and reactivity ~ Stability The product is stable (ambient temperature) . ~ Conditions to avoid With epoxide at high temperture (about 50°C) . ~ Materials to avoid Oxdizing agents, Strong acids, Amines 11 .Toxicological information ~ Acute oral toxicity LD50(oral rat) : >1000mg/kg * Skin irritation Acute dermal irritation : * Eye irritation Acute eye irritation ' * Sensitisation Buehierdelayed contact * Mutagenicity Bacterial mutagenicity * Mutagenicity Not tested Not tested hypersensitivity test(Ames test) Chromosomal aberration test ~ Sub-acute toxicity Toxicity study in the rat : Positive Not tested : Non-mutagenic D20=0.45mg/ml : NOEL=200mg/kg/day(28-day test) 12.EcoloBical information 

# 000015 0000202004/01/28 11 :44:54 Page-3 p. 21 

### * Acute toxicity to fish LC50(killifish) : 28.7mg/I * Biodegradability study Full MITI Biodegradation : Not biodegraded * Bioaccumulation study Bioaccumulation in the carp : Lowaccumulation 13.Disposal considerations Material can be incinerated by adding controlled amounts to a chemical incinerator equipped with an after burner and scrubber . Do not release to water . 

### 14.Transport information ~ Recommendations on the Transport of Dangerous ~ RID/ADR : Not applicable ICAO/IATA : Not applicable IMDG Code : Not applicable ~ Further information : Follow all regulations containers have no rent or leak before loading. against fall, drop and physical damage. 15.Regulatory information CHEMICAL SUBSTANCES ACT(JAPAN) : Registered 16.0ther information None Goods : Not applicable. in your country. Confirm Protect containers

Product should be stored, handled and used in accordance with good industrial hygiene practices and in conformity with legal regulation. The information contained herein is based on the present state of our knowledge and is intended to describe our products from the point of view of safety requirements . It should therefore not be construed as guaranteeing specific properties .p. 22



# a © h°+

# ooooz9ll ' ' '



# ~100019 000024

# 00020 000025

# 000021 0000 26

# 1100022 000027

# 1100023 000028

# 1100024 

# oeoo~9

### [Receipt No. T02-4088

STUDYCODE: KOI-2884



## FINAL REPORT

MUTAGENICITY TEST OF USING MICROORGANISMS



### May 2003

Hita Laboratory Chemicals Evaluation and Research Institute

Japan

### KO1-2884 p.31 STATEMENT

I, the undersigned, hereby declare that this report provides a correct English translation ofthe final report, Study Code K01-2884, issued on May 20, 2003.



## 4,, % t,~ 13, 200

.3 Shozo Ogura Date Hita Laboratory Chemicals Evaluation and Research Institute, Japan

### KO1-2884 p.32

GU STATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan _Sponsor: 1- Title: Mutagenicity Test ofC- '(Using Microorganisms Study Code: KO1-2884 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) .

Management : Signed in original May 20, 2003

Yoshifumi Fujino, M

.S.

### KO1-2884 p.33 GLP STATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor: L Title: Mutagenicity Test off lUsing Microorganisms Study Code: KO1-2884 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September l, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health .and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) . I also confirmed that this report accurately reflected the raw data and the test data were valid. Study Director: __ Signed in original May 20, 2003 Shozo Ogura

### K01-2884 p.34

n UALITY ASSURANCE STATEMENT

Hita Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor: 

# L Title: Mutaeenicity Test of [ 2Using Microorganisms Study Code:_ K01-2884 This study was audited and inspected by Quality Assurance Section of Hita Laboratory, Chemicals Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported these results to the study director and the management are as follows. Dates of Reports to the Items of Inspections/Audits Dates of Inspections/Audits Study Director and Management Protocol A p ril 14, 2003 A p ril 15, 2003 Preparation ofTest Substance and Experimental May 1, 2003 May 1, 2003 Implementation Raw Data and Draft Rep

ort May 19, 2003 May 19, 2003 Re-inspection of Draft May 20, 2003 May 20, 2003 I Rep ort Final Rep

ort May 20, 2003 May 20, 2003 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations, Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March l, 2000) and with "OECD Principles of Good Laboratory Practice" 

### (November 26, 1997) . This study was conducted in accordance with "Standards for Toxicity Investigations" 

# '~0002~ 000034

### KO1-2884 p~ 35

(Ministry of Labor, Notification No.77, September 1, 1988 and Notification No.67, June 2, 1997), and "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Official Notification, February 8, 1999) "Notification on Partial Revision of Testing Methods Relating to the NewChemical Substances" (Notification No. 700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW & No. 2 (1997) of the Basic Industries Bureau, MITI, October 31, 1997) . I, the undersigned, hereby declare that this report provides an accurate description of the 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 20, 2003 Keiji Shiraishi, B

.S.

### KO1-2884 p, 36 Study Code: KO1-2884 Test Substance Code: HR5186 Sponsor Code: N-0360 TITLE Mutagenicity Test of t ]Using Microorganisms 

### SPONSOR 

# L J TESTINGFACILITY Hita Laboratory Chemicals Evaluation and Research Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan PURPOSE OF STUDY The ability of the test substance to induce mutations is investigated using Salmonella typhimuriumandEscherichia coli . 

### TESTING METHOD This study was conducted in accordance with "Standards for Toxicity Investigations" (Ministry of Labor, Notification No.77, September l, " 1988 and Notification No.67, June 2, 1997), and "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Official Notification, February 8, 1999) "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 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) &No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW &No. 2 (1997) of the Basic Industries Bureau, MITI, October 31, 1997) .

### KO1-2884 p.37



### GLP COMPLIANCE This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the 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 of the Pharmaceutical Affairs Bureau, MHW & No. 85 (1984) of the Basic Industries Bureau, MITI, March 31, 1984; revised on March 1 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) PERIOD OF STUDY Commencement of Study: April 10, 2003 Initiation of Experiment (Initiation ofTreatment of Dose Finding Test) : April 15, 2003 Completion of Experiment (Completion of Colony Count) : May 6, 2003 Completion ofStudy: May 20, 2003 STORAGE AND RETENTIONPERIOD OF DATA Raw data, protocol, letter of test request, questionnaire, final report, inspection data by QAU and other documentation records will be retained in the archives and the test substance will be retained at the test substance storage roomof Hita Laboratory for 10 years after the date of the notification specified under Item 1 of Article 57-3 of Industrial Safety &Health Law and after the date of the notification specified under Article 4, Paragraph 1, No.l, No.2 or No.3 of Notification on Testing Methods Relating to the New

Chemical Substances . The sponsor will communicate the date of the notification to Hita Laboratory. Treatment of data after termination of the retention period will be carried out with the approval of the sponsor. 

### RETENTIONOF ORIGINAL DOCUMENTS An original and its copy of protocol is prepared and retained in Hita Laboratory and retained in the sponsor, respectively. An original of final report is prepared and retained in Hita Laboratory. The sponsor will retain a copy of the final report including a statement by the management (a certification of identity with the original) . 

# ,100032 000037

### K01-2884 p.38

NAMES, ASSIGNED SECTIONS AND JOBASSIGNMENT OF STUDY DIRECTORANDPERSONNEL Study Director: Shozo Ogura

_ Mutagenicity Section, Hita Laboratory Study Staff: Shozo Ogura, Hifumi Iwashita Mutagenicity Section, Hita Laboratory (Preparation and treatment of the test substance, counting of revertant colonies) Management : Yoshifumi Fujino, M .S. Hita Laboratory Person in Charge of Storage: Shizuka Kouda General Affairs Section, Hita Laboratory Person in Charge ofTest Substance Management : Takaharu Hara, B .S. Analytical Chemistry Section, Hita Laboratory APPROVAL BY AUTHOR Study Director: \ Signed in original May 20, 2003 Shozo Ogura



# 003%3 000038

### KO1-2884 p. 39

TABLE OF CONTENTS

MATERIALS ANDMETHODS 1 . TEST SUBSTANCE ANDPOSITIVE CONTROLS . . .. . . . . . .. . . . . . . 2 . BACTERIAL STRAINS . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . .. .. 3. MEDIUM

ANDS9 MIX . . . . . .. . . . . . . . . . . .. .. . . . . .. . . .. . . .. . . . . . . .. . . . . . . . .. 4 . PRE-CULTURES . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . .. .. . . . . . .. 5 . PREPARATIONOF TEST SUBSTANCE ANDPOSITIVE CONTROLS . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . .. .. . 6. METHODS . . . . . . .. . . . . . . . . .. .. . . .. . . .. . .. . . . . .. . . . . . . .. . . . . . .. 7. OBSERVATIONANDCOLONY COUNTING ..8 . JUDGEMENT CRITERIA OF TEST RESULTS .. . . . . . . . .. . . . . .. .. . 3 4 5 5 6 7 7 FACTORS AFFECTINGRELIABILITY OF TEST . . . . . . .. . . . . . . . .. . . . 7 TEST RESULTS 1. DOSE FINDINGTEST-1 2 . DOSE FINDINGTEST-2 3 . MAINTEST . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . .. .. .

. . .. . . . . .. . . . . . . . . .. . . . . . . .. . .. . . . .. . . . . . . . .. . . . . . . .

. . . . . . . . . . .. .. . . . . . . . . . . . . . . . . .. . . . . . . .. .. . . . . .. . . . . 7 8 8

### KO1-286440

SUMMARY The ability of E 3to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix) . As a result, the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that of each negative control in all test strains. It is concluded that ~ ] has no ability to induce mutations under the present test conditions .

### K01-28V . 41 MATERIALS ANDMETHODS 1 . TEST SUBSTANCE ANDPOSITIVE CONTROLS 1 .1 Test Substance (Information Provided by The Sponsor) 1) Name Other Name: CAS No. : 2) Lot No. 0-6182 3) Supplier 4) Structural formula r--

(Molecular formula: -) 5) Purity 

### 99.9w/w% - 6) Names and concentrations of impurities Water 0

.1 w/w% 7) Physicochemical properties Appearance at ordinary temperature: Yellow-brown solid Molecular weight : (- 3 Stability: Stable Melting point: 48°C(DSC) Boiling point: - Vapor pressure: - Partition coefficient: - Hydrolysis: Not hydrolyzed Solubility: Oil soluble

### KO1-281W

. 42 Degree of solubility Water: ~1 mg/L DMSO : ? 10% Acetone: ? 10% Others : Chloroform >--10% 8) Storage conditions Stored at roomtemperature in a dark place (cabinet No.l in test substance storage room) . 9) Precautions Gloves, a mask, head cap and lab coat were worn. 1 .2 Positive Controls Name Manufacturer Lot No. A ppearance Puri ty Grade Wako Red-yellow Special AF-2"' Pure Chemical SEL1402 crystalline powder ° 99.0/° grade Industries, Ltd. W~° White crystalline Special NaN3'~ Pure Chemical TCH3448 powder ° 99.2% grade Industries, Ltd

. Yellowcrystalline ICR-191'3 Polysciences, Inc. 492624 p owder - _ Wako Yellow-green2AA"4 Pure Chemical TCM6741 brown powder 93.3% -

Industries, Ltd. * 1 : 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide *2: Sodium

azide *3: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HC1 *4: 2-Aminoanthracene 1) Storage conditions Stored in a cold dark place (positive control substance exclusive refrigerator in test substance storage room) . 2) Precautions Gloves, a mask; head cap and lab coat were worn. 

### 2. BACTERIAL STRAINS 2.1 Kinds of Bacterial Strains and Reason for Selection Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA were used. Salmonella typhimuriumstrains were supplied from Dr. B . N . Ames, University of California, on June 20, 1990 . Escherichia coli strain 

# ~~00037 ooo042

### was supplied from Dr. Taijiro Matsushima, Japan Bioassay Research Center, on January 7, 1997. These test strains have been recommended to use for the mutagenicity test using microorganisms in "Standards for Toxicity Investigations" and the bacterial reverse mutation test in "Notification on Partial Revision ofTesting Methods Relating to the New Chemical Substances" . 2.2 Storage The properties of the test strains with regard to amino acid requirement, sensitivity to ultraviolet ray, membrane mutation rfa, the presence or absence of plasmid pKM101 and negative and positive control values were examined. The spectrophotometric grade of dimethyl sulfoxide (DMSO, Lot No. LR170, not less than 99.0%in purity, DOJINDOLaboratories) was added to fresh overnight cultures of the test strains that had been determined to possess these characteristics at a ratio of 0 .9 to 10 . The mixture was divided into microtubes and was frozen as the stock cultures in an ultradeep freezer (MDF-291, SANYOElectric Co., Ltd .) at -80°C. 3. MEDIUM

ANDS9 MIX 3.1 Medium 1) Minimal glucose agar plate Tesmedia AN(Oriental Yeast Co., Ltd.) was used. Lot No. : ANI040AS (manufactured on January 17, 2003) 2) Soft agar A solution containing 0 .5 mM histidine and 0

.5 mM biotin for S 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. 0130004, Difco Laboratories) and 0 .5 w/v%NaCl at a ratio of 1 to 10. 

### 3.2 S9 Mix 1) Rat liver S9 An S9 (Lot No. 03021412, manufactured on February 14, 2003, Oriental Yeast Co., Ltd.) prepared from the liver of rats (body weight : 210 .9f8.7 g) administered phenobarbital and 5,6-benzoflavone was used. The S9 was cryopreserved in an ultra-deep freezer (MDF-291, SANYOElectric Co., Ltd.) until use. 2) Coniposition of S9 mix S9 mix was prepared using a cofactor for S9 mix (Lot No. 999301, Oriental Yeast Co., Ltd.) immediately before use. One milliliter of S9 mix consisted of 8 ~tmol MgC12, 33 ~imol KCI, 5 ~Lmol G-6-P, 4 ~tmol NADPH, 4 ~tmol NADH, 100 pmol sodium-phosphate buffer (pH 7.4) and 0 .1 mLofS9. 

# 000043

- 4 - sN)0038

### K01-20444

4 . PRE-CULTURES A20-~tL aliquot of each of the frozen stock cultures was inoculated to 10 mL of 2.5% Oxoid Nutrient broth No. 2 (Lot No. 218041, UNIPATHLtd.) in each L tube (27 mL in volume) . The culture was incubated at 37f0.5°C for 9 hours by shaking at 50 times/minute in a seesaw type of shaker (MONOSIN-IIA, Taitec Corporation) . A density of each bacterial culture was measured at 660 run by a spectrophotometer (Novaspec II, Pharmacia Biotech Ltd.) at the end of the incubation to calculate the number of viable cells ( x 109 cells/mL) . The number of viable cells obtained was shown as follows: - TA100 TA1535 WP2 uvrA TA98 TA1537 Dose finding test-1 2.5 2.4 4 .0 2.4 2 .4 Dose finding test-2 2.5 2.5 2 .5 Main test 2.5 2.4 4 .1 2

.4 2.4 5. PREPARATION OF TEST SUBSTANCE ANDPOSITIVE CONTROLS 5.1 Preparation ofTest Substance 1) 2)



### 3) 4) Solvent DMSO(Lot No. NJ151) Reason for selection ofsolvent The Solubility of the test substance was less than 1 mg/L in water (information provided by the sponsor) and more than 50 mg/mL in DMSO . The 50 mg/mL solution of the test substance in DMSO was observed no color change and no exothermic reaction until 2hours afterpreparation . Preparation method After DMSOwas added to the test substance to make a solution of 50 mg/mL, the solution was subsequently diluted with the same solvent to give each required concentration. Preparationtime The test substance solution was prepared immediately before use, kept under yellowlamps at room

temperature and used within 2 hours. 5.2 Preparation of Positive Controls 1) 2) Preparation method NaN3 was dissolved in distilled water (distilled water for injection, Lot No. K2G77, Otsuka Pharmaceutical Factory Inc.) . AF-2, ICR-191 and 2AAwere dissolved in DMSO(Lot No. LR170) . Storage conditions Positive control solutions were stored in an ultra-deep freezer (MDF-291, SANYO Electric Co., Ltd.) at -80°C. 

# 5 - ~'00039 000044

### K01-2880 .45

6 .



### 6.1

6.2

6.3

-S9mix +S9 mix TA100 TA1535 WP2uvrA TA98 TA1537

AF-2 NaN, AF-2 AF-2 ICR-191 0 .01 0

.5 0

.01 0

.1 0

.5 2AA 2AA 2AA 2AA 2AA 1 2 10 0.5 2 (Unit: 4g/plate) 6 .4 Dose Selection 1) 2) METHODS The tests were performed using a pre-incubation method with and without S9 mix. Three plates were used for the negative control group and two plates for the test substance groups and the positive control groups. The test code number, name of test strain, presence or absence of S9 mix and dose level were noted on each plate. Procedures After 0 .1 mL of the test substance solution or the positive control solution, 0 .5 mL of 0 .1 M sodiumphosphate buffer (pH7.4) or S9 mix and 0 .1 mL of the bacterial culture were added to a test tube, the mixture was shaken at 37±0.5°C for 20 minutes. Two milliliters of the soft agar was then added to the test tube, mixed with a voltex and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after an incubation at 37t0.5°C for 48 hours. Sterility The test substance solution at the highest dose and S9 mix were mixed with 2 mL of the soft agar and poured onto a minimal glucose agar plate in order to examine a bacterial contamination. The plate was incubated at 37±0.5°C for 48 hours. Negative Control and Positive Controls The solvent used in the tests was employed as a negative control and the following positive controls were used for each bacterial strain.

pg/plate and 313 ~tg/plate, respectively. Dose finding test-1 Atotal of 6 doses including 5,000 ~g/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4was set. Dose finding test-2 In the dose finding test-1, the growth inhibition was observed in TA100, TA1535 and TA1537 regardless of the presence or absence of S9 mix. Four or more effective doses not showing any growth inhibition were not obtained in TA100, TA1535 and TA1537 in a treatment without S9 mix. Therefore, the dose finding test-2 was carried out at a total of 6 doses at which the highest dose showed the growth inhibition and 5 lower doses diluted with a geometric progression of 4 . The highest doses in TA100, TA1535 and TA1537 were used 19.5 pg/plate, 78.1 

# 000045 '

# -00040

### 3) Main test Based on the results of the dose finding tests, a total of 6 doses including the highest dose showing the growth inhibition and 5 lower doses diluted with a geometric progression of 2 . The highest doses in the main test were used 19.5 ~Lg/plate in TA100 and 78.1 ~ig/plate in TA1535 and TA1537 in a treatment method without S9 mix, and 1,250 ~tg/plate in TA100, TA1535 and TA1537 in a treatment with S9 mix. In WP2 uvrA and TA98, a total of 6 doses including the highest dose and 5 lower doses diluted with a geometric progression of 2 were employed. As the highest dose ofWP2 uvrA and TA98, 5,000 ~Ig/plate at which the precipitation was observed was taken. 7. OBSERVATIONANDCOLONY COUNTING 7.1 Observation The precipitates of the test substance and the growth inhibition of the bacteria were observed with the unaided eye and a stereomicroscope, respectively. 7.2 Colony Counting The number of revertant colonies was counted using a manual counter or a colony analyzer (CV9000, Dennou-Giken Co., Ltd .) . 8 . JUDGEMENT CRITERIA OF TEST RESULTS The test substance wasjudged to be positive when the number of revertant colonies was increased two times or more that of the negative control in a dose-dependent manner and when reproducibility on the test results was shown. In all other cases, it was judged to be negative. Any statistical treatment methods were not used.

FACTORS AFFECTING RELIABILITY OF TEST There were no factors that might have affected the reliability ofthe test. TEST RESULTS 1 . DOSE FINDINGTEST-1 The test results and the dose-response curves were shown in Table 1 and Figs. 1 and 2, respectively. The bacterial growth inhibition was observed in TA100, TA1535 and TA1537 regardless of the presence or absence of S9 mix . For TA100, TA1535 and TA1537 in a treatment without S9mix, 4 or more effective doses not showing the growth inhibition were not obtained, namely, the growth inhibition appeared at 19.5 ~ig/plate or more in 

# 000046 - 7 - "00041.KU1-Z80447



### TA100, at 78.1 4g/plate or more in TA1535 and at 313 ~tg/plate or more in TA1537. For TA100, TA1535 and TA1537 in a treatment with S9 mix, the growth inhibition appeared at 1,250 ~ig/plate or more. The test substance precipitates were observed at 1,250 ~Lg/plate or more regardless of thepresence or absence of S9 mix. The number of the revertant colonies did not increase two times or more that of the negative control in all test strains. 2 . DOSE FINDINGTEST-2 The test results and the dose-response curves were shown in Table 2 and Fig. 3. The growth inhibition was observed at 19.5 4g/plate in TA100, at 78.1 ~Lg/plate in TAI535 and at 78.1 4g/plate or more in TAI537. The test substance precipitates were not observed at any dose levels. The number of the revertant colonies did not increase two times or more that of the negative control in TA100, TA1535 and TA1537 in a treatment without S9 mix. 3. MAINTEST The test results and the dose-response curves were shown in Table 3 and Figs. 4 and 5, respectively. The growth inhibition was observed at 9.77 ~Lg/plate or more in TA100 and 39.1 ~tg/plate or more in TA1535 and TA1537 in a treatment without S9 mix, and 625 4g/plate or more in TA100 and TA1535 and 1,250 4g/plate in TA1537 in a treatment with S9 mix. The test substance precipitates were observed at 625 gg/plate or more in a treatment without S9 mix and 1,250 ~LgJplate or more in a treatment with S9 mix. The number of the revertant colonies did not increase two times or more that of the negative control in all test strains regardless of the presence or absence of S9 mix. DISCUSSION ANDCONCLUSION The test substance wasjudged to be negative because the number of the revertant colonies in the test substance treatment groups in all test strains was less than two times that of the negative control regardless of the presence or absence of S9 mix. The number of the revertant colonies for the positive controls and the negative control was within a range of the historical data in Hita laboratory. It was confirmed that the test system was free frombacterial contamination, which indicates the test results to be valid. Fromthe above results, it was concluded thatc under the present test conditions. had no ability to induce mutations 

# 000047 -8- 

# ~'00042

### K01-28S448



### REFERENCES 1 . Green M

.H.L. and W

.J. Muriel (1976) Mutagen testing using Trp+ reversion in Escherichia coli . Mutation Res., 38: 3-32. 2. Maron, D

.M . and B .N. Ames (1983) Revised methods for the Salmonella mutagenicity test. Mutation Res., 113: 173-215.

## xu~:~~~4

Table 1 Dose finding test-1 Test substance: C Test dates From April 15, 2003 to April 17, 2003 With (+) or Test substance Number ofrevertant colonies/ late without (-) dose Base-pair substitution e Frameshift e S9 mix (pg/plate) TA 100 TA 1535 WP2 uvrA TA 98 TA 1537

Negative 93 11 36 18 9 control 93 (98) 11 (12) 47 (41) 22 (20) 10 (7)

107 15 41 21 3 4.88 gg ( 85) 16 (12) 

### 49 46 (48) 

### 22

14 (18) ~ (6) ' 19.5 195* (105) 8 (7) 

### 45 37 (41) 19 (19) 4 (6) -S9 mix 78.1 113* 111* (112) 5*

6* (6) 32

39 (36) 28

16 (22) 4

4 (4) 

### 313 96* (94) g* (6) 4~ (40) 20 13 (17) 2* ( 3) +1250 76*

104* (90) 1 ~* ( 0) 26 (32) 

### 16 12 (14) 1 * ( 1) -1-5000 g2* (87) ~* ( 1) 3~ (29) 15 (13) 1* ( 1) Negative 102 17 48 26 20 control 98 (99) 15 (15) 38 (43) 29 (30) 14 (17)

97 14 42 35 18 4.88 117 (111)

105 18

17 (18) 46

45 (46) 37

26 (32) 16

19 (18) 19.5 132 (125)

117 14

9 (12) 49

47 (48) 38

29 (34) 17

15 (16) +S9 mix 78.1 111 (126) 11 (1,5) 42 (42) 27 (31) 19 (14) 141 19 42 34 9 313 105

108 (107) 3 (4) 40 (41) 3~ (33) 

### 13 12 (13) +1250 194* (104) i* ( 1) 35 (39) 

### 24 21 (23) 5* (7) +5000 113*

12* (113) 1 ~* ( 1) 36 (35) 20 (18) 2 1* (2) Chemical AF-2 NaN3 AF-2 AF-2 ICR-191 Positive control Dose late 0.01 0.5 0.01 0.1 0.5 -S9 mix Number of 321 346 162 470 1055

revertant 306 (314) 393 (370) 217 (190) 453 (462) 1108 (1082) 

### colonies/ late Chemical 2AA 2AA 2AA 2AA 2AA Dose 1 2 10 0.5 2 Positive control late +S9 mix , Number of 454 207 859 428 301

revertant 534 (494) 215 (211) 822 (841) 414 (421) 307 (304) colonies/ late [Notes] Parenthesis shows a mean between plates.

+: The precipitate of the test substance. * : Observed bacterial growth inhibition. " AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide " NaN3: Sodiumazide " ICR-191 : 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HC1 - 2AA: 2-Aminoanthracene 

# 000049 

# ~00044

### KRi-5h84



## Table 2 Dose finding test-2 Test substance: C Test period From April 23, 2003 to April 25, 2003 With (+) or Test substance Number of revertant colonies/ late without (-) dose Base- pair substitution e Frameshift e S9 mix (p.g/plate) TA 100 TA 1535 TA 1537

Negative 106 15 13 control 120 (113) 14 (13) 8 (10)

114 11 8 0.0191 192 (101) - - 0.0763 18g (102) 18 (19) - 0.305 95 (96) i g (18) ~ (6) -S9 mix 1 .22 102 (104) 19 (19) 7 (6) 106 18 5 4 .88 110

113 (112) 17 (17) i~ (12) 

### 19.5 92*

* (89) 13 (9) 5 (5) 78.1 - 10* (9) 1 * (3) 313 - - 

### 3* 

### (3) * Chemical AF-2 NaN3 ICR-191 Positive control Dose late 0

.01 0.5 0.5 -S9 mix Number of



### revertant 2~~ (293) 3g0 (379) 966 (969) colonies/ late [Notes] " AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

" NaN3: Sodiumazide " ICR-191 : 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HC1 Parenthesis shows a mean between plates. *: Observed bacterial growth inhibition.

## Table 3 Main test

Test substance: 884

Test period FromMay 1, 2003 to May 6, 2003 With (+) or Number of revertantcolonies/ late without (-) Test substancedose late) / ( Base-)air substitution e Frameshife S9 mix pg p TA 100 TA 1535 WP2 uvrA TA98 TA 1537

Negative 106 6 50 28 6 control 111 (103) 14 (10) 39 (40) 21 (23) 9 (7)

93 10 30 19 6 0 .610 g9 (91) - - - - 1 .22 102

78 (90) - - - - 2.44 g~ (91) i~ (11) - - 4 (6) 4.88 193

04 (99) 6 (7) - - 8 (7) 9 .77 97*

99* (98) 8

14 (11) - - 9 (8) 19.5 91, (92) 11 (13) - - 16 (9) -S9mix 39 .1 - 5* (6) - - 0* I (9) 1 * , 78.1 - g* (7) - - g* (8)'' 156 - - 53 (53) 20 23 (22) - 313 - - 39 (46) 26 (19) - +625 - - 46 (53) 13 12 (13) - +1250 - - 57 50 (54) 11

14 (13) - +2500 - - 49 51 (50) 10

21 (16) - +5000 - - 4~ (48) 18 1 (10) - Negative 82 7 49 25 23 control 94 (95) 7 (7) 30 (41) 23 (25) 17 (19)

110 7 45 27 18 39.1 107

122 (115) 9 (8) - - i~ (16) 78.1 114

110 (112) 9 (7) - - i8 (16) 156 104

101 (103) 10 (6) 55 54 (55) 38

24 (31) 14

17 (16) +S9mix 313 86 (84) 3 4) ( 54 (54) 29 (32) 18 (17) 82 5 . 53 35 15 625 1 * ( 95) 4, (3) 69 (60) 31 (26) 12 (13)

88 21 13 +1250 101* (103) 2 * (2) 6~ (60) 31 (32) * 8 (8) 05* 1 , 33 * +2500 - - 60 60 (60) 22

24 (23) - +5000 - - 60 57 (59) 18

15 (17) - Chemical AF-2 NaN AF-2 AF-2 ICR-191 Positive control' Dose late 0

.01 0

.5 0.01 0 .1 0

.5 -S9 mix Number ofrevertant 220 218 ( ) 399 (391) 169 (179) 546 (544) 658 (675) colonies/plate 215 383 189 542 692 Chemical 2AA 2AA 2AA 2AA 2AA Positive control Dose 1 2 10 05 2 late . +S9mix Number of revertant 753 (743) 199 (208) 587 (633) 459 (459) 289 (305)

colonies/ late 733 216 678 458 321 [Notes]

" 2AA: 2-Aminoanthracene

Parenthesis shows a mean betweenplates. +: Theprecipitate of thetest substance. *: Observed bacterial growth inhibition. " 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 1~1i .3 0oYV 

# 000051

## ~u~_isx4



## Dose finding test-1

N N 

### a

. .. 0 0 U >~ > N 150 120 

### 90 60 30 9 8 0 O : TA100 0 : TA1535 O ~ WP2uvrA A : TA98 0 : TA1537

8-- 0 4.88 19 .5 78.1 313 1250 5000 (ug/plate) 

### Fig. 1 Dose-response curve without S9 mix

M ~1 . a. U

. ., O O U C CC > 150 120 90 60 30 9 o : MOO O : TA1535 O: YIP2uvn1 A : TA98 0 : TA1537

--o 0 4.88 19.5 78.1 313 1250 5000 (pg/plate) 

### Fig. 2 Dose-response curve with S9 mixKU~;JdP



## Dose finding test-2

C1,

. O O U cd U > U O~ TAI 00 p : TA1535 O ~ TA1531 150 120 90 60 30

0 0.0191 0 .0763 0.305 1 .22 4.88 19.5 78 .1 313 (pg/plate) 

### Fig. 3 Dose-response curve without S9 mix00 c-~

i O D

m T < N a L N C . 3

4 N

O O !Fw w



### m

. T o

0 0 N

N

N O m ~ v N O 6 D

m m 4Fa

N 1 O A 47 <F0 O O < H

. . .~

.

### 3 U N O U

~ .. N O QddD 

### w m c : ~p. 54

O O N

O N

0 .0 t~

.

4b .0 ti . 

### 3 U U O

m U i ^ O

Q tiD 

### w Ito m m

0 o a .

0 0 

### °' `° " ~ . ; ~l U~IJ'i 

### al7ajd .zad satuoioo juuj .zana-d alEid .zad saiuoioa luuI .zana-d -` O 

### C~

### KU-~1~-25884 

# HISTORICAL DATA (Tesmedia AN) Nagative Control : October, 2002-March, 2003 Positive Control : December 17, 2002-March, 2003 

## Negative Control (Mean ±2S.D .) -S9 mix +S9 mix TA100 TA1535 WP2uvrA TA98 TA1537 TA100 TA1535 WP2uvrA TA98 TA1537 Mean 102 10 29 26 8 109 10 32 36 18 ' S .D. ------------- 13

. . . . . . . . . 3

. . . . . . . . . . 6

. . . . . . . . . . . 7

. . . . . . . . . 3

. . . . . . . . . 14

. . . . . . . . . 3

. . . . . . . . . 7 ----------- 7

. . . . . . . . . 4

. . . . . . . . . Upper limit 128 16 41 40 14 137 16 46 50 26 Lower limi 76 4 17 12 2 81 4 18 22 10



## Positive Control (Meanf 3S.D.) -S9 mix +S9 mix TA100 TA1535 WP2 uvrA TA98 TA1537 TA100 TA1535 WP2uvrA TA98 TA1537 

### Mean 284 357 254 575 1034 540 160 758 511 207 S.D. ------------- 44

. . . . . . . . . 54

. . . . . . . . . 64

. . . . . . . . . . . 73

.. . . . . . . . . 227

. . . . . . . . . 100

. . . . . . . . . 25

. . . . . . . . . 136

. . . . . . . . . . . 65

. . . . . . . . . . 34

. . . . . . . . . Upperlimit 416 519 446 794 1715 840 235 1166 706 309 

### Lowerlimi 152 195 62 356 353 240 85 350 316 105

### ~ Receipt No. T02-4089

STUDY CODE: K06-0989



# FINAL REPORT

CHROMOSOMAL ABERRATIONTEST OF

C -1 USING CULTURED MAMMALIANCELLS



### July 2003

Hita Laboratory Chemicals Evaluation and Research Institute

Japan

### K06-0989 p. 57 STATEMENT

I, the undersigned, hereby declare that this report provides a correct English translation of the final report, Study Code K06-0989, issued on July 14, 2003.



## ~~~1, 2ao

,j Wakamatsu Shinya, KS. Date Hita Laboratory Chemicals Evaluation and Research Institute, Japan

oOO057

### K06-0989 p. 58 GLPSTATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan S onsor: . -1 Title: Chromosomal Aberration Test of E JUsiniz Cultured Mammalian

Cells Study Code: K06-0989 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29 ; 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No . 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) .

Management : Signed in original July 14, 2003 Yoshifumi Fujino, M

.S.

### K06-0989p~ 59 

### GLPSTATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor: CTitle : Chromosomal Aberration Test of r jUsing Cultured Mammalian 

### Cells Study Code: K06-0989 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September l, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) . I also confirmed that this report accurately reflected the raw data and the test data were valid. Study Director: Signed in original July 14, 2003 Syozo Ajimi, B

.S.

### K06-0989p~ 60

QUALITYASSURANCE 

### STATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan Sponso r: L Title: Chromosomal Aberration Test of F Using Cultured Mam malian 

### Cells Study Code: K06-0989 This study was audited and inspected by Quality Assurance Section of Hita Laboratory, Chemicals Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported these results to the study director and the management are as follows. Items of Dates ofInspections/Audits Dates ofReport of Inspections/Audits Inspections/Audits Protocol April 7, 2003 April 7, 2003 Preparation of Test 

### April 21, 2003 April 21, 2003 Substance and Main Test 

### Raw Data and Draft Report July 9, 2003 July 9, 2003 Re-inspections of Raw Data and Draft Report July 11, 2003 July 11, 2003 

### Final Report July 14, 2003 July 14, 2003 I, the undersigned, hereby declare that this study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the Test Related to the NewChemical Substances and of the Toxicity Investigations Related to the Designated Chemical Substances" (Notification No. 39 of the Planning and Coordination Bureau, Environment Agency, No. 229 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare &No. 85 (1984) of the Basic Industries Bureau, Ministry of International Trade and Industry, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" 

### (November 26, 1997) . 

# 71~,00 55 000060

### K06-0989 p~ 61

This study was also conducted in accordance with "Standard for Chromosomal Aberration Test using Cultured Mammalian Cells and Notification on Partial Revision of Results of Chromosomal Aberration Test in Cultured Mammalian Cells (Notification No. 652 of Labor Standard Bureau, Ministry of Labor (MOL), September 29, 1997)", "Procedures of Chromosomal Aberration Test Using Cultured Mammalian Cells and Evaluation of Test Results" (Ministry of Labor, Official Notification, September 16, 1999), and "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances" (Notification No. 700of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) &No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW

&No. 2 (1997) of the Basic Industries Bureau, MITI, October 31, 1997) . 1, the undersigned, hereby declare that this report provides an accurate description of the methods and procedures used in this study and that the reported results accurately reflect the raw data obtained. Section Chief, Quality Assurance: Siizned in original July 14, 2003 Keiji Shiraishi, B

.S.



# 1-11VM056 000061

### K06-0989 p' 62 Study Code: K06-0989 Test Substance Code: HR5186 Sponsor Code: N-0360 TITLE Chromosomal Aberration Test of r JUsing Cultured Mammalian Cells SPONSOR

TESTINGFACILITY Hita Laboratory Chemicals Evaluation and Research Institute, Japan 822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan PURPOSE OF STUDY The ability of the test substance to induce chromosomal aberrations was examined using Chinese hamster lung fibroblasts (CHL/IU cells) . 

### TESTING METHOD This study was conducted in accordance with "Standard for Chromosomal Aberration Test using Cultured Mammalian Cells and Notification on Partial Revision of Results of Chromosomal Aberration Test in Cultured Mammalian Cells (Notification No. 652 of Labor Standard Bureau, Ministry of Labor (MOL), September 29, 1997)", "Procedures of Chromosomal Aberration Test Using Cultured Mammalian Cells and Evaluation of Test Results" (Ministry of Labor, Official Notification, September 16, 1999), and "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 of Health and Welfare (MHW) & No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986; and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW &No. 2 (1997) of the Basic Industries Bureau, MITI, October 31, 1997) . 

### ~M3057 

# o000s2

### K06-0989 p~ 63

GLP COMPLIANCE This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), and "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the 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 of the Pharmaceutical Affairs Bureau, MHW & No. 85 (1984) of the Basic Industries Bureau, MITI, March 31, 1984; revised on March 1, 2000) and with "OECD Principles of Good Laboratory Practice" (November 26, 1997) . 

### PERIOD OF STUDY Commencement of Study: March 31, 2003 Initiation of Experiment (Initiation ofTreatment of Cell Growth Inhibition Test) : April 2, 2003 Completion of Experiment (Completion of Observation of Specimens) : June 4, 2003 Completion of Study: July 14, 2003 STORAGE ANDRETENTION PERIOD OF DATA Raw data, protocol, letter of test request, questionnaire, final report, inspection data by QAU, other documentation records and specimens will be retained in the archives and the test substance will be retained at the test substance storage room of Hita Laboratory for 10 years after the date of the notification specified under Item 1 of Article 57-3 of Industrial Safety & Health Law and after the date of the notification specified under Article 4, Paragraph 1, No. 1, No.2 or No.3 of Notification on Testing Methods Relating to the New Chemical Substances . The sponsor will communicate the date of the notification to Hita Laboratory. Treatment of data after termination of the retention period will be carried out 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 sponsor's consent is needed on the disposal of the specimens. 

### RETENTION OF ORIGINAL DOCUMENTS An original and its copy of protocol is prepared and retained in Hita Laboratory and retained in the sponsor, respectively. An original of final report is prepared and 

### retained in Hita Laboratory. The sponsor will retain a copy of the final report 

# ~'20058

### K06-0989 p~ 64

including a statement by the management (a certification of identity with the original) . NAMES, ASSIGNED SECTIONS ANDJOBASSIGNMENT OF STUDY DIRECTORANDPERSONNEL Study Director: SyozoAjimi, B .S. Mutagenicity Section, Hita Laboratory Study Staff Syozo Ajimi, B.S. Mutagenicity Section, Hita Laboratory (Preparation and treatment of the test substance, observation of specimens) Study Staff: Wakamatsu Shinya, M .S. Mutagenicity Section, Hita Laboratory (Observation ofspecimens) Study Staff: Junko Kawaguchi, M .S. Mutagenicity Section, Hita Laboratory (Observation of specimens) Management : Yoshifumi Fujino, M .S. Hita Laboratory 

### Person in Charge ofStorage: Shizuka Koda General Affairs Section, Hita Laboratory 

### Person in Charge ofTest Substance Management : Takaharu Hara, B .S. Analytical Chemistry Section, Hita Laboratory APPROVAL BY AUTHOR Study Director: Siened in orieinal July 14, 2003 Syozo Ajimi, B.S.

### K06-0989

TABLE OF CONTENTS 

### SUMMARY .. . . . . . . . . . . . . . . . . . . . . . .. .. . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 MATERIALSAND METHODS 

### 1 . TEST SUBSTANCE AND POSITIVE CONTROLS . . . .. . .. . . . . . .. .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 2 . CELLS ANDMEDIUM . . . . . . . .. .. . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . 3. S9 MIX . . . . . . . . . .. . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . 4. CELL PRE-CULTURE . . . . . . . . . . . . .. . . . . . .. . . . . .. .. . . .. . . . . .. . . . . . . . . . . . . 5. PREPARATION OF TEST SUBSTANCE ANDPOSITIVE CONTROLS 6. SHORT-TERM

TREATMENT 7. CONTINUOUS TREATMENT 

### 8. JUDGEMENT CRITERIA OF RESULTS ? 3 4 4 5 6 8 8 FACTORSAFFECTED RELIABILITY OF TEST . . . . . . . . . . . . . .. 8 TEST RESULTS 

### 1 . SHORT-TERM

TREATMENT ... .... . . . . . .. . 8 p. 65



### DISCUSSIONANDCONCLUSION . . . . . . . . . . . . . . . 10

### K06-0989

SUMMARY The ability ofC 3to induce chromosomal aberrations was investigated by using Chinese hamster lung fibroblasts (CHL/IU cells) . A chromosomal aberration test was carried out at 120, 160, 200 and 240 ~iglmL of the test substance for a short-termtreatment without S9 mix and at 120, 180, 240 and 300 ~tg/mL of the test substance for a short-term treatment with S9 mix. As a result of the test, the frequency of the cells with structural aberrations increased dose-dependently at 200 and 240 ug/mL in the treatments without S9 mix and 240 and 300 ~tg/mL in the treatments with S9 mix. 

### The above results indicate that ~ Dpossesses an ability to induce chromosomal aberrations under the present test conditions.

### K06-0989 p~ 67

MATERIALS AND METHODS l . TEST SUBSTANCE ANDPOSITIVE CONTROLS 1 .1 Test Substance (Information Provided by The Sponsor) 1) Name 

### L J Other Name: CAS No. : 2) Lot No. 0-6182 3) Supplier 

# [ C 7 4) Structural formula Representative structural formula

(Molecular tormula: -) 5) Purity 

### 99.9 w/w% 6) Names and concentrations of impurities Water 0

.1 w/w% 7) Physicochemical properties Appearance at ordinary temperature: Yellow-brown solid Molecular weight : Mn= [ I Mw= Stability: Stable Melting point: 48°C (DSC) Boiling point: - Vapor pressure: - Partition coefficient: - 

### Hydrolysis: Not hydrolyzed Solubility: Oil soluble



# ~;uoos~

-

### K06-0989 Degree ofsolubility Water : 1 mg/L DMSO : ? 10% Acetone: ? 10% Others: 

### Chloroform ? 10% 8) Storage conditions Stored at room temperature in a dark place (cabinet storage room) . 9) Precautions Gloves, a mask, head cap and lab coat were worn. 1 .2 Positive Controls 

### 1) Mitomycin C (MMC) Manufacturer : Lot No. . Appearance: Composition: 2)



### 3) 4) Grade: No. l in test substance

Kyowa Hakko Kogyo Co., Ltd. 375ABB royal purple crystals or crystalline powder 2 mg titer ofJ .P.(Japan Pharmaceutical) MMCand 48 mg ofJ .P. sodium

chloride in onevial for injection Cyclophosphamide, monohydrate (CPA) Manufacturer : Lot No. : Appearance: Purity: 

### Grade: Storage conditions Wako Pure Chemical Industries, Ltd. WAQ7031 white crystalline powder 99.0% for biochemistry MMC was stored at room temperature (cabinet No. 2 in the test substance storage room) and CPAat a cold dark place (refrigerator for positive controls in the test substance storage room) . Precautions Gloves, a mask, head cap and lab coat were worn. 

### 2. CELLS AND MEDIUM 2.1 Cell Line and Reason for Selection Chinese hamster lung fibroblasts (CHL/IU cells) were supplied by Health Science Research Resources Bank, Japan Health Sciences Foundation on April 17, 2002. The modal number of chromosomes was 25 per cell . 

# '~0063 The doubling time was p. 68



# 00006Sp. 69 

### K06-0989

about 15 hours. It was confirmed in Hita laboratory that the cells were mycoplasma free and the frequency of the spontaneous chromosomal aberration of the cells was below5% . 

### CHL/IU cells have been recommended the availability on in vitro chromosome aberration test in "Procedures of Chromosomal Aberration Test Using Cultured Mammalian Cells and Evaluation ofTest Results" and "The Notification on Partial Revision ofTesting Methods for New Chemical Substances" . 2.2 Storage Cells were stored in liquid nitrogen. 2.3 Mediumand Culture Condition 1) Medium Eagle's minimum

essential medium(Lot No. 474210, Nissui Pharmaceutical Co., Ltd.) was supplemented with 10 v/v%newborn calf serum(Lot No. 27020859, SankoJunyaku Co., Ltd.). 2) Culture condition Cells were cultured at 37f0.5°C approximately 100%humidity and 5% COz. 2.4 Subculture Cells were subcultured using 90 mm diameter culture dish (Nunc Co., Ltd.) twice a week. The passage number of the cells was below15 after receipt . 

### 3. S9 MIX 3.1 Rat Liver S 9 

### S9 (Lot No. 03011711, manufactured on January 17, 2003, protein content / S9: 24.7 mg/mL, Oriental Yeast Co., Ltd.) which was prepared fromrat livers (body weight of rats: 212.8t8.2 g) administered phenobarbital and 5,6-benzoflavone was used. The S9 was stored in an ultra-deep freezer (MDF-U481ATR, SANYO Electric Medica Systems Co., Ltd.) at -80°C until use. 3.2 Composition of S9 Mix One milliliter of S9 mix comprised 0 .3 mL S9, 5 pmol MgC12, 33 pmol KCI, 5 pmol G-6-P, 4 qmol NADP and 4 ~Lmol HEPES (pH 7.2) . S9 mix was prepared immediately before use. 4 . CELL PRE-CULTURE Five milliliters of a cell suspension of 1 .5x 104 cells/mL or 5x 103 cells/mL were seeded in a culture dish (Asahi Techno Glass Corporation) with 60 mmin diameter and the cells were cultured continuously for 2 or 3 days, respectively. 

# 00

- 4 -

### K06-0989 p~ 70

5. PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROLS 5.1 Preparation ofTest Substance 1) Solvent 

### Acetone (Lot No. DWP7546, purity 99.5%, special reagent, Wako Pure Chemical Industries, Ltd .) was used. 

### 2) Reason for selection ofsolvent Solubility of the test substance in water was less than lmg/L (information from the sponsor) . The test substance was soluble in DMSOat 500 mg/mL, but the test substance solution with DMSO was as highly viscous as it was difficult to be taken. The test substance was soluble in acetone at 500 mg/mL and the solution of 50 and 500 mgimL of the test substance in acetone was considered to be stable there fromthe facts being neither any change in color nor heating at room temperature within 2 hours after preparation. Therefore, acetone was preferably selected as a solvent in this test. 3) Preparation method The test substance was crushed into small particles by an agar mortar because it was provided as a solid state fromthe sponsor . After the test substance was weighed, acetone was added to the test substance and a vortex mixer and an ultra-sonicator was used to dissolve it. A required concentration series was prepared by dilution with the same solvent . 

### 4) Preparation time The test substance solutions were prepared immediately before use and were stored in an iced water to prevent a volatilization of acetone and used within one hour. 

### 5.2 Preparation of Positive Controls 1) Preparation method and storage Prior to use, the stock solutions of MMCand CPA were prepared with distilled water at 0.01 mg/mL and 1 mg/mL, respectively, and the solutions were stored at -80°C . 2) Preparation time and expiry in use 

### The stock solutions were thawed when needed and were used within one hour. The date of expiry of the frozen stock solutions of the positive controls was 6 months after preparation. 

# 000070

~M3065

- 5 -

### K06-0989

6. SHORT-TERM

TREATMENT 6.1 Cell Growth Inhibition Test 1) Test method 

### In a treatment without S9 mix, proliferating cells were treated for 6 hours using 3 mL of mediumincluding 30 pL of the test substance solution or the solvent. In a treatment with S9 mix, proliferating cells were treated for 6 hours using a total of 3 mL which comprised 2 .5 mL medium including 30 pL of the test substance solution or the solvent supplemented with 0 .5 mL of S9 mix. After treatment, the mediumwas removed from each dish, rinsed with 2 mL Ca'`+, Mg'`+-free phosphate buffered saline 3 times and it was cultured with 5 mL of fresh mediumfor 18 hours for recovery. Fifty microliters of 10 pg/mL Colcemid® were added to each cell culture 2 hours prior to harvesting. At the beginning and the end of the treatment, it was examined using the unaided eye whether the test substance precipitated or not in culture medium

. At the harvest time, culture mediumwas removed, cells were collected fromeach culture dish with 0.25%trypsin and the number of the cells was counted using Microcell Counter (CDA-500, Sysmex Corporation) to estimate the cell growth rate and the 50%cell growth inhibition concentration (ICso) as the negative control 100% . The cultured cells were processed separately for the chromosome preparation, collected by centrifuge at 1,000 rpm for 5 minutes and were treated hypotonically with 3 mL of 0.075 mol/L KCl at 37°C for 15 minutes . Following the hypotonic treatment, cells were fixed with a fixative (methanol : acetic acid= 3:1) . The cell suspension was dropped onto a microscope slide, dried and stained with 2°/o Giemsa solution. One chromosome slide was prepared in each dose. 

### 2) Dose levels A primary assessment of the treatments without and with S9 mix, which was employed a total of 9 doses at 19.5-5.000 pg/mL spacing a geometric progression of 2, showed a drastically dose-dependent reduction of the cell growth rate (not shown in Table) . Therefore, the cell growth rate was reassessed at a total of 9 doses ranging 60.0-540 [ig/mL spacing a same dose difference of 60 pg/mL. Duplicate cultures were used in each dose. 

### 3) Microscopic observation and scoring of chromosomal aberration Amount of mitotic metaphases in each treatment group was qualitatively examined. The frequencies of cells with chromosomal aberrations as shown belowwere scored by observing 50 metaphases per dose as well as examining mitotic metaphase index in only doses which can be considered in dose setting



# "'~~t~66 p. 71



### - 6 -

### K06-0989 of the chromosomal aberration test. (1) Structural aberration The number of metaphases with structural aberrations excluding gaps was recorded. A gap was defined as an achromatic region whose length was shorter than the width of a chromatid. 

### (2) Numerical aberration The number of polyploid showing triploid or more was scored. 6.2 Chromosomal Aberration Test 1) Test method Chromosomal aberration test was carried out by using as same procedure as the cell growth inhibition test. Fromthe background data in Hita Laboratory, 0.1 pg/mL of MMCand 6 pg/mL of CPA were chosen as the treatment doses of the positive controls in the treatments without S9 mix and with S9 mix, respectively. Four chromosome slides were prepared in each dose. 2) Dose levels In cell growth inhibition tests without and with S9 mix, the highest concentration of the test substance yielding mitotic metaphases sufficient to assess chromosomal aberration was 240 and 540 pg/mL, respectively. Therefore, 240 pg/mL as the highest dose and three lower doses, 200, 160 and 120 ~ig/mL were set spacing a same dose difference of 40 ug/mL in the treatments without S9 mix. In the treatments with S9 mix, the cell growth rate at 300-540 pg/mL showed the almost value, and the frequency of cells with chromosomal aberrations there doses were not difference. Therefore, 300 pg/mL as the highest dose and three lower doses, 240, 180 and 120 ug/mL were set spacing a same dose different of 60 pg/mL, so that dose-dependability might be acquired. 

### 3) Microscopic observation and scoring of chromosomal aberration All chromosome slides were coded and microscopically observed in a blinded manner. (1) Structural aberration Two hundred metaphases per dose, a metaphase containing chromosomes 25f2, were examined. On scoring and categorizing structural aberrations, a total number and its frequency (%) ofcells with aberrations as well as the number of chromosomal aberration and its frequency in each aberration criteria were tabulated. Gaps were additionally scored separately from the structural aberration criteria. p. 72



# 0000'72

- -7

### K06-0989 (2) Numerical Aberration The number and the frequency of numerical aberration showing triploid or more were examined in 200metaphases per dose. 

### 7. CONTINUOUS TREATMENT Because a short-termtreatment of the chromosome aberration test showed a positive result, a continuous treatment method was canceled despite a completion of the cell growth inhibition test. The data of the cell growth inhibition test were not included in this report . 8. JUDGEMENT CRITERIA OF RESULTS The test substance was judged to be positive, when the frequency of cells with structural or numerical aberrations in the test substance treatment groups showed a dose-related increase ranging 10% or more, or when the frequency showed a reproducible increase ranging 5% . or more. Other cases werejudged to be negative. Any statistical methods or analyses were not used in this study. In a case of positive result, a D2o value indicating a concentration which will induce chromosomal aberration of 20°lo was calculated.

FACTORS AFFECTEDRELIABILITY OF TEST Therewere no factors which might affect the reliability of the test. TEST RESULTS 

### 1 . SHORT-TERM

TREATMENT 1 .1 Cell Growth Inhibition Test (Table I and Fig. 1) The 50%

cell growth inhibition concentrations were approximately 200 [tg/mL in the treatments without S9 mix and approximately 190 p.g/mL in the treatments with S9 mix. Precipitation of the test substance was observed at 120 pg/mL or more in the treatments without and with S9 mix at the beginning and the end of the treatment. The maximum doses, 240 ug/mL in the treatments without S9 and 300 pg/mL in the treatments with S9 mix, yielded a clear cytotoxicity showing the cell growth rate below50% . The frequency ofcells with structural aberration showed 8%and 10%

at maximum

in the treatments without and with S9 mix, respectively. p. 73



# ' 014068 0000'73

- 9 -

### K06-0989 p. 74

1 .2 Chromosomal Aberration Test (Table 2 and Fig. 2) 1) Without S9 Mix (1) Cell growth rate 

### The cell growth rates at 120, 160, 200 and 240 qg/mL of the test substance were 66.4, 54.7, 45.8 and 19.8%, respectively. (2) Precipitation of the test substance Precipitation of the test substance was observed at any dose levels at the beginning and the end ofthe treatment. (3) Frequency ofcells with structural aberration The frequencies of cells with structural aberration at 120, 160, 200 and 240 ug/mL of the test substance were 1 .0, 3.5, 8 .5 and 13.0%, respectively. On the other hand, the frequencies in the negative control and in the positive control (MMC) were 2.0% and 41 .0%, respectively. (4) Frequency of numerical aberration The frequencies of numerical aberrations were below5.0% in all treatment groups. (5) Dzovalue The D2o value in structural aberration criteria was estimated 0.50 mg/mL (S=0.022) . 2) With S9 Mix (1) Cell growth rate The cell growth rates at 120, 180, 240 and 300 pg/mL of the test substance were 67.0, 62.9, 58.7 and 22.9%, respectively. (2) Precipitation of the test substance Precipitation of the test substance was observed at any dose levels. at the beginning and the end of the treatment. ' (3) Frequency ofcells with structural aberration The frequencies of cells with structural aberration at 120, 180, 240 and 300 pg/mL of the test substance were 0, 2.0, 6 .0 and 18.5%, respectively. On the other hand, the frequencies in the negative control and the positive control (CPA) were 1 .0%

and 33.5%, respectively. (4) Frequency of numerical aberration The frequencies of numerical aberrations were below5.0% in all treatment groups . (5) D2ovalue The DZO value in structural aberration criteria was estimated 0 .45 mg/mI, (S=0.023) . 

# ~' ' ~1069 000074

- 9 -p. 75



### DISCUSSION ANDCONCLUSION 

### K06-0989

The frequencies of cells with chromosomal aberrations did not fluctuate markedly between two culture dishes, the frequencies in the negative control were below5%and the frequencies ofcells with structural aberrations excluding gaps in the positive control were over 20%, therefore, these results proved the test to be valid. A dose-dependent increase was found in the frequencies of the cells with structural aberrations in short-termtreatments with and without S9 mix. The above results indicate that ~ J' possesses an ability to induce chromosomal aberrations under the present test conditions. REFERENCES 1 . Toshio Sofuni (ed.) (1999) Data book of chromosomal aberration test in vitro. Revised edition, 1998 (inJapanese) . Life-science Information Center . 2. Japan Environmental Mutagenicity Society/Mammalian Mutagenicity Study Group (ed.) (1988) Atlas of chromosomal aberration induced with chemical substance (in Japanese) . Asakura Publishing Co., Tokyo.



# 000075

- 10 -p. 76

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, 10 100 1,000 10,000 Dose (gg/mL) Without S9 mix

100 \ U y C4 i O L a> U 50

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### With S9 mix

Fig. 1 Results ofcell growth inhibition test of C

- Short-termtreatment method - 000078K06-0989

0 N 0 t .a m 30-,

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### With S9 mix Fig. 2 Results of chromosomal aberration test of ~

- Short-termtreatment method - 

# 000079

,, s-I a ,ID074Control (Short-termtreatment, With S9 mix, Negative control)

Structural aberrations (Short-termtreatment . With S9 mix, 300 pg/mL)

a : Chromatid exchange 

### b : Chromatid break Photo 1 Chromosomal aberrations induced by ~ K06-0989 p. 80



# 000080

### Receipt No. D02-2~91

STUDY CODE: B11-0732



# FINAL REPORT

TWENTY-EIGHT-DAY REPEATED-DOSE ORAL TOXICITY STUDY OF

L IN RATS

July 2003

Hita Laboratory Chemicals Evaluation and Research Institute

Japan

### B11-0732 p' $2 STATEMENT

I, the undersigned, hereby declare that this report provides correct English translation of the final report (Study Code B11-0732, issued on July 25, 2003) .

Katsumi Miyata, M

.S., J .A.J .S .C.T. ]~Y 

### Date Hita Laboratory Chemicals Evaluation and Research Institute, Japan1311 -O -/j2 p.83



### GLP STATEMENT Hita Laboratory Chemicals Evaluation and Research Institute, Japan 

### Sponsor : 

# C Title: Twenty-Eight-Day Repeated-Dose Oral Toxicitv Studv of ~ - j in Rats Studv Code: B11-0732 1, the undersigned, hereby declare that this study was conducted in compliance with "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the 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 of the Pharmaceutical Affairs Bureau, MHW &No. 85 (1984) of the Basic Industries Bureau, MITI, March 31, 1984, revised on March 1, 2000)" and with "OECDPrinciples of Good Laboratory Practice (November 26, 1997)" . And, I confirmed that this report accurately reflects the rawdata obtained and that data ofthe study has reliability.

Study Director: Signed in original July 25, 2003

Kazutoshi Shinoda, Ph.D. Diplomate J .S.T.P.

### B11-0732 QUALITY ASSURANCE STATEMENT

Hita Laboratory 

### Chemicals Evaluation and Research Institute, Japan Sponsor: I J Title: Twenty-Eight-Dav Repeated-Dose Oral Toxicitv Study of L J in Rats Studv Code: B11-0732 This study was audited and inspected by Quality Assurance Section of Hita Laboratory, Chemicals Evaluation and Research Institute, Japan. The dates audited and/or inspected and the dates reported of these results to the study director and management are as follows. Items ofInspections and Audits Dates of Inspections and Audits Dates of Inspections and Audits Reports Protocol March 27, 2003 ~ March 28, 2003 ' Amendment to Protocol (No. l ) March 27, 2003 March 28, 2003 ' Documents ofAnimal Receipt March 31, 2003 March 31, 2003 Amendment to Protocol (No.2) March 31, 2003 March 31, 2003 Documents ofAnimal Grouping April 1, 2003 April 1, 2003 Preparation ofTest Substance April 2, 2003 April 3, 2003 Administration April 3, 2003 April 3, 2003 Amendment to Protocol (No.3) April 23, 2003 April 23, 2003 Autopsy May 1, 2003 May l, 2003 Trimming May 8, 2003 May 8, 2003 Clinical Chemistry Examination Data July 16, 2003 July 16, 2003 

### Pathological Data July 16, 2003 July 16, 2003 Animal Data July 16, 2003 July 16, 2003 Documents of Test Substance and Housing Conditions July 18, 2003 July 18, 2003 Draft of Physicochemical Report July 18, 2003 July 18, 2003 

### Draft ofFinal Report July 18, 2003 July 18, 2003 Reinspection of Draft Physicochemical Report July 18, 2003 July 18, 2003 ' , Reinspection of Draft Final _Report July 25, 2003 July 25, 2003 Final Report July 25, 2003 July 25, 2003 

### 1, the undersigned, hereby declare that this report provides an accurate description of the methods and procedures used in this study and that the reported results accurately reflect the raw data obtained. Section Chief, Quality Assurance: SiQned in original July 25, 2003 Keiji Shiraishi, B.S. p. 84t31 1-U / -) .L p. 85 Study Code: B11-0732 Test Substance Code: HR5186 Sponsor Code: N-0360 

### TITLE Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of [ ~ in Rats SPONSOR 

# C 7

TESTING FACILITY Hita Laboratory Chemicals Evaluation and Research 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) of the test substance by observing the functional and morphological changes in animals receiving repeated doses orally 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 of Testing Methods Relating to the NewChemical Substances (Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW

&No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986)", and with "Section 407, Repeated Dose Oral Toxicity-Rodent : 28-day or 14-day Study" prescribed in "OECD Guidelines for Testing of Chemicals (May 12, 1981)" . 

### GLP COMPLIANCE This study was conducted in conformity with "Concerning Testing Facilities Stipulated in Article 4 of the Order Prescribing the Items of the 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 of the Pharmaceutical Affairs Bureau, MHW & 

# 000085B11-07 32 p. 86

No. 85 (1984) of the Basic Industries Bureau, MITI . March 31, 1984, revised on March 1, 2000)" and with "OECD Principles of Good Laboratory Practice (November 26, 1997)" . 

### PERIOD OF STUDY Commencement of Study: Animal Receipt: Initiation of Examination (Initiation of Dosing) : Terminal Necropsy of Dosing Period: 

### Initiation of Recovery Period: Terminal Necropsy of Recovery Period: Termination of Examination (Termination of hematology) : Completion of Study: 

### March 24, 2003 March 25, 2003 April 3, 2003 March 1, 2003 March 1, 2003 March 15, 2003 June 27, '?003 July 25, 2003 LOCATION AND PERIOD FOR RETENTION OF RAW DATA AND SPECIMENS The rawdata, protocol, study contract documents, test substance information, final report, QA inspection records, other record documents and specimens will be stored in the archive of the Hita Laboratory of our organization, and samples of every lot of the test substance, in the test substance storage roomfor a period of 10 years fromthe date of receipt of the notification that they are applicable to No. 1, 2 or 3 of the first paragraph of A

. "-tlcle 4 of the Japanese Chemical Substances Control La,xJ . The sponsor will informthe Hita Laboratory of our organization of the date of receipt of the notification. 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 of the preparation permits evaluation with the sponsor's consent . 

### RETENTION OF ORIGINAL PROTOCOL ANDFINAL REPORT The original protocol and protocol amendment will be retained at Hita Laboratory. Acopy of the document will be retained by the sponsor. The original final report will be retained at Hita Laboratory. A copy of the final report with the relevant certificate of Management in Chemicals Evaluation and Research Institute, Japan will be retained by the sponsor. ^ `f T081 . Q000BG

### 1311-Uli_ 11

AUTHORANDPERSONNEL CONCERNEDWITH STUDY Study Director : Kazutoshi Shinoda, Ph.D . . Diplomate J .S .T.P. (Planning and management of the study, evaluation of the results, report creation, and over all responsible for the technical conduct of the study) 

### Study Staff: Takayuki Koga, S.L.A.T., J .A.C.S.C.T. (Quarantine, acclimation and housing management of animals, preparation and administration of the test substance, clinical observation, body weights and food intakes measurements, and responsible for the animal examination) 

### Person in charge of Pathologic Examination: Kazutoshi Shinoda, Ph.D ., Diplomate J.S.T.P. (Necropsy, collection of tissues, organ weight measurements, histopathological examinations, and responsible for the histopathology) Person in charge of Clinical Chemistry: Keiji Shiraishi, B.S. (Hematological and blood chemical examinations, urinalysis, and responsible for the biochemistry of the specimens) 

### Person in charge of Chemical Analysis: Takeharu Wada, M

.S. (Confirmation for stability of the test substance, homogeneity and stability of the formulations, and responsible for the physicochemical analysis) 

### AUTHORAPPROVAL Study Director: Sianed in oriEinal July 25, 2003 Kazutoshi Shinoda, Ph.D . Diplomate J .S.T.P. General Toxicology Section Hita Laboratory

~ fl ~,~c7~ p. 87



# D0On$'7

### B11-073'2 p. 88

TABLE OF CONTENTS 

### SUMMARY . . . . . . . . . . .. . . . . . . . . . . . . .. . ... . .. . .. . .... ... . . . . .

..... . . . . . . . . . . . .. .... . . . . . . .. . . . . .. . . . . .... .. 1 MATERIALS ANDMETHODS 1 . TEST SUBSTANCE .. .. .. . . .. .. .. . . . . . . .. . . . . . . . .. . . . . . . . . . . . .. . . .. . . . . . . .. .. . . .. . . . . .

. . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . , 2. ANIMALS . . . .. . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 3. HOUSING CONDITIONS . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . .. 3 4 . GROUPING . . . . . . . .. . . . .. . . . . . . . .. . . .. . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . .. .. . . . . .. .. . . .. . . . . . 4 5. STABILITY OF TEST SUBSTANCE 6. PREPARATION OF TEST SUBSTANCE .7 . ADMINISTRATION . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . 7 8. OBSERVATIONS . . . .. .. . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 7 9 . STATISTICAL ANALYSIS . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . . .. . . . . . . 11 FACTORS THAT MIGHT HAVE AFFECTED THE STUDY . . . . . . . . . . 12, RESULTS 1 . CLINICAL SIGNS . .. .. .. . . . .. . . . .. . . . . . . . . . . . . . . . .. .. . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2. BODY WEIGHTS . . . . . . . . . . . . . . . . . . .-- . .--- . . . . . . . . . . . . . . . . . . .. .. . . .. . . . . . . . . . . . .. . . . . . 13 3. FOOD INTAKES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . .. . . . . 13 4. HEMATOLOGICAL EXAMINATIONS . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. 13 5. BLOOD CHEMICAL EXAMINATIONS . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . 14 6. URINALYSES . . . . .---. . . . . .. . . .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . .. .. . . . . . . . 14 7. ORGAN WEIGHTS . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . .. . .. 14 8. NECROPSY . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 14 9. HISTOPATHOLOGICAL EXAMINATIONS . . . . . . . . . . . . . . . . . . . . . . . . 15 

### DISCUSSION .. .. .... .. ... . . . . . ... . .. .... .. .... . ..... . ... . . . . . . . . . . . . . . . . . . ...... .... . ... . . .. . . ... . .. . 15

### 1311-073' p.89 TABLE OF CONTENTS (continued) FIGURES 1 Body weights . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . .. . . . . . . . . . .. . . . . .. .. .. . . . . . . . . . 1 2 Food intakes . .. .. . . . .. . . . . . . . . . .- .- .-- .-- . . . . . .. . . .. . . . . . . . . .. . . . . . . . . . . .. . . . . . .. .. . . . . . . . . . 3 TABLES 1 Summary of clinical signs . . . . . . . . .. . . . . . . . . . .. .. . . . . . . . . . . . . .. . . . . .. . .. .. . . . . .. . . . . . 1 Summary of body weights . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . .. . . .. . . . . . . .. . . . . . 2 3 Summary of food intakes . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. .. . . . .. . . . . . . . . . . . . . . . . . 4 4 Summary of hematological examinations .5 Summary of blood chemical examinations . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . g 6 Summary of urinalyses .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . 10 7 Summary of absolute organ weights . .. . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . 11 8 Summary of relative organ weights .. .. . . . . . . . . .. .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . 12 9 Summary of macroscopic examinations . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ; 10 Summary of histopathological examinations . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 14 

### ADDENDA --- ------ - -- - - -- -- -- 1 Clinical signs of individual animals . . . .. . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . .. . . . . . 1 2 Body weights of individual animals . .. . . . . . . . . . . . . . .. .. . . . . .. . . . . . . . . . . .. . . . . . 5 3 Food intakes of individual animals . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . 8 4 Hematological data of individual animals . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 11 5 Blood chemical data of individual animals . . . . . . . . . . . . .. . .. . . . . .. . . . . . . . . . 15 6 Urinalytic data of individual animals . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 19 

### 7 Absolute organ weights of individual animals .. . . . . . . .. . . . . . . . . . . . . . . . . . 21 8 Relative organ weights of individual animals . . . . . . . . . .. . . . . . . . . . . . . . . . . . '23 9 Pathological findings of individual animals APPENDIX . .. . . . . . . .. . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . PHYSICOCHEMICAL REPORT

### 1311-0732 p.90

SUMMARY A 28-day repeated oral dose toxicity study of [ ]followed by a 14-day recovery study was performed in groups of 6 male and 6 female Crj : CD (SD) IGS rats at S weeks ofage. The high dose was set at 1,000 mg/lcg/day, and altogether 3 doses including 200 and 40 mg/kg/day were employed. Recovery groups were also set for the 1,000 mg/kg and vehicle control groups. In the present study, neither deaths nor any adverse effects of the treatment with the test substance on the body weight or food intakes during the dosing period or on the hematology, blood chemistry, urinalysis, organ weights, necropsy or histopathology at completion of the dosing period, were observed. Concerning the general condition, although soft stool and diarrhea were observed in males and females and mucous stool in females in the 1,000 mg/kg groups, these signs were not observed in the recovery study. Based on these results, it is considered that L Iffects the Pastrointestinal tract, and the NOEL of~ ~in rats under the present study conditions is estimated to be 200 mg/kg/day since soft stool and diarrhea were observed in males and females and mucous stool in females in the 1,000 mg/kg groups.



# 000090

- 1 -

### B11-0732 p'91

MATERIALS AND METHODS 1 . TEST SUBSTANCE (INFORMATION PROVIDED BY THE SPONSOR) 1 .1 Name 

### Other Name: CAS No. : - 1 .2 Lot No. 0-6182 1 .3 Supplier 

### 1 .4 Structural Formula Typical structural formula .--

(Molecular formula: -) 1 .5 Purity 99.9 w/w°lo 1 .6 Names and Concentration of Impurities Water: 0

.1 w/w% 1 .7 Physicochemical Properties Appearance at Ordinary Temperature: Molecular Weight : Mn= 

### Mn= L Stability: stable Melting Point: 48°C (DSC) Boiling Point: - Yellow-brown solid



# IMOSC 

# -I - ooo09a.

### B11-0732 p~ Vapor Pressure: - Partition Coefficient : - Hydrolyzability: none Solubility: oil soluble Degree of Solubility 

### Water: less than 1 mg/L DMSO : more than 10% Acetone: more than 10% Others : chloroform

; more than 10°.% 1 .8 Storage Conditions Stored in the dark place at room temperature (cabinet I in the test substance storage room) . 

### 1 .9 Handling Precaution Gloves, mask, cap and lab coat were put on. 2 . ANIMALS Crj : CD (SD) IGS rats (SPF) were obtained fromCharles River Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan) . Animals were quarantined and acclimatized, and healthy animals with favorable weight gains were allocated to groups to ensure homogeneity of mean body weights using body weight-stratified randomization for the study. At the onset of the treatment, the animals were 5 weeks old with body weight ranges of 145.4-164.3 g and 116.2-141 .1 g for males and females, respectively. Animals were identified by means of ear tags . 

### 3. HOUSING CONDITIONS The barrier-system animal roomwas maintained at a stable temperature (21-25°C) and relative humidity (40-70%) with 10-15 air changes per hour and artificial light-dark cycle of 12-12 hours (light on: 7:00 and light off: 19:00) . The actual temperature and humidity were 22.0-23.9°C and 46?-63.5%, respectively . The rats were housed in hanging stainless steel cages with wire-mesh floor at 5 animals/cage (260 WX380 Dx 180 H min, TOKIWA KAGAKU KIKAI CO ., LTD .) for quarantine and acclimation, and at 1 animal/cage (165 WX300 Dx150 H mm, TOKIWA KAGAKU KIKAI CO .. LTD.) for after grouping. Trays wer-changed once before grouping, once on day 2 after grouping, everyday fromday 6

- 3 - 

# -f, ~

J 0~~ 000092,

### B11-0732

to day 2 (recovery), and thereafter twice weekly. Feeders, cages and racks were changed once at the grouping and the termination of the dosing period for the recovery group. The racks and cages were identified by individual cards. p. 93

The animals had free access to an MF pelleted diet (lot no. 021112 and 030207, Oriental Yeast, Co.. Ltd .) and water (chlorinated) fromthe Hita City supply via automatic watering system with sipper tubes. The diet and housing materials were autoclaved at 121°C for 30 min prior to use. Analysis of the diet was performed in Japan Food Research laboratories, and the analytical data were provided by the manufacturer. The tested parameters met the requirements according to the "Toxic Substances Control Act of US-EPA" . Contaminants in drinking water were analyzed annually for all of 46 parameters according to the water regulations of the "Notification No. 69 of Environmental Health Bureau, MHW (December 1, 1993)" and annually for 32 parameters in Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan. Contaminants in the water were inside the stated ranges. No environmental deviations, which might affect the study results, were noted during the quarantine, acclimation or study periods. 4. GROUPING Grouping was as follows: Group Vehicle control Vehicle control (recovery) Low dose Intermediate dose High dose High dose (recovery) Dose Volume Concentration (mg/kg/day) (mL/kg) (w/v°ro) Male No. of Animals (Animal No.)

Female 0 10 0 6( 1 - 6 ) 6( 37 - 0 10 0 6( 7 - 12) 6(43- 40 10 0.4 6(13- 18) 6(49- 200 10 2.0 6 ( 19 - 24 ) 6 ( 55 - 1,000 10 10.0 6 ( 25 - 30 ) 6 ( 61 - 1 .000 10 10.0 - 6(31 - 36) 6(67- 42 ) 48 ) 54 ) 60 ) 66 ) 72 ) 

### Rationale for dosage selection: A 14-day repeated oral dose toxicity study was performed at 4 doses of 50, 250, 500 and 1,000 mg/kg/day. As a result, the suspected adverse effects of the test substance on the clinical conditions were observed in the 500 mgikg and higher 

# l f~ z ~,~0~~ 000093

### B1 1-0732 p"4

dose groups, and on the blood chemistry, necropsy and histopathology in the 1,000 mg/kg group. Accordingly, the high dose in the present study was set at 1,000 mg/kglday and lower doses of 200 and 40 mg/kg were set at a common ratio of 5, giving 3 doses. Recovery groups were also set for the 1 .000 mg/kg and vehicle control groups . 

### 5. STABILITY OF TEST SUBSTANCE Stability of the test substance during the dosing period was confirmed in Hita laboratory. The test substance was analyzed with IR spectrumto confirmthe stability before and after the dosing period. 

### IR spectrumof the test substance within 4,000 cm-1-400 cm-1 was compared with the IR spectrumprovided by the sponsor before dosing to determine the identity of the test substance with fourier transform infrared spectrophotometer (FTS 135, Nippon Bio-Rad Laboratories) . After dosing period, the test substance was also analyzed with infrared spectrophotometer to confirmthe stability. 6 . PREPARATION OF TEST SUBSTANCE 6 .1 Vehicle 0.5 w/v%

carboxymethyl cellulose sodiumsalt (CMC-Na) aqueous solution CMC-Na (lot no. LDG1638, Wako Pure Chemical Industries, Ltd.) Purified water (lot no. A152, Takasugi Pharmaceutical Co., Ltd.) 6.2 Preparation 

### When the preparation method of the test substance was investigated, the test substance did not dissolved in olive oil, but the suspended state of the substance in a vehicle of 0

.5 w/v% CMC-Na aqueous solution was found to be good. Therefore, 0

.5 w/v% CMC-Na aqueous solution was used as the vehicle. The preparations were conducted according to the following preparation method. The test substance was accurately weighed and ground in an agate mortar and then suspended in 0

.5 w/v% CMC-Na aqueous solution while adding the vehicle gradually to prepare suspensions at concentrations of 10.0, 2.0 and 0 .4 w/v% . These were prepared once weekly. 

# Q0009413 1 1-U /j2 p. 95



### 6.3 Homogeneity and Stability Tests The homogeneity and stability tests were performed in Hita Laboratory in these preparation method. Test-substance formulations of 10.0 and 0.05 w/v% were stable at roomtemperature for 7 days and showed Qood homogeneity. In the homogeneity test, top, middle and bottom layers of 10.0 and 0.05 w/v% formulations were taken (n=3) just after preparation. The samples were mixed with tetrahydrofuran and filtrated IDISMIC 13JP, Tokyo Roshi Kaisha), and quantitatively analyzed by high performance liquid chromatography (HPLC) . The homogeneity of formulations was confirmed with a relative standard deviation in each layer. In the stability test, the homogeneity samples were stored at room temperature. Middle layer of the sample was taken (n=3) after 3 days and 7 days. These samples were diluted with tetrahydrofuran and Fltrated (DISMIC 13JP, Tokyo Roshi Kaisha), and the samples were quantitatively analyzed by HPLC to confirm the stability as compared to the concentrations immediately after preparation (results of the homogeneity study) . 

### Analytical Conditions (1) Apparatus (HPLC1) Interface: Pump: Autosampler : UV-VIS Detector : Column Oven: Data Processor : 

### (2) Analytical Conditions Column: Model D-7000IF (Hitachi) Model L-7100 (Hitachi) Model L-7200 (Hitachi) Model L-7400 (Hitachi) Model L-5020 (Hitachi) D-7000 HPLC SystemManager (Hitachi) Shodea Asahipak GF-3) 10HQ7.6 mm I .D.x300 mm Column Temperature: Mobile Phase: FlowRate: Wave Length: Injection Volume: 

### 40°C Tetrahvdrofuran 1 mL/min 254 nm 10 uL

### B11-0732 p. 96

7. ADMINISTRATION The test substance was repeatedly administered daily in the morning by oral gavage using a syringe (Terumo Corporation) connected to a Neiaton catheter (Terumo Corporation) for 28 days . Thereafter, a 14-day recovery period was set. 

### 8. OBSERVATIONS Concerning the numbering of day and week, the day of the initiation of dosing was regarded as day 1, the day before initiation of dosing as day -1 and the week of initiation of dosing as week 1 . The day after the last dosing was regarded as day 1 (recovery) and the week of the initiation of recovery as week 1 (recovery) . 8.1 Clinical Signs During the dosing period, the general condition was observed 3 times a day, i .e., before dosing (in the morning), during and after dosing, and in the afternoon, daily from day 1 to day 28. During the recovery period, the general condition was observed once a day in the morning daily from day 1 (recovery) to day 14 (recovery) . 

### All individual results summarized weekly are presented in addendum and group results in tables divided into the dosing and recovery periods. 8.2 Body Weights Body weight was measured on day -2 (at allocation to groups) before initiation of dosing, on days 1, 3, 8, 12, 17, 21, 26 and 28 during the dosing period, and on days 1, 5, 10 and 14 (recovery) during the recovery period. 

### In addition, immediately before necropsy, body weights were measured for calculation of relative organ weights. 8.3 Food Intakes Food consumption was measured once before initiation of dosina, on days 3, 8, 15, 22 and 28 during the dosing period, and on days 4. 8 and 14 (recovery) during the recovery period.

### BI I -073? p. 97

8.4 Hematological Examinations Blood or plasma samples obtained by blood sampling from the abdominal aorta under ether anesthesia after overnight fasting (16 to 20 hr) at completion of the dosing period (excluding the recovery groups) and at completion of the recovery period were determined for the following items. As anticoagulant, 3 .2% sodium citrate aqueous solution was used for the determination of prothrombin time and activated partial thromboplastin time, and EDTA-2K for other measurements .

Parameters 2) White Blood Cell Count (WBC) (x 10 _ /pL) 3) Hemoglobin Conc. (Hb) (g/dL) 4) Hematocrit Value (Ht) (%) 5) Mean Corpuscular Volume (MCV) a 1) Red Blood Cell Count (RBC) (x 10 /pL)

) Method Systemfor detecting change in electrical resistance Systemfor detecting change in electrical resistance Noncyanhemoglobin method 

### RBCx MCV 103 Systemfor detecting change in electrical resistance 6) 7) 8) Mean Corpuscular Hemoglobin (MCH) Mean 

### (pg) Hb RBC x 10 Corpuscular Hemoglobin Conc (MCHC)(g/dL) Hb x 101 Ht 9) 10) 11) 12) Platelet Count (Platelet) (X 104/pL) Reticulocyte Count (Reticulo) (%a) Prothrombin Time (PT) (sec) Activated Partial Thromboplastin Time (APTT) (sec) Differentiation of Leukocytes (%) Stab Neutrophils (N-Band) Segmented Neutrophils (N-Seg) Eosinophils (Eosino) Basophils (Baso) Lymphocytes (Lymph) Monocytes (Mono) Systemfor detecting change in electrical resistance New methylene blue staining Magnetic sensor system Magnetic sensor system Wright staining



### l ) - 8) Hematology Analyzer, CELL-DYN3500, Abbott Laboratories 9), 12) Automatic Blood Cell Analyzer, MICROX HEG-120A. OMRONCorporation 10), 11) Coagulometer, KC-10A, AMELUNG



# _ "F',~~~ 00009'7ts 1 1-U p. 98



### 8.5 Blood Chemical Examinations Serumsamples were separated fromblood samples collected at the same times as those described in section 8.4, and the following items were determined in the obtained serum

samples. Parameters Method 1) GOT (IU/L) 2) GPT (IU/L) 3) Alkaline Phosphatase (ALP) (IU/L) 4) Cholinesterase (ChE) (IU/L) ) y-GTP (IU/L) 6) Total Cholesterol (T-Cho) (mg/dL) 7) Triglyceride (TG) (mg/dL) 8) Glucose (mg/dL) 9) Total Protein (T-Protein) (g/dL) 10) Albumin (g/dL) 11) 12) 13) 14) 15) 16) 17) A/G Ratio Blood Urea Nitrogen (BUN) Creatinine Total Bilirubin (T-Bil) Calcium(Ca) Inorganic Phosphorus (IP) Sodium(Na) 18) Potassium(K) 19) Chloride (CI) 1) - 3), 6)-8), 12), 13), 15), 16) 4), 5), 9), 10), 14) 17) - 19) 

### 8.6 Albumin T - Protein-Albumin (mg/dL) Urease~GIDH method (mg/dL) Creatininase~F-DAOS method (mg/dL) Azobilirubin method (mg/dL) OCPC method (mg/dL) Fiske-Subbarowmethod (mEq/L) Crown-Ether membrane

electrode method (mFq/L) Crown-Ether membrane

electrode method (mEq/L) Coulometric titration method 7170Automatic UV method (Method based on JSCC)

UV method (Method based on JSCC) p-Nitrophenyl phosphate method Butyrylthiocholine iodide method L-y-Glutamyl-p-nitroanilide method COD~ADPS method GPO-ADPS glycerol blocking method Hexokinase~G-6-PDH method Biuret method Bromocresol green method

Analyzer, Hitachi Ltd. 7150 Automatic Analyzer, Hitachi Ltd. PVA-aIII, A & T 

### Urinalyses Urinalysis was performed once (day 28) during the dosing period (excluding the recovery group) and once (day 14 (recoverv)) during the recovery period. Urine samples (accumulated for approximately 16 hr) collected in individual metabolic cages (150 WX200 DX263 H mm) were determined for the urinary

- 9 - 

# ;! I' M93 000098volume and color, while pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen were determined using the test paper (N-Multistix(D, Bayer Medical) . 

### 8.7 Pathological Examinations 1) Necropsy All animals were subjected to the detailed gross necropsy. 2) Organ Weights The weights of the following organs were measured in all animals. The relative organ weight was also calculated based on the body weight at the time of necropsy. 

### Liver, kidneys, testes, ovaries, brain, spleen and adrenals 3) Histopathological Examinations (1) The following organs and tissues were taken. Category Organs and Tissues Respiratory system trachea, lungs Digestive system esophagus, stomach, intestine (duodenum to rectum), pancreas, liver Cardiovascular system heart Urinary system kidneys, urinary bladder Reproductive system testes, epididymides, prostate, seminal vesicle Nervous system Hematopoietic and lymphatic (with coagulating gland), ovaries, uterus, vagina brain (cerebrumand cerebellum) bone marrow (femur), mesenteric lymph node, thymus systems spleen, Endocrine system pituitary gland, thyroids (with parathyroids), adrenals Special sense organ eye ball 

### Collected organs/tissues were fixed with 10%neutralized buffered formalin. However, testes and epididymides were fixed in Bouin's solution.

### Bl l-0732 p" 00 (2) For macroscopic lesions, the following organs/tissues were collected. Group (animal no.) Or~ians and tissues 40 mg/kg group (no. 54) skin (3) Light microscopic examinations were performed for the following organs and tissues after embedding in paraffin, sectioning and hematoxylin and eosin staining Group Vehicle control group

Vehicle control group (recovery) 200 mg/kg group 1,000 mg/kg group 1,000 mg/kg group (recovery) OrRans and Tissues stomach (forestomach and glandular stomach), intestine (duodenum

. jejunum, ileum, cecum, colon, rectum), liver, heart, kidneys, spleen, adrenals glandular stomach (males only), adrenals (females only) glandular stomach (males only) stomach (forestomach and glandular stomach), intestine (duodenum, jejunum, ileum, cecum, colon, rectum), liver, heart, kidneys, spleen, adrenals giandular stomach (males only), adrenals (females only) (4) The following organs/tissues were examined as macroscopic lesions, .

Group OrQ ans and Tissues 40 mg/kg group (no. 54) 200 mg/kg group (no. 22) skin liver 

### 9 . STATISTICAL ANALYSIS Data regarding body weights (excluding those at the time of necropsy), food intakes, hematological examinations, blood chemical examinations, urine volume and organ weights were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test.