U.S . ENVIRONMENTAL PROTECTION AGENCY 

PREMANUFACTURE 

NOTICE 

Date of receipt 

,:PA 

Enter the total number of pages 

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 

TS 0-1 0 4 A 

You must provide all information requested in this form to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates if you do not 

have actual data . 

Before you complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from the Toxic 

Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603) . 

If a user fee has been remitted for this notice (40 CFR 700.45), 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, HQ Accounting 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 of the new 

chemical substance, even if you claim the identity as confidential. You may authorize 

another person to submit chemical identity information for you, but your submission will 

not be complete and the review will not begin until EPA receives this information. A 

letter in support of your submission should reference your TS user fee identification 

number . You must submit an original and two copies of this notice including all test data. 

If you claimed any infomiation as confidential, a single sanitized copy must also be 

submitted. 

Part II-HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

If there are several manufacture, processing, or use operations to be described in Part II, 

sections A and B of 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 with the corresponding section heading . In Part III, list these 

attachments, any test data or other 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 for you to described 

pollution prevention and recycling information you may have regarding the new 

substance . 

So-called "binding" boxes are included throughout this form for 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 of consent orders or Significant New Use 

Rules. Except in the case of exemption 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 itself prohibit the submitter from later deviating from the information (except 

chemical identity) reported in the form. 

CONFIDENTIALITY CLAIMS 

You may claim any information in this notice as confidential . To assert a claim on the 

form, mark (X) the confidential box next to the information that you claim as 

confidential . To assert a claim in an attachment, circle or bracket the information you 

claim as confidential . If vou claim information in the notices as confidential, You nmst 

also provide a sanitized version of the notice, (including attachments) . For additional 

instructions on claiming information as confidential, read the Instructions Manual. 

® Mark (x) if any information in this notice is claimed as confidential. 

TEST DATA AND OTHER DATA 

You are required to submit all test data in your possession or control and to provide a 

description of all other data known to or reasonably ascertainable by you, if these 

data are related to the health and environmental effects on the manufacture, 

processing, distribution in commerce, use, or disposal of the new chemical 

substance. Standard literature citations may be submitted for data in the open 

scientific literature . Complete test data (written in English) not summaries of data, 

must be submitted if they 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 of the 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 (40 CFR Part 720) . 

Test Data (Check Below any included in this notice) 

Environmental fate data ® Yes " Other data ® Yes 

" Health effects data ® Yes Risk assessments 

" Environmental effects data ® Yes Structure/activity relationships 

PhysicaVChemical Properties` ® Yes Test data not in the possession 

or control of the submitter 

' A physical and chemical properties worksheet is located on the last page of this form . 

TYPE OF NOTICE (Check Only One) 

® 

a 

PMN (Premanufacture Notice) 

INTERMEDIATE PMN (submitted in sequence with final product PMN) 

SNUN (Significant New Use Notice) tv 

r~. 

c~a 

tTMEA (Test Marketing Exemption Application) 

; rl 

LVE (Low Volume Exemption) @ 40 CFR 723 .50(c)(I)~ 

N 

LJ 

r`-' i ' t 

LOREX (Low Release/Low Exposure Exemption) L 40 CFR 723 :59(W) 

LVE Modification 

IS THIS A CONSOLIDATED PMN? ® 

J 

LOREX Modification , 

Yes C,IT 

N 

# of chemicals ' 

(Prenotice Communication # required, enter # on page 3) 

EPA FORM 7710-25 (Rev . 5-95) Replaces previous editions of EPA Form 7710-25. 

+ 
Public reporting burden for this collection of information 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 of this collection of information, 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 

of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION 

I certify that to the best of my knowledge and belief: 

I I . The company named in Part I, section A, subsection 1 a of this notice form intends to manufacture or import for a 

commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, 

', Section B. 

2. All information provided in this notice is complete and truthful as of the date of submission. 

3 . 1 am submitting with this notice all test data in my possession or control and a description of all other data known to or 

reasonably ascertainable by me as required by §720.50 of the Premanufacture Notification Rule. 

Additional Certification Statements : 

If you are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNIJN, check the following user fee certification 

statement that applies: 

® The Company named in Part I, Section A has remitted the fee of $2500 specified in 40 CFR 700.45(b), or 

F-1 The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined @ 40 CFR 

700.43) in accordance with 40 CFR 700.45(b), or 

0 The Company named in Part I Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of $100 in 

accordance with 40 CFR 700 .45(b) . 

If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(c)(1) or a Low release 

and low exposure exemption (LoRex) application in accordance with 40 CFR 723 .50(c)(2), check the following certification 

statements : 

0 The 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 the terms of 40 CFR 723 .50. 

0 The manufacturer is familiar with the terms of this section and will comply with those terms; and 

F-1 The new chemical substance for which the notice is submitted meets all applicable exemption conditions . 

0 If this application is for an LVE in accordance with 40 CFR 723.50(c)(1), the manufacturer intends to commence 

manufacture of the exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day 

review period. 

The accuracy of the statements you make in this notice should reflect your best prediction of the anticipated facts regarding the chemical substance 

described herein . Any knowing and willful misinter p retation is subject to criminal penalty pursuant to 18 USC 1001 . Confidential _ 

Signature and title of Authorized Official (Original Signature Required) Date 

, 

Signature of agent -(if applicable) Date 

X 

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

~'" ~ ` 
Part I -- GENERAL INFORMATION 

Section A -- SUBMITTER IDENTIFICATION -~ Confidential 

I a. Person Name of authorized official Position 

Submitting 

Notice (in U.S .) 

Company 

Mailing address (number and street) 

City, State, ZIP Code 

b . Agent (if Name of authorized official Position 

applicable) 

Company 

Mailing address (number and street) 

City, State, ZIP Code Telephone Area Code Number 

c . If you are submitting this notice as part of a joint submission, mark (X) this box. 

Joint Submitter (if Name of authorized official Position 

applicable) 

Company 

Mailing address (number and street) 

City, State, ZIP Code Telephone Area Code Number 

2 . Technical Name of authorized official Position 

Contact (in 

X U.S .) 

Company 

Mailing address (number and street) 

City, State, ZIP Code Telephone Area Code Number 

3 . If you 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 . If you previously submitted an exemption application for the chemical 

substance covered by this notice, enter the exemption number assigned by Mark (X) -~ ® 

EPA. If you previously submitted a PMN for 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 of Notice - Mark (X) Only Only 3. ~ Both 

Binding Option Q Binding Option 

Mark (X) 

Mark Q the "Confidential" box next to an subsection you claim as confidential 

EPA FORM 7710-25 (Rev . 5-95) Page 3 
p. 4 

Part I -- GENERAL INFORMATION -- Continued 

Section B -- CHEMICAL IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) name of the substance based on 

the ninth Collective Index 9CI of CA nomenclature rules and conventions. 

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

Complete either item 1 (Class 1 or 2 substances) or 2 (Polymers) as appropriate . Complete all other items . 

If another person will submit chemical identity information for you (for either Item I or 2), mark (X) the box at the right . ConfiIdenti the name, company, and address of that person in a continuation sheet . - 10 E] dential 

I . Class 1 or 2 chemical substances (for definitions of class 1 and class 2 substances, see the Instructions Manual) 

a. Class of substance - Mark (X) 1 0 Class 1 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 1 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 I (CAS Inventory Expert Service - a copy of the Identification ~ Method 2 (Other Source) 

report obtained from the CAS Inventory Expert Services must be 

submitted as an attachment to this notice) 

d . Molecular formula and CAS 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 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 of composition and the typical composition (where appropriate) . (4) Provide a correct representative or partial chemical structure 

diaeram. ac comnlete as can be known. if one can he reasonahlv ascertained. 

® Mark (X) this box if you attach a continuation sheet. 

EPA FORM 7710-25 (Rev . 5-95) Page 4 
p. 5 

Part I -- GENERAL INFORMATION -- Continued 

Section B -- CHEMICAL IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) narne of the substance based on 

the ninth Collective Index (9C[) of CA nomenclature rules and conventions . 

Mark (X) the "Confidential" box next to any item you claim as confidential 

Complete either item I (Class I or 2 substances) or 2 (Polymers) as appropriate. Complete all other items. 

If another person will submit chemical identity information for you (for either Item I or 2), mark (X) the box at the right. ConfiIdentify the name, company, and address of that person in a continuation sheet. 0 dential 

I . Class 1 or 2 chemical substances (for definitions of class I and class 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 of the Identification ~ Method 2 (Other Source) 

report obtained from the CAS Inventory Expert Services must be 

submitted as an attachment to this notice) 

d . Molecular formula and CAS Registry Number (if a 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 of composition and the typical composition (where appropriate) . (4) Provide a correct representative or partial chemical structure 

diaeram. as comnlete as can he known. if one can he reasonahlv ascertained 

® Mark (X) this box if you attach a continuation sheet. 

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

000005 
03/0512004~ p. 6 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 oC rhc polymer you intend to manufacture. 

Indicate moximum weight percent oClnvn, molecular weight spccics (not including residual monomer;, rr_ictants, or so)vents) below 500 and 

below 1,000 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) maximum wcig}tt ^/o below 500 molecular weigiht : 

iii) mnximum we~ght °,~ belnw l 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 item you claim as confidential 

(1) - Provide thc spccrtic chemical name and CAS Rcgistry Number (ii a number cxists) of caelt 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 if you 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 TSC A Cherntcal 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 if you atrach a continuation sheet, 

c Me= identify which method you used to develop or obtatrt the specified chemical identity information reported in this notice (check one) 

Method 1 (CAS Inventory Lxperr $,ice - a. copy of the identification report Q Method 2 (otlter source) )( 

obtained from CAS Inventory Expett Service must be submittcd as 

as attaclhmcnt to tl)is notice' 

d. The cuiTrntly 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 if you at.lach a continutation shrct. FPA FORM 771a25 (Rev . 5.g5) Page 5 
Page 5A 000007 
0 .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 of polyn'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) ?lclow 500 and 

bclow 1,000 absolute mnlccular weight of that cninpo .,ftion 

Describe tile methods of mcGsurcmcnt or the bnsjc for your C9timStCC. CrPC ~ Other [:] . (Specify) 

i) lowcat ntlmbcr avcrsgc molccu1ar Weight : i 

ii) maximum weight % below 500 tttolecular weight : 

iii) maximum wclghl "/o hclow 1000 molecular weip-_lht 

Mark (X) this box if you attach a continuation ilicct, 

b . You must make separate 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;) of each 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 of Vic polymer 

description on the TSCA C?,rmical Substa ncc Inventory 

(il - Mark (X) tliis column if entrica in colamn: (3) and (4) are confidcntial, 

(G) - Indicate tile maximum wcight peircnt of cach monomer err other roactant tlhat may lrc present ns a rcsidual in the polymer as manufactumd for 

commercial purposc.a . 

r7Mark X this column if cnt m colomn (,6 is cnnfidential . 

Monomer or other rcsctatnt and CAS Registry Number Confi- Typical ldantity CanC " Maximum Con Citlentia1 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 if you 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 of the tdcnti ficslttott report a Mctliod 2 (other sourcc) )( 

obtained from CAS 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 you attach a continutation sheet. 

LPA FORM 7710-25 (Rev . 5-95) Page 5 B 
Page 5C 

000009 
p. 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 if available. If there are unidentified impurities, enter "unidentified." 

(b) - Estimate the maximum weight % of each impurity. If there are unidentified i mpurities, estimate their total weight %. 

Provide the 

Impurity and CAS Registry Number 

(a) 

Maximum 

percent 

(b) 

Confidential 

% X 

- % 

% 

% 

% 

% 

% 

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

X 

6. Generic chemical name - If you claim chemical identify as confidential, you must provide a generic name for your substance that reveals 

the specific chemical identity of the new chemical substance to the maximum extent possible . Refer to the 

TSCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing generic names. 

~ Copolymer of phenol and aromatic hydrocarbon 

Mark (X) this box if you attach a continuation sheet: 

7 . Byproducts - Describe any byproducts resulting from the manufacture, processing, use, or disposal of the new chemical substance. Provide the CAS Registry 

Number if available. 

, Byproduct 

(1) 

CAS Registry Number 

(2) 

Confidential 

X 

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

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

000010 
p. 11 

Part I -- GENERAL INFORMATION -- Continued 

Section C -- PRODUCTION, IMPORT, AND USE 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 of production . Estimates should be on 100% new chemical 

substance basis. For a Low Volume Exemption application, if you choose to have your notice reviewed at a lower production volume than 10,000 

k /yr, specify the volume and mark (x) in the bindin g box. If anted, you are bound to 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 of the category of use, the percent of production 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 new chemical 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 of total production for the first three years devoted to each category of use. 

(5) -- Mark (X) this column if entry in column (4) is confidential business information (CBI). 

(6) -- Estimate the percent of the new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for 

commercial purposes at sites under your control associated with each category of use. 

(7) -- Mark (X) this column if entry in column (6) is confidential business information (CBI). 

(8) -- Indicate % of product 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 of substance 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 pol yester 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 of the 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 if you attach a continuation sheet . 

b. Generic If you claim any category 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 of reasonable 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~k 1xl 

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 you attach hazard information. 

EPA FORM 7710-25 (Rev . 5-95) Page 7 

000011 
p. 12 

Part II-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER Mark (X) the "Confidential" box next to any item you 

claim as confidential 

Complete section A for each type of manufacture, 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 

there are further industrial processing or use operations after import. You must describe these operations . See instructions manual 

1 . Operation description 

Confidential 

a. Identity -- Enter the identity of the site at which the o peration 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 if you attach a continuation sheet. 

b. Type -- 

Mark (X) DManufacturing aProcessing E]Use 

c. Amount and Duration -- Complete 1 or 2 as app ropriate 

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) Diagram the major unit operation steps and chemical conversions . Include interim storage 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 of all starting materials and 

feedstocks (including reactants, solvents, catalysts, etc .), and of all products, recycle streams, and wastes . Include cleaning chemicals (note frequency if not 

used daily or per batch .) . 

(3) Identify by number the points of release, including small or intermittent releases, to the environment of the new chemical 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 AND ENVIRONMENTAL 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 form of the new chemical substance, 

number of works exposed, and duration of activity. Mark (X) the "Confidential" box next to any item you 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 if entry 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 new chemical substance (e .g ., solid: crystal, granule, powder, or dust) and % new chemical substance (if part of a mixture) 

at the time of exposure. 

(7) --Mark (X) this column if entry in column (5) is confidential business information (CBI) . 

(8) --Estimate the maximum number of workers involved in each activity for all sites combined . 

(9) -- Mark (X) this column if entry in column (8) is confidential business information (CBI) . 

(10) and (11) -- Estimate the maximum duration of the activity for any worker in hours per day and days per year . 

(12) -- Mark (X) this column if entries in columns (10) and (11) are confidential busines s information (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 if you attach a continuation sheet . 

3. Environmental Release and Disposal -- You must make separate confidentiality claims for the release number and the amount of the new chemical substance 

released and other release and disposal information . Mark (X) the "Confidential" box next to each item you claim as confidential . 

(1) --Enter the number of each release point identified in the process description, part 11, section A, subsection l d(3) . 

(2) --Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology (in kg/day or kg/batch) . 

(3) --Mark (X) this column if entries 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 from that release point . 

(5) -- a. Describe control technology, if any, and control efficiency that will be used to limit the release of the new substance to the environment. For releases 

disposed of on 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 if entries in columns (4) and (5) are confidential business information (CBI) . 

(7) -Identify the destination(s) of releases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or 

NPDES numbers of the POTW (Publicl Owned Treatment Works). Mark (X) if the POTW name or NPDES # is confidential business information (CBI) . 

Release Amount of new substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) 

Number released release 

(1) r (2a) (2b) 

(3 

e.g . dack air 

(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 

000013 
p. 14 

Part II-- HUMAN EXPOSURE 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 any processing or use activities after import . See the Instructions Manual . Complete a separate 

section B for each type ojprocessing, or use operation involving the new chemical substance. If the same operation is performed at more than one site describe the 

typical operation common to these sites . Identify additional sites on a continuation sheet . 

1 . Operation Description -- To claim information in this section as confidential, circle or bracket the specific information that you claim as 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 substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all 

products, recycle streams, and wastes . Include cleaning chemicals (note frequency if not used daily or per batch) . (3) -- Identify by number the points of release, 

including small or intermittent releases, to the environment of the new chemical substance. (4) Please enter the # of sites (remember to identify the locations of 

these sites on a continuation sheet) : 

# of sites 

® Mark (X) this box if you attach a continuation sheet . 

2. Worker Exposure/Environmental Release 

(1) --From the diagram above, provide the letter for each worker activity. Complete 2-8 for each worker activity described. 

(2) -- Estimate the number of workers exposed for all sitgs combined . 

(4) -- Estimate the typical duration of exposure per worker in (a) hours per day and (b) days per year . 

(6) -- Describe physical form of exposure and % new chemical substance (if in mixture), and any protective equipment and 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 diagram above, 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 of release 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 limit the release of the new substance to the environment. 

(14) -- Identify byproducts which may result from the operation . 

(3 ) , (5), (8), (11 , (13) and (15) --Mark (X) this column if an of the p roceeding 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 you attach a continuation sheet, 

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

00003.4 
Page 1 0A 000015 
p. 16 

OPTIONAL POLLUTION PREVENTION INFORMATION 

To claim information in this section as confidential circle or bracket the specific information that you claim as confidential. 

In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce 

or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal 

of the PMN substance. Please include new information pertinent to pollution prevention, including source 

reduction, recycling activities and safer processes or products available due to the new chemical substance. 

Source reduction includes the reduction in the amount or toxicity 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 from wastes that would 

otherwise be treated or released as air emissions or water discharges, or land disposal . Descriptions of pollution 

prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance 

with existing regulatory requirements and can be either quantitative or qualitative. The EPA is 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 PMN substance to potential 

alternatives may be provided. 

All information provided in this section will be taken into consideration during the review of this 

substance. See Instructions Manual and Pollution Prevention Guidance manual for guidance and 

examples. 

Describe the expected net benefits, such as (1) an overall reduction in risk to human health or the environment; (2) a reduction in 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 of production 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. 

0 Mark (X) this box if you attach a continuation sheet. 

EPA FORM 7710-25 (Rev . 5-95) Page 11 

000016 
p. 17 

Part III -- LIST OF ATTACHMENTS 

Attach continuation sheets for sections of the form and 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 of the form to 

which it relates, if appropriate . 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 claim any information in an attachment as confidential . You must include with the sanitized copy of the notice form a sanitized 

version of any attachment in which you claim information 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 

NMR spectrum 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 

PMN fee documentation 383 

CASIES Report 384,384A 

CMA electronic PMN documentation 385 

In vivo Micronucleus Test 386-410 

~ Mark (X) this box if you attach a continuation sheet . Enter the attachment name and number . 

EPA FORM 7710-25 (Rev . 5-95) Page 12 
p. 18 

PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

To assist EPA's review of physical and chemical properties data, please complete the following worksheet for data you provide and 

include it in the notice . Identify the property measured, the page of the notice on which the property appears, the value of the property, 

the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential . The physical 

state of the 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 (% PMN substance in -). You are not required to 

submit this worksheet ; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential 

information is properly protected. You should submit this worksheet as a supplement to your submission of test data . This worksheet 

is not a substitute for submission of test data. 

Property Mark Page Value Measured o Confdential (x) if number Estimate Mark (x) 

(a) provided (b) (c) (M or E) tdl 

X 15 

Physical state of neat substance X (s - (1) - ( ) 

Vapor pressure 

Temperature °C Torr 

Density/relative density /cm3 

Solubility 

@ Temperature °C g/L 

Solvent 

Solubility in water Temperature °C 

X 15 

Melting temperature (softening point) 60-130 °C M 

Boiling / sublimation tem erature torr pressure 

oc 

X 17,18 M 

Spectra: infrared, nmr 

Dissociation constant 

Particle size distribution 

Octanol / water partition coefficient 

Henry's Law constant 

Volitalization ftom water 

Volitalization from soil 

H concentration 

Flammability 

Ex lodability 

Adsorption / coefficient 

' X 119-24 M ' 

Other - Specify: gpc data 

ATTACHMENT 

oO0018 

PAGE 13 
2004/01/28 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 000019 
2004/01/28 11 :44:54 r ' ~ 

Pag2-Z 

p. 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 000020 
2004/01/28 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 : Low accumulation 

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°+ 
ooooz9 
ll 

' 

' 

' 

~100019 000024 
00020 000025 
000021 000026 
1100022 000027 
1100023 000028 
1100024 

oeoo~9 
[Receipt No . T02-4088 

STUDY CODE : 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 

of the 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 New Chemical 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 New Chemical 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 

nUALITY 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 April 14, 2003 April 15, 2003 

Preparation of Test 

Substance and Experimental May 1, 2003 May 1, 2003 

Implementation 

Raw Data and Draft Report May 19, 2003 May 19, 2003 

Re-inspection of Draft May 20, 2003 May 20, 2003 I 

Report 

Final Report 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 New Chemical 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 
KO 1-2884 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 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). 

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 

TESTING FACILITY 

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 

typhimurium and Escherichia 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 of Treatment of Dose Finding Test) : April 15, 2003 

Completion of Experiment 

(Completion of Colony Count): May 6, 2003 

Completion of Study: May 20, 2003 

STORAGE AND RETENTION PERIOD 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 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.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. 

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 

including a statement by the management (a certification of identity with the 

original). 

,100032 000037 
K01-2884 p.38 

NAMES, ASSIGNED SECTIONS AND JOB ASSIGNMENT OF STUDY 

DIRECTOR AND PERSONNEL 

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 of Test 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 
KO 1-2884 p. 39 

TABLE OF CONTENTS 

MATERIALS AND METHODS 

1 . TEST SUBSTANCE AND POSITIVE CONTROLS . . . . . . . . . . . . . . . . . 

2. BACTERIAL STRAINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

3 . MEDIUM AND S9 MIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

4. PRE-CULTURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

5. PREPARATION OF TEST SUBSTANCE AND POSITIVE 

CONTROLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

6 . METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

7. OBSERVATION AND COLONY COUNTING .

. JUDGEMENT CRITERIA OF TEST RESULTS . .8. . . . . . . . . . . . . . . . . . . 

3 

4 

5 

5 

6 

7 

7 

FACTORS AFFECTING RELIABILITY OF TEST . . . . . . . . . . . . . . . . . . . . 7 

TEST RESULTS 

1. DOSE FINDING TEST-1 

2. DOSE FINDING TEST-2 

3. MAIN TEST 

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

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

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

7 

8 

8 
KO 1-28 6440 

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 AND METHODS 

1 . TEST SUBSTANCE AND POSITIVE 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 

99.9 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: (-

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 room temperature 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. Appearance Purity Grade 

Wako Red-yellow Special 

AF-2"' Pure Chemical SEL 1402 crystalline powder 

° 

99.0 /° 

grade 

Industries, Ltd. 

W~° White crystalline Special 

NaN3'~ Pure Chemical TCH3448 powder 

° 

99 .2% grade 

Industries, Ltd. 

Yellow crystalline 

ICR-191'3 Polysciences, Inc. 492624 

powder 

- _ 

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 typhimurium strains 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 of Testing 

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, 

DOJINDO Laboratories) 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, SANYO Electric Co., Ltd.) at -80°C . 

3 . MEDIUM AND S9 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, SANYO Electric 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 . 

- 4 000043 

- 

sN)0038 
K01-20444 

4. PRE-CULTURES 

A 20-~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, UNIPATH Ltd.) 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 findin g 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 AND POSITIVE CONTROLS 

5 .1 Preparation of Test Substance 

1) 

2) 

3) 

4) 

Solvent 

DMSO (Lot No. NJ 151) 

Reason for selection of solvent 

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 2 hours after preparation. 

Preparation method 

After DMSO was 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. 

Preparation time 

The test substance solution was prepared immediately before use, kept under 

yellow lamps 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 2AA were 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 

-S9 mix 

+S9 mix 

TA100 TA1535 WP2 uvrA 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 sodium phosphate buffer (pH 7.4) 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 

A total of 6 doses including 5,000 ~g/plate as the highest dose and 5 lower doses 

diluted with a geometric progression of 4 was 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 of WP2 uvrA and TA98, 5,000 ~Ig/plate at which 

the precipitation was observed was taken. 

7. OBSERVATION AND COLONY 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 was judged 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 of the test. 

TEST RESULTS 

1 . DOSE FINDING TEST-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 S9 mix, 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. 
KU 1-Z 80447 

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 

the presence 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 FINDING TEST-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 

TAI 535 and at 78.1 4g/plate or more in TAI 537. 

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 . MAIN TEST 

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 AND CONCLUSION 

The test substance was judged 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 from bacterial contamination, which indicates the test results to be valid. 

From the 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 of revertant col onies/ late 

without (-) dose Base-pair substitution e Framesh ift 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: Sodium azide 

" 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 : Sodium azide 

" 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 : 

8 8 4 

Test period From May 1, 2003 to May 6, 2003 

With (+) or Number of revertant colonies/ late 

without (-) Test substance dose 

/ late) ( 

Base- ) air substi tution e F rames hife 

S9 mix pg p TA 100 TA 1535 WP2 uvrA TA 98 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) 

-S9 mix 39.1 - 5* (6) - - 1 0* *(9) I ,

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) 1 0 (6) 55 

54 (55) 38 24 (31) 14 17 (16) 

+S9 mix 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) 1 05* , 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 of revertant 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 0 5 2 late . 

+S9 mix 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 between plates . 

+: The precipitate of the test 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 mix 
KU~; 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 mix 
00 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 

4 

Fa 

N 

1 

O 

A 

47 

< 

F0 

O 

O 

< 

H 

...~ 

. 

3 

U 

N 

O 

U 

~.. 

N 

O 

Q 

ddD 

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 ~I J' 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 WP2 uvrA TA98 TA1537 TA100 TA1535 WP2 uvrA 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 (Mean f 3S .D.) 

-S9 mix +S9 mix 

TA100 TA1535 WP2 uvrA TA98 TA1537 TA100 TA1535 WP2 uvrA 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 ABERRATION TEST OF 

C -1 

USING CULTURED MAMMALIAN CELLS 

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 

GLP STATEMENT 

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 New Chemical 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 

GLP STATEMENT 

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 New Chemical 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 

QUALITY ASSURANCE 

STATEMENT 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

Sponsor: L 

Title: Chromosomal Aberration Test of F Using Cultured Mammalian 

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 of Inspections/Audits Dates of Report 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 New Chemical 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 ~,0 0 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 . 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). 

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 

TESTING FACILITY 

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 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). 

~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 of Treatment 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 AND RETENTION 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 AND JOB ASSIGNMENT OF STUDY 

DIRECTOR AND PERSONNEL 

Study Director: Syozo Ajimi, 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 of specimens) 

Study Staff: Junko Kawaguchi, M.S . 

Mutagenicity Section, Hita Laboratory 

(Observation of specimens) 

Management: Yoshifumi Fujino, M.S . 

Hita Laboratory 

Person in Charge of Storage: Shizuka Koda 

General Affairs Section, Hita Laboratory 

Person in Charge of Test 

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 

MATERIALS AND METHODS 

1 . TEST SUBSTANCE AND POSITIVE 

CONTROLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

2. CELLS AND MEDIUM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

3 . S9 MIX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

4 . CELL PRE-CULTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

5 . PREPARATION OF TEST SUBSTANCE 

AND POSITIVE CONTROLS 

6 . SHORT-TERM TREATMENT 

7. CONTINUOUS TREATMENT 

8. JUDGEMENT CRITERIA OF RESULTS 

? 

3 

4 

4 

5 

6 

8 

8 

FACTORS AFFECTED RELIABILITY OF TEST . . . . . . . . . . . . . . . 8 

TEST RESULTS 

1 . SHORT-TERM TREATMENT . . . . . . . . . . . . . . . 8 

p. 65 

DISCUSSION AND CONCLUSION . . . . . . . . . . . . . . . 10 
K06-0989 

SUMMARY 

The ability of C 3 to 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-term treatment 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 AND POSITIVE 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 of solubility 

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 of J.P .(Japan Pharmaceutical) MMC and 48 

mg of J.P . sodium chloride in one vial 

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 CPA at 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 

00006S 
p. 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 below 5%. 

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 of Test Results" and "The Notification on Partial 

Revision of Testing Methods for New Chemical Substances" . 

2.2 Storage 

Cells were stored in liquid nitrogen . 

2 .3 Medium and 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, 

Sanko Junyaku 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 below 15 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 from rat 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 .5 x 104 cells/mL or 5x 103 cells/mL were 

seeded in a culture dish (Asahi Techno Glass Corporation) with 60 mm in 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 of Test Substance 

1) Solvent 

Acetone (Lot No. DWP7546, purity 99 .5%, special reagent, Wako Pure 

Chemical Industries, Ltd.) was used. 

2) Reason for selection of solvent 

Solubility of the test substance in water was less than lmg/L (information from 

the sponsor). The test substance was soluble in DMSO at 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 from the 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 from the 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 MMC and 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 medium including 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 medium was 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 medium for 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 medium was removed, cells were 

collected from each 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 

below were 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 . 

From the background data in Hita Laboratory, 0 .1 pg/mL of MMC and 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 (%) of cells 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 200 metaphases per dose. 

7. CONTINUOUS TREATMENT 

Because a short-term treatment 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 were judged 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 AFFECTED RELIABILITY OF TEST 

There were 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 below 50%. The frequency of cells 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 of the treatment. 

(3) Frequency of cells 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 below 5.0% in all treatment 

groups. 

(5) Dzo value 

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 of cells 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 below 5 .0% in all treatment 

groups . 

(5) D2o value 

The DZO value in structural aberration criteria was estimated 0.45 mg/mI, 

(S=0.023) . 

~' ' ~1069 000074 

- 9 - 
p. 75 

DISCUSSION AND CONCLUSION 

K06-0989 

The frequencies of cells with chromosomal aberrations did not fluctuate markedly 

between two culture dishes, the frequencies in the negative control were below 5% and 

the frequencies of cells 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-term treatments 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 (in Japanese). 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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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. 

]~YDate 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 
1311 -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 "OECD Principles of Good Laboratory Practice (November 26, 1997)" . 

And, I confirmed that this report accurately reflects the raw data obtained and that data 

of the study has reliability. 

Study Director: Signed in original July 25, 2003 

Kazutoshi Shinoda, Ph.D . 

Diplomate J.S .T.P . 
B 11-0732 

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 of Inspections 

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 of Animal Receipt March 31, 2003 March 31, 2003 

Amendment to Protocol (No .2) March 31, 2003 March 31, 2003 

Documents of Animal Grouping April 1, 2003 April 1, 2003 

Preparation of Test 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. 84 
t31 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 New Chemical 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 & 

000085 
B 11-07 3 2 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 raw data, 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 room for a period of 10 years from the 

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 inform the 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 AND FINAL REPORT 

The original protocol and protocol amendment will be retained at Hita Laboratory. 

A copy 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 

AUTHOR AND PERSONNEL CONCERNED WITH 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) 

AUTHOR APPROVAL 

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 
B 11-073'2 p. 88 

TABLE OF CONTENTS 

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 

MATERIALS AND METHODS 

1 . TEST SUBSTANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 

2 . ANIMALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

3 . HOUSING CONDITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 

4. GROUPING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 

5 . STABILITY OF TEST SUBSTANCE 

6 . PREPARATION OF TEST SUBSTANCE 

. ADMINISTRATION .7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 

Summary of blood chemical .5 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 

8 Relative organ weights of individual animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . '2 3 

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 

of age . 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 from Charles 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 room was 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 

once before grouping, once on day 2 after grouping, everyday from wer-changed day 6 

- 3 - 

-f, ~ J0 ~~ 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) from the 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 
B 1 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 spectrum to confirm the stability before 

and after the dosing period. 

IR spectrum of the test substance within 4,000 cm-1-400 cm-1 was compared with 

the IR spectrum provided 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 confirm the stability . 

6. PREPARATION OF TEST SUBSTANCE 

6.1 Vehicle 

0 .5 w/v% carboxymethyl cellulose sodium salt (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. 

Q00094 
13 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 room temperature 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 (HP LC 1) 

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 System Manager (Hitachi) 

Shodea Asahipak GF-3) 10HQ 7 .6 mm I.D.x300 mm 

Column Temperature : 

Mobile Phase : 

Flow Rate : 

Wave Length: 

Injection Volume : 

40°C 

Tetrahvdrofuran 

1 mL/min 

254 nm 

10 uL 
B 11-0732 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. 
B I 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 

System for detecting change 

in electrical resistance 

System for detecting change 

in electrical resistance 

Noncyanhemoglobin method 

RBC x MCV 

103 

System for detecting change 

in electrical resistance 

6) 

7) 

8) 

Mean Corpuscular Hemoglobin (MCH) 

Mean 

(p g) 

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) 

System for 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. OMRON Corporation 

10), 11) Coagulometer, KC-10A, AMELUNG 

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

8 .5 Blood Chemical Examinations 

Serum samples were separated from blood 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-Subbarow method 

(mEq/L) Crown-Ether membrane 

electrode method 

(mFq/L) Crown-Ether membrane 

electrode method 

(mEq/L) Coulometric titration method 

7170 Automatic 

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 000098 
volume 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 (cerebrum and cerebellum) 

bone marrow (femur), mesenteric lymph node, 

systems spleen, thymus 

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. 
B l 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 OrQans 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.