r 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

PRE MANUFACTURE 

NOTICE 

Date of receipt 

AGENCY USE ONLY 

~l'V- r '- ....... I 

lllllllli/ll/11/lll//llilllll/i/ll/!! IIIII llliiiJ!/1 Iiiii II/I! II i/11111 ill I 

51040UuOt340/!:; 

/ 

in the Premanufacture Notice 

}~,b~ 

• You must provide all information requested in · Make reasonable estimates if you do 

not have actual data. 

• Before you complete this form, you should read the "Instructions Manual for Prernanufacture 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). 

• 1f 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, ~chis sent to EPA, HQ Accounting Operations Branch (PM-226), P.O. 

360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee. 

Part I-GENERAL INFORMATION 

You must provide the CWTently correct Chemical Abstracts (CA) Name of the new 

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

another person to! • ·· • • • ·• · ··· · " ··--·~--you, but your submission will 

not be complete ar )ceives this information. A 

letter in support oJ ~ rs user fee identification 

number. You m~ ~ his notice including all test 

data. 1f you clain ""' , gle sanitized copy must also 

be submitted. ~ 

•rt II-HUMAJ 

~ .tf there are several 

sections A and B c 

Part III -LIST C 

Attach additiOnal ' 

each continuation · 

attachments, any I< 

notice. 

OPTIONAL INFO 

You may include ;u 

substance. Ou pag 

pollution preventioJ 

substance. 

0 

u 

~ 

~ ~ 

t: 

·&RELEASE 

ns to be described in Part II, 

1eded. 

era question fully. Label 

ing. In Part III, listthese 

nnation included in the 

ider in evaluating the new 

i for you to described 

regarding the new 

So-called "binding" ~ r I\ or you to indicate your 

willingness to be bo \1 ..J. ; section, such as use, 

production volume, intended to reduce delays 

that routinely accom _ •r Significant New Use 

Rules. Except in the ca.~e of exemption applications (such as TMEA, L VE, LOREX) 

where certain infonnation 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 you claim information in the notices as confidential you must 

lso vrovide a sanitized version of the notice. (including attachments). For additional 

1Structions 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 conunerce, use, or disposal of the new chemical 

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

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

data, must be submitted if they do not appear in the open literature. You should 

clearly identity 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 oftest data and other data. Data should be submitted according to 

the requirements of §720.50 of the Premanufacture Notification Rule (40 CFR Part 

720). 

Test Data (Check Below any included in this notice) 

Environmental fate data 0 Yes Other data 0 Yes 

Health effects data 181 Yes Risk assessments 

Environmental effect~ data 0 Yes Structure/activity relationships 

Physical/Chemical Properties* 181 Yes Test data not in the possession 

or contrul of the submitter 

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

TYPE OF NOTICE (Check Only Oue) 

~ 

0 

D 

D 

D 

D 

D 

PMN (Premanufacture Notice) 

INTERMEDIATE PMN (submitted in sequence with final product~) :;e:: 

SNUN (Significant New Use Notice) 

TMEA (Test Marketing Exemption Application) 

L VE (Low Volume Exemption)@ <W CFR 723.50( c )(1) 

N 

LOREX (Low Release/Low Exposure Exemption)@ <W CFR 723.SQ~)(2) 

L VE Modification LOREX Modification 

IS THIS A CONSOLIDATED PMN? 

D 

D Yes 

# of chemicals 

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

I f1 ~"~ ~-rr-·--r--~ -~r-rrr· 

('J:) ',, 

""0 I ll 

'""!",) ) 

·-·-{ !) 
/-

Public reporting burden for this collection of information is estimated to average 110 hours per response, including time for reviewing 

instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of 

{ "ormation. Send comments regarding the burden estimate or any other aspect of this collection of information, including suggestions for \ 

'-1educing 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: 

1. The company named in Part I, section A, subsection la 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. I 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, Intennediate PMN, Consolidated PMN, or SNUN, check the following user fee certification 

statement that applies: 

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

D 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 

D 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)(l) or a Low release 

and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check the following certification 

statements: 

D 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. 

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

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

D If this application is for an L VE 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 ofthe anticipated facts regarding the chemical substance 

described herein. Any knowilll! and willful misinterpretation is sub· ect to criminal penaltv pursuant to 18 USC 1001. 

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

Signature of agent - (jf applicable) Date 

Confidential 

X 

X 

- ------------------------------------------------------~------------------------~----------~ 

EPA FORM 7710-25 (Rev. 5-95) Page2 
Person 

'\ Submitting 

/ 

Notice (in U.S.) 

b. Agent(if 

applicable) 

Joint Submitter (if 

applicable) 

2. Technical 

Contact (in 

U.S.) 

Company 

street) 

City, State, ZIP Code 

Name of authorized official 

Company 

Mailing and street) 

Code 

Name of authorized official 

Company 

street) 

City, State, ZIP Code 

Name of authorized official 

Company 

street) 

City, State, ZIP Code 

3. If you have had a prenotice communication (PC) concerning this notice 

and EPA assigned a PC Number to the notice, enter the number. 

for the chemical substance covered by this notice, enter the notice number 

assigned by EPA. 

I. 

Type of Notice - Mark (X) 

EPA FORM 7710-25 

D 

Manufacture 

Only 

Binding Option 

Mark 

Telephone 

Position 

Telephone 

Position 

Telephone 

D 

(Rev. 5-95) 

Import 

Only 

Mark (X) 

if none 

Mark (X) 

if none 

Mark (X) 

if none 

Binding Option 

Mark 

I 

I 3. I D 

X 

X 

Both 

Page3 
\ 

I. Class 

a. Class l or Class 

c. 

d. 

Chenucal name correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. 

For Class l substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name orCA Preferred Name must 

be provided, which ever is appropriate based on CA 9CI nomenclature rules and conventions). 

Please identify which method you used to develop or obtain the specified 

D Method 1 (CAS Inventory Expert Service- a copy ofthe Identification 

report obtained from the CAS Inventory Expert Services must be 

CAS# 

identity reported in 

D Method 2 (Other Source) 

e. For a class l substance, provide a complete and correct chemical structure diagram. For a 2 substance- (l) List 

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 

dia.rram. as como Jete as can he known. if one can he reasonahlv ascertained. 

this box if you attacl! a continuation sheet. 

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

'''"'!""'' 

on 

X 

Page4 
\ 

a. Indicate the number-average weight of the lowest molecular weight composition of the polymer you intend to manufacture. 

c. 

Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and 

below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement or the basis for your estimates: GPC Other (Specify) 

i) lowest number average molecular weight: 

ii) maximum weight% below 500 molecular weight: 

iii) maximum weight %below 1000 molecular weight: 

attach a continuation sheet. 

You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the 

"Confidential" box next to any item you claim as confidential 

(1) 

(2) -

(3) -

(4) -

(5) -

(6) -

Provide the specific chemical name and CAS Registry Number (if a number exists) of each monomer or other reactant used in the manufacture of 

the polymer. 

Mark (X) this column if entry in column (I) is confidential. 

Indicate the twical weight percent of each monomer or other reactant in the polymer. 

Mark (X) the identity column if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer 

description on the TSCA Chemical Substance Inventory. 

Mark (X) this column if entries in columns (3) and ( 4) are confidential. 

Indicate the maximum weight percent of each monomer or other reactant that may be present as a reSidual in the polymer as manufactured for 

conunercial purposes. 

this 

Conli-

% % 

% % 

% % 

% % 

% % 

Method I (CAS Inventory Expert Service- a copy ofthe identilication report 

obtained from CAS-Inventory Expert Service must be submitted as 

as attachment to this 

Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings similar polymers. 

attach a continuation sheet. 

X 

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

., .. , '['~~ --

IdentifY each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial purpose. Provide 

the CAS Registry Number if available. If there are unidentified enter "unidentified." 

Estimate the maximum % of each If there estimate their total %. 

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

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

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

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 hwentory, i 985 Edition, Appendix B for guidance on developing generic names. 

Maximum 

percent 

% 

% 

% 

% 

% 

% 

% 

dential 

X 

Confidential 

X 

of the new chemtcal substance. Provide the CAS Registry 

EPA FORM 7710-25 (Rev. 5-95) PageJ 
Part I -- GENERAL INFORMATION-- Continued 

Section C --PRODUCTION, IMPORT, AND USE INFORMATION: 

' 

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

\ Production volume-- Estimate the maximum production volume during the first 12 months 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 kg/yr, s~ifY the volume and mark (x) in the binding box. lf granted, 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) dmtial 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. (I)-- Describe each intended category of use of the new chemical substance by function and application .. 

(2) -- Mark (X) this cohunn if entry column (l) is confidential business information (CBI). 

(3) -- Indicate your willingness to have the information provided in column ( l) 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 infonnation ( 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 infonnation(CBI). 

Category of use ( 1) CBI Binding Produc- CBI %in CBI % of substance expected per use CBI 

Option tion% Form- (8) 

(by function and application i.e. a dispersive dye Mark(x) ulation Site- Con-* Indus- Com- Binding 

for finishing polyester fibers) (2) (3) (4) (5) (6) (7) limited sumer trial mercia! Option (9) 

-- X X X X 

% 

- X % X X X 

X % X X X 

% % 

% % 

% % 

% % 

* lf you have idmtified 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 idmtity in the consumer product. 

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

b. Generic lfyou claim any category of use description in subsection 2a as confidmtial, mter a gmeric description of that category. Read the 

use Instructions Manual for examples of gmeric use descriptions. 

description A crosslinking agent for solvent based inks. A polymerization monomer. A water scavenger for 

anhydrous polymers. 

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

3. Hazard Infonnation -- Include in the notice a copy of reasonable facsimile of any hazard warning statemmt, 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 equipmmt or practices Option 

Mark(x) 

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. M.S.D.S. 

I 

EPA FORM 7710-25 (Rev_ 5-95) Pagef 
( 

/ 

"-· 

next to any item you 

'1mplete section A for each type of pn>eessung, or use operation involving the new substance at industrial sites you 

ntrol. hnporters do not have to complete this for operations outside the U.S.; however, you may still have reporting requirements if 

there are further industrial · or use "'''.,."'t."''" · You describe these · See instructions manual 

Site address (number and 

City, County, State, ZIP code 

the same operation occur at more than one site, enter the number 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. 

b. 

I. Batch 

Mallillnutn kg/batch (100% new chemical 1 Hours/batch 

substance) 1 

I 

2. Continuous 

Process description Mark (X) to indicate your willingness to have your process description binding. 

: Batches/year 

1 Batches/year 

I 

(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.). 

Confidential 

(2) Provide the identity, the approximate weight (by kg/day 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. 

box if you attach a continuation sheet. 

EPAFORM7710-25 (Rev. 5-95) Pagef 

..... , .... ~·-
Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued 

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, 

I number of works exposed, and duration of activity. Mark (X) the "Confidential" box next to any item you claim as confidential. 

I, . (I) -Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling, cleaning, etc.) in whidl workers may be exposed to the substance. 

(2) - Mark (X) this column if entry in column (I) 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 business information CBI). 

Worker activity CBI Protective Equipment/ Binding Physical f01ms(s) Binding CBI #of CBI Maximum duration CBI 

(i.e., bag dumping, filling drums) Engineering Controls Option and%new Option Workers Hrs/day Days/yr 

Mark(x) substance Mark(x) Exposed 

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) 

NA 

0 Mark (X) this box 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 II, section A, subsection Id(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, fhgitive 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 

eadl 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 colmlllls ( 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 disdlarges or 

NPDES numbers ofthe POTW_(Publicly Owned Treatment Works). Mark (X) ifthe POTW name or NPDES #is confidential business information (CBI). 

Release !Unountofnewsubstance CBI Media of Control tedmology and efficiency (you may wish to optionally attach efficiency data) CBI 

Number released release 

(1) (2a) (2b) e.g. stack air I Bin<ling 

(3) (4) (5a) I Mark (X) (5b) (6) I 

1 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

I 

(7) Mark (X) the 0 POTW providename(s) below: CBI 0 Navigable 0 Other - Specify provide NPDES # CBI 

!----

destination(s) of waterway 

releases to water. 

,L.J Mark (X) this box if you attadl a continuation sheet. 

EPAFORM7710-25 (Rev. 5-95) PageiO 
Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS 

_!::omplete section B for typical processing or use operations involving the new cllemical substance at sites you do not control. Importers do not have to complete this 

( 'ion for operations outside the U.S.; however, you must report any processing or use activities after import. Seethe Instructions Manual. Complete a separate 

, Jon B for each type of processing, or use operation involving the new chemical substance. 

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

If the same operation is performed at more than one site describe the 

1. Operation Description - To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. 

(I)-- Diagram the major unit operation steps and cllemical conversions, including interim storage and transport containers (specifY- e.g. 5 gallon pails, 55 gallon 

drums, rail cars, tank trucks, etc). On the diagram, identity by letter and briefly describe eacll worker activity. (2) -- Provide the identity, the approximate weight 

(by kg/day or kg'batcll, on an I 00% new cllemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all 

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

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

these sites on a continuation sheet): 

- #of sites 

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

2. Worker Exposure/Envirorunental Release 

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

(2) --Estimate the number of workers exposed for all sites 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 cllemical substance (if in mixture), and any protective equipmart and atgineering controls, if any, used to 

protect workers. 

(7) --Estimate the percent of the new substance as formulated when packaged or used as a fmal product. 

(9)- From the process diagram above, mter the number of eacll release point. Complete 9-13 for each release point identified. 

(I 0) -- Estimate the amount of the new substance released (a) directly to the mvironment or (b) into control technology to the enviromnmt (in kg! day or kg'batcll). 

( 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 lintit the release of the new substance to the environmmt. 

( 14) - Identity byproducts whicll may resuh from the operation. 

(3), (5), (8), II), (13) and (15)- Mark (X this column if any of the proceeding entries are confidential business information (CBI). 

Letter #of CBI Duration CBI Protective Equip. I o/o in CBI Release Amount of CBI Media of Release CBI 

of Workers of Engineering Controls/ Form- Number New &Control 

Act- Exposed Exposure Physical Form and% ulation Substance Technology 

ivity new substance Released 

(I) (2) (3) (4a) I _(4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (II) (12) (13) 

X I X X I X X I I 

I I 

I I 

X I X X I 

I I X X I I 

I I 

X I X X I X X I I 

I I 

I I 

X I X X I X X I I 

I I 

I I 

( 

I 

- 14)- Byproducts: (15) 

X 

\ 

-, X Mark (X) this box if you attacll a continuation sheet, 

EPAFORM7710-25 (Rev. 5-95) Page I~ 

''"''!~'~~ 

I 
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued 

Section B -INDUSTRIAL SITES CONTROLLED BY OTHERS 

Complete section B for typical processing or use operations involving the new dJ.emical substance at sites you do not control. hnporters do not have to complete this 

'ion for operations outside the U.S.; however, you must report any processing or use activities after import. Seethe Instructions Manual. Complete a separate 

, ion B for each type of processing, or use operation involving the new chemical substance. 

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

If the same operation is performed at more than one site describe the 

1. Operation Description - To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. 

(I)- Diagram the major unit operation steps and dJ.emical 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 eadJ. worker activity. (2) -- Provide the identity, the approximate weight 

(by kg/day or kglbatdJ., 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 dJ.emicals (note frequency if not used daily or per batdJ.). (3)- IdentifY by number the points of release, 

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

these sites on a continuation sheet): 

- #of sites 

X Mark (X) this box if vou attadJ. a continuation sheet. 

2. Worker Exposure/Environmental Release 

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

(2)- Estimate the number of workers exposed for all sites 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 dJ.emical 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 fmal product. 

(9) - From the process diagram above, enter the number of eadJ. release point. Complete 9-13 for eadJ. release point identified. 

( 1 0) - Estimate the amount of the new substance released (a) directly to the environment or (b) into control tedJ.nology to the environment (in kg/day or kglbatdJ.). 

(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 tedJ.nology, if any, that will be used to limit the release of the new substance to the environment. 

(14) --IdentifY byproducts whim may resuh from the operation . 

. (3), (5), (8), 11 ), _(13) and ( 15) - Mark 0 this column if anv of the proceeding entries are confidential business information (CBI). 

Letter #of CBI Duration CBI Protective Equip. I %in CBI Release Amount of CBI Media of Release CBI 

of Worl<ers of Engineering Controls! Form- Number New & Control 

Act- Exposed Exposure Physical Form and % ulation Substance Technology 

ivity new substance Released 

(1) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (11) (12) (13) 

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EPA FORM 7710-25 (Rev. 5-95) Page 1~ 

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Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued 

Section B - INDUSTRIAL SITES CONTROLLED BY OmERS 

Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. hnporters do not have to complete this 

(" · 'ion for operations outside the U.S.; however, you must report any processing or use activities after import. Seethe Instructions Manual. Complete a separate 

ion B for each type of processing, or use operation involving the new chemical substance. Ifthe same operation is performed at more than one site describe the 

1:ypical operation common to these sites. Ident.izy 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. 

(I)- 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, ident.izy by letter and briefly describe each worker activity. (2) --Provide the identity, the approximate weight 

(by kg! day or kg/batch, on an I 00% 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) -- Ident.izy bynumberthepoints of release, 

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

these sites on a continuation sheet): 

- #of sites 

.-

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

2. Worker Exposure!Enviromnental Release 

(I) -- 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 sites 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 fmal product. 

(9) - From the process diagram above, enter the number of each release point. Complete 9-13 for each release point identified. 

(I 0) -- 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 resuh from the operation. 

(3), (5), (8), 11 ), (13) and (15)- Mark 0\ this column if any of the proceeding entries are confidential business information (CBI). 

Letter #of CBI Duration CBI Protective Equip. I %in CBI Release Amount of CBI Media ofRelease CBI 

of Wolkets of Engineering Controls/ Form- Number New & Control 

Act- Exposed Exposure Physical Form and% ulation Substance Technology 

ivity new substance Released 

(I) (2) (3) (4a). I (4b)_ (5) (6) (7) (8) (9) (lOa) I (JOb) (11) (12) (13) 

X I X X I X X I I 

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"-14) -- Byproducts: (15) 

X 

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

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

··--~· ···r-- ·--·~ .. ''"'j''"" 
Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued 

Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS 

£omplete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers donothaveto completethis 

'on for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate 

\ .on B for each type of processing, or use operation involving the new chemical substance. 

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

If the same operation is performed at more than one site describe the 

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 Jetter 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 

-

( 

\ "- X. 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 sites 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 ofthenew substance as formulated when packaged or used as a fmal product. 

(9) - From the process diagram above, enter the number of each release point. Complete 9-13 for each release point identified. 

(1 0) - 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 resuh from the operation. 

(3), (5), (8), 11 ), (13) and (15)- Mark (X this column if any of the proceeding entries are confidential business information (CBI). 

Letter #of CBI Duration CBI Protective Equip. I %in CBI Relea•e Amount of CBI Media of Release CBI 

of Workers of Engineering Controls/ Form· Number New &Control 

Act- Exposed Exposure Physical Form and % ulation Substance Technology 

ivity new substance Released 

(I) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (JOb) (11) (12) (13) 

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X 

l X Mark (X) this box if you attacl:t a continuation sheet, 

EPA FORM 7710-25 (Rev. 5-95) Page 1~ 

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Part ll-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued 

Section B - INDUSTRIAL SITES CONTROLLED BY OTHERS 

~omplete section B for typical processing or use operations involving the new d:temical substance at sites you do not control. Importers do not have to complete this 

( •ion for operations outside the U.S.; however, you must report any processing or use activities after import. Seethe Instructions Manual. Complete a separate 

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

~~.rpical 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. 

(I)-- Diagram the major unit operation steps and d:temical 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 ead:t worker activity. (2) - Provide the identity, the approximate weight 

(by kg/day or kglbatd:t, on an 100% new d:temical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all 

products, recycle streams, and wastes. Include cleaning d:temicals (note frequency if not used daily or per batd:t). (3)- IdentifY by number the points of release, 

including small or intermittent releases, to the environment of the new d:temical substance. (4) Please enter the# of sites (remember to identifY the locations of 

these sites on a continuation sheet): 

- #of sites 

" 

X Mark (X) this box if you attad:t a continuation sheet. 

2. Worker Exposure/Environmental Release 

(I)-· From the diagram above, provide the letter for ead:t worker activity. Complete 2-8 for ead:t worker activity described. 

(2) -- Estimate the number of workers exposed for all sites 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 d:temical 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 ead:t release point. Complete 9-13 for ead:t release point identified. 

(I 0) •· Estimate the amount of the new substance released (a) directly to the environment or (b) into control ted:tnology to the environment (in kg/day or kglbatd:t). 

(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 ted:tnology, if any, that will be used to limit the release ofthe new substance to the environment. 

(I4) -IdentifY byproducts whid:t may resuh from the operation. 

(3), (5), (8), 11 ), (13) and (15) --Mark(); this column if any of the proceeding entries are confidential business infonnation (CBI). 

Letter #of CBJ Duration CBI Protective Equip. I %in CBI Release Amount of CBI Media ofRelease CBJ 

of Workers of Engineering Controls/ Form- Number New &Control 

Act- Exposed Exposure Physical Fonn and % ulation Substance Technology 

ivity new substance Released 

(I) (2) (3) (4a) I (4b) (5) (6) (7) (8) (9) (lOa) I (lOb) (11) (12) (13) 

X I X X I X X I I 

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X I X X I X X I I 

I I 

I I 

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_ J4) ··Byproduct.~: (15) 

X 

1 X Mark (X) this box if you attad:t a continuation sheet, 

EPA FORM 7710-25 (Rev. 5-95) Page /Of 
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OPTIONAL POLLUTION PREVENTION INFORMATION 

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

,r.,_ this section you may provide information not reported elsewhere in this form regarding your efforts to reduce 

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

( 

( 

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 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 greater overall risk to human health or the environment. 

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

EPA FORM 7710-25 (Rev. 5-95) Pagepl-;l.. 

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Part III -- LIST OF ATTACHMENTS 

t1ch continuation sheets for sections of the form and test data and other data (including physical/chemical properties and 

~ucture/activity information), and optional information after this page. Clearly identify the attachment and the section of the form to 

which it relates, if appropriate. Nwnber consecutively the pages of the attachments. In the colwnn below, enter the inclusive page 

nwnbers 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 

pagenuml>erOO 

Index to the "Substance and Study Reports" 36 

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

I 

·-~-Biodegradation Test ofV-OSH in Carp 77-155 

( 

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3-Bacterial Reverse Mutation Test ofV-OSH 

4-Chromosomal Aberration Test ofV-OSH Using Cultured Mammalian Cells 

5-Twenty-eight-day Repeated-Dose Oral Toxicity Study of V -OSH in Rats 

6-Measurement of Molecular Weight Distribution of V-05H 

7-Study on "Safety Evaluation Flow Scheme for Polymer" of V-OSH 

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

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

156-178 

179-203 

204-289 

290-305 

306-340 

Confidential 

X 

X 

X 

X 

X 

X 

X 

X 

Page.Ji? ;2'} 
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PHYSICAL AND CHEMICAL PROPERTffiS 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. IdmtifY the property measured, the page of the notice on which the property appears, the value of the 

physical 

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

state of the neat substance should be provided. These measured properties should be for the neat ( 100% pure) cl!emical 

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 

This worksheet 

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

is not a substitute for submission of test data. 

Property Mark Page Value Measured or Confi-

(X) if number Estimate dential 

(a) provided (b) (c) Mark (X) 

_(1-!_orE) (d) 

Physical 

X 

state of neat substance (s) X (I) (g) 

Vapor pressure 

_@_ Tem~rature oc Torr 

Density/relative density g/cm3 X 

Solubility 

X 

@ Temperature 20 oc 

Solvent giL 

Solubility 

X 

in water_@_ Tet11pe_rature 20 oc giL 

Melting temperature oc 

Boiling I sublimation temperature@ torr _.pressure oc 

Spectra GPC, 

X 

Infra Red 

Dissociation constant 

Particle size distribution 

Octanol/ waterpartition coefficient 

Henry's Law constant 

Volitalization from water 

Volitalization from soil 

_pH@_ concentration 

Flammability X 

Explodability 

Adsotption I coefficient 

Other - Specify 

ATTACHMENT 

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Original copy of information for the substance and study reports 

CDBiodegradation test ofV-05H by microorganisms 

Results Percentage biodegradation by BOD 0% 

Percentage biodegradation by HPLC 1% 

@Biodegraaatton test ofV-05H in carp 

Results 48-hour LC50 value >100mg/L 

Bioconcentration factors Level 1 < 12 

Level2 <129 

@Bacterial Reverse Mutation Test ofV-05H 

Results no reverse mutagenic potential . 

@Chromosomal Aberration Test of V~05H Using Cultured Mammalian 

Cells 

Results no chromosomal aberration 

@'1\venty-eight-day Repeated-Dose Oral Toxicity Study of V-05H in Rats 

Results NOEL=200mg/kg/day 

®Measurement of molecular weight distribution of V -05H 

Results Mn=4120 Mw=16500 

CVStudy on "Safety Evaluation Flow Scheme for Polymer" ofV-05H 

Results No difference was observed between those measured before 

the test and those measured after the test. 

--r...--
Accepted number 

Test number 

$96-3013 

13013 

FINAL REPORT 

Biodegradation test ofV-OSH by microorganisms 

March 31, 1997 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 
Sponsor 

Title 

Test number 

STATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

U

Kurume Research Laboratories] 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Biodegradation test ofV-OSH by microorganisms 

13013 

I, the undersigned, hereby declare that this report provides a correct English translation of 

the Final Report (Test No.13013, issued on March 31, 1997). 

However, sponsor's address, 11 1-18-1 Nishiarai Sakaecho, Adachi-ku, Tokyo, 123, Japan 11 

in the original; was changed to 11 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan 11, and CAS 

number was added to section 11.1 on the text by sponsor's request. 

Date J ClY\\At\Yf i 8", .J.-Vv 2 

~. _A<AclJ- Study director 

oshihtsa Sudo 

,-.~ • • -~• ~~ --·· •-r- --r·---- ; I 

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Sponsor 

Title 

Test number 

GLPSTATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Biodegradation test ofV-05H by microorganisms 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" (March 

31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination Bureau, 

Environment Agency, Yak:uhatu No.229, Pharmaceutical Affairs Bureau, Ministry ofHealth and 

Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of International Trade and 

Industry, Japan) and "OECD Principles of Good Laboratory Practice" (May 12, 1981). 

Date March 31, 1997 

Management Signed in original 

Hiroshi Katsuura 
. ' 

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Sponsor 

Title 

Test number 

GLPSTATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Biodegradation test ofV-05H by microorg~isms 

13013 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" 

(March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination 

Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry 

of Health and Welfare, and 59 K.ikyoku No.85, Basic Industries Bureau, Ministry of 

International Trade and Industry, Japan) and "OECD Principles of Good Laboratory 

Practice" (May 12, 1981). 

This GLP statement was issued as supplement to the GLP statement issued on March 

31, 1997 because ofthe amendment and addition of the Final Report. 

Date June 5, 1998 

Study director Signed in original 

Hiroshi Tadokoro 

TlrT 
Sponsor 

Title 

QUALITY ASSURANCE STATEMENT 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

NISSHINBO INDUSTRIES, INC. 

Biodegradation test ofV-05H by microorganisms 

Test number 13013 

Study audits and inspections of this test were carried out and the results were reported to the 

management and the study director by Quality Assurance Unit ofKurume Research Laboratories, 

Chemical Biotesting Center, Chemicals Inspection & Testing Institute, Japan as follows. 

Date of audit Date of report Date of report 

or inspection to management to study director 

January 31, 1997 January 31, 1997 January 31, 1997 

February 12, 1997 February 14, 1997 February 14, 1997 

March 17, 1997 March 17, 1997 March 17, 1997 

February 26, 1997 March 19, 1997 March 19, 1997 

March 12, 1997 March 19, 1997 March 19, 1997 

March 17, 1997 March 19, 1997 March 19, 1997 

March 18, 1997 March 19, 1997 March 19, 1997 

March 31, 1997 March 31, 1997 March 31, 1997 

It has been assured that the final report accurately describes the test method used, that 

details in the report are in compliance with the protocol and Standard Operating Procedures and 

that the final report reflects the raw data. 

Date March 31, 1997 

Quality Assurance Unit, Head Signed in original 

Susumu Uchitomi 
( 

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Sponsor 

Title 

QUALITY ASSURANCE STATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Biodegradation test ofV-05H by microorganisms 

Test number 13013 

Study audit of the corrected parts in the Final Report was carried out and it was 

confirmed that the correction has no problem. The result was reported to the management 

and study director as follows. 

Date of audit Date of report Date of report 

or inspection to management to study director 

June 5, 1998 June 5, 1998 June 5, 1998 

This statement was issued as supplement to the quality assurance statement issued on 

March 31, 1997. 

Date June 5, 1998 

Quality Assurance Unit, Head Signed in original 

Susumu Uchitomi 

- ... --· .. ~~ ·-- ~- ~--~----:-~r 
CONTENTS 

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page 

SUMMARY 1 

1. Title 2 

2. Sponsor 2 

3. Testing facility 2 

4. Objective 2 

5. Test method 2 

6. GLP Standards to comply with 3 

7. Test period 3 

8. Personnel 3 

9. Preparation of final report 3 

10. Approval of final report 3 

11. Test substance 4 

12. Preparation of activated sludge 6 

13. Performance ofbiodegradation test 8 

14. Validity of test conditions 14 

15. Results 14 

16. Factors possibly affecting accuracy 16 

17. Storage oftest substance, raw data, etc. 16 

18.Remarks 16 

19. Contents of tables and figures 17 

Tables and figures 

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SUMMARY 

1. Title 

Biodegradation test ofV-05H by microorganisms 

2. Biodegradation test 

2.1 Conditions of cultivation 

( 1) Concentration of test substance 

(2) Concentration of activated sludge 

(as the concentration of suspended solid) 

(3) Volume oftest solution 

(4) Cultivation temperature 

(5) Cultivation duration 

2.2 Measurement and analysis 

100mg!L 

30mg!L 

300mL 

25+1 oc 

28 days 

(1) Measurement of biochemical oxygen demand (BOD) by means of a closed system 

oxygen consumption measuring apparatus 

(2) Dete~ination of test substance by means of high performance liquid chromatography 

(HPLC) 

3. Results 

(1) Percentage biodegradation by BOD 

(2) Percentage biodegradation by HPLC 

4. Conclusion 

0%, 

2%, 

0%, 

0%, 

0% 

0% 

average 0% 

average 1% 

The test substance was not biodegraded by microorganisms under the present test 

conditions. 

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FINAL REPORT 

Test No. 13013 

1. Title 

Biodegradation test ofV-OSH by microorganisms 

2. Sponsor 

3. Testing facility 

4. Objective 

Name 

Address 

Name 

Address 

Tel 

Management 

NISSHINBO INDUSTRIES, INC. 

1-2-3, Onodai, Midori-ku Chiba, 267-0056, 

Japan 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

19-14, Chuomachi Kurume 

Fukuoka 830, Japan 

(0942) 34-1500 

Hiroshi Katsuura 

This test was performed to evaluate the biodegradability of V -OSH by microorganisms. 

5. Test method 

This test was conducted according to the "Method for Testing the Biodegradability of 

Chemical Substances by Microorganisms" stipulated in the "Testing Methods for New 

Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, 

Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health 

and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry of International Trade 

and Industry, Japan) and "Ready Biodegradability : 301C, Modified MITI Test (I)" (Revised 

July 17, 1992) in the OECD Guidelines for Testing of Chemicals. 

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( 

c 

6. GLP Standards to comply with 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances 

(hereafter referred to as "GLP standards")" (March 31, 1984, Revised November 18, 1988, 

Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yak:uhatu No.229, 

Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyok:u No.85, Basic 

Industries Bureau, Ministry of International Trade and Industry, Japan) and "OECD Principles 

of Good Laboratory Practice" (May 12, 1981 ). 

7. Test period 

(1) Start of test January 31, 1997 

(2) Start of cultivation with activated sludge February 12, 1997 

(3) Termination of cultivation with activated sludge March 12, 1997 

(4) End of test March 31, 1997 

8. Personnel 

Study director Y oshihisa Sudo 

Test operator Hisako Haruguchi 

Staff for cultivation of activated sludge Y oshiharu Ohshima 

Person in charge of storage of test substance and raw data Akira Kobayashi 

9. Preparation offmal report 

Date March 31, 1997 

Name Hisako Haruguchi 

10. Approval of final report 

Study director Date March 31, 1997 

Name Signed in original 

Y oshihisa Sudo 

- 3- U1 '~' 

( 

11.5 Confirmation of test substance 

Two infrared (IR) spectra of the test substance provided by the sponsor and this 

laboratory were confirmed to be identical (see Fig.6 and Reference). 

11.6 Physicochemical property*' 

Appearance Brown viscous substance 

* 1 Information supplied by the sponsor. 

11.7 Storage and stability 

(1) Storage condition 

Cold storage place. 

(2) Stability 

The test substance was stable during the cultivation, as shown by the fmding 

that IR spectra of the test substance before the start and after the termination of the 

cultivation were identical (see Fig.6). 

- 5 -

·~------ .. ,_. --.....-=oc"""" --=--- C""OT -·-- •"--~- =-·~ r:-
( 

( 

12. Preparation of activated sludge 

12.1 Sludge sampling sites and date 

(1) Sampling sites 

On-site sludge sampling was carried out at the following 10 locations in Japan: 

Fushikogawa city sewage plant (Sapporo-shi Hokkaido) 

Kashima industry sewage plant (Kashima-gun Ibaraki) 

Nakahama city sewage plant (Osaka-shi Osaka) 

Ochiai city sewage plant (Shinjuku-ku Tokyo) 

Kitakami river (Ishinomaki-shi Miyagi) 

Shinano river (Nishikanbara-gun Niigata) 

Yoshino river (Tokushima-shi Tokushima) 

Lake Biwa (Otsu-shi Shiga) 

Hiroshima bay (Hiroshima-shi Hiroshima) 

Dookai bay (Kitakyushu-shi Fukuoka) 

(2) Date December, 1996 

12.2 Sludge sampling method 

( 1) City sewage 

Return sludges from sewage plants were collected. 

(2) Rivers, lake and sea 

Surface water and surface soil which are in contact with the atmosphere were 

collected. 

12.3 Mixing of fresh and old activated sludge 

The filtrate (5 L) of the supernatant of the activated sludge used at that time was 

mixed all together with each of the filtrate (500 mL) of the supernatant of a newly 

collected sludge (10 sources). The mixed filtrate (10 L) was aerated"2 after the pH value 

of the mixture was adjusted to 7.0+ 1.0. 

*2 Prefiltered open air was used. 

-6-

I • nr r 
( 

I 

12.4 Cultivation 

Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant 

corresponding to about 113 of the whole volume was removed. Then an equal volume of 

dechlorinated water was added to the remaining portion. This mixture was aerated, and 

then a previously decided amount of synthetic sewage"3 was added to the mixture so that 

the concentration of the synthetic sewage was O.l(WIV)% in the volume of the 

dechlorinated water added. This procedure was repeated once every day. Cultivation 

was carried out at 25 + 2 °C. 

*3 Synthetic sewage 

' Glucose, peptone. and potassium dihydrogenphosphate were dissolved in 

dechlorinated water to obtain 5(W IV)% of the solution for each components. 

The pH ofthe solution was adjusted to 7.0+ 1.0 with sodium hydroxide. 

12.5 Control and use 

During cultivating, the appearance of the supernatant, setting of the sludge, 

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

were checked and adjusted if necessary. Microflora in the activated sludge was 

microscopically observed and the sludge with no abnormal symptoms was used for the 

test. 

12.6 Inspection of activity and date of initiation ofuse of activated sludge 

(I) Inspection of activity 

Activity of the sludge was assessed using a reference substance and the relation 

between new and old activated sludge was taken into account. 

(2) Date of initiation of use January 21, 1997 

-7-

,·-·rrrr-
I 

\ 

"'-

13. Performance ofbiodegradation test 

13 .1 Preparation for test 

( 1) Measurement of concentration of suspended solid 

Method In accordance with Japanese Industrial Standards (JIS) K 0102-1993 

section 14.1 

Date February 10, 1997 

Result Concentration of suspended solid in the activated sludge was 4700 

mg/L. 

(2) Preparation of basal culture medium 

Each 3 mL of solution A, B, C and D, which are prescribed in JIS K 0102-1993 

section 21, were made up to 1000 mL with purified water (Takasugi Seiyaku Co., 

Ltd.), and then the pH ofthis solution was adjusted to 7.0. 

(3) Reference substance 

Aniline (reagent grade, Showa Chemicals Inc. Lot No. SF 2347B) was used as a 

reference substance. 

13.2 Preparation of test solutions 

The following test solutions were prepared and cultured under the conditions 

described in section 13.3. 

(1) Addition oftest substance or aniline 

(a) Test solution (water+ test substance) (n=l, Vessel No.6) 

In one test vessel, 30 mg of the test substance was accurately weighed and 

added to 300 mL of purified water, so that the concentration reached 100 mg/L. 

(b) Test solution (sludge+ test substance) (n=3, Vessel No.2, 3 and 4) 

In each test vessel, 30 mg of the test substance was accurately weighed and 

added to the basal culture medium (300 mL - a volume of the activated sludge 

inoculated), so that the concentration reached 100 mg/L. 

(c) Test solution (sludge+ aniline) (n=1, Vessel No.5) 

In one test vessel, 29.5 flL [30.0 mg = 29.5 flL X 1.022 g/cm3 (density)] of 

aniline was added into the basal culture medium (300 mL - a volume of the 

activated sludge inoculated), so that the concentration reached 1 00 mg!L. 

(d) Test solution (control blank) (n=l, Vessel No.1) 

In one test vessel, nothing was added to the basal culture medium (300 mL 

a volume of the activated sludge inoculated). 

-8-

r1 r1 
I 

\ 

( 

\ 

(2) Inoculation of activated sludge 

The activated sludge cultivated under the conditions described in section 12., 

was added to each test vessel (b), (c) and (d), so that the concentration of the 

suspended solid reached 30 mg!L. 

13.3 Instruments and conditions of cultivation 

(I) Instruments for cultivation 

Closed system oxygen consumption measuring apparatus 

(Coulometer: Ohk:ura Electric Co., Ltd.) 

(Data sampler: Asahi Techneion Co., Ltd.) 

Vessel 300 mL in volume 

Absorbent for carbon dioxide 

Soda lime No.1 (for absorption of carbon dioxide, 

Wako Pure Chemical Industries, Ltd.) 

Stirring method 

Each test solution was stirred by a magnetic stirrer. 

(2) Conditions of cultivation 

Cultivation temperature 

Cultivation duration 

.Room 

-9-

25+1 oc 

28 days 

Apparatus room No.5 II 

1 "Ti'f r 
13.4 Analysis of test solution 

After the termination of the cultivation, the test substance in the test solutions was 

determined. 

13 .4.1 Pretreatment of test solutions for analysis 

After the termination of the cultivation, the test solution (water+ test substance), 

the test solutions (sludge + test substance) and the test solution (control blank) were 

pretreated for high-performance liquid chromatography (HPLC) analysis of the test 

substance as follows : 

I 

l Test solution 300 mL 

~chloroform 40 mL (graduated cylinder) 

• Irradiation with ultrasonic waves 

(about 30 seconds) 

~chloroform 50 mL (graduated cylinder) 

~sodium chloride 90 g (even balance) 

• Shaking (about 10 minutes) 

I Water layer II Chloroform layer I ~-------.--------~ 

• Filtration and dehydration (IPS filter paper) 

• Filling up to 100 mL (chloroform, 

volumetric flask) 

• Taking out 10 mL (transfer pipet) 

• Dryness (rotary evaporator, about 40 °C, 

purge with nitrogen) 

~Tetrahydrofuran about 5mL (komagome pipet) 

• Irradiation with ultrasonic waves 

(about 15 seconds) 

• Filling up to 10 mL (tetrahydrofuran, 

volumetric flask) 

I Sample for HPLC analysis I 

- 10-
13.4.2 

( 

( 

Quantitative analysis 

Detennination of test substance 

The samples for HPLC analysis were analyzed under the following conditions. 

The concentration of the test substance in the sample for HPLC analysis was 

proportionally calculated by comparing the peak area on the chromatogram of the 

sample for HPLC analysis with that on the chromatogram of 300 mg!L of the standard 

solution (see Table-3 and Fig.4). 

The lowest detectable peak area of the test substance was regarded as 5000 J.l V ·sec 

considering the noise level, which corresponded to 3.2 mg/L of the test substance. 

(1) Analytical conditions 

Instrument 

Pump 

Detector 

Column 

Eluent 

Flow rate 

Measurement wave length 

Sample size 

Sensitivity 

Detector output 

(2) Preparation of standard solution 

High-perfonnance liquid chromatograph 

Hitachi, Ltd. type L-6000 

Hitachi, Ltd. type L-4000 

Shodex GPC KF-804L 

30 em X 8.0 mm I.D. stainless steel 

Tetrahydrofuran 

l.OmL/min 

250 nm (see Fig.5) 

20J!L 

0.4 V/AU 

The standard solution to detennine the concentration of the test substance in 

the sample for HPLC was prepared as follows. 

100 mg of the test substance was accurately weighed and dissolved in 

tetrahydrofuran to obtain 1000 mg!L solution. 300 mg/L standard solution was 

then prepared from this solution by dilution with tetrahydrofuran. 

(3) Calibration curve 

75.0, 150 and 300 mg/L of standard solutions were prepared by the same 

method as described in (2) .. These solutions were analyzed according to the 

analytical conditions described in (1). 

A calibration curve was drawn based on the relation between the peak area 

on the chromatograms and the respective concentrations (see Fig.2). 

- 11-
13.4.3 Recovery test and blank test 

Each two test solutions (water+ test substance) and two test solutions (sludge+ test 

substance) for recovery test were prepared according to the methods described in 13.2. 

The test solutions were pretreated in accordance with the method described in 13.4.1, 

then analyzed according to the procedures and analytical conditions described in 13.4.2. 

A test solution for blank test was prepared according to the method described in 

13.2. The test solution for blank test was analyzed in the same way as the recovery test. 

As for the blank test, no peak was appeared around the peak of the test substance on the 

chromatogram. Two individual recovery rates and their averages on the analytical 

procedure are shown below. The average recovery rates were used as correction factor, 

for the determination of the test substance in the analytical samples (see Table-2 and 

Fig.3). 

Recovery rate of the test solutions (water+ test substance) 

93.9 %, 97.2% average 95.6% 

Recovery rate of the test solutions (sludge+ test substance) 

98.1 %, 98.1 % average 98.1 % 

- 12-

I -:- II II 
( 

13.5 Calculation of percentage biodegradation 

The percentage biodegradation was calculated by the following equations and 

expressed in whole numbers. 

(1) Percentage biodegradation by BOD 

Percentage biodegradation (%) = 

BOD-B 

TOD X 100 

BOD 

B 

TOD*4 

Biochemical oxygen demand in the test solution 

(sludge+ test substance) (experimental) (mg) 

Biochemical oxygen demand in the control blank 

(experimental) (mg) 

Theoretical oxygen demlllld required when the test substance 

was completely oxidized (theoretical) (mg) 

*4 TOD was calculated with molecular formula CI96Hz4oNJo02 (n = 14). 

(2) Percentage biodegradation by HPLC 

Percentage biodegradation (%) = 

Sw- Ss 

Sw X 100 

Ss 

Sw 

Residual amount of the test substance in the test solution 

(sludge+ test substance) (experimental) (mg) 

Residual amount of the test substance in the test solution 

(water + test substance) (experimental) (mg) 

13.6 Treatment of numerical values 

Values were rounded off in accordance with JIS Z 8202-1985 reference 3 rule B. 

- 13-

,--.--... ~- .. ~- ~ ...... ..- ,.._. .. -- .......... =----=- ... ~·-- -
14. Validity of test conditions 

Percentage biodegradations of aniline calculated by the BOD values was 64 % and 72 % 

at the 7th and 14th day, respectively. It was concluded that this test conditions were valid (see 

Table-1 and Fig .I). 

15. Results 

15.1 Appearances of test solutions 

Appearances of test media in cultivation vessels were as follows. 

Test solution Appearance 

Water + test substance Test substance was not dissolved. 

At the initiation 

of cultivation 

Sludge + test substance Test substance was not dissolved. 

Water + test substance Test substance had not been dissolved. 

At the completion 

of cultivation Test substance had not been dissolved. Sludge+ test substance Growth of the sludge was not observed. 

- 14-
( 

\ 

15.2 Analytic results oftest solutions 

Analytic results of the test solution after 28 days were as follows. 

(Water+ 

test (Sludge + test substance) Theoretical 

substance Table Fig. amount 

Vessel-6 Vessel-2 Vessel-3 Vessel-4 

BOD*6 mg 0.7 0 0 0 89.7 1 

Residual amount and mg 30.6 29.8 30.7 31.2 30.0 

percentage residue ----- -------------- ------------- .......................... ------------- --------------- 3 of test substance 

(HPLC) % 102 99 102 104 -

*6 The value of control blank was subtracted from the values of the test solutions (sludge+ 

test substance). 

15.3 Percentage biodegradation 

Percentage biodegradations after 28 days were as follows. 

Percentage biodegradation (%) 

Method Table 

Vessel-2 Vessel-3 Vessel-4 Average 

BOD 0 0 0 0 1 

HPLC 2 0 0 1 3 

15.4 Conclusion 

The test substance was not biodegraded by microorganisms under the present test 

conditions. 

- 15-

1 

4 
16. Factors possibly affecting accuracy 

No adverse effects on the reliability of this test were noted. 

17. Storage of test substance, raw data, etc. 

17.1 Test substance 

About 5 g of the test substance is sealed in a store vessel and stored in a storage 

room in this laboratory for the period designated by article 32 of the GLP standards. 

17.2 Raw data and materials 

Raw data used for the preparation of the final report such as notebooks and results 

of measurements and observations, protocol, a record of the request of the test from the 

sponsor, a record of information about the test substance presented by the sponsor, etc. are 

stored with the final report in the same manner as the test substance. 

18. Remarks 

18.1 Instruments used for test 

18.2 

Fourier transform infrared spectrophotometer: 

Shimadzu Corporation type FTIR-8200PC 

Closed system oxygen consumption measuring apparatus : 

seepage 9 

High-performance liquid chromatograph : 

see page 11 

Electronic analytical balance : Sartorius type BP21 OS 

pH meter: Toa Electronics Ltd. type HM-50S 

Ultraviolet and visible spectrophotometer : 

Reagents used for analysis 

Tetrahydrofuran (HPLC grade): 

Chloroform (reagent grade): 

Sodium chloride (reagent grade): 

- 16-

Shimadzu Corporation type UV-2200A 

KANTO KAGAK.U 

Kishida Chemical Co., Ltd. 

MANAC Incorporated 

-..,.~~~.,..II ""~~"11---,.--! ir:-!Tr -
19. Contents of tables and figures 

Contents of tables 

Table-1 Calculation table for percentage biodegradation by BOD 

Table-2 Calculation table for recovery rate by HPLC 

Table-3 Calculation table for percentage biodegradation by HPLC 

Contents of figures 

Fig. 1 Chart of BOD 

Fig.2 Chromatograms ofHPLC analysis for calibration and calibration curve 

Fig. 3 Chromatograms ofHPLC analysis for recovery test 

Fig. 4 Chromatograms ofHPLC analysis for regular test 

Fig. 5 UVspectrum 

( Fig. 6-1 IR spectrum of test substance measured before cultivation 

\ 

Fig. 6-2 IR spectrum of test substance measured after cultivation 

Reference IR spectrum supplied by sponsor 

- 17-

--- - ·· .. -·-~~- ~- ··- ,- · ~ --'"'lr I 'if"T 
( 

\ "-

Table 1 

Calculation table for percentage biodegradation by BOD. 

Test No. 13013 Cultivating Duration: 28 days 

7th day 14th day 21st day 28th day Vessel Mean 

No. BOD Deg. BOD Deg. BOD Deg. BOD Deg. Deg. (mg) (%) (mg) (%) (mg) (%) (mg) (%) % 

~ 59.5 64 68.7 72 71.2 72 73.3 72 

rn 1.6 - 3.9 - 6.3 - 8.0 -

~ 0.8 0 2.8 0 4.7 0 6.1 0 0 

~ 0.9 0 2.8 0 4.8 0 5.9 0 

@ 2.4 1 4.7 1 6.9 1 8.0 0 

[§] 0.0 - 0.2 - 0.7 - 0.7 -

Deg. : Percentage biodegradation 

Vessel no. ~ : Sludge + Aniline 

Vessel no. rn : Control blank [B) 

Vessel no. ~ ~@ : Sludge + Test substance 

Vessel no. [§] : Water + Test substance 

- Test substance of 30 mg was added. 

Chart of BOD . 

. Fig. 1 

Deg. = [ BOD - B ] / [ TOD ] X 100 (%) 

TOD of test substance : 89.7 (mg) 

CJ...eaH24oN3o02 + 285 02- 196 C02 + 120 H20 + 30 N02 

285 02 / CJ...aaH24oN3o02 = 9119.66 / 3048.20 = 2.99 

TOO = 30.0 X 2.99 = 89.7 (mg) 

TOO of aniline : 90.3 (mg) 

CaH7N + 8. 75 02 - 6 C02 + 3.5 H20 + N02 

8. 75 02 / CaH7N = 279.99 / 93.13 = 3.01 

TOO = 30.0 X 3.01 = 90.3 (mg) 

19 9 7 . 0 3 . 12 ::..c..N a=m=e'---._(-~.-f ,--'f-1'--'---'ctr<..-· :...,sft-~~A_e-h_; ___ _ 
Table-2 Calculation table for recovery rate by HPLC 

Test No 13013 

( 

i 

Sample description A D E F 

Standard solution 300mg!L 516947 

Water+ test substance -1 485506 28.2 93.9 

Water + test substance -2 502551 29.2 97.2 95.6 

Sludge+ test substance -1 507254 29.4 98.1 

Sludge + test substance -2 507125 29.4 98.1 98.1 

Control blank n.d. 

Amount of test substance added : 30 (mg) 

A: Peak area (uV·sec) 

B: Final volume: 100 (mL) 

C: Ratio of portion used for analysis : 3001300 

D: Recovery amount (mg) 

D = G x (A I A(Standard)) x ( B I C) I 1000 

E: Recovery rate (%) 

E = D I 30 (mg) x 100 

F: Average recovery rate(%) - G: Concentration of standard solution : 300 (mg!L) 

See Fig. 3 

March 19, 1997 Name 
Table-3 Calculation table for percentage biodegradation by HPLC 

Test No 13013 

Sample description A E F G H 

Standard solution 300mg!L 476005 

[6] Water+ test substance 463693 30.6 102 

[2] Sludge + test substance 464259 29.8 99 2 

[3] Sludge + test substance 477205 30.7 102 0 1 

[4] Sludge+ test substance 485813 31.2 104 0 

[1] Control blank n.d. 

-

Amount of test substance added : 30 (mg) 

A : Peak area (p.V·sec) 

B : Final volume : 100 (mL) .. C: Ratio of portion used for analysis : 300/300 

D: Recovery rate: 95.6 (%)(Water+ test substance) 

98.1 (%) (Sludge +test substance) 

E: Residual amount of test substance (mg) 

E =I x (A I A(Standard)) x ( B I C) I ( D I 100) I 1000 

F: Percentage residue (%) 

F = E I 30 (mg) x 100 

G: Percentage biodegradation (%) 

G = {( E(Water +test substance)- E(Sludge +test substance)) 

I E(Water +test substance)} x 100 

H: Average percentage biodegradation(%) 

I : Concentration of standard solution : 300 (mg!L) 

See Fig. 4 .. 

March 19, 1997 

11 I I I 
( 

Fig.1 Chart of BOD 

Test No. 13013 ( Test substance 

Apparatus · · · · · · · · · · · · · · · · · · · · · · · · · · · 

Cultivating conditions: 

Concentration 

Test substance · · · · · · · · · · · ·· · · ·· · Reference substance( aniline ) · · Activated sludge · · · · · · · · · ··· · · · · 

V- ot-H 

No. .CM-22 

100 (mg/.Q) 

100 (mg/.Q) 

30 (rng/.Q) 

Temperature · · · · · · · · · · · · · · · · · · · · ·· · Duration · · · · · · · · · · · · · · · · · · · · · · · · · · 25 ± 1' c 28days(Feb.12~Mar.12,1997) 

Note: 

-t>O 80- e .._, 

'0 

c:: 

~ 

e 

<l) 

0 60- c:: 

<l) 

t>O 

>. 

>< 

0 

-

~ 

(,) 40-

·-e 

<l) 

.c:: 

(,) 

0 

·-

CQ 

20-

0 

Regular condition 

III-

!ZJ--

[]---

@1······ 

~-·­

[§]-··-

f 

I 

I 

i 

I ... _ .... ·····..:.:.:·.::,:· .. ····· · · ............. · __ 1 @][2)~ 

i -..:.:.:;:--::··::··::··~~· .. _-;.:. __ .::~., .. -.. _ .. _ .. _ .. _ .. ____ .. ____________ -[§] 

7 I I I I 1

1

4 I I I I I I 

21 28 

Cultivation Time ( 0~28thday) 

1997.03.12 Name /-1 · 1-fttv--~ke.hi 
Standard solution 300 mg/L Operating date : Mar. 13. 1997 

PKNO Time Area Height Cone. 

1 (min) (.uV ·sec) (.uV)' (%) 

I 7. 94 5212Q;;l 4090 100.00 

Total 521603 100.00 

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

Standard solution !50 mg/L Operating date : Mar. 13. 1997 

PKNO Time Area Height Cone. 

(min) (.uV·sec) (.uV) (%) 

1 7. 89 244955 1954 100.00 

Total 244955 100.00 

1 

~"--v 

0. 0 5. 0 10. 0 15.0 Operator : Hisako Haruguehi 

Standard solution 75. 0 mg/L Operating date : Mar. 13. 1997 

PKNO Time Area Height Cone. 

(min) (.uV·sec) (.uV)' (%) 

1 7. 89 132320 1057 100.00 

Total 132320 100. 00 

I 

~ 

0.0 5.0 10.0 15.0 Operator : Hisako Haruguehi 

Solvent blank Operating date : Mar. 13. 1997 

PKNO Time Area Height Cone. 

(min) (.uV·sec) (.uV) (%) 

Total 0 0. 00 

Peak position 

~ 

...,,q. 

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

Fig. 2 - I Chromatogram of HPLC analysis for calibration curve. 

Date : Mar. 13. 1997 Name 

]"':" :T ,II 
Test No. 13013 

Fig. 2- 2 Calibration curve and conditions ofHPLC analysis for V-OSH 

March 19, 1997 

( .f( 06 
I <:::H 1~0. I .:lU I ;;! 

Standard solution 300 mg/L Operating date : Mar. 13, 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·see) (t.tV) (%) 

1 7. 88 516947 4017 100. 00 

1 Total 516947 100. 00 

A.· 

-"--v- .. 

0.0 5. 0 10.0 15. o(min) Operator -: Hisako Haruguchi 

Water t test substance- 1 Operating date : Mar. 13, 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·see) (t.tV) (%) 

I 7. 95 485506 3850 100.00 

Total 

I 485506 100. 00 

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

Water t test substance- 2 Operating date : Mar. 13, 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·see) (t.tvr (%) 

I 7. 93 502551 4006 100.00 

I Total 502551 100. 00 

0. 0 5.0 

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

Fig. 3 - l Chromatogram of HPLC analysis for recovery test. 

Date : Mar. 13. 1997 Name H-f-.1~~; 

( 

"- f1 b7 

- <--<~F 0 • -•• • __ ,.._ ,..._- .. -_.,..--··· ..,...._.._--=-- ~--=·-~~ 0 
lest No. 13013 

Sludge + test substance - I Operating date : Mar. 13, 1997 

PKNO Time Area Height Cone. 

(min) (p.V·see) (p.V) (%) 

1 7. 91 507254 3990 100.00 

Total 507254 100.00 

1 

~ ( 

\ 

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

Sludge + test substance - 2 Operating date : Mar. 13, 1997 

PKNO Time Area Height Cone. 

(min) (p.V·see) (p.V) (%) 

I 7.95 507125 3983 100.00 

Total 507125 100.00 

1 

- A 

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

Control blank Operating date : Mar. 13. 1997 

PKNO Time Area Height Cone. 

(min) (p.V·see) (p.V). (%) 

Total 0 0. 00 

Peak position 

\/) \ c.l' 

~~ 

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

Fig.3-2 Chromatogram of HPLC analysis for recovery test. 

Date : Mar. I 3. I 997 Name 

( 
Standard solution 300 mg/L Operating date : Mar. 18, 1997 

PKNO Time Area Height Cone. 

(min) (tJ.V·see) (tJ.V) (%) 

7. 98 476005 3830 100.00 

Total 476005 100.00 

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

[ 6 ] Water + test substance Operating date : Mar. 18, 1997 

PKNO Time Area Height Cone. 

(min) (tJ.V•see) (tJ.V) (%) 

8. 06 463693 3771 100.00 

Total 463693 100.00 

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

[ 2 ] Sludge + test substance Operating date : Mar. 18. 1997 

PKNO Time Area Height Cone. 

(min) (tJ.V·see) (tJ.vr (%> 

7. 90 464259 3721 100.00 ( Total 464259 100.00 

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

Fig. 4 - l Chromatogram o.f HPLC analysis for regular test. 

Date : Mar. 19. 1997 Name 
( 

\ 

( 

0. 0 

o. 0 

o. 0 

[ 3 ] Slu~ge + test substance 

5. 0 10,0 

[ 4] Sludge + test substance 

5. 0 

[ I ] Control blank 

10.0 

Peak position 

~ 

V\' d . 

I 'tV. I "'"'VI oJ 

Operating date : Mar. 18, 1997 

PKNO Time Area Height Cone. 

(min) (t.t.V·sec) (t.t.V) {%) 

7. 97 477205 3756 100. 00 

Total 477205 100.00 

15.o(min) Operator : Hisako Haruguehi 

Operating date : Mar. 18, 1997 

PKNO Time 

(min) 

7. 94 

Total 

Area 

(t.t.V·sec) 

485813 

485813 

Height Cone. 

(t.t.V) (%) 

3798 100.00 

100.00 

15. o(min) Operator : Hisako Haruguehi 

Ope.rat ing date : Mar. 1 B. 1997 

PKNO Time Area Height Cone. 

(min) (t.t.V·sec) (t.t.Vl' (%) 

Total 0 0. 00 

5.0 10.0 15.o(min) Operator Hisako Haruguehi 

Fig. 4 - 2 Chromatogram of HPLC analysis for regular test. 

Date : Mar. 19, 1997 Name 
.; 

! 

e 

. ") 

" 

' 

• 1 

' 

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Fig.6-1 IR spectrum of test substance measured before 

the cultivation 

Sample: 

Method: 

Date 

Name 

13013 

Neat 

Jan. 14, 1997 

f-J ~ t-lv.-rvq-~ ; 
( 

I : 

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4000.0 3000.0 2000.0 1500.0 1000.0 

Fig.E3-~ IR spectrum of test substance measured after 

the cultivation 

Sample: 

Method: 

Date 

Name 

13013 

Neat 

May 27, 1997 

H . f-jo..KL~I,., ., 

--· ·-·r---~-

Jr 73 

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( 

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ls.o•co~· • ;aoo l&oo ••oo l200 looo 2soo 2500 2•oo 2200 2000 190o noo 11oo 1500 1500 1400 noo 1200 1100 1000 9oo aoo roo Goo 5co •oo 

Wavenumber[cm-1) 

V-OSH ~~: 4 cm-1 in.ri@ll:!£: 17 

7;l"t~'1ii-~:/: Cosine Bit: 96/11/06 10:36 

Reference IR spectrum supplied by the sponsor 
( 

Amendment and addition to Final Report 

1. Title 

2. Test number 

3. Content 

4. Reason 

5. Content of correction 

6.Date 

7. Approval 

. Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Biodegradation test ofV-05H by microorganisms 

13013 

CD 11.2 Chemical structure, etc .• , Molecular weight (page 4) 

® 11.2 Chemical structure, etc .• , Polymerization degree 

(page 4) 

CD The molecular weight was about 30000 when average of 

polymerization degree was n = 180. 

® The polymerization degree was ~leared. 

CD " Molecular weight 

" Molecular weight 

® " n = 2-800 " is added. 

June 5, 1998 

Date 

About 3000 "is corrected to 

About 30000 ". 

June 5, 1998 

Study director Signed in original 

Y oshihisa Sudo 

fl 7S 
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I i 1 ! r- I~--
Accepted number 

Test number 

S97-3112 

43112 

FINAL REPORT 

Bioaccumulation test ofV-05H in carp 

February 6, 1998 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

~~ 77 

r--
( 

\ 

( 

I 

Sponsor 

Title 

Test number 

STATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Bioaccumulation test ofV-05H in carp 

43112 

I, the undersigned, hereby declare that this report provides a correct English translation 

of the Final Report {Test No.43112, issued on February 6, 1998). 

However, sponsor's address, 11 1-18-1 Nishiarai Sakaecho, Adachi-ku, Tokyo, 123-0843, 

Japan 11 in the original, was changed to 11 1-2-3, Onodai, Midori-ku Chiba, 267-0056, Japan 11 

and CAS number was added to section 10.1 on the text by sponsor's request. 

Date "3tti'IJW1 ;&, ..2 oo.l 

Study director 

r1J1~-vc Y asuko Matsunobu 

r· 
.•i 

'I 

I 

Sponsor 

Title 

Test number 

GLP STATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Bioaccumulation test ofV-OSH in carp 

43112 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" 

(March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination 

Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry 

of Health and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of 

International Trade and Industry, Japan) and "OECD Principles of Good Laboratory 

Practice" (May 12, 1981). 

Date February 6, 1998 

Management Signed in original 

Hiroshi Katsuura 
( 

( 

Sponsor 

Title 

Test number 

GLPSTATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Bioaccumulation test ofV -OSH in carp 

43112 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances" 

(March 31, 1984, Revised November 18, 1988, Kanpogyo No.39, Planning and Coordination 

Bureau, Environment Agency, Yakuhatu No.229, Pharmaceutical Affairs Bureau, Ministry 

of Health and Welfare, and 59 Kikyoku No.85, Basic Industries Bureau, Ministry of 

International Trade and Industry, Japan) and "OECD Principles of Good Laboratory 

Practice" (May 12, 1981). 

This GLP statement was issued as supplement to the GLP statement issued on 

February 6, 1998 because of amendment and addition to Final Report. 

Date June 5, 1998 

Management Signed in original 

Hiroshi Tadokoro 
-

QUALITY ASSURANCE STATEMENT 

Sponsor 

Title 

Test number 

NISSHINBO INDUSTRIES, INC. 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Bioaccumulation test ofV-05H in carp 

43112 

Study audits and inspections of this test were carried out and the results were reported to 

the management and the study director by ·Quality Assurance Unit of Kurume Research 

Laboratories Chemical Biotesting Center Chemicals Inspection & Testing Institute, Japan as 

follows. 

Item of audit Date of audit Date of report Date of report 

or inspection or inspection to management to study director 

Protocol November 14, 1997 November 14, 1997 November 14, 1997 

November 18, 1997 November 20, 1997 November 20, 1997 

December 16, 1997 December 24, 1997 December 24, 1997 

Test Conditions December 19, 1997 December 24, 1997 December 24, 1997 

January 8, 1998 January 13, 1998 January 13, 1998 

January 9, 1998 January 13, 1998 January 13, 1998 

Raw Data and February 6, 1998 February 6, 1998 February6, 1998. Final Report 

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

details in the report are in compliance with the protocol and Standard Operating Procedures 

and that the final report reflects the raw data. 

Date February 6, 1998 

Quality Assurance Unit, Head Signed in original 

Susumu Uchitomi 

... ~!'""T I --·r--~--- I 1 ·; 
I 

I 

\ 

( 

QUALITY ASSURANCE STATEMENT 

Sponsor 

Title 

Test number 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

NISSHINBO INDUSTRIES, INC. 

Bioaccumulation test ofV-05H ester in carp 

43112 

Study audit of the corrected parts in the Final Report was carried out and it was 

confirmed that the correction has no problem. The result was reported to the management 

and study director as follows. 

Date of audit Date of report Date of report 

to management to study director 

June 5, 1998 June 5, 1998 June 5, 1998 

This statement was issued as supplement to the quality assurance statement issued on 

February 6, 1998. 

• 

Date June 5, 1998 

Quality Assurance Unit, Head Signed in original 

Susumu Uchitomi 
CONTENTS 

page 

SUMMARY 1 

1. Title 2 

2. Sponsor 2 

3. Testing facility 2 

4. Objective 2 

5. Test method 2 

6. GLP Standards to comply with 3 

7. Test period 3 

8. Personnel 3 

( 

" 9. Preparation of final report 3 

10. Test substance 4 

11. Substance supplied by the sponsor 5 

12. Performance of acute toxicity test 6 

13. Performance ofbioaccumulation test 9 

14. Results 17 

15. Factors possibly affecting accuracy 18 

16. Storage of test substance, raw data, etc. 18 

17. Remarks 19 

18. Contents of tables and figures 20 

( Tables and figures 

\ fr <61 
( 

( 

\ 

SUMMARY 

1. Title 

Bioaccumulation test ofV-05H in carp 

2. Test conditions 

2.1 Acute toxicity test 

(1) Test fish 

(2) Duration of exposure 

(3) Exposure method 

2.2 Bioaccumulation test 

Orange-red killifish (Ot:yzias latipes) 

48 hrs. 

Semi static system 

(Renewal of test water, at every 8 - 16 hrs.) 

(1) Test fish Carp (Cyprinus carpio) 

(2) Nominal concentrations of test substance 

High exposure level (Level 1) 0.5 mg/L 

Low exposure level (Level 2) 0.05 mg/L 

(3) DUration of exposure 8 weeks 

( 4) Exposure method Continuous flow system 

( 5) Analytical method High performance liquid chromatography 

3. Results 

(1) 48-hour LC50 value 

(2) Bioconcentration factors 

4. Stability of test substance 

>100mg/L 

Levell 

Level2 

< 12 

<129 

The test substance was confirmed to be stable under storage and test conditions. 

- 1 -
FINAL REPORT 

1. Title 

Bioaccumulation test ofV-OSH in carp 

2. Sponsor 

3. Testing facility 

4. Objective 

Name 

Address 

Name 

Address 

Phone 

Management 

Test No. 43112 

NISSHINBO INDUSTRIES, INC. 

1-2-3, Onodai, Midori-ku Chiba, 267-0056, 

Japan 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

19-14, Chuomachi Kurume 

Fukuoka830,Japan 

(0942) 34 - 1500 

Hiroshi Katsuura 

This test was performed to evaluate the bioaccumulation potential ofV -OSH in carp. 

5. Test method 

This test was conducted according to the "Method for Testing the Degree of 

Accumulation of Chemical Substances in Fish Body" stipulated in the "Testing Methods for 

New Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination 

Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry 

of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry of 

International Trade and Industry, Japan), and "Bioaccumulation : 305C, Degree of 

Bioconcentration in Fish" in the OECD Guidelines for Testing of Chemicals (May 12, 1981). 

-2-
( 

6. GLP Standards to comply with 

This test was conducted in compliance with Good Laboratory Practice Standards for 

industrial chemicals, "Basic standards to be observed by testing facilities in conducting tests 

stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New 

Chemical Substances and Study on Harmful Effects of Designated Chemical Substances 

(hereafter referred to as "GLP standards")" (March 31, 1984, Revised November 18, 1988, 

Kanpogyo No.39, Planning and Coordination Bureau, Environment Agency, Yakuhatu 

No.229, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 59 Kikyoku 

No.85, Basic Industries Bureau, Ministry of International Trade and Industry, Japan) and 

"OECD Principles of Good Laboratory Practice" (May 12, 1981 ). 

7. Testperiod 

(1) Start of test 

(2) Initiation of exposure 

(3) Termination of exposure 

( 4) End of test 

8. Personnel 

Study director 

Test operator 

Staff for fish care 

Person to conduct of fish acute toxicity test 

Person in charge of storage oftest substance and raw data 

9. Preparation of final report 

Date 

Signature 

- 3-

November 14, 1997 

November 27, 1997 

January 22, 1998 

February 6, 1998 

Y asuko Matsunobu 

YukaKida 

Y asuro Kawashima 

Y asuro Kawashima 

Kuniichi Kajiwara 

Tadashi Ohki 

February 6, 1998 

Signed in original 

Y asuko Matsunobu 

rrr 1 : 

:1 I 
( 

l 

12. Performance of acute toxicity test 

12.1 Test method 

The test was performed in accordance with Japanese Industrial Standard (TIS K 

0102-1993 section 71.), "Testing methods for industrial waste water, Acute toxicity test 

with fish". 

12.2 Test fish 

(1) Species Orange-red killifish (Oryzias latipes) 

(2) Supplier 

Nakashima fish farm 

(Address: Daimyojin Nagasu-cho Tamana-gun, Kumamoto 869-0100, Japan) 

(3) Conditions for fish care 

The fish were visually examined upon receiving and those demonstrating any 

abnormalities were removed. The fish were reared for 19 days in a flow through 

system following an external disinfection. 

( 4) Conditions for acclimatization 

After rearing, the fish were transferred to an acclimatizing aquarium and 

acclimatized there after the second external disinfection. The fish those 

demonstrating any abnormality were removed and reared for 34 days in a flow 

through·system at the temperature of25 ± 2 °C. The fish were checked for health 

conditions and reared another 53 days after the external disinfection. 

(5) Weight average 0.17 g 

(6) Length average 2.7cm 

(7) Certification 

Fish of the same lot No. TF0-970820 which passed the HgC12-test"2 were 

subjected to the test. 

*2 Kenji Tabata, Journal ofWater and Waste .1.1, 1297-1303 (1972) 

-6-
( 

12.3 Dilution water for test 

(1) Origin 

Ground water from the premises of Kurume Research Laboratories. 

(2) Water quality assessment 

The dilution water for test was taken out on August 1, 1997, and it was 

analyzed and measured (once every six months in this laboratory). The results are 

shown in Reference 1. 

It was confirmed that the dilution water met the ministerial ordinance of the 

Ministry of Health and Welfare (December 21, 1992), water quality criteria for 

fisheries (Shadanhozin Nihon Suis~igen Hogokyokai, March 1983), and OECD 

Guidelines for Testing of Chemicals 210, Fish, Early-life Stage Toxicity Test (July 

17, 1992). 

12.4 Test conditions 

(1) Test tank Round glass vessel 

(2) Volume of test water 4 L/ level 

(3) Temperature of test water 

( 4) Concentration of dissolved oxygen in test water 

At initial exposure 8.1 mg/L 

At termination of exposure 7.1 mg/L 

(5) pH of test water 

At initial exposure 7.7 

At termination of exposure 7. 7 

(6) Number offish 10 /level 

(7) Duration of exposure 48 hrs. 

(8) Exposure method Semi static system 

(Renewal of test water, at every 8- 16 hrs.) 

( f1 1ro 

-7-
( 

12.5 Preparation of stock solution 

( 1) Dispersant 

HC0-40 (Hydrogenated castor oil) 

(2) Preparation 

Test substance and HC0-40 (100 times amount of the test substance) were 

dissolved in tetrahydrofuran. Ion-exchan~ed water of the about same quantity as 

the tetrahydrofuran was added. After tetrahydrofuran was evaporated, the mixture 

was diluted with ion-exchanged water to prepare 1000 mg/L stock solution. 

12.6 Performance oftest 

(1) Place 214 LC50 room 

(2) Date November 17, 1997- November 19, 1997 

12.7 Estimation of 48-hour LC50 value 

The 48-hour LC50 value was estimated by the Doudoroff method. 

12.8 Result oftest 

48-hour LC50 value > 100 mg/L (see Fig.3) 

- 8-

r·~-~·. -~ ~-···· ·- ---···- -~~~ -~·~-
( 

\ 

13. Performance ofbioaccumulation test 

13.1 Test fish 

(1) Species Carp (Cyprinus carpio) 

(2) Supplier 

Sugishima fish farm 

(Address: 123-2 Gunchiku Ichibancho, Yatsushiro-shi, Kumamoto 866-0024, 

Japan) 

Date received September 17, 1997 

(3) Conditions for fish care 

The fish were checked visually at the delivery and removed those 

demonstrating any abnormalities. The fish were reared for 1 day in a flow 

through system following an external disinfection. 

(4) Conditions for acclimatization 

After rearing, the fish were medicated to eliminate parasites and transferred to 

an acclimatizing aquarium. After the second external disinfection, they were 

acclimatized. The fish those demonstrating any abnormalities during this period 

were removed and reared for 51 days in a flow through system at the temperature 

or'25 ± 2 °C. The fish were then transferred to test tanks and reared at the same 

temperature in the flow through system for another 16 days, following the external 

disinfection. 

(5) Weight, length and lipid content at initial exposure 

Weight average 23.6 g 

Length average 9.8 em 

Lipid content average 3. 7 % 

Measured values ofLotNo. TFC-970917 

Date of determination November 6, 1997 

(6) Feeding 

Feed Pelleted feed for carp 

Manufacturer Nippon Formula Feed Mfg. Co., Ltd. 

Feeding amount and interval 

Amount corresponding to about 2% of total body weight was fed twice 

a day in halves. 

On the day before fish sampling, no feed was given. 

13.2 Dilution water for test 

Same as 12.3. 

-9-

~-·~-.~~ 
( 

13.3 Conditions of test and circumstances 

(1) Supply of test water 

Flow through system assembled at this laboratory was used. 

(2) Test tank 

100-L glass tank 

(3) Flow rate of test water 

2 mL/min for stock solution and 400 mL/min for dilution water ; 579 

liters/day for test water were supplied. 

( 4) Stock solution bottle 25-L glass bottle 

(5) Temperature of test water 

(6) Concentrations of dissolved oxygen in test tank 

Levell 6.4-7.6 mg/L (see Fig.ll) 

Level2 7.3-7.9 mg/L (see Fig.12) 

Control 6.6-7.8 mg/L (see Fig.13) 

(7) Number of fish (at initial exposure) 

Level 1 and 2 11 

· Control 

(8) Duration of exposure 

(9) Place 

13.4 Preparation of stock solutions 

( 1) Dispersant 

Same as 12.5 (1). 

(2) Preparation 

·Level 1 

5 

8 weeks 

213 Aquatron room 

100 mg/L stock solution was prepared in the same way as 12.5 (2). 

• Level2 

10 mg/L stock solution was prepared in the same way as 12.5 (2). 

·Control 

250 g of HC0-40 was dissolved in ion-exchanged water and the solution 

was filled up to 25 L. 

f1 13 - 10-
( 

13.5 Test concentrations 

Based on preliminary test results for 48-hour LC50 and analytical detection limits, 

test concentrations of the test substance were decided on as follows. The control was 

set as a blank test. 

Levell 

Level2 

0.5 mg/L (as the test substance) 

0.05 mg/L (as the test substance) 

13.6 Observation, measurement, etc. 

(1) Observation oftest fish 

Condition of test fish was observed visually twice a day. 

(2) Flow rate of test water 

Flow rate of stock solution and dilution water were measured with graduated 

cylinder and recorded once a day. · 

(3) Temperature of test water 

Temperature of test water was measured with alcohol thermometer and 

recorded once a day. 

(4) Concentration of dissolved oxygen in test tank 

· Concentration of dissolved oxygen in test tank was measured with dissolved 

oxygen probe and recorded twice a week. 

(5) The others. 

In test period, excreta of carp, dirt on test tank, etc. were removed once about 

a day. 

- 11-
( 

/ 

\ 

13.7 Analysis of test water and fish 

13.7 .1 Frequency of analysis 

The concentrations of the test substance in the test water for both level 1 and 2 

were analyzed 16 times ; twice a week intervals (n=l). The concentrations in the 

test fish for both levels were analyzed 4 times ; in weeks 2, 4, 6 and 8 (n=2). The 

control fish were analyzed at the initiation and the termination of exposure (n=2). 

13.7 .2 Pretreatment for analysis 

(1) Test water 

Aliquot of test water was taken from each test tank, 

Levell 40mL 

Level 2 400 mL 

and pretreated for high-performance liquid chromatography (HPLC) analysis as 

follows: 

I I Water layer I 

I Test water I 

~water for recovery test 360 mL 

(graduated cylinder) (only Level 1) 

~sodium chloride 120 g (even balance) 

~Ethyl acetate 100 mL (graduated cylinder) 

• Shaking for 10 min 

I Ethyl acetate layer 

• Filtration and dehydration (IPS filter paper) 

• Evaporation to dryness 

(rotary evaporator, about 40 °C, nitrogen purge) 

• Filling up to 2 mL (tetrahydrofuran, 

volumetric flask) 

I Sample for HPLC analysis .I 

- 12-
( 

(2) Test fish 

I 

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

follows: 

I Test fish I 

• Measurement of weight and body length 

• Chopping into pieces 

~Tetrahydrofuran 100 mL (graduated cylinder) 

• Homogenization (polytron, ca. 1 min) 

• Washing (tetrahydrofuran 20 mL) 

• Centrifugation ( 4000 x g, 5 min) 

I Residue I I Supernatant I 

• Filtration (absorbent cotton) 

• Filling up to 150 mL (tetrahydrofuran, volumetric flask) 

• Taking out 5 mL (transter pipet) 

• Column chromatography*3 

I Supernatant I 

• Filling up to 5 mL (tetrahydrofuran, volumetric flask) 

I Sample for HPLC analysis I 

*3 Conditions of column chromatography 

Sep-Pak Silicagel 

(Conditioning: Tetrahydrofuran 10 mL) 

Loading Whole volume of the solution was loaded. 

Test substance was eluted with the loaded solution. 

- 13-
( 

13.7 .3 Quantitative analysis for test substance 

The samples for HPLC analysis in pretreatment were analyzed by HPLC under 

the following analytical conditions. The concentration of the test substance in the 

sample for HPLC analysis was determined based on a comparison of the peak height 

on the chromatogram of the sample solution with that of a standard solution (see 

Tables-4, 5, Fig.6 and Tables-7, 8, 9, Figs.8, 9, 10). 

(1) Analytical conditions 

Instrument 

Pump 

Detector 

Column 

Eluent 

Flow rate 

Measurement wave length 

Sample size 

Detector output 

(2) Preparation of standard solution 

High-performance liquid chromatograph 

Shimadzu Corporation type LC-6A 

Shimadzu Corporation type SPD-6A 

Asahipak GS-31 OH 

25 em x 7.6 mmi.D. stainless steel 

Tetrahydrofuran (containing 10 mmol/L 

sodium perchlorate monohydrate) 

l.OmL/min 

260 nm (see Fig.14) 

100 f.!L 

0.8 V/AU 

The standard solution to determine the concentration of the test substance in 

the sample solutions was prepared as follows. 

100 mg of the substance supplied by the sponsor was dissolved in 

tetrahydrofuran to obtain 1000 mg/L solution of the test solution. 10 mg/L 

standard solution was then prepared from this solution by dilution with 

tetrahydrofuran. 

(3) Calibration curve 

5.0, 10 and 20 mg/L standard solutions were prepared by the same method 

as described in (2). These solutions were analyzed according to· the analytical 

conditions described in (1). A calibration curve was drawn based on the relation 

between the peak height on the chromatograms and the respective concentrations. 

In consideration of the noise level, the lowest detectable peak height of the 

test substance was regarded as 300 J..lV, which corresponded to 0.89 mg/L of the 

test substance (see Fig.4). 

- 14-

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I 

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/ 

\. 

( 

13.7 .4 Recovery and blank test 

(1) Method 

A specified amount of the test substance was added to test water and fish 

homogenate for the recovery test, followed by pretreatment described in 13.7.2. 

The blank test was also conducted in the same manner without the test substance. 

All the recovery and blank tests were carried out in duplicate. 

(2) Results of recovery test 

In the blank test, the chromatogram of HPLC had no peak interfering with 

determination of the test substance concentration. The duplicate recovery rates 

and the average of them in the pretreatment are shown below (see Tables-3, 6 and 

Figs.5, 7). The average recovery rate was used as correction factors for the 

determination of the test substance concentrations in the analytical samples. 

For analysis of test water (20 p.g test substance added) 

86.4 %, 84.9% average 85.6% 

For analysis of test fish (1500 p.g test substance added) 

77.7 %, 75.1% average 76.4% 

- 15-
i 

\ 

13.7.5 Calculation of test substance concentration m sample and minimum limit of 

determination 

(1) Calculation of test substance concentration in test water 

The equation in Tables-4, 5 was used to obtain the concentrations, and they 

were rounded off to 3 figures. 

(2) Determination limit of test substance in test water 

This determination limit*4 was calculated based on that in 13.7.3 (3) as 

follows. 

Levell 

Level2 

0.052 mg/L 

0.0052 mg/L 

(3) Calculation of test substance concentration in test fish 

The equations in Tables-7, 8, 9 were used to obtain the concentrations. 

But the measured concentrations of test substance in .test fish were less than 

minimum determination limit oftest substance. 

(4) Determination limit oftest substance in test fish 

Based on the determination limit 13.7.3 (3), the determination limit*4 of the 

test substance in test fish was calculated as follows, assuming fish weight of30 g. 

5.8 p.g/g 

*4 Minimum determination limit of test substance (mg/L or p.g/g) 

= 

A 

B 

100 X 

where 

CxE 

D 

A: Minimum determination limit of test substance on the calibration curve 

(mg/L) 

B : Recovery rate (%) 

C: Sampling volume of test water (mL) or weight offish (g) 

D: Final volume of sample solution (mL) 

E: Ratio of the portion, used for analysis to whole volume 

Results were rounded off to 2 figures. 

- 16-
( 

13.8 Calculation ofbioconcentration factors (BCFs) 

The measured concentrations of test substance in test fish were less than 

minimum determination limit of test substance. 

From the minimum limit of detection of the test substance in 13.7.5 (4), BCFs 

could be obtained for cases of a BCF exceeding the followings : 

level 1 12 

level2 129 

13.9 Treatment of numerical values 

Values were rounded off in accordance with JIS Z 8202-1985 reference 3 rule B. 

14. Results 

14.1 Concentrations oftest substance in test water 

The measured concentrations of the test substance in test water are shown in 

Table-1. 

Each average concentration of the test substance in test water was maintained at 

more than 90 % of each the nominated concentration. 

Table-I Measured concentrations of test substance in test water (average value 

at times from start of exposure) 

(Unit: mg/L) 

2 weeks 4weeks 6weeks 8 weeks Table Fig. 

Levell 0.473 0.460 0.465 0.468 4 

6 

Level2 0.0486 0.0457 0.0451 0.0452 5 

- 17-
I 

\ 

i 

'\_ 

14.2 Bioconcentration factors 

BCFs are shown in Table-2. 

BCFs in Table-2 plotted against duration of exposure are shown in Fig.1 and 2. 

BCFs of the test substance were less than 12 atLevel1 and less than 129 at Level2. 

Table-2 BCFs 

2 weeks 4weeks 6weeks 8 weeks Table Fig. 

<12 <12 <t2 <t2 

Levell 7 8 

<t2 <t2 <t2 <t2 

<t29 <t29 <t29 <129 

Level2 8 9 

<t29 <129 <t29 <129 

14.3 Results of test fish observation 

No abnormality in behavior or appearance was noted. 

15. Factors possibly affecting accuracy 

No adverse effects on the reliability of this test were noted. 

16. Storage of test substance, raw data, etc. 

16.1 Test substance 

About 5 g of the test substance is sealed in a store vessel and stored, for the period 

designated by article 32 of the GLP standards, in a storage room in this laboratory. 

16.2 Raw data and materials 

Raw data used for the preparation of the final report such as notebooks and results 

of measurements and observations, protocol, a record of the request of the test from the 

sponsor, a record of information about the test substance presented by the sponsor, etc. 

are stored with the final report, for the period designated by article 32 of the GLP 

standards, in a storage room in this .laboratory. 

- 18-
I 

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17. Remarks 

17.1 Instruments, apparatuses, special apparatuses and reagents, etc. for the test 

(1) Instruments for fish care 

Micro quantitative pump for supplying stock solution : 

Tokyo Rika Kikai Co., Ltd. type GMW 

Instrument for measuring concentration of dissolved oxygen : 

IIJIMA ELECTRONICS CORPORATION 

type F-102 

(2) Instruments, apparatuses, special apparatuses and reagents 

Instruments and apparatuses 

High performance liquid chromatograph : 

Electronic analytical balance : 

see page 14 

Sartorius type 1702MP8 

Sartorius type 1404MP8 

Metler Toledo type PB602 

Infrared spectrophotometer : Shimadzu Corporation type FTIR-8200PC 

Ultraviolet and visible spectrophotometer : 

Rotary evaporator : 

Mechanical shaker : 

Homogenizer (polytron): 

Centrifuge : 

Special apparatuses 

Sep-Pak Silicagel: 

Reagents 

Shimadzu Corporation type UV-2200A 

Tokyo Rika Kikai Co., Ltd. type N-1 

Iuchiseieido Co., Ltd. type MS-1 

Kinematica 

Kubota Syouji Co., Ltd type 6900 

Nihon Millipore Ltd. 

Tetrahydrofuran (HPLC) : Kanto Chemical Co., Inc. 

Ethyl acetate (reagent grade): Kanto Chemical Co., Inc. 

Sodium perchlorate monohydrate (reagent grade) : 

Wako Pure Chemical Industries, Ltd. 

Sodium chloride (reagent grade): MANAC Incorporated 

HC0-40 : Nikko Chemicals Co., Ltd. 

- 19-

·1--r I IT 
( 

\ 

18. Contents oftables and figures 

Contents oftables 

Table-I 

Table-2 

Table-3 

Table-4 

Table-5 

Table-6 

Table-7 

Table-8 

Table-9 

Reference 1 

Measured concentrations oftest substance in test water (see text) 

BCFs (see text) 

Calculation table for recovery and blank test (analysis oftest water) 

Calculation table for analysis oftest water (Levell) 

Calculation table for analysis oftest water (Level2) 

Calculation table for recovery and blank test (analysis of test fish) 

Calculation table for analysis of test fish (Level 1) 

Calculation table for analysis of test fish (Level2) 

Calculation table for analysis of test fish (Control) 

Analytical results of dilution water 

/~ (OJ 

-20-
( 

( 

' 

Contents of figures 

Fig. 1 Correlation between exposure period and bioconcentration factor 

(Levell) 

Fig. 2 Correlation between exposure period and bioconcentration factor 

(Level2) 

Fig. 3 Concentration-mortality curve 

Fig. 4 Analytical chart and calibration curve ofHPLC analysis 

(analysis of test water) 

Fig. 5 

Fig. 6 

Fig. 7 

Fig. 8 

Fig. 9 

Fig.lO . 

Fig.ll 

Fig.l2 

Fig.l3 

Fig.14 

Fig.l5-l 

Fig. I 5-2 

Reference 2 

Recovery and blank test (analysis of test water) 

Analysis of test water 

Recovery and blank test (analysis of test fish) 

Analysis oftest fish (Level 1) 

Analysis oftest fish (Level2) 

Analysis of test fish (Control) 

Concentration of dissolved oxygen (Level 1) 

Concentration of dissolved oxygen (Level 2) 

Concentration of dissolved oxygen (Control) 

UV spectrum of test substance 

IR spectrum of test substance measured before the initiation of 

exposure to fish 

IR spectrum of test substance measured after the termination of 

exposure to fish 

IR spectrum supplied by the sponsor 

-21 -
Table-3 Calculation table for recovery and blank test 

(Analysis oftest water) 

Sample description A B c 

Standard 10mg!L 3457 

Recovery a 2986 1 2 

Recovery b 2935 1 2 

Standard l Omg!L 3477 

Blank a n.d. l 2 

Blank b n.d. 1 2 

( a. b : indh-idual sample ) 

A : Peak height (fl V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 

C : Final volume (mL) 

D :Amount of blank in test water (J.tg) 

E : Amount of test substance recovered (J!g) 

E= P x ( A(t) I A(std) ) I B x C - D ... 

F: Recovery rate(%) 

F=E/Q><lOO 

P : Concentration of test substance in standard solution (mg/L) 

Q: Amount of test substance added (20!lg) 

See Fig. 5 

D 

-

-

-

-

January 26. I 998 Name 

Test No 43112 

E F 

17.3 86.-1-% 

17.0 "84.9% 

Average 

85.6% 

-

-

f7 I (15 

--r~ 
: 

\ 

Table-4-1 Calculation table for analysis of test water 

(Levell) 

Sample description A I 

Standard lOmg!L 3227 

Test water at 2nd day 2450 0.443 

Standard 10mg!L 3156 

Test \Vater at 7th day 2834 0.524 

Standard lOmg!L 3180 

Test water at 8th day 2568 0.471 

Standard 10mg!L 3033 

Test water at 13th day 2358 0.454 

Standard 1 Omg!L 3160 

Test water at 16th day 2346 0.433 

Standard lOmg!L 3098 

Test water at 20th day 2264 0.427 

Standard 1Om giL 3559 

Test water at 24th day 2815 0.462 

Standard 1 Omg!L 3577 

Test water at 26th day 2837 0.463 

A : Peak height (J.l V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis l 

C : Final volume 2mL 

F : Recovery rate 85.6% 

H: Volume of test ·water taken out 40mL 

I: Concentration of test substance in test water (mg!L) 

I = P x ( A(t) I A(std) ) I B x C I F x 100 I H 

J: Average concentration of test substance in test water (mg!L) 

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

n : Number of test water analysis 

I (l) : First analysis oflest water I (n) : Last analysis of test water 

P : Concentration of standard solution lOmg!L 

See Fig. 6 

January 21. 1998 

Test No 43ll2 

J 

0.484 

0.473 

0.459 

-

0.460 

Name ~ ·?pkcv 

~1 . ( 0 b 
: 

\ 

( 

\"'-- -

Table-4-2 Calculation table for analysis of test water 

(Levell) 

Sample description A I 

Standard I Omg!L 3669 

Test water at 29th day 2745 0.437 

Standard 1Om giL 3566 

Test ·water at 33rd day 2734 0.448 

Standard IOmg/L 2897 

Test \Vater at 39th day 2700 0.544 

Standard l Omg/L 2840 

Test water at 41st day 2323 0.478 

Standard l Om giL 3178 

Test water at 44th day 2587 0.475 

Standard IOmg!L 3229 

Test water at 48th day 2492 0.451 

Standard IOmg!L 3300 

Test water at 51st day 3014 0.533 

Standard l Om giL 3220 

Test water at 55th day 2495 0.452 

A : Peak height (J.L V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 1 

C : Final volume 2mL 

F : Recovery rate 85.6% 

H: Volume of test water taken out 40mL 

I: Concentration oftesi substance in test water (mg/L) 

I = P x ( A(t) I A(std)) I B x C IF x IOO/ H 

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

J = ( I( I) + ... + l(n) ) I n 

n : Number of test water analysis 

I (l): First analysis of test water I (n) :Last analysis of test water 

P : Concentration of standard solution lOrn giL 

See Fig. 6 

January 27, 1998 

Test No 43II2 

J 

0.456 

0.465 

0.465 

. 0.468 

Name J6 ~/. 

f1 
( 

\ 

( 

\ 

Table-5-1 Calculation table for analysis oftest water 

(Level2) 

Sample description A I 

Standard 1 Omg!L 3227 

Test water at 2nd day 2635 0.0477 

Standard lOmg!L 3156 

Test water at 7th day 2369 0.0438 

Standard lOmg!L 3180 

Test water at 8th day 2685 0.0493 

Standard 10mg!L 3033 

Test ·water at 13th day 2778 0.0535 

Standard lOmg!L 3160 

Test water at 16th day 2674 0.0494 

Standard 1 Omg!L 3098 

Test water at 20th day 2189 0.0413 

Standard 1Om giL 3559 

Test water at 24th day 245-l 0.0403 

Standard lOmg!L 3577 

Test water at 26th day 2464 0.0402 

A : Peak height (JL V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 1 

C : Final volume 2rnL 

F : Recovery rate 85.6% 

H: Volume of test water taken out 400mL 

I : Concentration oftest substance in test water (mg/L) 

I = P x ( A(t) I A(std) ) I B x C IF x 100 I H 

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

J = ( I( l) + ... + I( n) ) I n 

n : Number of test water analysis 

I (I) : First analysis of test v.:ater I (n) : Last analysis of test \Vater 

P : Concentration of standard solution lOmg/L 

See Fig. 6 

January 2 L 1998 

Test No 43112 

J 

0.0458 

0.0486 

0.0475 

-

0.0457 

,, . ('j _/, 12. - Name X/. ~k_ 

(rfi 
( 

\. 

( 

( 

Table-5-2 Calculation table for analysis oftest water 

(Level2) 

Sample description A I 

Standard lOmg/L 3669 

Test water at 29th day 2481 0.0395 

Standard 1Om giL 3566 

Test water at 33rd day 2650 0.0434 

Standard 1 Omg/L 2897 

Test water at 39th day 2269 0.0457 

Standard lOmg/L 2840 

Test water at 41st day 2276 0.0468 

Standard 10mg/L 3178 

Test water at 44th day 2112 0.0388 

Standard lOmg/L 3229 

Test \Vater at 48th day 2447 0.0442 

Standard 1Om giL 3300 

Test water at 51st day 2711 0.0480 

Standard 1Om giL 3220 

Test water at 55th day 2876 0.0521 

A : Peak height (/l V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis l 

C : Final volume 2mL 

F : Recovery rate 85.6% 

H: Volume oftest water taken out 400mL 

I :Concentration of test substance in test water (mg/L) 

I = p X ( A(t) I A(std)) I B X c IF X 100 I H 

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

J = ( I( l) + ... + l(n) ) I n 

n: Number of test water analysis 

I ( l) : First analysis of test water I (n) : Last analysis of test water 

P : Concentration of standard solution lOrn giL 

See Fig. 6 

January 2 7, 1998 

Test No 43112 

J 

0.0448 

0.0451 

0.0446 

-

0.0452 

Name JG ~?1/ 

Pt (1J9 
Table-6 Calculation table for recovery and blank test 

(Analysis oftest fish) 

Sample description A B c 

Standard 1Om giL 3310 

Recovery a 2571 5/150 5 

Recoveryb 2485 5/150 5 

Standard 10mg!L 3404 

Blank a n.d. 5/150 5 

Blank b n.d. 5/150 5 

( a, b : indhidual sample ) 

A : Peak height (!1 V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 

C : Final volume (mL) 

D : Amount of blank in test fish (!lg) 

E : Amount of test substance recovered (!lg) 

E= P x ( A(t) I A(std) ) I B x C • D 

F: Recovery rate(%) 

F=E/Qx 100 

P : Concentration of test substance in standard solution (mg/L) 

Q: Amount of test substance added (1500J.tg) 

See Fig. 7 

D 

-

-

. 

. 

January 26, 1998 Name 

Test No 43112 

E F 

1170 77.7% 

1130 75.1% 

Average 

76.4% 

-

-

-

f1 //D 
i 

\ 

Table-7 Calculation table for analysis of test fish 

(Levell) 

Sample description A D G 

Standard 10mg!L 3169 

Test fish after 2 weeks a n.d. 1 25.3 

Test fish after 2 weeks b n.d. 1 25.8 

Standard 1Om giL 3619 

Test fish after 4 weeks a n.d. 1 35.6 

Test fish after 4 weeks b n.d. 1 27.8 

Standard 1Om giL 3235 

Test fish after 6 weeks a n.d. 1 31.4 

Test fish after 6 weeks b n.d. 1 32.5 

Standard 1Om giL 3055 

Test fish after 8 weeks a n.d. 1 37.9 

Test fish after 8 weeks b n.d. 1 33.5 

(a, b : individual sample ) 

A : Peak height (p.V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 51150 

C : Finai volume 5mL 

D : Dilution factor 

E :Average concentration of blank in analysis of control OJlgfg 

F : Recovery rate 76.4% 

G: Weight of test fish (g) .. 

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

K = { P x ( A(t) I A(std)) I B x D x C I G- E } IF x 100 

H : Average concentration of test substance in test water (mg!L) 

J: BCF 

J = KIH 

P : Concentration of test substance in standard solution 10m giL 

See Fig. 8 

Test No. 43112 

K H J 

- 0.473 - - 0.473 -

-· 0.460 - - 0.460 -

- 0.465 - - 0.465 -

- 0.468 -

- - 0.468 -

January 23, 1998 Name JC; . 1j«Ail / 

f1 (/ ( 
( 

/ 

I 

Table-8 Calculation table for analysis of test fish 

(Level2) 

Sample description A D G 

Standard 10mg!L 3182 

Test fish after 2 weeks a n.d. 1 27.5 

Test fish after 2 weeks b n.d. 1 23.2 

Standard 10mg!L 3600 

Test fish after 4 weeks a n.d. 1 31.2 

Test fish after 4 weeks b n.d. J, 27.9 

Standard 10mg!L 3220 

Test fish after 6 weeks a n.d. 1 32.9 

Test fish after 6 weeks b n.d. 1 34.1 

Standard lOmg!L 3042 

Test fish after 8 weeks a n.d. 1 31.9 

Test fish after 8 weeks b n.d. 1 33.6 

(a, b: individual sample) 

A : Peak height (p.V) 

A(std) : Standard solution A(t) : Sample 

8 : Ratio of portion used for analysis 51150 

C : Final volume SmL 

D :Dilution factor 

E : Average concentration of blank in analysis of control Opg/g 

F : Recovery rate 76.4% 

G :Weight of test fish (g) 

K : Concentration of test substance in test fish (ug/g) 

K = { P x ( A(t) I A(std)) I 8 x D x C I G- E } IF x 100 

H :Average concentration of test substance in test water (mg!L) 

J: 8CF 

J = Kl H 

P : Concentration of test substance in standard solution lOmg!L 

See Fig. 9 

K 

-

-

-

-

-

-

-

-

January 27, 1998 Name 

TestNo 4311? - H J 

0.0486 -

0.0486 -

0.0457 - 0.0457 -

0.0451 - 0.0451 -

0.0452 -- 0.0452 -

-
( 

Ffable-9 Calculation table for analysis of test fish 

(Control) 

Sample description A 

Standard lOmg!L 3216 

Before the initiation of exposure a n.d. 

Before the initiation of exposure b n.d. 

' 

Standard lOmg!L 3043 

After the termination of exposure a n.d. 

After the termination of exposure b n.d. 

( a, b : individual sample ) 

A : Peak height (Jl.V) 

A(std) : Standard solution A(t) : Sample 

B : Ratio of portion used for analysis 51150 

C : Final volume SmL 

E : Amount of blank in analysis of control (Jl.g) 

E= P x ( A(t) I A(std) ) I B x C 

G : Weight of test fish (g) 

I : Concentration of blank in test fish (uglg) 

l = EIG 

E 

-

-

-

-

P : Concentration of test substance in standard solution 10m giL 

See Fig. 10 

January 23, 1998 Name 

Test No 4311? - G I 

20.7 -

20.4 -

36.6 -

32.1 -

F1 /13 
/ 

\ 

( 

I 

\ 

Reference 1 Analytical results of dilution water 

Sampling date August 1. 1997 

Item Unit Measured value Standard value Detection limit 

Total hardness (Ca. Mg) mg/L 121 < 300* 1 

Suspended solid mg/L < 1. 0 < 20* 3 1 

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

Total organic carbon mg/L 2. 0 < 2q 0. 1 

Chemical oxygen demand mg/L < 0. 5 < 5* 2 0.5 

Residual chlorine mg/L < 0. 01 < 0. 02* 2 0. 01 

Ammonia nitrogen mg/L 0. 01 < 1*2 0. 01 

Total cyan mg/L < 0. 01 n. d. • 2 0. 01 

Alkalinity mg/L 90.5 -

Electric conductivity 11 s/ ern 360 -

Organic phosphorus mg/L < 0. 1 n. d.· 2 0.-1 

Alkylmercury mg/L < 0. 0005 n. d. • 2 0.0005 

Mercury mg/L < 0.0005 < 0. 0005. 2 0.0005 

Cadmium mg/L < 0. 005 < 0. 01. 2 0.005 

Cr 6

• mg/L < 0. 02 < 0. 05 * 2 0.02 

Lead mg/L < 0.005 < o. 1 * 2 0.005 

Arsenic mg/L 0. 002 < o. 05. 2 0.002 

[ron mg/L 0.06 < t. o· 2 0. 01 

Copper mg/L < 0.005 < 0. 005. 2 0.005 

*1 Ministerial ordinance of the Ministry of Public Welfare No. 69 (Revised December 

21. 1992) 

*2 Water quality criteria for fisheries (Shadanhozin Nihon Suisansigen Hogokyokai, 

March 1983) 

*3 OECD Guideline for Testing of Chemicals 210, Fish, Early-life Stage Toxicity 

Test 

fr 111 

·---r---· 
\ 

/ 

\ 

... 

0 

0 

<5 

Test No. 43112 

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

0 2 4 6 8 

Exposure period (weeks) 

Eight data at 2, 4, 6 and 8 weeks were lower than detection limit. 

Correlation between exposure period and bioconcentration factor (Level 1) 

The minimum calculation limit ofbioconcentration factor. 

2 4 6 8 

Exposure period (weeks) 

Eight data at 2, 4, 6 and 8 weeks were lower than detection limit. 

Fig. 2 Correlation between exposure period and bioconcentration factor (Level 2) 

February 3, 1998 ·Name 

((1 f15' 
I 

I 

\ 

I 

\ 

100 

/"""'\ 

~ '--' 

>. 

......, 

__, 

ro 

......, 50 r.... 

0 

~ 

F i g. 3 

T e s t No. 4 3 1 1 2 

48hourLC50= >100 •~ 

10 100 

C o n c e n t r a t o n (mg/L) 

Cumulative Mortality 

Concentrati or. Cumulative Mortality (%) 

C•IL> r-------~--------~--------~ 2 hours 24 hours 48 hours 

Control 0 0 

1 0 0 0 0 

Concentration-Mortality Curve 

1000 

Date : Nov.21,1997 Name:J(__J(_~ 

p1 //k 
Solvent Operating date : Oct. 2~ 1997 

PKNO Time Area Height Cone. 

(min) (ttV·sec) (t.LV} (%) 

Total 0 0. 00 

( 

\ ?eak position 

lad-

" 

0. 0 2. 5 5.0 7. 5 10.0 Operator : Yuka Kida 

Standard solution 5. 0 mg/L Operating date : Oct. 22, 1997 

PKNO Time Area Height Cone. 

(min) (J.LV·sec) (J.LV) (%} 

I 3. 65 73576 1683 10<;>. 00 

Total 73576 100.00 

1 

~ I \?: ~( : 

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

Standard solution 10 mg/L Operating date : Oct.22.1997 

PKNO Time Area Height Cone. 

(min) (ttV·sec) (J.LV) (%) 

1 3. 65 145993 3266 100. 00 

Total 145993 100.00 

1 

~ \ ( 

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

Standard solution 20 mg/L Operating date : Oct. 22. 1997 

1 PKNO Time Area Height Cone. 

(min) (J.LV·sec) (J.LV) (%) 

1 3. 65 295293 6756 100. 00 

Total 295293 100. 00 

\I 

0. 0 2. 5 5. 0 7. 5 10. 0 Operator : Yuka Kida 

Fig. 4 - I Analytical chart of HPLC analysis for calibration curve. 

Date : Oct. 22. 1997 Name : 

(h //7 

r 
( 

\ 

( 

/ 

8000 

4000 

0 

Conditions of HPLC 

Instrument 

Sample 

Solvent 

Injection vol. 

Column 

Size 

Eluent 

Flow rate 

Detector 

Wave length 

Output 

Rec. range 

y = 336 X 

r = 1.000 

10 

Concentration (mg!L) 

Shimadzu LC-6A, SPD-6A 

V-05H 

Tetrahydrofuran 

lOO,uL 

Asahipak GS-310H 

25cm x 7.6mm LD. 

• 

20 

lOmmoi/L NaCI04 • H20 in tetrahydrofuran 

l.OmL!min. 

uv 

260nm 

O.SV/AU 

SmV 

Test No. 43112 

Fig. 4- 2 Calibration curve and conditions of HPLC analysis for Y-05H 

' 

January 28, 1998 Name }0 ·1f~~-

t4 1~ 
( 

\ 

Standard solution 10 mg/L Operating date : Oct. 29, 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·sec) (ttY) (%) 

3. 65 144809 3457 100.00 

Total 144809 100.00 

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

Recovery test from test water a Operating date : Oct. 29. 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·sec) (ttY) (%) 

1 3. 64 132185 2986 11. 94 

2 6. 02 975148 17515 88.06 

Total 1107334 100.00 

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

Recovery test from test water b Op,erating date : Oct. 29. 1997 

PKNO Time Area Height Cone. 

(min) (t.tV·sec) (t.tV) (%) 

1 3. 64 132315 2935 13. 53 

2 6. 01 845548 19035 86. 47 

Total 977863 100. 00 

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

Fig. 5 - I Recovery and blank test (analysis of test water) 

Date : Oct. 29, 1997 Name 

Or 111 
( 

\ 

Standard solution 10 mg/L Operating date : Oc~ 2~ 1997 

PKNO Time Area Height Cone. 

(min) (JJ.V·sec) (JJ.V) (%) 

1 3. 64 144662 3477 100. 00 

Total 144662 100. 00 

I 

~ ~ ( 

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

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

v PKNO Time Area Height Cone. 

(min) (JJ.V·sec) (JJ.V) (%) 

Total 0 0. 00 

'v 

PMi position 

1 n.~ 

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

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

v PKNO Time Area Height Cone. 

(min) (JJ.V·sec) (JJ.V) (%) 

Total 0 0. 00 

v Peak position 

1M 

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

Fig. 5-2 Recovery and blank test (analysis of test water) 

Date Oct. 29, 1997 Name 

-T""lF"'__,...-lT,_I-----·---r-- --
Standard solution 10 mg/L Operating date : Nov. 28, 1997 

PKNO Time Area Height Cone. 

(min) (/l.V·see) (/l.V) (%) 

1 3. 65 141570 3227 100.00 

Total 141570 100. 00 

1 

~ ,,--

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

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

I 

PKNO Time Area Height Cone. 

(min) (/l.V·see) (/l.V) (%) 

v 1 3. 65 117831 2450 10.40 

2 6. 02 1015634 20080 89. 60 

Total 1133466 100.00 

~ ~ 

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

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

v PKNO Time Area Height Cone. 

(min) (/l.V·see) (/l.V) (%) 

1 3. 64 114007 2635 8. 58 

2 6. 03 1214822 21182 91. 42 

Total 1328828 100.00 h) ~ 

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

Fig. 6 - I Analysis of test water 

Date Nov. 28, 1997 Name 

( 

r?7 I~ r 
Standard solution 10 mg/L Operating date : Dec. 3, 1997 

PKNO Time Area Height Cone. 

(min) (tLV ·sec) (tLV) (%) 

3. 64 143004 3156 100.00 

Total 143004 100. 00 

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

Test water at 7th day (Level I ) Operating date : Dec. 3, 1997 

PKNO Time Area Height Cone. 

(min) ( tLV· sec) (tLV) (%) 

1 3. 64 128030 2834 10. 26 

2 6. 02 1120233 19358 89. 74 

Total 1248263 100. 00 

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

Test water at 7th day (Level 2) Op~rating date : Dec. 3, 1997 

PKNO Time Area Height Cone. 

(min) (tLV·sec) (tLV) (%) 

1 3. 64 108234 2369 7. 92 \ 

' 2 6.02 1258936 20156 92.08 

Total 1367169 100. 00 

0. 0 2. 5 5.0 7. 5 10. o!min) Operator Yuka Kida 

Fig. 6 - 2 Analysis of test water 

Date Dec. 3. 1997 Name X; 7:fvh&1r 

( 

; ·--,-T~ 
Standard solution 10 mg/L Operating date : Dec. 4, 1997 

PKNO Time Area Height Cone. 

(min) (ttV·sec) (ttV) (%) 

I 3. 64 146744 3180 100.00 

Total 146744 100. 00 

I 

-- ~ - f0. 0 2.5 5. 0 7. 5 10. o(min) Operator : Yuka Kida 

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

v PKNO Time Area Height Cone. 

(min) (tJ.V·sec) (tJ.V) (%) 

I 3. 63 120174 2568 10. 86 

2 6. 01 986385 18524 89. 14 

Total 1106559 100. 00 

h) 

11 

~ 

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

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

v PKNO Time Area Height Cone. 

(min) ( tJ.V ·sec) (tLvr (%) 

I 3. 63 115034 2685 10. 21 

2 6. 01 1011497 18085 89. 79 

Total 1126531 100.00 

~ L3 

I 

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

Fig. 6-3 Analysis of lest water 

Date Dec. 4. 1997 Name 

/)-) 
( 

\ 

( 

0. 0 

0. 0 

0.0 

Fig. 

Standard solution 10 mg/L 

2. 5 5. 0 

Test water at 13th day (Level l ) 

2. 5 5. 0 

Test water at 13th day (Level 2) 

2. 5 5.0 

6 - 4 Analysis of test water 

PKNO 

7. 5 10. o(min) 

PKNO 

1 

2 

7. 5 10. o(min) 

PKNO 

1 

2 

7. 5 10. o(min) 

Date Jan. 27. 1998 

Operating date : Dec. 9. 1997 

Time Area Height Cone. 

(min) (p.V·sec) (p.V) (%) 

3. 64 144074 3033 100. 00 

Total 144074 100. 00 

Operator : Yuka Kida 

Operating date : Dec. 9. 1997 

Time Area Height Cone. 

(min) (p.V•sec) (p.V) (%) 

3. 64 127481 2358 9. 47 

6. 02 1218105 19960 90. 53 

Total 1345586 100.00 

Operator : Yuka Kida 

Op~rating date : Dec. 9. 1997 

Time Area Height Cone. 

(min) (p.V·sec) (p.V} (%) 

3. 63 127429 2778 12. 60 

6.02 883918 16906 87. 40 

Total 1011347 100. 00 

Operator Yuka Kida 

Name J0 1fclttv 
i 

i" 

Standard solution 10 mg/L 

PKNO 

I 

1 

0. 0 2. 5 

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

Test water al 16th day CLeve I I ) 

\ PKNO 

\1'1 I 

2 

w ~ 

' 10. o(min) 0. 0 2. 5 5.0 7. 5 

Tes l water at 16th day CLeve I 2 ) 

v \ PKNO 

1 

2 

~~ ~ 

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

Fig. 6-5 Analysis of lest water 

Date Dec. 12, 1997 

r··~~-·· ~~~··· ·~ ---·~ ~---• ··~ ~~-

Operating date : Dec. 12. 1997 

Time Area Height Cone. 

(min) (tLV·se~) (tLV) (%) 

3. 63 143749 3160 100. 00 

Total 143749 100. 00 

Operator : Yuka Kida 

Operating date : Dec. 12. 1997 

Time Area Height Cone. 

(min) (tLV·sec) (tLV) (%) 

3. 63 104912 2346 10. 55 

6.00 889327 16125 89.45 

Total 994238 100. 00 

Operator : Yuka Kida 

Operating date : Dec. 12, 1997 

Time Area Height Cone. 

(min) (tLV·sec) (tLV) (%) 

3. 51 108453 2674 11. 71 

6. 00 817464 14469 88. 29 

Total 925918 100. 00 

Operator : Yuka Kida 

Name 

;~1. /)5 

T 
( 

i 

\ 

\ 

0.0 

0. 0 

0.0 

Fig. 

Standard solution tO mg/L 

2. 5 5. 0 

Test water at 20th day (Level l ) 

2. 5 5. 0 

Test water at 20th day (Level 2 ) 

2. 5 5.0 

6 - 6 Analysis of test water 

PKNO 

7. 5 10. o{min) 

PKNO 

I 

2 

7. 5 10. o(min) 

PKNO 

1 

2 

7. 5 10. o(min) 

Date Dec. I 6, 1997 

Operating date : Dec. 16, 1997 

Time Area 

(min) (IJ.V· sec) 

3. 51 142180 

Total 142180 

Height 

{IJ.V) 

3098 

Operator : Yuka Kida 

Cone. 

(%) 

100.00 

100.00 

Operating date : Dec. 16, I 997 

Time Area Height Cone. 

{min) {JJ.V·sec) {JJ.V) (%) 

3. 61 I 15137 2264 6. 28 

5. 96 1717224 55245 93. 72 

Total 1832361 100.00 

Operator : Yuka Kida 

Operating date : Dec. 16. 1997 

Time Area Height Cone. 

(min) {JJ.V·sec) (JJ.V) {%) 

3. 61 113389 2189 8. 19 

5. 98 1271517 17968 91. 8 I 

Total 1384906 100. 00 

Operator Yuka Kida 

Name J6 1fw&Ct./ 

,... -Tr 
Standard solution 10 mg/L 

PKNO 

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

Test water at 24th day (Level l ) 

PKNO 

1 

2 

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

Test water at 24th day (Level 2 ) 

PKNO 

1 

2 

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

Fig. 6 - 7 Analysis of test water 

Date Dec. 24. 1997 

( 

t~v. -.wl I C.. 

Operating date : Dec. 24. 1997 

Time Area 

(min) (.uV·sec) 

3. 64 146007 

Total 146007 

Height Cone. 

(.uV) (%) 

3559 100.00 

100. 00 

Operator : Yuka Kida 

Operating date : Dec. 24, 1997 

Time Area Height Cone. 

(min) (.uV· sec) (.uV) (%) 

3. 63 133499 2815 12.50 

6. 03 934589 15719 87.50 

Total 1068088 100. 00 

Operator : Yuka Kida 

Operating date : Dec. 24. 1997 

Time Area Height Cone. 

(min) (.uV·sec) (.uV) (%) 

3. 62 112463 2454 5. 63 

6. 01 1884706 51752 94.37 

Total 1997170 100.00 

Operator Yuka Kida 

Name 26. ~tV 

/)7 

I' 
i 

\ 

\ 

0.0 

0. 0 

0. 0 

Fig. 

Standard solution 10 mg/L 

2.5 5. 0 

Test water at 26th day (Level I ) 

2.5 5. 0 

Test water at 26th day (Level 2) 

2. 5 5. 0 

6 - 8 Analysis 0 f test water 

PKNO 

7. 5 10. o(min) 

PKNO 

1 

2 

7. 5 10. o(min) 

PKNO 

1 

2 

7. 5 10. o(min) 

Date Dec.24.1997 

Operating date : Dec. 24. 1997 

Time Area Height Cone. 

(min) (p.V ·sec) (tLV) (%) 

3. 64 150547 3577 100. 00 

Total 150547 100.00 

Operator : Yuka Kida 

Operating date : Dec. 24. 1997 

Time Area Height Cone. 

(min) (p.V·sec) (p.V) (%) 

3. 63 140173 2837 12.21 

6. 02 1007894 15759 87. 79 

Total 1148068 100.00 

Operator : Yuka Kida 

Operating date : Dec. 24. 1997 

Time Area Height Cone. 

(min) (p.V·sec) (p.V} (%) 

3. 62 115472 2464 8. 97 

6.02 1172136 18481 91.03 

Total 1287608 100. 00 

Operator Yuka Kida 

Name /6, ~/ 
'''-"• ......... , 14 

Standard solution 10 mg/L Operating date : Dec. 26, 1997 

PKNO Time Area Height Cone. 

(min) (J.LV· sec) (J.LV) (%) 

3. 64 154609 3669 100.00 

Total 154609 100. 00 

( 

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

Test water at 29th day (Level I ) Operating date : Dec. 26. 1997 

PKNO Time Area Height Cone. 

(min) (p.V·sec) (p.V) (%) 

I 3. 62 134928 2745 6.85 

2 6. 01 1833528 53354 93. 15 

Total 1968456 100.00 

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

Test water at 29th day (Level 2 ) Operating date : Dec. 26. 1997 

PKNO Time Area Height Cone. 

(min) (J.LV·sec) (p.Vt (%) 

( I 3. 63 124289 2481 6. 19 

2 6. 01 1883837 53549 93.81 

Total 2008127 100. 00 

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

Fig. 6 - 9 Analysis or test water 

Date Dec. 26. 199 7 Name !'/.~ 

I"·~···~~-····~---····-~~~ ~~----·· ,- i rr 
( 

\. 

Standard solution 10 mg/L 

PKNO 

I 

I 

~ ~ 

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

Test water at 33rd day (Level l ) 

v PKNO 

I 

2 

1 

~ 

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

Test water at 33 rd day (Leve I 2 ) 

PKNO 

1 

2 

1 

0.0 2.5 5. 0 7. 5 10. o{min) 

Fig. 5 -10 Analysis of test water 

Date Dec. 29, 1997 

Operating date : De~ 2~ 1997 

Time Area Height Cone. 

(min) (ttV·sec) (ttV) (%) 

3.64 153058 3566 100. 00 

Total 153058 100. 00 

Operator : Yuka Kida 

Operating date : Dec. 29, 1997 

Time Area Height Cone. 

(min) (ttV·sec) {ttV) (%) 

3. 63 125594 2734 6. 92 

6. 01 1689912 49161 93. 08 

Total 1815506 100. 00 

Operator : Yuka Kida 

Op~rating date : Dec. 29. 1997 

Time Area Height Cone. 

(min) (ttV·sec) {tLV) (%) 

3. 62 123274 2650 5. 59 

6. 01 2082429 53542 94. 41 

Total 2205703 100. 00 

Operator : Yuka Kida 

Name 

J3ra 
Standard solution 10 mg/L Operating date : Jan. 4, 1998 

PKNO Time Area Height Cone. 

(min) ( t.LV ·sec) (uV) (%) 

I 3.62 156979 2897 100. 00 

Total 156979 100. 00 

1 

~ J 

. v 

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

Test water at 39th day (Level 1 ) Operating date : Jan. 4. 1998 

PKNO Time Area Height Cone. 

(min) (uV·sec) (uVl (%) 

I 3.60 147272 2700 6.60 

2 5. 96 2085439 58955 93. 40 

Total 2232711 100.00 

I 

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

Test water at 39th day (Leve I 2 ) Operating date : Jan. 4. 1998 

PKNO Time Area Height Cone. 

(min) (uV·sec) (uVJ (%) 

I 3.60 118367 2269 4. 68 

2 5. 96 2408287 54860 95.32 

Total 2526654 100.00 

1 

v 

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

Fig. 6 -11 Analysis or test water 

Date Jan. 4, 1998 Name 

~1 /)/ 

I 'Ill. 
( 

( 

\ 

Standard solution 10 mg/L 

PKNO 

I 

I 

~ ' ~Q c.. =: 

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

Test water at 41st day (Level I ) 

\ PKNO 

I 

2 

I 

I 

~ 

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

Test water at 41st day (Level 2 ) 

\] PKNO 

I 

2 

1 

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

Fig. 6-12 Analysis of test water 

Date Jan. 6. 1998 

t'C.:;,\. HV .. 6tVII&. 

Operating date : Jan. 6. 1998 

Time Area Height Cone. 

(min) (.uV·see) (,uV) (%) 

3. 62 157951 2840 100.00 

Total 15795.1 100.00 

Operator : Yuka Kida 

Operating date : Jan. 6. 1998 

Time Area Height Cone. 

(min) (.uV·see) (.uV) (%) 

3. 61 131561 2323 7. 05 

5. 96 1733559 52604 92. 95 

Total 1865120 100. 00 

Operator : Yuka Kida 

Operating date : Jan. 6. 1998 

Time Area Height Cone. 

(min) (.uV·see) (.uV) (%) 

3. 61 131096 2276 6. 10 

5.96 2019023 54202 93. 90 

Total 2150120 100. 00 

Operator : Yuka Kida 

Name 

!3~ 
/ 

\ 

Standard solution 10 mg/L 

1 

~ 0. 0 2. 5 5. 0 

Test water at 44th day (Level l ) 

~ 0. 0 2. 5 5. 0 

Test water at 44th day (Level 2 ) 

1 

0. 0 2. 5 5. 0 

Fig. 6 -13 Analysis of test water 

PKNO 

1 

7. 5 10. o(min) 

PKNO 

1 

2 

~ 

7. 5 10. o(min) 

PKNO 

1 

2 

~ 

7. 5 10. o(min) 

Date Jan. 9. 1998 

Operating date : Jan. 9. 1998 

Time Area Height Cone. 

(min) (t.LV·secl (t.LV) (%) 

3. 63 130726 3178 100.00 

Total 130726 100. 00 

Operator : Yuka Kida 

Operating date : Jan. 9, 1998 

Time Area Height Cone. 

(min) (t.LV·see) (t.LV) (%) 

3. 63 133432 2587 7. 12 

5. 97 1739760 50230 92. 88 

Total 1873192 100.00 

Operator : Yuka Kida 

Operating date : Jan. 9. 1998 

Time Area Height Cone. 

(min) (t.LV·see) (t.LV) (%) 

3. 62 120648 2112 5. 70 

5. 97 1995270 55649 94.30 

Total 2115917 100. 00 

Operator : Yuka Kida 

Name 

/1} 
( 

\ 

Standard solution 10 mg/L 

PKNO 

1 

1 

;= t?:~ : c- I 

0. 0 2. 5 5.0 7. 5 10. o(min) 

Test water at 48th day (Level I ) 

~ PKNO 

1 

2 

1 

1 ~ 

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

Test water at 48th day (Leve I 2 ) 

\ PKNO 

1 

2 

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

Fig. 6 -14 Analysis of test water 

Date Jan. 13, 1998 

Operating date : Jan. 13. 1998 

Time Area Height Cone. 

(min) (tLV·see) (tJ.V) (%) 

3. 62 146080 3229 100. 00 

Total 146080 100. 00 

Operator : Yuka Kida 

Operating date : Jan. 13. 1998 

Time Area Height Cone. 

(min) (tJ.V·see) (tJ.V) (%) 

3. 61 128316 2492 6. 71 

5. 97 1782742 54044 93. 29 

Total 1911058 100. 00 

Operator : Yuka Kida 

Operating date : Jan. 13, 1998 

Time Area Height Cone. 

(min) (tJ.V·see) (tJ.V) (%) 

3. 61 115673 2447 5. 83 

5.97 1868253 53133 94. 17 

Total 1983927 100. 00 

Operator : Yuka Kida 

Name 

/3Y 
Standard solution 10 mg/L Operating date : Jan. 16. 1998 

PKNO Time Area Height Cone. 

(min) (tLV·see) (tLV) (%) 

1 3. 62 144170 3300 100.00 

Total 144170 100.00 

1 

0. 0 2. 5 

~ 5. 0 

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

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

"'\) PKNO Time Area Height Cone. 

(min) (tLV·see) (tLV) (%) 

1 3. 60 121842 3014 9. 78 

2 5. 96 1124603 20346 90. 22 

~ ~ 

Total 1246445 100.00 

-

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

Test water at 51st day (Level 2 ) Operating date : Jan. 16. 1 998 

PKNO Time Area Height Cone. 

(min) (tLV·see) (.uV) (%) 

1 3. 60 123895 2711 7. 85 

2 5. 97 1454769 22387 92. 15 

~ Total 1578663 100. 00 

0. 0 2. 5 5.0 7. 5 10. o!min) Operator : Yuka Kida 

Fig. 6 -15 Analysis of test water 

Date Jan. 16. 1998 Name 

." -

(35 
( 

( 

\. 

( 

Standard solution 10 mg/L 

1 

__.}--- ~ 

0.0 2~ 5 5.0 

Test water at 55th day (Level l ) 

~ 

1 

__) 

. 0. 0 2. 5 5. 0 

Test water at 55th day (Level 2 ) 

v 

1 

) 

0. 0 2. 5 5.0 

Fig. 6 -16 Analysis of test water 

PKNO 

A 1 

\ 

7 .. 5 10. o(min) 

PKNO 

1 

2 

~ 

7. 5 10. o(min) 

PKNO 

1 

2 

~ 

7. 5 10. o(min) 

Date Jan. 20. 1998 

Operating date : Jan. 20, 1998 

Time Area Height Cone. 

(min) ( p.V ·sec) (.uV) (%) 

3. 61 146036 3220 100.00 

Total 146036 100.00 

Operator : Yuka Kida 

Operating date : Jan. 20. 1998 

Time Area Height Cone. 

{min) {p.V·sec) (.uV) (%) 

3. 60 127197 2495 6. 41 

5. 95 1857047 53123 93.59 

Total 1984244 100.00 

Operator : Yuka Kida 

Op~rating date : Jan. 20. 1998 

Time Area Height Cone. 

{min) {p.V· sec) (p.Vj (%) 

3. 60 121077 2876 6. 05 

5. 96 1879872 54306 93. 95 

Total 2000950 100. 00 

Operator : Yuka Kida 

Name 

/?6 
Standard solution 10 mg/L Operating date : Nov. 6. 1997 

PKNO Time Area Height Cone. 

(min) (J.LV·see) (t.LV) (%) 

3. 64 142834 3310 100. 00 

Total 142834. 100. 00 

I ' 

\ 

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

Recovery test from test fish a Operating date : Nov. 6, 1997 

PKNO Time Area Height Cone. 

(min) (J.LV·sec) (t.LV) (%) 

1 3. 64 100297 2571 4. 01 

2 5. 35 2398622 106240 95. 99 

Total 2498919 100.00 

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

Recovery lest from test fish b Operating date : Nov. 6, 1997 

PKNO Time Area Height Cone. 

(min) (J.LV·see) (tLVt (%) 

1 3. 64 100565 2485 5. 10 

2 5. 35 1869761 74968 94.90 

Total 1970326 100. 00 

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

Fig. 7 - I Recovery and blank test (analysis of test fish) 

Date : Nov. 6, 1997 Name 

·.\.._ f1 
Standard solution 10 mg/L Operating date : Nov. 6, 1997 

PKNO Time Area Height Cone. 

(min) (t!V·see) (tLV) (%) 

3. 64 142228 3404 100. 00 

Total 142228 100. 00 

( 

\ 

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

Blank test of test fish a Operating date : Nov. 6, 1997 

PKNO Time Area Height Cone. 

(min) (tLV·see) (tLV) (%) 

Total 0 0. 00 

Peak. position 

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

B I ank test of test fish b Op~rating date : Nov. 6, 1997 

PKNO Time Area Height Cone. 

(min) (tLV·sec) (tLV) (%) 

( Total 0 0. 00 

Peak position 

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

Fig. 7-2 Recovery and blank test (analysis of test fish) 

Date : Nov. 6, 1997 Name 
( 

( 

Standard solution 10 mg/L 

0.0 2. 5 5. 0 7. 5 10.0 

Test fish after 2 weeks (Level 1-a) 

Peak. position 

l~ 

0. 0 2. 5 5. 0 7. 5 10.0 

Test fish after 2 weeks (Level !-b) 

Peak position 

ta~ 

0.0 2. 5 5. 0 7. 5 10. 0 

Fig. 8 - I Analysis of test fish (Level I ) 

Operating date : Dec. 11, 1997 

PKNO Time Area 

(JLV ·sec) 

141045 

Height Cone. 

(min) 

3. 62 

Total 141045 

Operator : Yuka Kida 

(JLV) {%) 

3169 100. 00 

100. 00 

Operating date : Dec. 11. 1997 

PKNO Time Area Height Cone. 

(min) (JLV·sec) (.uV) (%) 

Total 0 0. 00 

Operator : Yuka Kida 

Operating date : Dec. 11. I 997 

PKNO Time Area Height Cone. 

(min) (tLV·sec) (.uV1 (%) 

Total 0 0. 00 

Operator Yuka Kida 

Date : Dec. 11, 1997 Name 

13 r 
Standard solution 10 mg/L Operating date : Dec. 25. 1997 

PKNO Time Area Height Cone. 

(min) (.u.V·sec) (.u.V) (%) 

1 3. 64 151972 3619 100. 00 

1 Total 151972 100. 00 

~ - ' 

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

Test fish after 4 weeks CLeve I 1-a) Operating date : Dec. 25. 1997 

PKNO Time Area Height Cone. 

(min) (.u.V·sec) (.u.V) (%) 

Total 0 0. 00 

Peak position l n.d. 

f'--J\ 

0. 0 2. 5 5.0 7. 5 10. o(min) Operator : Yuka Kida 

Test fish after 4 weeks CLeve I 1-b) Operating date : Dec. 25. 1997 

( ~ ~ PKNO Time Area Height Cone. 

(min) (.u.V·sec) (.u.V) (%) 

Total 0 0. 00 

Peak position 1 •. ~ 

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

Fig. 8 - 2 Analysis of test fish C Level I ) 

Date : Dec. 25. 1997 Name 

r 
0. 0 

0. 0 

0.0 

Standard solution !0 mg/L 

2. 5 5. 0 

Test fish after 6 weeks (Level 1-a) 

Peak position 

1 n.d. 

2. 5 5. 0 

Test fish after 6 weeks (Level !-b) 

Peak position 

7. 5 

7. 5 

nv. ""t\oJI IL. 

Operating date : Jan. 8. 1998 

PKNO Time Area Height Cone. 

(min) {.uV·see) (t.tV) (%) 

3. 62 136345 3235 100.00 

Total 136345 100. 00 

IO.o(min) Operator : Yuka Kida 

Operating date : Jan. 8. 1998 

PKNO Time Area Height Cone. 

(min) (t.tV·see) (t.LV) (%) 

Total 0 0. 00 

IO.o(min) Operator : Yuka Kida 

Operating date : Jan. 8. 1998 

PKNO Time Area Height Cone. 

(min) (uV·see) (t.tVT (%) 

Total 0 0. 00 

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

Fig. 8-3 Analysis of test fish (Level 1 ) 

Date : Jan. 8. 1998 Name 

/f{( 
Standard solution \0 mg/L Operating date : Jan. 22, 1998 

PKNO Time Area Height Cone. 

(min) (ttV·see) (ttV) {%) 

I 3. 66 147124 3055 100.00 

I Total 147124 100.00 

~ \_ 

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

Test fish after 8 weeks (Leve I 1-a) Operating date : Jan. 22. 1998 

PKNO Time Area Height Cone. 

(min) (ttV·secl (ttV) (%) 

Total 0 0.00 

Peak position l •. ~ 

I 

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

Test fish after 8 weeks (Leve I 1-b) OP.erating date : Jan. 22. 1998 

PKNO Time Area Height Cone. 

(min) (f.LV•see) (t.LV) (%) 

( Total 0 0. 00 

Peak position 

1 n.d. 

) I 

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

Fig. 8-4 Analysis of test fish (Level I ) 

Date : Jan. 22, 1998 Name 

. ' 1---"'1[ ·-;-rr-
Standard solution 10 mg/L Operating date : Dec. 11. 1997 

PKNO Time Area Height Cone. 

(min) (t.LV·sec) (t.LV) (%) 

1 3. 62 140595 3182 100. 00 

Total 140595 100.00 

1 

~ \. 

\ 

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

Test fish after 2 weeks (Leve I 2-a) Operating date : Dec. 11, 1997 

~ PKNO Time Area Height Cone. 

(min) (t.LV·sec) (t.LV) (%) 

Total 0 0.00 

Peak uosition 

~~ ' 

1~~ 

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

Test fish after 2 weeks (Level 2-b) Operating date : Dec. 11, 1997 

PKNO Time Area Height Cone. 

(min) (t.LV·secl (t.LV) (%) 

( Total 0 0.00 

Peak position 

l~~ ~ 

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

Fig. 9 - I · Analysis of test fish (Level 2) 

Date : Dec. 11. 1997 Name 
Standard solution 10 mg/L Operating date : Dec. 25, 1997 

PKNO Time Area Height Cone. 

(min) ( JLV ·sec) (JLV) (%) 

1 3. 64 158534 3600 100. 00 

Total 158534 100. 00 

1 

~ -

( 

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

Test fish after 4 weeks CLeve I 2-a) Operating date : Dec. 25, 1997 

PKNO Time Area Height Cone. 

(min) (JLV·sec) (JLV) (%) 

Total 0 0. 00 

Peak position 1 n.d. ~ 

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

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

\ PKNO Time Area Height Cone. 

(min) (JLV ·sec) (tM (%) 

( Total 0 0.00 

Peak position 

1 n.d. ~ 

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

Fig. 9 - 2 Analysis of test fish (Level 2) 

Date : Dec. 25. 1997 Name 

lT I. 
i 

\ 

Standard solution 10 mg/L 

PKNO 

1 

1 

0. 

:-

0 

~ 2. 5 5.0 

~ 7.5 

~ 10. o(min) 

Test fish after 6 weeks (Leve 1 2-a) -

PKNO 

Peak. position l n.d. f'--..---. 

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

Test fish after 6 weeks (Leve 1 2-b) 

PKNO 

Peak position 1 .. ~ 

I 

0. 0 2. 5 5.0 7. 5 10. o(min) 

Fig. 9 - 3 Analysis of test fish (Level 2) 

Date : Jan. a. 199a 

Operating date : Jan. a. 199a 

Time Area Height Cone. 

(min) (tLV· sec) {/.tV) (%) 

3. 62 123327 3220 100. 00 

Total 123327 100. 00 

' 

Operator : Yuka Kida 

Ope rat i.ng date : Jan. a. 199a 

Time Area Height Cone. 

(min) (uV·sec) (uV) (%) 

Total 0 0. 00 

Operator : Yuka Kida 

Operating date : Jan. a. 199a 

Time Area Height Cone. 

(min) (uV·sec) (uVt (%) 

Total 0 o. 00 

Operator : Yuka Kida 

Name 

~1 /vf5 
( 

\ 

0. 0 

0. 0 

0. 0 

Standard solution 10 mg/L 

2. 5 5. 0 7. 5 

Test fish after 8 weeks (Level 2-a) 

Pea! position 

2. 5 5. 0 7. 5 

Test fish after 8 weeks (Level 2-b) 

Peak position 

1 n.d 

Operating date : Jan. 22. 1998 

PKNO Time Area 

(min) (ttV·see) 

He i gh t Cone. 

(ttV) (%) 

3. 66 136682 3042 100. 00 

Total 136682 100. 00 

10.o(min) Operator : Yuka Kida 

Operating date : Jan. 22. 1998 

PKNO Time Area Height Cone. 

(min) (ttV·see) (ttV) (%) 

Total 0 0.00 

10. o(min) Operator : Yuka Kida 

Operating date : Jan. 22. 1998 

PKNO Time Area Height Cone. 

(min) (ttV·see) (ttV) (%) 

Total 0 0. 00 

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

Fig. 9 - 4 Analysis of test fish (Level 2) 

Date : Ja~ 2~ 1998 Name 

·-;-,- r 
Standard solution 10 mg/L Operating date : Nov. 26, 1997 

PKNO Time Area Height Cone. 

(min) (JLV· sec) (JLV) (%) 

1 3. 63 145789 3216 100.00 

Total 145789 100.00 

1 

~ \I 

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

Before the initiation of exposure ( Control-a ) Operating date : Nov. 26. 1997 

PKNO Time Area Height Cone. 

(min) (JLV·sec) (JLV) (%) 

Total 0 0. 00 

Peak position 

1M ~ 

0. 0 2.5 5. 0 7.5 10. o(min) Operator : Yuka·Kida 

Before the initiation of exposure ( Control-b ) Operating date : Nov. 26. 1997 

( ~ PKNO Time Area Heig~t Cone. 

(min) (JLV· sec) (JLV) (%) 

Total 0 0. 00 

\ 

I 

Peak. position 

1 ~~ 

0. 0 2.5 5.0 7. 5 10. o(min) Operator : Yuka Kida 

Fig. 10 - I Analysis of test fish (Control ) 

Date : Nov. 26. 1 997 Name 
Standard solution 10 mg/L 

PKNO 

0.0 2. 5 5. 0 7. 5 10. o(min) 

After the termination of exposure ( Control-a ) 

v PKNO 

Peak positiOn l n.d 

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

After the termination of exposure ( Control-b ) 

PKNO 

( 

\ 

Peai: position 1 n.d. 

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

Fig.I0-2 Analysis of test fish ( Control 

Date : Jan. 23. 1998 

Operating date : Jan. 23. 1998 

Time Area 

(min) (t.tV·sec) 

3. 65 151819 

Total 151819 

Height 

(tLVl 

3043 

Operator : Yuka Kida 

Cone. 

(%) 

100. 00 

100. 00 

Operating date : Jan. 23. 1998 

Time Area Height Cone. 

(min) (uV·sec) (uVl (%) 

Total 0 0. 00 

Operator : Yuka Kida 

Operating date : Jan. 23. 1998 

Time Area Height Cone. 

(min) (t.LV ·sec) (t.LV} (%) 

Total 0 0. 00 

Operator Yuka Kida 

Name 10 r!uk 

-~~-v 

r 
Test No. 43112 ,........ 

....l 10 ~ -=- § 

Ql) .. • • » • .....___._ .--- X • .-----... .......,. 0 

"0 • • ~ 

> 

0 5 

"' "' :a 

...... 

0 

.g 

~ 

... 

-

a 0 ~ 

0 0 10 20 30 40 50 60 

u 

E:-..-posure period (days) 

Fig. 11 Concentration of dissolved oxygen (Level 1) 

~ 10 

~ 

a 

Ql) ._.__ .. __..__ . . __. ·...-• • • • • • • ~· • 0 

"0 

0 

> 

0 5 Vl 

Vl 

:a 

...... 

0 

-.g 

~ 

... ' 0 0 \ 

' ~ 

~ 

0 0 10 20 30 40 50 60 

u 

Exposure period (days) 

Fig. 12 Concentration of dissolved oxygen (Level 2) ,..... 

....l 

'eb 10 -

s, 

0 • Ql) 

>. • • • .....__. :.-: ·-- • ·--- ...... • • • 0 ... "0 

0 

> - 0 5 Vl 

Vl 

:a 

...... 

0 

.:2 

~ u 0 ~ 

0 0 10 20 30 40 50 60 u 

Exposure period (days) 

Fig. 13 Concentration of dissolved oxygen (Control) 

January 28, 1998 Name k>. ~.£?/ 

fi ft{ I 
co 

M 

l{) 

"' I 

0 

0 

N 

1-

~ ""' 

:r: 

v 

z 

Q 

1-

~ 

0 ""' 

"-

0 ""' 

u 

:::> 

N 

0 

~ 

;:f 

s: 

V) 

( ID '. 

co 

M 

''"> 

"' 0 

0 

N 

( 1-

ex: 

< 

:r: 

v 

z 

Q 

1-

< 

Fig. I 4 

Test No. 

Sample 

Date 17'17. 7. 16 

V- o/iH 

Solvent 1e tra hvdr-c furan. I 

Reference 1e tHA b 11dro fu ran I 

Photometric Mode _ __,A~bs"--------

Cell lOmm X lOmm(quartz) 

Wave Length I ?D liM JM nl't\. 

Scale Limit -(),/ /.0 

@VIS 

Instrument S&!;nttdz.u uv- J..lbO A 

Slit(Spectral Band)Width .>..0 nm 

Scan Speed H;ddleSampling Pitch Auto 

Note. _-l.lt/~V~_-Ea'-"'b~s~o.Lro!Lt!<!i~on!.!...£s<l<p!32el<.ct!<!r..leu~m!L.._ 

Chemicals Inspection & Testing Institute, Japan Chemical Biotesting Center (1997. 500&) 

------------------------- PARAMETERS OF SPECTRUM 1997/87/16 18:39:18 

MEASURING MODE ABS 

SAMPLING PITCH(Delta A) AUT0(8.1 nm) 

SCAN SPEED MIDDLE 

SLIT WIDTH 2.8 nm SAMPLE NAI~E 43112 

ANALYST Y.KIDA 1Jfi~ 

------------------------~~~:~~-~--~~~~~-~~~~~-------~~~~!~~~--~~~-- -<: = = = = = = = = 00 = ...,. ~ = ~ r-: ...,., ~ = ~ 

= = = = I 

= = 

(V'.) 

cq =.. = = c: 

= = = = = = ~ 

= 00 = """" C-.J 

= r- ..,., ~ 

= = = = = I 

f1 I )D 

V> 

:r: 

~ )> 

0 

N 

c 

n 

0 

;Q 

., 

0 

;Q 

)> 

-i 

6 

z 

n 

:c 

)> 

;Q 

-i 

0 "' 

? 

'() 

"' w 

(X) 
( 

/ 

~ i 

j ! I ' i 

i I ! I : l 100.0 -: ----------- ---- -~--- -~----------- "'---- ~-------, 1-----------:--: 

-i ....-' I i fA,. ~ ' · (' ~~; •1 ' j 

, / " : 11ir~, ~~ • . ; : r~ : . ;~, }; ~ ~ . i 

n J ·, . /\. . 1

· i ; ,.,; · ·~ ·\ tt~: ! t , ; ~~ t \ ri I! i! hi! 

! .dlflh~ ,./" i : I ~ i . ; 1 ~ : I I I :/' i j I I \,;! ! i .• , ~ : ~~ 

so o -. ~"ft~!( :~t.; \,\~ 1· !; 1tr: ~~~i h, 1: v1~· iiviL~t t·'~i 1· -.~. "-~----r· ,_ __________ ,T ________ ,_4 4--• ~-;--1--T-"' __ . -'-'-"' _ .. _4·- . . . . ' • I' . •1'' tl .• ll ;-1 'I I • 

~: ~} ! 1!~ ;it /if!! \il;! 1

1

ii ~ !! !r! i ~ .! 

J i 1

; 

1

:1 ji J~~l:llii !Hi: ,!1; 11 !tii; ·: 

~ : ji i ·~~ :i!\il\1 illl1 IIi 1,1 \ Iiiii : ij :IIi l/ ; ! 

i ' -~1 . ! : :~' \ 1 ji , j ; f !I I !. II I I : ! _J I J! ; ·~~.' :; ;1~1 • I i ~ I :, ' 't i ' ' 

so.o ~ -----------rr-------r;~------------: ,,r

1

. --! ·rr---rr,,-w-~--~ 

i !/ ! J! I I I I I ij ' 'I 

-; •!i i ,,. ' ! I \ ' ' I li I . 

! •!; I I ' I' I) I ' ,, ' I -i i! ; / ! i i I I I ~ 'i ; I 

40. 0 J -----------:1 ---------;-i!- -----------: ------r ! ~- -: -----_1, -:. -- 2 - -I 

! . . H !'l 1 !; I ; ! 1 c-. il: v , 1 

, st· 11 ~ . • , : ...; r. 11 1 . I • • 

20. 0 j -----------:- ---------t -:- -: ----------:- ----------~ -----------~ --I 

4000.0 3000.0 2000.0 1500.0 1000.0 

! 

-500.0 

1/cm 

Fig. 15-1 ~R spectrum of test substance measured before 

the initiation of exposure to fish 

Sample:V-05H(43112) 

Method:KBr tablet 

Date :Oct.21. 1997 

Name : J6. Jrhcv 

f 7 !. 5 I 

1 TT 
_; 

J ~ i I j 

1oo. o -+-----------.------:..---- _~_---------- ~----------- ~------ r---- ~- -' ~ r ' I I l '!' l . "' 

II,T ; • . f: I t~ 1 .; i I, 'I: ~ t \ . . :{'1 . :: ;,"1' 

l ~"'~""\ : ' \ ! J\ i\ : I \ t I i!l I .I 'I < : : 

80. 0 ~~~- - - ~ - A ;; p -~ --!'- ---+ --_!- ---__ -/- ~ -: _\1_ - ~- - - + ~' ----- -}~, i --pLI ~ !H, :II - - -,f ~ ~ ~ ; rv l : ! ., ; r \ i i ll: ! I l'j i! i !I'll'\ li fl:i : ' 'r i · 1• \ 1 ; 11 ; {;. ,, 1 /• 1 : .,. II :1~·; i ...J ,. ~· • ;· ". \1 i I., I' I • 

..J :J ; I' \ ,,· 'J ' 'I l I r I I ' , ' ~~~ 0 ~I ' I I t ' I ~ f I : l ! :j I II I H ! 

so. o ~ - - - - - - - - - -t .- -1- ------:- --! -~- ------r ~ -i- -~ r r ----f : lt 'r rl 1 r II- r -t -: ~ ~~ ; : : 1 :,1 i 1

1

1 i, 1 : ~ '

1

,; 1

1

' 111 1· 0 1 ...! tl I '1 I " , l B ~ I I I I ' I 

' I I ' : i !! i 1

1

1

,, o!l il Jl! ' i ii 11' i ,: ' ! ...... l . . . 1

1 l I "' i ,. p I II I ! : I 

40 0 

~ -----------:-r-----n------ -rl\l,,~r~/[J :----~nlm-- n 

I Pi : J :n a r 1\ I \ ! 1i : i ,, ; ; 20.0 ---;------------li'j'-------- -·,- T---------- -., J . - -:; j7 I-.,---- - -~-;:-- '--· - Ui ,; : i' I! I I !! I 

J 11 : !1 : :1 l

1

1 : · II : : .:1 \II' . \' J . , .~ . I ~ I 

. \,. ! 

4000.0 3000.0 2000.0 1500.0 

' I 

1000.0 -500. 0 

1/cm 

Fig. 15-2 IR spectrum of test substance measured after 

the termination of exposure to fish 

Sample:V-05H(43112) 

Method:KBr tablet 

Date :Jan.27. 1998 

Name : ;t.. ·~-v 
100.1 ~-;-;-:-:--:-:--r~:-:7<;-r;-;-;-r;--;-;-;-;-;-:-;;~;-;-r;-;;-;-;--;-;"ii;-;-:-;-rr;-:-rrr--;-;"iiTTOi:rr;-::orr;-;-;-;r.-;-;-n-:;r-;"7T:-rr;,..,.,-rTT:"!7rr:-:-no;--:-:-;-;-r.-r.,.,.,-,--:-:--:-:-.,..,..,.., 

::~:[!:!!~II J!ll+UI !Ill !!!~ !! 111~1111 !

1

!! ''!' !!!! Willi ll'~ll II I J.+l~l1 mJ ~I~ illll. 

friTTlfi iilllllllirr1~ 11111111111111 11111111111111111 w u '' J 11111m·n111 H n 11i111111 90

•

0 

i1!i !Pi Hilli'' !lil 1111 l.l''lllllll 'I li11 1 

r.-:+r .;.: ~. ·.;..:.~ J, , d ~~, ~,,, .•.• _ • , " I I _ I' , I .· 11"11 I • I ! J . w· , 'lllllll'll il it -J{tt J_n ~· ' I-ll! !Ill ,.h !I'' 

: ·rr ; i I! ~!!I ; ! 'I !! ! I; 111'1'1 w t 'II! I' I I . Tl i I I: I" II I ! I I II! m,,! II q ,. r !! II I II :·:;: ;: ::1! ··!! :;;, llllii. 11 !!, I ill!.:! ,,I! t!ll II I I li II i 1 II I il · I_, dn II ao.o ! : H : ~:: H !I ! ll! ! ! II !' I !j !I 'Ill i i! I l I !j l'j ! I i I! I ! I ld I! '1 ! ! II I i I Ullli j IIIII! I 'Ill ! I p·' 'I 'II '.;..:.:. .:.: ' I •• 't . !I' I. I i I i ! i I Ill i IiI ' I 1· I iiI ! i I ' I I II i i ! i I li l i i i ! ,, I i. !I l.llr. 

! :: i 'Tl1! :! ! ! ! l! I :! 1

11 !i 1' !, l!!! H 1'~1 ! ! I 1.111!'111! I 1 !! l,jl !11'1 

1

1 '1 11'1

1 u! 'j, ~, I ! '1lll i I ;

1

'1 ill ,i Ill,, 1,11 r! ~lll 1, 11 1

1

1 .•.•.. , ..• ! ....... 1 .. 1 ••. "'' • '.,! 1·.· rill 1. 11 11 •• , • :1 ,,,, 1 :11 ,1!11 1 1 , 

7o.o '_;; ;; ' :t _;q •:r: :ill !:1! jql iii! ! I iiljllll lllj 11!1 'Ill II II ijli ''ll "I'' 'II' I iii I 'II., iiJij'll !iii II II 

%T ~,;r ;! :H :o!r :1!! i1H !:,~ ;!l r:., 1 1 !t,, .1" :j 1 pr! 1:11 i' 1 11 1111 11 1 i: 1 : 1 , 1.1 11 1 tl!! I' .. ;-~ ----:·: -; :-: :~;!- ;::-r ~~·r! ·HH-:-lT: tr+H-f ;·:JI·II-~ ~~:ttll. rHfl m-Il'' H" i+h rm,,· -:+·l't f+(t -:-r,1

j -:-m, -j"l·,·r· T·!,l- '!·rti· :!-·~·t : :! ',·; ;,!_!:!;_'!;!it,' ii,,l! lid H!l, H',·, i ·,iii! !I:! 1

j

11lrl i, ,,,·1 :1' ·I'll! d. iiI !j·,·l il 1, !,1,, ,.,· ,,,,- ,id ,-,,·,, ! 1 , ;: 1 ,, ':,,. ,, o. I ,,J ••• •• • '' I,,, f,:j II ••' I I o o .1 I ,J t ftol ioo 1 1 , , !fit 60

'

0 

::;; ::~: !:i*' ::il !:i'1· i!il Lii :iii !j'l: !iii i_w:·i !1 1

: il'l'l il'll ;ij'l i!!'11!!:! l'i i!ll :,11 '''f' iiii !:il ... •' •; ..... tl '"' '"!"I ''II I 0: II''L' ''1-UJJ" I [I I I if 'I "I '''+ '•JJ.'··· I'P' ol I ,, .. ,,. ! ;: • :;i .j;: ::;; ::t: i:l !JJ.l ;~ . ;:1u •rl li~:; ft I !II·,, I titt'' -ji!!: i! l ' 1 :; ql! 'H"I t 1;:; I• I 

;·;-a .. "!- ::-: -·;·;; ·;-r;--r:·;. l :t·, ; 'ji rrrr 1 ''i' ii1"o 'fi1"i . II. ·t; ,,·,rtt,- H,-H I il II I l"iTTjll :Ti·t 11,. '1'1 !rjT -:~~, t;"( 1"'("!<· 11'1 • i ·:: '·i' •l :·:: ::!. ~-,i ''I I ·I ;:1' ·''I I'll "I ill I' i' 'II .. 1' :·::'II :!:1 'i :r I :!IJ 1: I :. ; -;; ::: ::1 lilt ::11 it;,!! ::i: ild :!: It, 1: ii :.1 !i:; !!,, !lil :!n i .. iii;!;! ilr !!:1 id! • 

50

'

0 

: : : . ; i : ; : i ; I ; . ' ; I ; ! I ! : ij i ! ! i I ;; i I i : ! ,, i li'l ii "I I ill i i I ! i 'ill jl i I llj' j ,. ! II I : ,, i I i I! ! ! II ! Ill I i ! I i I II ! i l i i i '! 

.. i~ .. J -·- .:;+; .L.LL~.~~~!.ri+!i l! J.U-!~-t !l~~m';n lnTii m·lnL :, 1-H-~ :! : Iii! 1*t _!,r_li!! _i_:Jlj_li! Uli . : ! : i! . ::! . ii r :: l :; i I ! l r ! !! ,, I! IT i iT'i ii 1 i! i 'lllTi I fl I p-n: I' ! ! I ! (!111TI"il1 ijl!! I111 l i I I i I' iTI1 1 nrrl · .. · :; :!: ;:;1 ·:~! l:;! !i;l iiL !! i J! l!!i Iii !!•1 I dil i I .!I 1111 rill i! .. ,l!l !·!i !I. iiI iilj !! i 

•o.o i J : i .. if , • I i i !iiI i iII P II i! i I I! II I d I iiI i l-!11 d I II I Hi 1111111 ! IIIII I !I Uuill Ul HI I ! Ill II I! !i H I liT 36.0 •ooo 3eoo 3600 3•oo noo 3000 2aoo 2600 2•oo 2200 2000 t9oo uoo 1100 1600 uoo uoo 1300 ·1200 1100 1000 9oo aoo 100 600 500 •oo 

"t;.,. :1 Jt..~ : V-OSH 

5H~: 4 cm-1 

7 ;f-')1"-( i:f-:.- 3 :,;: Cosine 

Reference 2 

Wavenumbcr[cm-1) 

l.li~[§]~: 

8{~: . 17 

96/11/06 10:36 

IR spectrum supplied by the sponsor 

/ 
( 

( 

( 

\ 

Amendment and addition to Final Report 

1. Title 

2. Test number 

3. Content 

4. Reason 

Kurume Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & 

Testing Institute, Japan 

Bioaccumulation test ofV-05H in carp 

43112 

CD 10.2 Chemical structure, etc. molecular weight (Page 4) 

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

CD Molecular weight is about 30000 when average degree of 

polymerization is n = 180. 

® The polymerization degree was cleared. 

5. Content of correction and addition 

6. Date of correction 

7. Preparation 

CD " Molecular weight about 3000 " is corrected to 

" Molecular weight about 30000 ". 

® " n = 2 - 800 " is added. 

June 5, 1998 

Date June 5, 1998 

Study director Signed in original 

Y asuk:o Matsunobu 
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Receipt No. T96-2575 

R~ort No. T-4743 

STUDY CODE: K01-1861 

FINAL REPORT 

BACTERIAL REVERSE MUTATION TEST OF 

V-OSH 

January 1997 

Hita Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & Testing Institute 

Japan 

f1 /)C 
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STATEMENT 

I, the undersigned, hereby declare that this report provides a correct English translation 

ofthe final report (Study Code K01-1861, issued on January 28, 1997). 

Chemicals Inspection & Testing Institute, Japan (CITI) formally changed its name to 

Chemicals Evaluation and Research Institute, Japan (CERI) as ofNovember 1, 1999. 

The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, 

Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to 

"NISSHINBO INDUSTRIES, INC. R & D CENTER" and "1-2-3, Onodai, Midori-ku 

Chiba, 267-0056, Japan", respectively by Sponsor's request. 

CAS No. 157299-02-0 oftest substance was added by Sponsor's request. 

In the structural formula, n=180 was changed to n=2-800. 

Molecular weight of 3,000 was changed to 30,000. 

J~ !~, 2002 

Shozo Ogura 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

Date 

TC 
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QUALITY ASSURANCE STATEMENT 

Hita Laboratory, 

Chemicals Evaluation and Research Institute, Japan 

Sponsor: NISSIDNBO INDUSTRIES, INC. R&D CENTER 

Title: Bacterial Reverse Mutation Test of V-OSH 

Study code: KO 1-1861 

This report was audited by the Quality Assurance Section. 

I, the undersigned, hereby declare that this report reflects the 

original Japanese report. 

Section Chief, Quality Assurance 

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

(Signature) ~ii!:Ei>:._ • 

Keiji Shiraishi, B.S. 
( 

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Sponsor: 

Title: 

GLP STATEMENT 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

NISSHINBO INDUSTRIES, INC. R & D CENTER 

Bacterial Reverse Mutation Test ofV-05H 

_l(,.Ul-l~bl 

Study Code: KOl-1861 

I, the undersigned, hereby dec_lare that this study was conducted in compliance with 

"Standards to be observed by Testing Institutions for Toxicity Investigations" (Japan's 

Ministry of Labor, No.76, September 1, 1988), and "Concerning Testing Facilities 

Stipulated in Article 4 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 (EA), No. 229 of the Pharmaceutical Affairs Bureau, Ministry of 

Health and Welfare (MHW) & No. 85 (1984) of the BasicJndustries Bureau, Ministry 

of International Trade and Industry (MIT!), March 31, 1984; Notification No. 233 of the 

Planning and Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, 

MHW & No. 823 (1988) ofthe Basic Industries Bureau, MITI, revised on November 18, 

1988). 

Management: Signed in original January 28, 1997 

Shigetaka Yamane, Ph.D. 
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K.Ul-lH6l 

QUALITY ASSURANCE STATEMENT 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

Sponsor: NISSHINBO INDUSTRIES, INC. R & D CENTER 

Title: Bacterial Reverse Mutation Test ofV-05H 

Study Code: K01-1861 

This study was audited and inspected by Quality Assurance Section of Hita Research 

Laboratories, Chemical Biotesting Center, Chemicals Inspection & Testing Institute, 

Japan. The dates audited and/or inspected and the dates reported these results to the 

study director and management are as follows. 

Dates of Dates of Reports to Dates of Reports to 

Inspections/ Audits Study Director Management 

November 26, 1996 November 29, 1996 December 3, 1996 

December 3, 1996 December 3, 1996 December 3, 1996 

January 27, 1997 January 28, 1997 January 28, 1997 

January 28, 1997 January 28, 1997 January 28, 1997 

I, the undersigned, hereby declare that this report provides an accurate description 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 January 28, 1997 

Keiji Shiraishi, B.S. 

f1 /to 
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TITLE 

Bacterial Reverse Mutation Test ofV -05H 

SPONSOR 

NISSHINBO INDUSTRIES, INC. R & D CENTER 

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

TESTING FACILITY 

.li....Ul-l~bl 

Study Code: K01-1861 

Test Substance Code: HR3392 

Sponsor Code: N-680 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

822, 3-chome, Ishii-machi, Hita, Oita 877, Japan 

PURPOSE OF STUDY 

The mutagenic potential of the test substance is examined using Salmonella 

typhimurium and Escherichia coli. 

TESTING METHOD 

This study was conducted in accordance with "Standards for Toxicity Investigations" 

(Japan's Ministry of Labor, No.77, September 1, 1988), 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. 

1 03 9 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)". 

GLP COMPLIANCE 

This study was conducted in compliance with "Standards to be observed by Testing 

Institutions for Toxicity Investigations" (Japan's Ministry of Labor, No.76, 

September 1, 1988), and ••concerning Testing Facilities Stipulated in Article 4 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, MIT!, March 31, 1984; Notification No. 233 of the Planning and 

?1 (£. ( 
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KOI-1861 

Coordination Bureau, EA, No. 38 of the Environmental Health Bureau, MHW & No. 

823 ( 1988) of the Basic Industries Bureau, MITI, revised on November 18, 1988). 

PERIOD OF STUDY 

Commencement of Study: November 27, 1996. 

Commencement of a Dose-Range-Finding Test: November 28, 1996 

Completion of Observation: 

Completion of Study: 

December 5, 1996 

January 28, 1997 

LOCATION AND PERIOD FOR RETENTION OF DATA 

Data and the remaining test substance will be retained in the archives and test 

substance storage room, respectively, of Hita Research Laboratories for I 0 years 

following the date of the notification specified under Item 1 of Article 57-2 of 

Industrial Safety & Health Law. Date of the notification will be communicated 

from the Sponsor to Hita Research Laboratories. After termination of the retention 

period, any measures taken will be done so with the approval of the Sponsor. 

AUTHOR AND PERSONNEL CONCERNED WITH STUDY 

AUTHOR 

Study Director: Signed in original 

Tsunehiko Inai, B.S. 

Mutagenicity Section 

Hita Research Laboratories 

PERSONNEL CONCERNED WITH STUDY 

Study Staff: Shozo Ogura 

Person in charge of Storage: Shizuka Kouda 

January28, 1997 
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K01-1861 

TABLE OF CONTENTS 

SUMMARY 1 

MATERIALS AND METHODS 

I. TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 2 

2. BACTERIAL STRAINS 

3. MEDIUM AND S9 MIX 

4. PRE-CULTURES OF TEST STRAINS 

5. PREPARATION OF TEST SUBSTANCE AND POSITIVE 

4 

5 

6 

CONTROL SUBSTANCES ···················································· 6 

6. METHODS 7 

7. OBSERVATION AND MEASUREMENT ···························· 8 

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

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 8 

RESULTS 9 

CONCLUSION 9 

REFERENCES 9 

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

(/1 /£) 
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KOl-1861 

SUMMARY 

The mutagenicity of V-05H was examined in Salmonella typhimurium strains TAtOO, 

TA1535, TA98 and TA1537 and Escherichia coli strain WP2 uvrA using a preincubation method with and without metabolic activation system (S9 mix). 

The results showed that the number of the revertant colonies was less than two times 

that of negative control in all of the test strains with and without S9 mix. 

The number of revertant colonies in the negative and positive controls were confirmed 

to be within a range of the historical data in our laboratory. 

Based on the above results, V -05H was considered to have no reverse mutagenic 

potential under the condition the present study. 

f1 /£i 

-1-
f;/~5 
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Partition coefficient: -

Solubility: Soluble in oil 

Degree of solubility: 

Water: <5 w/v% (measured in our laboratory) 

DMSO: <5 w/v% (measured in our laboratory) 

Acetone: > 10 w/v% (measured in our laboratory) 

Others: 

8) Storage conditions 

Stored within a desiccator in a cold and dark place. 

9) Precautions 

KOl-1861 

Gloves, a mask, a head cap and a lab coat were worn. Hand-washing and 

gargling were carried out at the end of handling. 

1.2 Positive Control Substances 

1) 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) 

Manufacturer: 

Lot No.: 

Properties: 

Purity: 

Grade: 

2) Sodium azide (NaN3) 

Manufacturer: 

Lot No.: 

Properties: 

Purity: 

Wako Pure Chemical Industries, Ltd. 

LEN0571 

Reddish-:-orange crystalline powder 

99.5% 

Special grade 

W ako Pure Chemical Industries, Ltd. 

DLP2438 

White crystal 

99.4% 

Grade: Special grade 

3) 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine · 2HCI 

(ICR-191) 

Manufacturer: Polysciences, Inc. 

Lot No.: 412795 

Properties: Yell ow crystalline powder 

Purity: 

Grade: 

4) 2-Aminoanthracene (2AA) 

Manufacturer: 

Lot No.: 

Properties: 

Purity: 

Grade: 

Wako Pure Chemical Industries, Ltd. 

DLR7869 

Yellowish-green-brown powder 

95.7% 

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5) Storage conditions 

Stored in a cold and dark place. 

6) Precautions 

Gloves, a mask, a head cap and a lab coat were worn. 

2. BACTERIAL STRAINS 

2.1 Kinds of Bacterial Strains 

K01-1861 

Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537 were 

obtained from Dr. B. N. Ames, University of California, on June 20, 1990. 

Escherichia coli strain WP2 uvrA was obtained from Japan Bioassay Laboratoy on 

April6, 1995. 

S. typhimurium strains TAlOO, TA1535, and E. coli strain WP2 uvrA were used 

for detection of base-pair substitution mutagens, and S. typhimurium strains T A98 

and T A 153 7 for the detection of frameshift mutagens. 

2.2 Storage 

After 0.045 ml of dimethyl sulfoxide for spectrophotometric grade (DMSO; Lot 

No.CF103, Purity >99%, DOJINDO Laboratories) was added to 0.5 ml of the 

bacterial suspension, the mixture was stored at -80oC in an ultra-deep freezer 

(MDF-291, SANYO Electric Co., Ltd.) until use. 

2.3 Characteristics of Strains and Date of Characterization 

1) Characteristics of strains 

Mutations in 

amino acid 

Mutations in Mutation in R-factor 

Strains excision- membrane (pKM 101) 

synthesis genes repair genes 

Salmonella typhimurium 

TA1535 hisG46 l::::.uvrB rfa 

TA1537 hisC3076 l::::.uvrB rfa 

TA98 hisD3052 !::::.uvrB rfa + 

TAIOO hisG46 l::::.uvrB rfa + 

Escherichia coli 

WP2 uvrA trp l::::.uvrA + 

The amino acid requirement was determined as histidine for S. typhimurium 

strains and tryptophan for E. coli strain. The presence or absence ofR-factor was 

determined by ampicillin resistance, and mutations in membrane and DNA repair 

were determined by sensitivity to crystal violet, and UV, respectively. 

(fl 1~7 -4-
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K01-1861 

2) Date of characterization 

Strains Date of characterization 

Salmonella typhimurium TA1535 July 18, 1996 

TA1537 June 7, 1996 

TA98 June 7, 1996 

TA100 October 18, 1996 

Escherichia coli WP2 uvrA April 18, 1996 

3. MEDIUM AND S9 MIX 

3.1 Medium 

3.2 

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

2) 

1) 

An aliquot of 30 ml of medium consisting of the following components was 

poured into a plastic dish. 

Components Amount contained in one liter 

20xVogel-Bonner Minimum medium E 

40 w/v% Glucose 

50 ml 

50ml 

Agar 15 g 

(1) Agar: Bacto-Agar (Lot No. 90800JA, Difco Laboratories) 

(2) Date of manufacture: Dose-range-finding test: November 8, 1996 

Main test: November 25, 1996 

Soft agar 

A solution containing 0.5 mM histidine and 0.5 mM biotin for Salmonella 

typhimurium strains or 0.5 mM tryptophan for E. coli strain was added to a soft 

agar solution containing 0.6 w/v% agar (Bacto-Agar, Lot No. 71892AJB, Difco 

Laboratories) and 0.5 w/v% NaCl at a ratio of 1 : 10. 

S9mix 

Rat liver S9 (Kikkoman Co., Ltd.) 

Induction method: Phenobarbital (30 mg/kg x 1 time, 60 mg/kg x 3 times) 

and 5,6-benzoflavone (80 mg/kg x 1 time) were 

intraperitoneally administered to male 7weeks old SpragueDawley rats (body weight 194-232 g). 

Lot No.: RAA-352 (manufactured on September 27, 1996 and 

purchased on October 9, 1996) 

Storage conditions: Stored at -80 oC in an ultra-deep freezer (MDF-291, 

SANYO Electric Co., Ltd.). 

(// I G ~ -5-
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1 

2) Cofactor for S9 mix (Oriental Yeast Co, Ltd.) 

Lot No.: 999602 

Storage conditions: Stored at -20oC in a bio freezer 

(GS-2603, Nihon Freezer Co., Ltd.). 

3) Composition of S9 mix 

K01-1861 

One milliliter of S9 mix comprised 8 J.tmol MgCh, 33 J.tmol KCl, 5 J.tmol G-6-

p, 4 J.tmol NADPH, 4 J.tmol NADH, 100 J.tmol of sodium-phosphate buffer (pH 

7.4) and 0.1 ml ofS9. 

4. PRE-CULTURES OF TEST STRAINS 

After 20 J.tl of each working stock was inoculated to 10 ml of Nutrient Broth No. 2 

(OXOID Ltd., Lot No. OI6 572I2) in L tubes (27 ml volume), it was incubated at 37 

±0.5°C for 8 hours by shaking at 50 times/minute in a Monod shaker (MONOSINll A, Taitec Corporation). The number of viable cells at the end of the incubation, 

which was calculated from the value of O.D. at 660 nm measured by 

spectrophotometry (NOVASPEC, LKB Japan) was shown as follows. 

TA100 TA1535 WP2 uvrA TA98 TAI537 

Dose-range-finding test 2.1 3.1 2.7 1.2 2.4 

Main test 1.4 3.7 3.I 1.2 2.2 

( x I 09 viable cells/ml) 

5. PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROL 

SUBSTANCES 

5.1 Preparation ofthe Test Substance 

I) Preparation method 

The test substance was added to acetone (Lot No. DLP4532) and dissolved to 

make a I 0 w/v% solution for irradiating with ultrasonic wave for 5 min, and it 

was diluted with the same solvent to give each required concentration. 

2) Stability of test substance solution 

Denaturation of the test substance solution in color and exothermic reaction 

was not observed until 2 hours after preparation. 

3) Preparation time 

The test substance was prepared just before use and used within 0.5 hours. 

The test substance solutions were kept at room temperature until use. 

-6-

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KOl-1861 

5.2 Preparation of Positive Control Substances 

1) Preparation method 

NaN3 was dissolved in distilled water (Lot No. K6B74, Otsuka Pharmaceutical 

Factory Inc.). AF-2, ICR-191 and 2AA were dissolved in DMSO (Lot No. 

CF103). 

2) Preparation time and storage condition 

The working solutions were prepared every 3 months and stored at -80oC m an 

ultra-deep freezer (MDF-291, SANYO Electric Co., Ltd.). 

6. METHODS 

The test was carried out inS. typhimurium TA1535, TA1537, TA98 and TAIOO as 

well as E. coli WP2 uvrA using the pre-incubation method with and without a 

metabolic activation system. Three plates were used for the negative control group, 

and two plates for a dose of the test substance and for the positive control group. 

The test code number, presence or absence of S9 mix and dose levels were noted on 

the plates. 

6.1 Procedures 

After 0.05 ml of the test substance solution (0.1 ml of the positive control), 0.5 ml 

ofO.l M sodium phosphate buffer (pH 7.4) or S9 mix, and 0.1 ml of the bacterial 

culture were added to a test tube, and the mixtures was shaken for 20 min at 37± 

0.5°C. Two ml of the soft agar was then added to each tube, and the mixture 

poured onto a minimal glucose agar plate. After incubation at 3 7±0.5°C for 48 

hours, the number of revertant colonies was counted. 

In the sterility test, 0.1 ml of each bacterial culture, the test substance solution, S9 

mix and 0.1 M sodium phosphate buffer (pH 7.4) were smeared on a minimal 

glucose agar plate, which was incubated at 37±0SC for 48 hours. 

Acetone was used as a negative control, and the following positive controls were 

used corresponding to the tester strains. 

TA100 TA1535 WP2 uvrA TA98 TA1537 

AF-2 NaN3 AF-2 AF-2 ICR-191 

S9 mix(-) 0.01 0.5 0.01 0.1 

2AA 2AA 2AA 2AA 2AA 

S9 mix(+) 1 2 10 0.5 2 

(f.!g/plate) 

f? /70 

-7-
K01-1861 

6.2 Dose Selection 

1) Dose-range-finding test 

The test was carried out at seven dose levels consisted of the highest dose of 

5,000 f..lg/plate and 6 following doses of 1,000, 500, 100, 50, 10, and 5 f..lg/plate. 

2) Main test 

Based on the results of the dose-range-finding test, growth inhibition and 

increases in the revertant colonies were not observed. Therefore, a main test 

was carried out at 5,000 J..tg/plate as the highest dose and 4lower doses of2,500, 

1,250, 625, and 313 f..tg/plate diluted with a geometric progression of2. 

7. OBSERVATION AND MEASUREMENT 

7.1 Observation 

Appearance of revertant colonies (size and number of colonies), deposition of the 

test substance, and the growth inhibition were examined with a stereo microscope. 

7.2 Measurement 

The number of revertant colonies was counted with a manual counter or a colony 

analyzer (CA-9, Toyo~sokki Co., Ltd.). The number of revertant colonies 

obtained using a colony analyzer was corrected by a supplementary coefficient. 

Each plate was measured three times, and the average of these three measurements 

was adopted as the number of the revertant colonies on each plate. The mean 

value was calculated for each dose from the number of plates set up, and the 

decimal point rounded off. 

8. JUDGEMENT CRITERIA OF TEST RESULTS 

The test substance was judged positive when the number of revertant colonies 

increased two times or more than that of the negative control with a dose-dependent 

manner, and also when reproducibility of the test results was determined. In all 

other cases, it was judged negative. Statistical procedures were not applied. 

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 

There were no unexpected circumstances which might have affected the quality of the 

study and no deviation from protocol. 

01/71 

-8-
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K01-1861 

RESULTS 

The test results showed that the number of revertant colonies for all of the test strains 

was less than two times that in the negative control at any of the dose levels regardless 

of the presence or absence of S9 mix. 

Opaque oily deposition of the test substance were observed at more than 1,000 Jlg/plate 

with and without S9 mix, however, these did not interfere with measurement of the 

revertant colonies. 

The present . study was confirmed to be valid because the positive control substances 

induced mutations to the tester strains, and the number of revertants in the positive 

controls and the negative control was within the range of background data in our 

laboratories. The sterility test demonstrated that the test system was free from bacterial 

contamination. Therefore, there were no fluctuations which affected the test results. 

CONCLUSION 

In conclusion, V -05H was considered to have no reverse mutagenic potential under the 

present test conditions. 

REFERENCES 

1. Ministry of Labor (1991) Guidebook on Mutagenicity Tests using Micro-organisms, 

New Edition (in Japanese) published by Japan Industrial Safety and Health 

Association, 

2. Ministry of Health and Welfare (1992) Guideline for toxicity testing of chemicals, 

revised edition, published by the Chemical Daily Co., Ltd. 

3. Green M.H.L. and W.J. Muriel (1976) Mutagen testing using Trp+ reversion in 

Escherichia coli. Mutation Res., 38: 3-32. 

4. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella 

mutagenicity test. Mutation Res., 113: 173-215. 

vYJ 17Y 

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K01-1861 

Dose-range-finding test 

Test substance: V -05H 

With(+) or Test substance Number of revertants per plate 

without(-) dose Base-pair substitution type Frameshift type 

S9mix (1-!g/plate) TA 100 TA 1535 WP2 uvrA TA98 TA 1537 

Negative 115 8 21 31 13 

102 (115) 12 (11) 29 (24) 25 (29) 13 (13) control 127 12 22 30 13 

5 143 (134) 14 (11) 33 (32) 30 (32) 7 (8) 124 7 31 34 9 

10 143 (145) 13 (14) 27 (29) 36 (33) 13 (13) 146 14 31 29 13 

50 127 (131) 19 (18) 30 (32) 29 (30) 9 ( 9) -S9 mix 134 16 33 31 9 

100 113 (128) 11 (12) 29 (28) 24 (24) 19 (19) 143 13 27 24 18 

500 116 (129) 22 (18) 28 (26) 37 (33) 21 (20) 142 13 24 28 19 

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

• 5000 I19 (122) I4 (I2) 27 (28) 37 (37) IS (I4) I25 10 29 37 I2 

Negative I23 8 33 39 22 

control 122 (122) 14 (11) 33 (34) 29 (37) I8 (19) 

121 12 36 42 16 

5 II9 (130) 6 ( 9) 26 (28) 29 (34) 19 (18) I40 I1 30 39 16 

10 142 (147) 8 (1 0) 28 (31) 33 (34) 14 (16) 152 12 34 34 ' 18 

50 I29 (130) I2 (II) 35 (34) 47 (39) 25 (26) +S9 mix 130 IO 32 30 27 

100 138 (125) 11 (1 0) 32 (34) 39 (40) 25 (24) 1I2 9 35 40 23 

500 122 ( 13I) 11 (10) 37 (34) 39 (39) 22 (21) 140 8 3I 38 19 

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

• 5000 126 ( 135) 15 (13) 33 (33) 44 (43) 24 (23) I43 IO 33 41 22 

Chemical AF-2 NaN3 AF-2 AF-2 ICR-I91 

Positive control Dose O.OI 0.5 O.ot 0.1 I (Jlg/plate) -S9 mix Number of 505 303 (3I4) 111 (124) 55I (535) 2792 (521) (2804) revertants/plate 536 324 137 519 2815 

Chemical 2AA 2AA 2AA 2AA 2AA 

Positive control Dose 1 2 IO 0.5 2 

+S9 mix (!lg/plate) 

Number of 834 (833) 138 (144) 423 (448) 316 (307) 137 (145) revertants/plate 831 150 472 297 152 

[Notes] Parenthesis shows the mean of each plate. 

A: Deposition of test substance. 

• AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 

• NaN3: Sodium azide 

• ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine· 2HC1 

· 2AA: 2-Aminoanthracene 

-1- Pi !73 
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K01-1861 

Main test 

Test substance: V -05H 

With(+) or Test substance Number of revertants per plate 

without(-) dose Base-pair substitution type 

S9mix (Jlg/plate) TA IOO TA I535 

Negative I38 13 

131 (134) I6 (I5) control 133 I6 

313 166 (153) 10 (11) 139 12 

625 126 (130) 11 (15) -S9 mix 134 18 

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

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

... 5000 140 (138) 14 (I6) 135 18 

Negative 140 11 

129 (134) 14 (14) control 134 18 

313 120 (133) 16 (16) 145 16 

625 126 (130) I6 (13) +S9 mix 133 9 

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

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

... 5000 147 (137) 16 (17) 127 17 

Chemical AF-2 NaN3 

Positive control Dose 0.01 0.5 (J.l.g/plate) -S9 mix Number of 510 328 (326) revertants/plate 501 (506) 324 

Chemical 2AA 2AA 

Positive control Dose 1 2 

+S9 mix (Jlg/plate) 

Number of 770 (772) I57 (155) revertants/ {>late 774 I 53 

[Notes] Parenthesis shows the mean of each plate. 

A.: Deposition of test substance. 

• AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 

· NaN3: Sodium azide 

WP2 uvrA 

24 

20 (24) 

27 

25 (25) 25 

26 (27) 27 

30 (30) 29 

22 (22) 22 

19 (20) 21 

21 

28 (26) 

30 

27 (28) 28 

26 (27) 28 

22 (21) 19 

23 (21) 19 

30 (29) 27 

AF-2 

0.01 

109 (Ill) 113 

2AA 

10 

482 (537) 591 

Frameshift type 

TA 98 TA I537 

25 II 

27 (23) IO (12) 

18 14 

34 (30) 10 (13) 25 16 

23 (28) 16 (18) 32 19 

25 (23) 15 (15) 21 15 

25 (28) 16 (17) 30 17 

32 (29) 15 (13) 25 11 

30 25 

37 (38) 15 (20) 

46 19 

38 (38) 22 (28) 37 33 

39 (37) 22 (22) 34 22 

36 (40) 21 (24) 44 27 

39 (38) 34 (27) 37 19 

43 (45) 27 (26) 47 24 

AF-2 ICR-191 

0.1 1 

512 2671 

527 (520) 2566 (2619) 

2AA 2AA 

0.5. 2 

277 (273) 138 ·(142) 269 145 

• ICR-I91: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino ]acridine· 2HCI 

· 2AA: 2-Aminoanthracene 

- 2-
Dose-range-finding test K01-1861 

<ll 

....., 

cd 

.....-i 

150 

~ 100 <ll 

c:::>. 

"' <ll 

..... 

c: 

0 

.....-i 

0 

u 

....., 

c: 

~ 5 ~ > 

<1) 

~ 

C1J ...., 

cd 

...... 

Q. 

.... 

<1) 

Q. 

<lJ "' ..... 

g 

...... 

0 

u 

.._; 

;;; 

...., 

.... 

<1) 

> 

(!) 

0::: 

10 

50 

5 10 50 100 500 1000 

O:TA 100 

D:TA 1535 

..6.:WP2uvrA 

5000 

(J.Lg/plate) 

Fig. 1 Dose-response curve without S9 mix: Base-pair substitution type 

5 

Fig. 2 

10 50 100 500 1000 

O:TA 100 

D:TA 1535 

..6.:WP2uvrA 

5000 

(J.Lg/plate) 

Dose-response curve with S9 mix: Base-pair substitution type 

-3- f1 /7J 
( 

aJ 

....., 

a:l 

...-i 

0.. 

.... 

aJ 

0.. 

"' aJ 

...... 

§ 

...-i 

0 

<..> 

~ 

....., ~ 

.... 

OJ 

> aJ 

~ 

( 

aJ 

.... 

a:l 

....... 

0.. 

.... 

aJ 

c.. 

"' C1l 

...... 

c:: 

0 

....... 

0 

u 

....., 

c:: 

a:l 

....... 

.... 

C1l 

> 

(l) 

~ 

Dose-range-finding test KOl-1861 

40 

30 

20 

10 

5 

Fig. 3 

4 

Fig. 4 

10 50 100 500 1000 

O:TA 98 

D:TA 1537 

5000 

(.ug/pl at e) 

Dose-response curve without S9 mix: Frameshift type 

10 50 100 500 1000 

Dose-response curve with S9 mix: Frameshift type 

-4-

5000 

(Jlg/p 1 at e) 

f7 17L 

O:TA 98 

D:TA 1537 ' 
CJ 

...., 

<d 

....... 

0.. 

t... 

C1J 

0.. 

Cll 

C1J 

...... 

c:: 

0 

....... 

0 

(.J 

...., 

...., ~ 

t... 

C1J 

> 

(l) 

c:: 

Q) 

...., 

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....... 

0.. 

Q) 

0.. 

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Q) 

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§ 

....... 

0 

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...., 

c:: 

<d ...., 

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> 

C) 

= 

Main test K01-1861 

zoo O:TA 100 

D:TA 1535 

.6.;\'IPZuvrA 

150 

100 

50 

0·~----.---------~----------~--------~~--------~ 

313 625 1250 2500 5000 . (J.Lg/plate) Dose-response curve without S9 mix: Base-pair substitution type 

150 

100 

50 

Fig. 5 

O:TA 100. 

D:TA 1535 

.6.;\'IP2uvrA 

0~----.-----------~--------~----------~----------. 

313 625 1250 2500 5000 

(.ug/plate) 

Fig. 6 Dose-response curve with S9 mix: Base-pair substitution type · 

-5- ~1 /77 
Cl.) 

....., 

cd 

.-t 

c. 

'-

Cl.) 

c. 

Cll 

Cl.) 

...... 

::::: 

0 

.-t 

0 

c.J 

....., 

c::: 

cd 

....., 

'-

Cl.) 

:> 

Cl.) 

~ 

Cl.) 

....., 

cd 

...-< 

0.. 

Cl.) 

c.. 

Cll 

Cl.) 

...... 

5 

....... 

0 

c.J 

....... 

c::: 

cd 

....., 

Cl.) 

:> 

Cl.) 

c::: 

Main test 

30 

20 

10 

313 

Fig. 7 

40 

30 

20 

10 

625 1250 2500 

Dose-response curve without S9 mix: Frameshift type 

K01-1861 

O:TA 98 

D:TA 1537 

5000 

(JLg/plate) 

O:TA 98 

D:TA 1537 

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

313 625 1250 2500 

Fig. 8 Dose-response curve with S9 mix: Frameshift type 

-6-

5000 

(JLg/p 1 at e) 

f7 /7~ 
( 

( 

STUDY CODE: K06-0612 

FINAL REPORT 

Receipt No. T97-2707 

Report No. T-5194 

CHROMOSOMAL ABERRATION TEST OF 

V-OSH 

USING CULTURED MAMMALIAN CELLS 

March 1998 

Hita Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & Testing Institute 

Japan 
( 

/ 

\~ 

STATEMENT 

I, the undersigned, hereby declare that this report provides a correct English translation 

of the final report (Study Code K06-0612, issued on March 23, 1998). 

Chemicals Inspection & Testing Institute,Japan (CITI) formally changed its name to 

Chemicals Evaluation and Research Institute,Japan (CERI) as on November 1, 1999. 

The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, 

Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to 

"NISSHINBO INDUSTRIES, INC. R & D CENTER" and "1-2-3, Onodai, Midori-ku 

Chiba, 267-0056, Japan", respectively by Sponsor's request. 

CAS No.157299-02-0 of test substance was added by Sponsor's request. 

In the structural formula, n=2-800 was added. 

Molecular weight of 3,000 was changed to 30,000. 

Wakamatsu Shinya, M.S. 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

b /~ / 2.0l72 

Date 

f1 ( ~() 

~~-
( 

( 

QUALITY ASSURANCE STATEMENT 

Hita Laboratory, 

Chemicals Evaluation and Research Institute, Japan 

Sponsor: NISSHINBO INDUSTRIES, INC. R&D CENTER 

Title: Chromosomal Aberration Test of V-OSH Using Cultured Mammalian Cells 

Study code: K06-0612 

This report was audited by the Quality Assurance Section. 

I, the undersigned, hereby declare that this report reflects the 

original Japanese report. 

(Date) J~ l~, ..2..o~2. -;:;:)\ 

....... - . ,/, ~ . (Signature) ~~--~ Section Chief, Quality Assurance 

Keiji Shiraishi, B.S. 

fr l1l 

Tl 
Sponsor: 

Title: 

GLPSTATEMENT 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

NISSHINBO INDUSTRIES, INC. R & D CENTER 

Chromosomal Aberration Test of V -05H Using Cultured Mammalian 

Cells 

Study Code: K06-0612 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Concerning Testing Facilities Stipulated in Article 4 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 (EA), No. 229 of the Pharmaceutical 

Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 85 (1984) of the Basic 

Industries Bureau, Ministry of International Trade and Industry (MITI), March 31, 1984; 

Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the 

Environmental Health Bureau, MHW & No. 823 (1988) of the Basic Industries Bureau, 

MITI, revised on November 18, 1988) and with "Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice" (May 12, 

1981). 

Management: Signed in original March 23, 1998 

Yoshifumi Fujino, M.S. 
I 

\ 

QUALITY ASSURANCE STATEMENT 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

Sponsor: NISSHINBO INDUSTRIES, INC. R & D CENTER 

Title: Chromosomal Aberration Test of V -05H Using Cultured Mammalian 

Cells 

Study Code: K06-0612 

This study was audited and inspected by Quality Assurance Section of Hita Research 

Laboratories, Chemical Biotesting Center, Chemicals Inspection & Testing Institute, 

Japan. The dates audited and/or inspected and the dates reported these results to the 

study director and management are as follows. 

Dates of Dates of Reports to Dates of Reports to 

Inspections/ Audits Study Director Management 

December 11, 1997 December 12, 1997 December 12, 1997 

January 12, 1998 January 12, 1998 January 12, 1998 

January21, 1998 January 22, 1998 January 22, 1998 

March 4, 1998 March 4, 1998 March 4, 1998 

March 9, 1998 March 9, 1998 March 9, 1998 

March 17, 1998 March 17,1998 March 17,1998 

March 23, 1998 March 23, 1998 March 23, 1998 

I, the undersigned, hereby declare that this report provides an accurate description 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 March 23, 1998 

Keiji Shiraishi, B.S. 

f1 (<6} 
Study Code: K06-0612. 

Test Substance Code: HR3539 

Sponsor Code: N-680 

TITLE 

Chromosomal Aberration Test ofV-05H Using Cultured Mammalian Cells 

SPONSOR 

NISSHINBO INDUSTRIES, INC. R & D CENTER 

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

TESTING FACILITY 

Rita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

822, 3-chome, Ishii-machi, Hita, Oita, 877-0061, Japan 

PURPOSE OF STUDY 

The ability of the test substance to induce chromosome aberration is investigated 

using Chinese hamster lung fibroblast cells (CHL cells). 

TESTING METHOD 

This study was conducted in accordance with "Notification on Partial Revision of 

Testing Methods Relating to the New Chemical Substances (Notification No. 700 of 

the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 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)". 

GLP COMPLIANCE 

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) ofthe Basic Industries Bureau, MITI, March 31, 1984; Notification No. 

233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental 

Health Bureau, MHW & No. 823 (1988) of the Basic Industries Bureau, MITI, 

revised on November 18, 1988) and with "Organisation for Economic Co-operation 
( 

/ 

\ 

and Development Principles of Good Laboratory Practice" (May 12, 1981). 

PERJOD OF STUDY 

Commencement of Study: 

Initiation of Preliminary Test: 

Completion of Observation: 

Completion of Study: 

December 11, 1997 

December 17, 1997 

March 13, 1998 

March 23, 1998 

LOCATION AND PERJOD FOR RETENTION OF RAW DATA AND SPECIMENS 

Data and specimens, and the test substance of this study will be retained in the 

archives and the test substance storage room, respectively, of Hita Reseach 

Laboratories for 10 years following the date of completion of study. After 

termination of the retention period, any measures taken will be done with the 

approval of the Sponsor. However, specimens that are liable to deteriorate 

markedly will be retained only for the period the quality of specimens permits 

evaluation, and the abandonment ofthem will be taken with approval of the sponsor. 

AUTHOR AND PERSONNEL CONCERNED WITH STUDY 

AUTHOR 

Study Director: Signed in original 

Nahoko Yoshida, B.S. 

Hita Research Laboratories 

Mutagenicity Section 

March 23, 1998 
( TABLE OF CONTENTS 

SUMMARY 

MATERIALS AND METHODS 

1. TEST SUBSTANCE AND POSITIVE CONTROL 

SUBSTANCES 

2. CELLS AND MEDIUM 

3. S9MIX 

4. CELL CULTURE ................................................... . 

5. PREPARATION OF THE TEST SUBSTANCE 

1 

2 

4 

4 

5 

AND THE POSITIVE CONTROL SUBSTANCES ················ 5 

6. PREPARATION OF SPECIMENS ················ 5 

7. DETERMINATION OF DOSE LEVELS ················ 6 

8. CHROMOSOMAL ABERRATION TEST ················ 6 

9. OBSERVATION AND SCORING ················ 7 

10. JUDGEMENT CRITERIA OF RESULTS ················ 8 

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 8 

TEST RESULTS 

1. CELL GROWTH INHIBITION TEST 

AND CELL DIVISION INHIBITION TEST 

2. CHROMOSOMAL ABERRATION TEST 

CONCLUSION 

REFERENCES 

TABLES AND FIGURES 

8 

9 

10 

10 

1-6 

T I 
I 

\_ 

( 

SUMMARY 

The ability of V -05H to induce the chromosomal aberration was investigated using 

Chinese hamster lung fibroblast cells (CHL cells) without S9 mix (direct method) and 

with S9 mix (metabolic activation method). 

From the results of the cell growth inhibition test and the cell division inhibition test, 

chromosomal aberration test was carried out at 156, 313 and 625 f.!g/ml for the 24, 48 htreatment group by the direct method and the 18 h-recovery group without S9 mix, and 

at 1,250, 2,500 and 5,000 f.lg/ml for the 18 h-recovery group with S9 mix by the 

metabolic activation method. As the result, the test substance induced no 

chromosomal aberrations within the dose range, which included the doses producing 

cytotoxicity in the direct method and the metabolic activation method. On the other 

hand, Mitomycin C and Cyclophosphamide monohyderate obviously induced 

chromosomal aberrations in positive control groups. 

Based on the above results, it is concluded that V -05H induce no chromosomal 

aberration with and without metabolic activation under the conditions of the present 

study. 

-1-
T. 
( 

( 

\ 

Melting point: 

Boiling point: 

. Vapor pressure: 

Partition coefficient: -

Solubility: Soluble in oil 

Degree of solubility: Water: <50 mg/mL(measured in our laboratory) 

8) Storage conditions 

DMSO: <50 mg/mL(measured in our laboratory) 

Acetone: > 500 mg/mL(measured in our laboratory) 

Others: 

Stored in the sealed container at the cold and dark place. The hightemperature and humidity was avoided. 

9) Precautions 

Goggles, gloves, a mask, a head cap and a lab coat were worn. Hand-washing 

and gargling were carried out at the end of handling. 

1.2 Positive Control Substances 

1) Mitomycin C (MMC) 

Manufacturer: 

Lot No.: 

Appearance: 

Composition: 

Kyowa Hakko Kogyo Co., Ltd. 

158AGB 

royal purple crystals or crystalline powder 

It includes 2 mg (titer) of Japanese Pharmacopoeia 

(J.P.) MMC and 48 mg of J.P. sodium chloride in 

one vial 

2) Cyclophosphamide monohydrate (CPA) 

Manufacturer: 

Lot No.: 

Appearance: 

Purity: 

3) Storage conditions 

Wako Pure Chemical Industries, Ltd. 

PTL7433 and LEE7264 

white crystalline powder 

98.8% and 98.9% 

MMC and CPA were stored at room temperature and at a cold and dark place, 

respectively. 

4) Precautions 

Gloves, a mask, a head cap and a lab coat were worn in handling. 

- 3 - fl t~r 
( 

2. CELLS AND MEDIUM 

2.1 Cell Line and Reason for Selection 

Chinese hamster lung fibroblasts (CHL cells, clone No. 11), which were supplied 

by the National Institute of Health Science (old name: National Institute of 

Hygienic Sciences) on September 28, 1988, were used. The modal number of 

chromosomes is 25, and the doubling time is about 15 hours. The passage 

number was 18 at receipt. Cells were used at 39 passage number in the 

chromosomal aberration test. 

CHL cells are widely used in in vitro chromosomal aberration tests because they . 

have quite high sensitivity to chemical mutagens and a large amount of back 

ground data available. 

2.2 Medium and Water 

Eagle's minimum essential medium (Lot No. 388706 and 398711, NISSUI 

PHARMACEUTICAL CO., LTD.) supplemented with newborn calf serum (Lot 

No. NBQ07, MITSUBISHI CHEMICAL CORPORATION) at a rate of 10 v/v% 

was used, which is referred to hereafter as 10% NCS/MEM. 

The medium was prepared using distilled water obtained with a glass still 

(WG220, YAMATO SCIENTIFIC CO., LTD.). 

3. S9 MIX 

3.1 Rat Liver S9 (Kikkoman Corporation) 

Induction method: Male Sprague-Dawley rats aged 7 weeks old (Body weight; 

RAA-371:205~244 g, RAA-372:186~225 g) were 

Lot No.: 

intraperitoneally administrated phenobarbital (30 mg/kgx 1 

time, 60 mg/kgx3 times) and 5,6-benzoflavone (80 mg/kgx1 

time). 

RAA-371 (date manufactured: October 24, 1997; date 

purchased: November 12, 1997) and RAA-372 (date 

manufactured: October 31, 1997; date purchased: December 3, 

1997) 

Storage condition: Stored at -80°C (Deep freezer, ULT-1285, REVCO CO., 

LTD.) 

3.2 Composition ofS9 mix 

One milliliter of S9 mix consists of 4 J.tmol of HEPES (pH 7 .2), 5 J.tmol of MgCh, 

33 J.tmol ofKCl, 5 J.tmol ofG-6-P, 4 J.tmol ofNADP and 0.3 ml ofS9. 

3.3 S9 mix/MEM 

A halfmL ofS9 Mix was added to 2.5 mL of 10% NCS/MEM. 

- 4-
I 

( 

( 

4. CELL CULTURE 

4 Cells suspension of 1.5x104 or 0.5x10 cells/mL was seeded five milliliter of 10% 

NCS/MEM in a 60 mm diameter Petri dish (IWAKI GLASS CO., LTD.). 

Monolayer cells in logarithmic growth phase after cultivation for 2 or 3 _days were 

applied to the tests. 

Cells were cultured in a C02 incubator, which was set at 37±0.5°C, almost 100% 

humidity and 5% C02 atmosphere_ 

5. PREPARATION OF THE TEST SUBSTANCE AND THE POSITIVE CONTROL 

SUBSTANCES 

5.1 

1) 

Test Substance 

Preparation 

Acetone (Lot No. TPN6909, Wako Pure Chemical Industries, Ltd.) was 

selected as a solvent, because degree of solubility of the test substance was less 

than 50 mg/ml in distilled water and DMSO and more than 500 mg/ml in 

acetone. The test substance was dissolved in acetone to make the original 

solution. The original solution was diluted with the same solvent to make the 

required concentrations. 

2) Preparation time 

Test substance solution was freshly prepared just before use, and it was used 

within 2 hours. 

5.2 Added Amount of Test Substance Solution 

5.3 

The test substance solution was added to the medium to become 1%. 

Positive Control Substances 

1) Preparation 

Positive control substances were dissolved in distilled water. 

2) Preparation time 

Positive control substance solutions were freshly prepared just before use, and 

they were used within 2 hours. 

6. PREPARATION OF SPECIMENS 

0.075 M KCl was added to the cells, which were removed from each dish with 2 ml 

of 0.25% trypsin and collected by centrifugation, and hypotonic treatment was 

carried out at 37°C for 15 min. Cell suspension fixed by Carnoy's solution 

(methanol: acetic acid=3:1) was dropped onto microscope slides and dried, and then 

stained with 2% Giemsa solution. One specimen and four specimens per dose (two 

specimens per dish) were made in the cell division inhibition test and the 

chromosomal aberration test, respectively. 

- 5-

---~-=~~_..-.-.. .. -=..--- -=--~-- .... --...---~-··"~' ... _..... ----, 
7. DETERMINATION OF DOSE LEVELS 

The 50% growth inhibition concentration of the test substance was determined by 

the cell growth inhibition test. The highest dose, at which chromosome was 

observed, was determined by the cell division inhibition test. A concentration 

equivalent to 5 mg/ml was employed as the maximum dose when no cytotoxicity 

was observed. 

7.1 Cell Growth Inhibition Test 

For the treatment by the direct method, the cells in logarithmic growth phase were 

treated with 5 ml of the 10% NCS/MEM including the test substance solution for 

24 and 48 hours. 

For the treatment by the metabolic activation method; the cells were treated with 3 

ml of 10% NCS/MEM (the treatment group without S9 mix) or S9 mix/MEM (the 

treatment group with S9 mix) including the test substance solution for 6 hours. 

The medium was removed after treatment. The cells were rinsed three times 

with 2 ml of Ca2

+, Mg2

+-free phosphate-buffered saline [PBS(-)], and 

subsequently the cells were cultured with 5 mL of 10% NCS/MEM for 18 hours 

on the group with and without S9 mix. 

Colcemid was added to the medium to give a final concentration of 0.1 Jlg/ml 2 

hours before the end of incubation in either treatment method. 

After removal of the medium at the end of incubation, the cells were detached 

with 2 ml of 0.25% trypsin and the number of cells was counted using a Microcell 

Counter (F-500, Toa Medical Electronics Co., Ltd.) to obtain cell growth rate(%). 

The medium was changed 2 hours before the end of incubation because 

precipitation ofthe test substance prevented observing specimens. 

7.2 Cell Division Inhibition Test 

Specimens of cells were made which were used in the cell growth inhibition test 

and dividing cells were observed. Chromosome aberration was observed 

preliminarily. 

8. CHROMOSOMAL ABERRATION TEST 

8.1 Dose Levels 

Test substance: Three dose levels were made by a geometric progression of 2, 

and limit was maximum dose from the result of the cell growth 

inhibition test and the cell division inhibition test. 

-6-
( 

'· 

Positive control substances: The concentrations of the positive control substances 

were selected as follows according to our 

background data. 

8.2 Method 

Direct method: MMC 0.05 )!g/ml 

Metabolic activation method: 

Without S9 mix: MMC 0.1 )!g/ml 

With S9 mix: CPA 10 )!g/ml 

Cultures and treatment of cells were done and specimens were made by the same 

procedure as that in the cell growth inhibition test. 

A solvent-treated group was set in a negative control, and MMC or CPA-treated 

groups were set in a positive control. 

8.3 Number of Dishes and Identification 

Two dishes were used for each dose. The study code number, treatment method 

and dose level were written on the dishes. 

9. OBSERVATION AND SCORING 

The number of cells with structural aberrations of chromatid or chromosome (such 

as gaps, breaks or exchanges) and numerical aberrations (polyploid, endoreduplication) were examined by observing 100 metaphases for each dish. An 

aberration cell was defined had structural or numerical aberrations. Two hundreds 

of metaphases for each group were observed and incidence of aberration cells were 

calculated. 

The incidence of structural aberration with or without gaps (larger than a width and 

less than twice width of chromatid) was calculated. 

All specimens were coded and observed in a blind manner. 

- 7-
10. JUDGEMENT CRITERIA OF RESULTS 

Structural aberrations (including gaps) and numerical aberrations were judged as 

follows according to their incidences: negative (-) in less than 5%; falsely positive 

(±)in 5% or more but less than 10%; and positive(+-+++) in 10% or more. The 

positives were subdivided into ( +) for an incidence in 10% or more but less than 

20%; (++)in 20% or more but less than 50%; and(+++) in 50% or more. The test 

substance was judged to be positive for the induction of chromosomal aberrations 

was dose-related or reproducible. 

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 

There were no unexpected situations which might have affected the test results and no 

deviation from protocol. 

TEST RESULTS 

The incidence of aberrant cells was not markedly different between two dishes. The 

incidence of cells with aberration did not exceed 5% in the negative control. The 

incidence of cells with structural aberrations excluding gap was 1 0% oi more in the 

positive control. There were no fluctuations in the test conditions. The tests were 

therefore considered to be valid. 

1. CELL GROWTH INHIBITION TEST AND CELL DIVISION INHIBITION TEST 

(Table 1, and Fig. 1) 

The cell growth inhibition test and the cell division inhibition test were performed at 

doses shown in Tables 1. In the cell growth inhibition test, 50% cell growth 

inhibition concentrations of the test substance were about 630 J.lg/ml in 24 hours 

treatment group, and about 400 Jlglml in 48 hours treatment group by the direct 

method, as 100% in the solvent control. However, the 50% cell growth inhibition 

concentration was not able to obtained even at 5,000 J.lg/ml with S9 mix because of 

the weak cytotoxicity of the test substance. In the examination used acetone as a 

solvent, the cell growth rate was the lowest at 625 J.tg/ml, and increased above 1,250 

J.lg/rnL because of the bad suspension of the test substance in the medium by the 

direct method or without S9 mix by the metabolic activation method. Therefore, 

dose determination test was performed to used 0.5% sodium carboxymethyl 

cellulose (CMC) solution as a solvent. However, cell growth inhibition and 

division inhibition were not observed all the doses, because of the bad suspension of 

the test substance in the medium. Based upon the above results, acetone was 

- 8-
I 

1 

! 

( 

( 

( 

\, 

selected as the solvent. The results of the observation of specimens, increase 

remarkable the chromosomal aberrations were not observed at all doses by either the 

direct method or the metabolic activation method. The maximum dose was 625 

J.lg/ml in the direct method and without S9 mix by the metabolic activation method, 

5,000 J.lg/ml in S9 mix in the chromosomal aberration test. Based upon the above 

results, the following 3 dose levels were set in the chromosomal aberration test. 

24 hours treatment by the direct method: 156, 313 and 625 Jlg/ml 

48 hours treatment by the direct method: 156, 313 and 625 Jlg/ml 

Without S9 mix by the metabolic activation method: 156, 313 and 625 J.lg/ml 

With S9 mix by the metabolic activation method: 1 ,250, 2,500 and 5,000 Jlg/ml 

2. CHROMOSOMAL ABERRATION TEST 

2.1 Direct Method (Table 2 and Fig. 2) 

1) 24 hours treatment 

2) 

The incidence of cells with structural aberrations include gap was 0.5, 0.5 and 

1.0% at 156, 313, 625 J.lg/ml, respectively. On the other hand, the incidence 

in the solvent control was 2.5%, which was within the normal range, and the 

positive with MMC induced the structural aberration at 57.0%. 

The incidence of numerical aberration was less than 5% in all treatment groups. 

48 hours treatment 

The incidence ofcells with structural aberrations include gap was 1.0, 0.5 and 

1.0% at 156, 313, 625 J.lg/ml, respectively. On the other hand, the incidence 

in the solvent control was 1.0%, which was within the normal range, and the 

positive with MMC induced the structural aberration at 55.0%. 

The incidence of numerical aberration was less than 5% in all treatment groups. 

- 9- (/1 /1> 
1. 

2.2 Metabolic Activation Method (Table 3 and Fig. 3) 

1) Without S9 mix 

The incidence of cells with structural aberrations include gap was 1.0, 1.0 and 

2.0% at 156, 313, 625 ~g/ml, respectively. On the other hand, the incidence 

in the solvent control was 0.5%, which was within the normal range, and the 

positive with MMC induced the structural aberration at 36.5%. 

The incidence of numerical aberration was less than 5% in all treatment groups. 

2) With S9 mix 

The incidence of cells with structural aberrations include gap was 0.5, 1.0 and 

1.0% at 1,250, 2,500, 5,000 ~g/ml, respectively. On the other hand, the 

incidence in the solvent control was 0.5%, which was within the normal range, 

and the positive with CPA induced the structural aberration at 88.0%. 

The incidence of numerical aberration was less than 5% in all treatment groups. 

CONCLUSION 

In the chromosomal aberration test using CHL cells, V -05H induced no structural 

aberration or numerical aberration within the dose range including the cytotoxicity by 

either the direct method or the metabolic activation method. 

It is concluded that V -05H induce no chromosomal aberration in the presence or 

absence of metabolic activation under the present test conditions. 

REFERENCES 

1. Ministry of Health and Welfare (1992) Guideline for toxicity testing of chemicals, 

revised edition. published by the Chemical Daily Co., Ltd. 

2. Ishidate, M.J r. ( 1979) Chromosomal aberration test in vitro for chemical mutagens 

in our environment (in Japanese). Tissue Culture, 5:115-122. 

3. Ishidate, M.Jr. ( ed.) (1987) Data book of chromosomal aberration test in vitro. L.I.C. 

Inc., Tokyo. 

4. Japan Environmental Mutagenicity Society/Mammalian Mutagenicity Study Group 

(ed.) (1988) Atlas of chromosomal aberration induced with chemical substance (in 

Japanese). Asakura Publishing Co., Tokyo. 

5. Yoshida, M. (1982) Shape of chromosome and chromosomal aberration (in 

Japanese). Metabolism, 19:427-438. 

- 10 -

- T Lo0<• • -- =--·---···-~ -...... -- --<> ........ ,.-.---....,..,-~, .......... ----·---· ' 0 
i 

\. 

6. Yoshida, M. (1983) Chromosome analysis in cultured cells (in Japanese). Cell 

Technology, 2:1352-1360. 

- 11 -
_j 

I 

l 

l 

I 

\ 

1 I 

l 

J 

I 

j 

~ 

-...... 

~ 

Table I Results ofthe dose determination test ofV-OSH 

Concentration of the test substance(l!g!ml) 

24 hours 

treatment 

Direct 

method 

48 hours 

treatment 

without S9 mix 

Metabolic 

activation 

method 

with S9 mix 

---- -- ---- --- -

*I Mean value for two dishes. 

*2 Judgement 

Growth rate 'I(%) 

Index amount of mitotic metaphase'2 

Chromosomal numerical 

aberrations •J (%) structural 

Concentration of the test substance(J!g/ml) 

Growth rate 'I (%) 

Index amount ofmitotic metaphase'2 

Chromosomal numerical 

aberrations'3(%) structural 

Concentration of the test substance(J!g/ml) 

Growth rate 'I (%) 

Index amount of mitotic metaphase '2 

Chromosomal numerical 

aberrations '

1(%) structural 

Concentration of the t~st substance(J-Lg/ml) 

Growth rate ' 1 (%} 

Index amount of mitotic metaphase '2 

Chromosomal numerical 

aberrations '

1(%) structural 

++ : abundant, + : a few • ± : rare • - : none 

*3 50 cells/dose were observed. 

*Precipitation of the test substance was observed in culture medium. 

0 313 

100 85.3 

++ + 

0 0 

4 2 

0 313 

100 65.3 

++ + 

2 0 

0 0 

0 313 

100 91.1 

++ ++ 

2 0 

2 0 

0 313 

100 80.4 

++ ++ 

0 0 

0 0 -

* 625 * * * 

1,250 2,500 5,000 

50.2 72.1 83.3 90.8 

+ + ++ ++ 

0 2 0 2 

4 2 4 0 

* * * * 

625 1,250 2,500 5,000 

16.1 3D.4 54.0 82.9 

+ + ++ ++ 

2 2 0 0 

0 4 0 0 

* * * * 

625 1,250 2,500 5,000 

74.5 81.3 85.7 91.1 

++ ++ ++ ++ 

0 4 0 0 

2 2" 0 0 

* * 625 1,250 * * 

2,500 5,000 

65.1 72.3 69.0 59.6 

++ ++ ++ ++ 

0 0 2 0 

0 0 0 0 
• 

I 

l 

_J 

j 

I l l 

N I 

l 

j I 

-l 

l 

1 

l 

-1 

~ 

.........._ l 

~ - \ 

l 

I 

\ 

Name of test substance: Y-05H Table 2 Results of chromosomal aberration test (Without metabolic activat

I 

I 

Treatment Concent- Number of cells umber and the percenta1esl%l ·of cells shmdn2 s

Treatment time(h) ration Observed Number of Judge- Gap Chromatid-type Chromosome-t }'pe 

(~g/ml) IDolypJoids ment 

g ctb cte csb cse 100 0 

0 3 n ·o 0 

24 0 lllO 0 0 __l I 0 0 

Solvent 200 0( 0. 0 Of 0. 0 ....ti 2.0 II IU 0( 0 .{) 0( 0.0 

(Acetone) tOO 0 0 I 0 0 0 

48 0 100 t' 

200 l ( 0. 5 0( 0 0 0 I 0 

100 0 0 0 I( 0,5 0 0.0 t( 0.5 0( 0.0 

0 0 I -0 156 * Too 0 - 0 

200 0( 0.0 0 0 0 0 

100 0 Q Of 0.0 OJ 0 0 I 0.5 01 0. 0 0( 0.0 

_Q 0 0 0 

24 JIJ * 100 0 - 0 I 0 0 0 

-filo100 0 01 0.0 ot 0 o.o· I ( 0. 5 ol o.o O( 0.0 0( 0.0 

625 * 100 0 - 1 c 0 0 

0 __Q 0 0 

Test 200 or o· o 0( 0.0 t ( 0 5 ( 0.5 0( 0.0 0( 0. 0 

Substance 

156 * 100 

100 

2 

I - 0 0 0 0 0 

2oo 31 1.5 0 0 0 I 

100 2 

01 0. 0 01 0 0 t I 0 5 O( 0.0 I ( 0. 5 

48 Jl:l* tOO 0 - . 0 0 0 0 0 0 

Jl. 0 n I 

20o 2 f 1.0 

100 t 0( o. 0.0 0( 0.0 . 0( 0.0 O( 0.0 l ( 0. 5 

625 ' 100 2 - _1 0 0 0 0 

200 31 .1.5 0 0 0 0 

100 0 Zl 1.0 nt JL_O_ O( 0.0 O( 0. 0 0( 0._0_ 

24 0.05 100 0 - t 13 40 0 0 

I a 53 0 I 

Positive loo Of 0 .o 2( 1.0 3U JB ._i 93 ( 4 6 5 O( 0.0 I ( 0. 5 

control 100 I 0 18 56 0 1 

(HHC) 48 0.05 

200 

tOO 0 

t ( 0.5 - I 26 34 0 I 

-g: 

l ( 0 5 44( 22.0 01 4' .0 0( 0.0 2( I. 0 

ctb: 

total 

chromatid 

number 

break 

and the 

cte: 

percentage 

chromatid 

of 

exchange 

cells except those which has only gaps tg: total number and the percentage of cells 

H/>IC: Hitomycin C csb: chromosome break cse: chromosome exchange Others: fragmentation e

t : Precipitation of the test substance. 
/~·-., 

w 

I 

i I 

I 

-l 

l 

\ 

~ l ~· 

I 

~ I 

: 

I 

I 

/--- 

Name of test substance: V-05H Table 3 Result~ of chromosomal aberration te~t (With metabolic activation) 

I 

Concent- Number of cells Number and the oercentaeesl%1 or eel s showinr sc

Treatment S9 mix ration Observed Number of Judge- Gap Chromatid-type Chromosome-type 

(!Jg/ml) jpolyploids ment 

100 I g ctb cte csb cse 

0 I 0 0 0 

- 0 100 0 0 0 0 0 0 

Solvent 200 II 0. 5 0 ( _0_. Q_ H _0._5_ Ol o o ol o o . 01 0.0 

(Acetone) 100 0 I 2 1 0 (} 

t 0 100 I 0 1 0 . 0 0 . 200 II 0. 5 

100 0 0 It 0 5 31 u I l 0. 5 . 01 0. 0 01 0 0 

2 0 -0 156 * 100 I - 0 0 

200 I ( 0. 5 O( 0.0 0 0 0 0 

100 0 0 2 ( I 0 01 O.o Ol 0 0 ot o:O 

0 I 0 0 

- 313 * 100 0 - 0 I 0 0 0 

200 01 0 0 Ot 

100 0 0 0 H 0. 5 l ( 0 5 01 (} .11 (}( 0 '0 

625 * 100 0 - 0 2 0 0 0 

0 I I 0 0 

res l 200 01 0 0 01 . 0.0 3l 1,5_ If II. 5 OT -oll or ·-o.o 

Substance 100 0 ' 

I, 250 * 100 0 I 0 0 0 

l . 

200 I ( 0. 5 0 0 0 0 0 

100 0 0( 0.0 II 0. 5 ot o:OC OT olf 6{ 0.0 

0 l 0 0 0 

t 2' 500 j 100 0 - 0 1 0 0 0 

200 01 0.0 

10 0 0( 0.0 2( 1.0 01 0. 0 Ol 0 0 Ol 0.0 

5. 000 * 10 I - 0 . _0_ 0 0 0 

201 II 0. 5 0 ·~ 0 0 0 

_Ot (LO H J.O 0( 0. 0 0( 0 0 0( 0 0 

Positive· 10 0 J Jl 31\ . I 

cont~ll - 0 .I 10 0 - 0 

0 6 32 ·o 0 

IHHC 20 0( 0 0 I l 0 5 IS( 9.0 62T11:0 11 o:s ot o o 

Positive 100 0 

cont~\1 

l 62 79 0 0 

t 10 100 0 . - 0 28 81 0 0 

I CPA 200 or o.o If L5 90f 45.0 I R?.{ Rl 0 · Of 0 0 01 o-n 

Amount 

of S9(5%), Treatment time of the test substance(6h), Recoven• time of cells after treatment of the test substanc·e(l8h

ctb: 

-g: total 

chromatid 

number 

break 

and the 

cte: 

percentage 

chromatid 

of 

exchange 

cells except those which has only gaps +g: total number and the percentage of cells 

~~C: Mitomycin C csb: chromosome break cse: chromosome exchange Others: fragmentation et

CPA: Cyclophosphamide, monohydrate 

• : Precipitati~h of the test substance. 
l ~ I 

l ~ i 

-- j I 

' 

I 

i 

..p.. 

I 

~ 

~ 

~ 

~ 

,.-_ 

Q ~ '--" 

Q) 

...... ~ I- ~ ...... ~ 0 

I0 

100 

50 

100 

,.-_ ~ Q 

'--" 

Q) 

...... ~ I- 50 ~ ...... ~ 0 

I0 

-----" 

10 100 1,000 10,000 

Concentration of the test substance ( J.l g/ml) 

24 hours treatment by the direct method 

10 100 1,000 10,00 

Concentration of the test substance ( J.l g/ml) 

48 hours treatment by the direct method 

.-~ " '-' 

Q) 

...... 

1'1 

I-

.J:l 

...... ~ 0 

).. 

0 

.-~ Q 

'--" ~ ...... 

100 

50 

100 

e 5o 

-E ~ 0 

).. 

0 

10 10

Concentration of the 

Without S9 mix by the m

10 10

Concentration of the 

Wit}l S9 mix by the met

Fig. 1 Results of the dose determination test ofV-05H 
(_ ~ 

"' 

5 

"' 

<... ·<... 

(1) 

.= 

"' 

"' 

E 

0 

en 

0 

E 

0 

<... 

~ 

.c: 

~ 

:>: 

"' 

<ll 

<.> 

._ 

0 

<1> 

'-' 

c:; 

(1) 

""" 

'-' 

..:: 

-en 

c:; 

0 

~ 

'-

"' 

'-

(1) 

..0 

"" 

E "' 

0 

0 "" 

E 

0 

"-

-= 

'-' 

-= 

~ 

:>: 

"' 

<1.1 

<.> 

.... 

0 

<1.1 

'-' 

c:; 

<ll 

""" 

"-' 

c:; 

-

30 

o numerical aberrations 

• structural aberrations (rg) 

20 

10 

0~ " 0 156 313" 

Concent rat Lon( IJ.g/ ml) 

24 hours treatment by direct method 

JO 

0 nume deal aberrations 

• structural aberrations (rg) 

20 

tO 

0\ 

0 156 313 

Concentration(IJ.g/ml) 

48 h.ours treatment by direct method 

Fig.2 Results of chromosomal aberration test of V-05H 

- 5-

KOS-0612· 

625 

625 
! / 

( ..., 

"' <= 

0 

_, 

... "' 

... 

QJ 

.0 

"' 

"' e 

0 

"' 0 

E 

0 

"-

<:; 

5 

:;: 

~ 

... 

""' ~ 

0 

QJ 

<= ""' <I> 

""' 

""' c:: 

/ 

\ ~ 

"' c: 

0 

_, 

'-

"' 

'-

QJ 

.0 

"' 

E "" 0 

"' 0 

El 

0 

... 

.<:: 

""' 

5 

;: 

-"' 

QJ 

""' ._ 

0 

QJ 

<.l 

c:: 

QJ 

'"0 

<.> 

c:: 

-

/ 

30 

20 

10 

JO 

20 

10 

o numerical aberrations 

• structural .aberrations ( tg) 

156 313 

Concent.rat ion( u.g/ml) 

Without S9 mix by metabo!Lc activation method 

o numerical aberrations 

• s.tructural aberrations (+g) 

1,250 2,500 

Concentration(u.g/ml) 

With S9 mix by metabolic activation method 

6Z5 

5,000 

Fig.3 Results of chromosomal aberration test of V-05H 

- 6-

~1 ;Jo 3 

KOS-0612 
STUDY CODE: Bll-0443 

FINAL REPORT 

ReceiptNo. D97-1763 

Report No. D-5317 

TWENTY-EIGHT-DAY REPEATED-DOSE 

ORAL TOXICITY STUDY OF 

V-OSH 

IN RATS 

May1998 

Hita Research Laboratories 

Chemical Biotesting Center 

Chemicals Inspection & Testing Institute 

Japan 
c 

( 

Bll-0443 

STATEMENT 

I, the undersigned, hereby declare that this report provides a correct English translation 

of the final report (Study Code B11-0443, issued on May 29, 1998). 

Chemicals Inspection & Testing Institute, Japan (CITI) formally changed its name to 

Chemicals Evaluation and Research Institute, Japan (CERI) as of November 1, 1999. 

The Sponsor's name and address, "NISSHINBO INDUSTRIES, INC." and "1-18-1, 

Nishiarai Sakaecho, Adachi-ku Tokyo, 123, Japan" in the original, were changed to 

"NISSHINBO INDUSTRIES, INC. R & D CENTER" and "1-2-3, Onodai, Midori-ku 

Chiba, 267-0056, Japan", respectively by Sponsor's request. 

CAS No.l57299-02-0 oftest substance was added by Sponsor's request. 

In the structural formula, n=2-800.was added. 

Molecular weight of3,000 was changed to 30,000. 

Madoka Tada, B.S. 

Junior Laboratory Animal Technician 

Hita Laboratory 

Chemicals Evaluation and Research Institute, Japan 

q~ /J' ' .,J-I)b.,L Q 

Date 
( 

( 

( 

QUALITY ASSURANCE STATEMENT 

Hita Laboratory, 

Chemicals Evaluation and Research Institute, Japan 

Sponsor: NISSHINBO INDUSTRIES, INC. R&D CENTER 

Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study of V-OSH in Rats 

Study code: Bll-0443 

This report was audited by the Quality Assurance Section. 

I, the undersigned, hereby declare that this report reflects the 

original Japanese report. 

Section Chief, Quality Assurance 

(Date) J~ t2", 2.c:>o2.. 

(Signature) ~ 

Keiji Shiraishi, B.S. 
t' 

( 

Sponsor: 

GLP STATEMENT 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

NISSHINBO INDUSTRIES, INC. R & D CENTER 

Bll-0443 

Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV-05H in Rats 

Study Code: B11-0443 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Concerning Testing Facilities Stipulated in Article 4 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 (EA), No. 229 of the Pharmaceutical 

Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 85 ( 1984) of the Basic 

Industries Bureau, Ministry of International Trade and Industry (MITI), March 31, 1984; 

Notification No. 233 of the Planning and Coordination Bureau, EA, No. 38 of the 

Environmental Health Bureau, MHW & No. 823 (1988) ofthe Basic Industries Bureau, 

MITI, revised on November 18, 1988) and with "Organisation for Economic Cooperation and Development (OECD) Principles of Good Laboratory Practice" (May 12, 

1981). 

Management: Signed in original May 29, 1998 

Yoshifumi Fujino, M.S. 
( 

( 

I 

I 

I Bll-0443 

QUALITY ASSURANCE STATEMENT 

Hita Research Laboratories, Chemical Biotesting Center, 

Chemicals Inspection & Testing Institute, Japan 

Sponsor: NISSHINBO INDUSTRIES, INC. R & D CENTER 

Title: Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV-05H in Rats 

Study Code: B11-0443 

This study was audited and inspected by Quality Assurance Section 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. 

Dates of Inspections Dates of Reports to Dates of Reports to 

and Audits Study Director Management 

October 28, 1997 October 31, 1997 October 31, 1997 

January 21, 1998 January 22, 1998 January 22, 1998 

January 27, 1998 January 27, 1998 January 27, 1998 

February 2, 1998 February 3, 1998 February 3, 1998 

February 3, 1998 February 3, 1998 February 3, 1998 

February 18, 1998 February 18, 1998 February 20, 1998 

March 3, 1998 March 4, 1998 March 4, 1998 

March 13, 1998 March 13, 1998. March 13, 1998 

March 23, 1998 March 24,. 1998 March 24, 1998 

April21, 1998 April21, 1998 April21, 1998 

April23, 1998 April 23, 1998 April23, 1998 

April 28, 1998 April 28, 1998 April 28, 1998 

May26, 1998 May 26, 1998 May 26, 1998 

May28, 1998 May 29, 1998 May 29, 1998 

May 29, 1998 May29, 1998 May 29, 1998 

I, the undersigned, hereby declare that this report provides an accurate description 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 29, 1998 

Keiji Shiraishi, B.S. 
( 

I 

\ 

Bll-0443 

Study Code: B11-0443 

Test Substance Code: HR3539 

Sponsor Code: N-680 

TITLE 

Twenty-Eight-Day Repeated-Dose Oral Toxicity Study ofV -05H in Rats 

SPONSOR 

NISSHINNBO INDUSTRIES, INC. R & D CENTER 

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

TESTING FACILITY 

Hita Research Laboratories, Chemical Biotesting Center 

Chemicals Inspection & Testing Institute, Japan 

822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan 

PURPOSE OF STUDY 

The purpose of this study is to define the type, severity and reversibility of 

toxicological signs and to determine the no-observed-effect-level (NOEL) ofthe test 

substance by observing the functional and morphological changes in animals 

receiving repeated doses for 28 days. 

TESTING METHOD 

This study was conducted in accordance with "28-day Repeated Dose Toxicity Study 

in Mammalian Species" prescribed in "Notification on Partial Revision 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 ofHealth and Welfare (MHW) & No. 1014 

(1986) of the Basic Industries Bureau, Ministry of International Trade and Industry 

(MITI), December 5, 1986)", and with "Section 407, Repeated Dose Oral ToxicityRodent: 28-day or 14-day Study" prescribed in "OECD Guidelines for Testing of 

Chemicals (May 12, 1981)". 
I 

\ 

i 

\ 

Bll-0443 

GLP COMPLIANCE 

This study was conducted in compliance with "Concerning Testing Facilities 

Stipulated in Article 4 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) ofthe Basic Industries Bureau, MITI, March 31, 1984; Notification No. 

233 of the Planning and Coordination Bureau, EA, No. 38 of the Environmental 

Health Bureau, MHW & No. 823 (1988) of the Basic Industries Bureau, MITI, 

revised on November 18, 1988) and with .. OECD Principles of Good Laboratory 

Practice" (May 12, 1981). 

PERIOD OF STUDY 

Commencement of Study: 

Animal Receipt: 

Start of Dosing: 

Terminal Autopsy of the Dosing Period: 

Start of the Recovery Period: 

Terminal Autopsy of the Recovery Period: 

Completion of Study: 

October 31, 1997 

January 27, 1998 

February 3, 1998 

March 3, 1998 

March 3, 1998 

March 17,1998 

May 29, 1998 

LOCATION AND PERIOD FOR RETENTION OF RAW DATA AND SPECIMENS 

Data and specimens, and each remaining lot of the test substance will be retained in 

the archives and test substance storage room, respectively, of Hita Research 

Laboratories for 10 years following the date of completion of study. After 

termination of the retention period, any measures taken will be done so with the 

approval of the Sponsor. Samples and specimens that are liable to deteriorate 

markedly will be retained only for as long as the quality of the preparation permits 

evaluation with the Sponsor's consent. 
( 

I 

,i 

' i 

AUTHOR AND PERSONNEL CONCERNED WITH STUDY 

AUTHOR 

Bll-0443 

Study Director: Signed in original May 29, I 998 

Takaaki Umano, M.S., D.V.M. 

General Toxicology Section 

Hita Research Laboratories 

PERSONNEL CONCERNED WITH STUDY 

Study Staff: Takako Muroi 

Junior Laboratory Animal Technician 

Person in charge of Pathologic Examination: 

Kanji Yamasaki, Ph. D., D.V.M. 

Person in charge of Clinical Chemistry: 

Keiji Shiraishi, B.S. 

Person in charge of Chemical Analysis: 

Takeharu Wada, M.S. 

Person in charge ofDivided Work: 

Teruhiko Kanabayashi, Ph.D. 

Toxicological pathologist, Diplomate J.S.T.P. 

Bio pathology Institute, Ltd. 
( 

( 

Bll-0443 

TABLE OF CONTENTS 

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

MATERIALS AND METHODS 

1. TEST SUBSTANCE ······················································ 2 

2. ANIMALS ······················································ 3 

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

4. GROUPING ······················································ 4 

5. PREPARATION OF TEST SUBSTANCE ································· 4 

6. DOSING ······················································ 5 

7. OBSERVATION AND MEASUREMENT ................................ 5 

8. STATISTICAL ANALYSIS ...................................................... 9 

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 9 

RESULTS 

1. CLINICAL SIGNS ...................................................... 10 

2. BODY WEIGHTS ...................................................... 10 

3. FOOD CONSUMPTION ...................................................... 10 

4. HEMATOLOGICAL EXAMINATIONS ................................ 10 

5. BLOOD CHEMICAL EXAMINATIONS ································ 11 

6. URINALYSIS ······················································ 11 

7. ORGAN WEIGHTS ······················································ 11 

8. AUTOPSY ······················································ 11 

9. HISTOPATHOLOGICAL EXAMINATIONS ································ 12 

DISCUSSION ······················································ 13 
FIGURES 

1. Mean body weights 

2. Mean food consumption 

TABLES 

1. Clinical signs 

2. Mean body weights 

3. Mean food consumption 

4. Hematology 

5. Blood chemistry 

6. Urinalysis 

7. Absolute organ weights 

8. Relative organ weights 

9. Gross pathological findings 

10. Histopathological findings 

PHOTOS 

1, 2 

ADDENDA 

1. Clinical signs 

2. Body weights in individual rats 

Bll-0443 

3 

······················································ 2 

...................................................... 5 

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

······················································ 9 

...................................................... 11 

······················································ 12 

...................................................... 13 

...................................................... 14 

······················································ 15 

................................ 1 

................................ 5 

3. Food consumption in individual rats ................................ 10 

4. Hematology in individual rats ................................ 13 

5. Blood chemistry in individual rats ································ 17 

6. Urinalysis in individual rats 

7. Absolute organ weights in individual rats 

································ 21 

................................ 23 

8. Relative organ weights in individual rats 

9. Pathological findings in individual rats 

APPENDIX 

································ 25 

································ 27 

PHYSICOCHEMICAL REPORT 
( 

Bll-0443 

SUMMARY 

A 28-day repeated-dose toxicity study of orally administered V -05H followed by a 14-

day recovery period was performed in groups of 6 male and 6 female Crj: CD (SD) rats 

at 5 weeks of age. Three dose levels were selected with the highest dose of 1 ,000 

mg/kg/day, in addition to 200 and 40 mg/kg/day. Recovery groups were prepared for 

the 1,000 mg/kg and vehicle control groups. 

In this study, there were no death according to the test substance administration. No 

abnormalities were noted in the clinical signs, body weight, food consumption during 

the dosing period and in the hematological examination, blood chemical examination, 

urinalysis, autopsy and histopathological examination at the end of the dosing period. 

In the organ weight, increased relative kidney weights were noted in the female of 1 ,000 

mg/kg group at termination of dosing period. 

In the recovery study, no abnormalities were noted according to the treatment. 

Based on these results, NOEL ofV-05H for rat was considered to be 200 mg/kg/day in 

this study. 

- 2-

/ 

I 

\ 

( 

\_ 

( 

\ 

1.8 

Stability: 

Melting Point: 

Boiling Point: 

Vapor Pressure: 

Partition Coefficient: 

Solubility: 

Degree of Solubility: 

Storage Conditions 

Bll-0443 

The structure changes by additional reaction in the presence 

of active hydride (carboxylic acid, alcohol, amine and 

others). The stability test was conducted in our laboratories, 

and confirmed that the test substance was stable during the 

dosing period. 

Soluble in oil 

Water: 

DMSO: 

Acetone: 

Others: 

Stored in the sealed container at the cold and dark place. The high-temperature 

and humidity was avoided. 

1.9 Precautions 

Goggles, glove, mask, head cap and lab coat were worn. Washing hands and 

gargle at the end of handling. 

2. ANIMALS 

Cij: CD (SD) rats (SPF) were obtained from Charles River Japan, Inc. (Hino 

Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, 

Japan). The animals were quarantined and acclimatized, and healthy animals 

with favorable weight gains were allocated to groups to ensure homogeneity of 

mean body weight using body weight-stratified randomization. The animals 

were 5 weeks old, and weighed 128.8-142.8 g for males and 111.1-125.7 g for 

females at the start of dosing. The animals were identified by ear-tagging. 

3. HOUSING CONDITIONS 

The barrier-system animal room was maintained at a temperature of 23±2°C and a 

relative humidity of 55±10% with 10-15 air changes per hour and artificial light 

for 12 hours (between 7:00 and 19:00). The animals were housed individually in 

a hanging stainless steel cage with wire-mesh floor ( 165 W x 300 D x 150 H mm, 

TOKIWA KAGAKU KIKAI CO., LTD.). The trays were changed twice a 

weekly, cages once weekly and racks once four weeks. The racks and cages were 

- 3-
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Bll-0443 

identified by individual cards. The animals had free access to MF pelleted diet 

(Oriental Yeast Co., Ltd.) and water (chlorinated) from the Hita City supply via 

automatic watering system with sipper tubes. The diet and housing materials 

were autoclaved at 121 oc for 30 min prior to use. Analysis of contaminants in 

both the diet and drinking water confirmed that they would not affect the test 

system. 

No environmental deviations, which might affect the study results, were noted 

during the quarantine, acclimation or study periods. 

4. GROUPING 

The grouping was as follows: 

Dose Volume Concent- No. of Animals (Animal No.} 

GrouE (m&kg/da~) (ml/kg) ration(%} Males Females 

Vehicle control 0 10 0 6( I - 6 ) 6 ( 37 - 42 ) 

Vehicle control (recovery) 0 10 0 6 ( 7 - 12 ) 6 ( 43 - 48 ) 

Low dose 40 10 0.4 6 ( 13 - 18 ) 6 ( 49 - 54 ) 

Intermediate dose 200 10 2.0 6 ( 19 - 24 ) 6 ( 55 - 60 ) 

High dose 1,000 10 10.0 6 ( 25 - 30 ) 6 ( 61 - 66 ) 

High dose (recovery) 1,000 10 10.0 6 ( 31 - 36 ) 6 { 67 - 72 ) 

Reason for dosage selection: 

A 14-day preliminary repeated-dose oral toxicity study was carried out at 3 doses 

of 50, 250 and 1,000 mglkg/day. As a result, no death occurred in all groups. 

Abnormalities were noted in organ weights in female of the 1 ,000 mg/kg group. 

Therefore high dose level at 1,000 mg/kg/day and two lower doses at 200 and 40 

mg/kg/day were set in the main study. Recovery groups were prepared for the 

1,000 mg/kg and vehicle control groups. 

5. PREPARATION OF TEST SUBSTANCE 

5.1 Preparation 

The test substance was weighed accurately and ground with dry ice to a fine 

powder in an agate mortar. The powder was mixed with gum arabic (5.0 w/v% of 

final cone., Lot No. WTP0132, Wako Pure Chemical Co., Ltd.) and purified water 

(Lot No. A060, Takasugi Pharmaceutical Co., Ltd.) to make 10.0 and 2.0 w/v% 

once weekly. The lowest concentration of 0.4 w/v% was diluted from 10.0 

w/v% prior to use. 

- 4- ~11 
( 

5.2 

6. 

Bll-0443 

Stability and Homogeneity Tests 

The stability and homogeneity of the test substance in above preparations were 

confirmed by our laboratories. 

DOSING 

Treatment by oral gavage was carried out daily using a Nelaton catheter 

(TERUMO CORPORATION) and a syringe (TERUMO CORPORATION) in the 

morning for 28 days, and the subsequent 14 days were used as a recovery period. 

7. OBSERVATION AND MEASUREMENT 

The day of the start of dosing was defined as day 1, and the day before as day -1. 

The week of the start of dosing period was defined as week 1. Also, the next day 

of the final dosing was defined as recovery day 1, and the week of the start of the 

recovery period as recovery week 1. 

7.1 Clinical Signs 

All animals were observed at least once daily. 

7.2 Body Weights 

All animals were weighed as follows: 

Before Dosing: day -1 (at the time of grouping) 

During the Dosing Period: day 1 (at the start of dosing), 3, 5, 8, 10, 12, 15, 

17, 19, 22, 24, 26 and 28 

During the Recovery Period: day 1 (at the start of the recovery period), 3, 5, 8, 

10, 12 and 14 

In addition, immediately before autopsy, body weight was measured for 

calculation of relative organ weight. 

7.3 Food Consumption 

Food consumption was measured as follows: 

Before Dosing: Once 

During the Dosing and Recovery Periods: Twice weekly 

- 5-
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B 11-0443 

7.4 Hematological Examinations 

All survivals were fasted overnight (16-20 hours) at the termination of dosing and 

recovery periods, and blood samples were collected from abdominal aorta under 

ether anesthesia. Sodium citrate was used as an anticoagulant for examinations 

of prothrombin time and activated partial thromboplastin time, and EDTA-2K for 

another items. 

Parameters Method 

4 3 

1) Red Blood Cell Count (RBC) (xJO /mm) System for detecting change 

in electrical resistance 

2) White Blood Cell Count (WBC) 

2 3 

(xiO /mm) System for detecting change 

in electrical resistance 

3) Hemoglobin Cone. (Hb) (g/dl) Oxyhemoglobin method 

4) Hematocrit Value (Ht) (%) System for detecting pulse 

5) Mean Corpuscular Volume (MCV) (Jlm3) __!!!__X 103 

RBC 

6) Mean Corpuscular Hemoglobin (MCH) (pg) Hb X 103 

RBC 

7) Mean Corpuscular Hemoglobin Cone. (MCHC) (%) Hb X 102 

Ht 

4 3 

8) Platelet Count (Platelet) (xiO /mm) System for detecting change 

in electrical resistance 

9) Reticulocytes Count (Reticulo) (o/oo) New methylene blue 

staining 

10) Prothrombin Time (PT) (sec) Magnetic sensor system 

11) Activated Partial Thromboplastin Time (APTT)(sec) Magnetic sensor system 

12) Differentiation of Leukocytes (%) Wright staining 

Stab Neutrophils (N-Band) 

Segmented Neutrophils (N-Seg) 

Eosinophils (Eosino) 

Basophils (Baso) 

Lymphocytes (Lymph) 

Monocytes(Mono) 

I) - 8) Microcell counter M-2000, Toa Medical Electronics 

9), 12) MICROX HEG-120A, OMRON 

1 0), 11) KC-1 OA, Amelung 

- 6-
B11-0443 

7.5 Blood Chemical Examinations 

( Sera were separated from the blood samples collected at the same time of the 

hematological examinations and examined for following items. 

Parameters Method 

1) GOT (IU/1) UV method (Method based on JSCC) 

2) GPT (IU/1) UV method (Method based on JSCC) 

3) Alkaline Phosphatase (ALP) (IU/1) p-Nitrophenyl phosphate method 

4) Cholinesterase (ChE) (lUll) Butyrylthiocholine iodide method 

5) y-GTP (IU/1) L-y-Glutamyl-p-nitroanilide method 

6) Total Cholesterol (T-Cho) {mg/dl) COD·DAOS method 

7) Triglyceride (TG) (mg/dl) GPO·DAOS method 

8) Glucose (mg/dl) Glucokinase-G-6-PDH method 

9) Total Protein (T-Protein) (g/dl) Biuret method 

10) Albumin (g/dl) Bromocresol green method 

11) AIG Ratio Albumin 

T -Protein - Albumin 

12) Blood Urea Nitrogen (BUN) (mg/dl) Urease indophenol method 

13) Creatinine (mg/dl) Jaffe's method 

I 14) Total Bilirubin (T-Bil) (mg/dl) Azobilirubin method \ 

' 15) Calcium (Ca) (mg/dl) OCPC method 

16) Inorganic Phosphorus (IP) {mg/dl) Fiske-Subbarow method 

17) Sodium (Na) (mEq/1) Crown-Ether membrane 

electrode method 

18) Potassium (K) (mEq/1) Crown-Ether membrane 

electrode method 

19) Chloride (Cl) (mEq/1) Coulometric titration method 

1)-10), 12)-16) 7150 Automatic Analyzer, Hitachi 

17)- 19) PV A- a III, A & T 

7.6 Urinalysis 

Sixteen-hour urine samples were collected in individual metabolic cages, from all 

animals at the termination of the dosing (day 28) and recovery (day 14) periods 

and examined for volume, color and additional items of pH, protein, ketone 

bodies, bilirubin, occult blood, glucose and urobilinogen using a test paper (NMultistix®, Bayer·Sankyo ). 

- 7-
( 

\ 

( 

( 

7.7 Autopsy 

Animals were subjected to a detailed gross necropsy. 

7.8 Organ Weight 

Following organs were measured in wet weight: 

Brain, Liver, Kidneys, Spleen, Adrenals, Testes, Ovaries 

7.9 Histopathological Examinations 

1) The following organs and tissues were preserved in 10% formalin: 

Bll-0443 

Brain (Cerebrum, Cerebellum), Hypothesis, Eyeball, Thyroid glands (with 

Parathyroid glands), Heart, Lung, Liver, Kidneys, Spleen, Adrenal glands, 

Stomach, Intestine (Duodenum to Rectum), Testes, Ovaries, Uurinary bladder, 

Bone marrow (Femur), Gross lesions. 

2) Light microscopic examinations were performed for the following organs and 

tissues after embedding in paraffin, sectioning and hematoxylin and eosin 

staining. The histological preparation was conducted by Bio Pathology 

Institute, Ltd. according to the assignment protocol. 

Terminal Autopsy of the Dosing Period 

Vehicle control and 1 ,000 mg/kg groups: 

Liver, Kidneys, Spleen, Heart, Stomach, Intestine (Duodenum, 

Jejunum, Ileum, Cecum, Colon, Rectum), Adrenal glands, Testes. 

200 mg/kg/day group: Kidneys (females only), Stomach (females only) 

Terminal Autopsy of the Recovery Period 

Vehicle control and 1,000 mg/kg/day groups: 

Kidneys (females only), Stomach (females only), Testes 

Gross Lesions 

Terminal Autopsy of the Dosing Period 

40 mg/kg group (Male, No. 15): Cerebrum 

Terminal Autopsy of the Recovery Period 

1,000 mg/kg group (Male, No. 33): Kidneys 

Vehicle control group (Female, No. 43): Skin 

- 8-
i 

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8. 

Bll-0443 

STATISTICAL ANALYSIS 

Data regarding body weights, food consumption, hematological examination, 

blood chemical examination, urine volume and organ weight were analyzed using 

Bartlett's test for homogeneity of variance. If the variances were homogeneous 

at a significance level of 5%, one way analysis of variance was performed. 

When there was a significant difference in this analysis, the difference between the 

vehicle control group and each of the treatment group was analyzed by Dunnett's 

test. 

If the variances were not homogeneous in the Bartlett's test, Kruskal-Wallis's test 

was used. When there was a significant difference in this test, the difference 

between the vehicle control group and each of the treatment group was analyzed 

by nonparametric Dunnett's test. 

UNEXPECTED SITUATIONS AND DEVIATIONS FROM PROTOCOL 

There were no unexpected situations which might have affected the test results and 

deviations from protocol. 

- 9-
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\ 

( 

RESULTS 

I. CLINICAL SIGNS (TABLE 1, ADDENDUM 1) 

1.1 During Dosing Period 

No abnormalities were noted in all groups. 

1.2 During Recovery Period 

Male: No abnormalities were noted in all groups. 

Female: Loss of hair (1/6) was noted in the vehicle control group. 

2. BODY WEIGHTS (FIG. I, TABLE 2, ADDENDUM 2) 

2.1 During Dosing Period 

No abnormalities were noted in all groups. 

2.2 During Recovery Period 

3. 

3.1 

3.2 

No abnormalities were noted in all groups. 

FOOD CONSUMPTION (FIG.2, TABLE 3, ADDENDUM 3) 

During Dosing Period 

No abnormalities were noted in all groups. 

During Recovery Period 

' 

No abnormalities were noted in all groups. 

4. HEMATOLOGICAL EXAMINATIONS (TABLE 4, ADDENDUM 4) 

4.1 At Termination of Dosing Period 

No abnormalities were noted in all groups. 

4.2 At Termination of Recovery Period 

Male: No abnormalities were noted in all groups. 

Bll-0443 

Female: Decreased reticulocytes count and increased eosinophils ratio were noted 

in the 1,000 mg/kg group. 

- 10 -

.... ---=---r--=-----r • ---- -
( 

\ ' 

( 

\. 

i 

Bll-0443 

5. BLOOD CHEMICAL EXAMINATIONS (TABLE 5, ADDENDUM 5) 

5.1 At Termination of Dosing Period 

Male: Decreased y-GTP activity was noted in the 40 mg/kg group. Decreased 

y-GTP activity and total protein level were noted in the 200 mg/kg group. 

Decreased total protein level was noted in the 1 ,000 mg/kg group. 

Female: Decreased GOT activity was noted in the 40 mg/kg group. 

5.2 At Termination of Recovery Period 

Male: Decreased albumin levels was noted in the 1,000 mg/kg group. 

Female: No abnormalities were noted in all groups. 

6. URINALYSIS (TABLE 6, ADDENDUM 6) 

6.1 At Termination of Dosing Period 

No abnormalities were noted in all groups. 

6.2 At Termination of Recovery Period 

No abnormalities were noted in all groups. 

7. ORGAN WEIGHTS (TABLES 7,8, ADDENDA 7,8) 

7.1 At Termination of Dosing Period 

Male: No abnormalities were noted in all groups. 

Female: Increased relative kidney weights were noted in the 1,000 mg/kg group. 

7.2 At Termination of Recovery Period 

No abnormalities were noted in all groups. 

8. AUTOPSY (TABLE 9, ADDENDUM 9) 

8.1 At Termination of Dosing Period 

Male: Blackish region of the mucosa in the glandular stomach (1/6) was 

observed in the vehicle control group. Partial defect of the cortex in the 

cerebrum (1/6) was observed in the 40 mg/kg group. 

Female: Pelvic dilatation of the kidney (1/6) and blackish region of the mucosa in 

the glandular stomach (1/6) were observed in the 1,000 mg/kg group. 

8.2 At Termination of Recovery Period 

Male: Apparent spottY pattern of the surface in the kidney (1/6) and small testis 

(1/6) were observed in the 1,000 mg/kg grouup. 

Female: Loss of hair in the skin (1/6) was observed in the vehicle control group. 

-II-
( 

( 

Bll-0443 

9. HISTOPATHOLOGICAL EXAMINATIONS (TABLE 10, ADDENDUM 9) 

9.1 At Termination of Dosing Period 

Male: Microgranuloma in the liver (+, 2/6), basophilic tubules (+, 116) and 

fibrosis(+, 116) in the kidney, and necrosis of the mucosa in the glandular 

stomach (+, 116) were observed in the vehicle control group. Partial 

defect of the cortex in the cerebrum (+, 111) was observed in the 40 

mg/kg group. Microgranuloma in the liver(+, 1/6) was observed in the 

1,000 mg/kg group. 

Female: Microgranuloma in the liver(+, 1/6) was observed in the vehicle control 

group. Basophilic tubules (+, 116), cyst formation (+, 116) and fibrosis 

(+, 1/6) in the kidney were observed in the 200 mg/kg group. Pelvic 

dilatation of the kidney (+, 1/6) and necrosis of the mucosa in the 

glandular stomach(+, 1/6) were observed in the 1,000 mg/kg group. 

9.2 At Termination of Recovery Period 

Male: Increased eosinophilic bodies in the kidney(++, 111) and focal atrophy of 

the seminiferous tubules in the testis(++, 1/6) were observed in the 1,000 

mg/kg group. 

Female: Atrophy of hair follicles in the skin(+, Ill) was observed in the vehicle 

control group. Cyst formation in the kidney(+, 116) was observed in the 

1,000 mg/kg group. 

- 12-
/ 

\ 

Bll-0443 

DISCUSSION 

The oral toxicity of V -05H was examined in male and female SD rats given a daily dose 

of 40, 200 and 1,000 mg/kg/day for 28 days followed by the 14-day recovery period. 

In the present study, no deaths were observed throughout the study period. No 

abnormalities were noted in the clinical signs, body weights, food consumption during 

the dosing period or hematological examinations and urinalyses at termination of dosing 

period, either. 

In the blood chemical examinations, decreased total protein levels were observed in 

male of the 200 and 1,000 mg/kg groups at termination of dosing period. This change 

was considered to be of no toxicological significance, because this was within the 

historical control data in our laboratories and it had no other related findings. 

In organ weight, increased relative kidney weights was observed in female of the 1 ,000 

mg/kg group at termination of dosing period. This finding was considered to be 

accidental one, since this was slight change and it had no related findings; absolute 

weight; blood chemical parameters, autopsy, and histopathological findings. But the 

elevation is suspectable change of the treatment. 

In autopsy, pelvic dilatation of the kidney, and blackish region of the mucosa in the 

glandular stomach were observed in female of the 1 ,000 mg/kg group at termination of 

dosing period. 

In histopathological examination, pelvic dilatation of the kidney, and necrosis of the 

mucosa in the glandular stomach were observed in female of the 1 ,000 mg/kg group at 

termination of dosing period. These changes were not considered related to 

administration of the test substance. Because they were limited to one each animal, 

and frequently found in normal rats. 

In the recovery study, the effect of the test substance noted in the dosing period was 

disappeared. At termination recovery period decreased albumin levels, apparent spotty 

pattern of the surface and increased eosinophilic bodies in the kidney, focal atrophy of 

- 13 -
( 

Bll-0443 

the seminiferous tubules in the testis were observed in male of the 1 ,000 mg/kg group, 

and increased eosinophils ratio and cyst formation in the kidney were observed in 

female of the 1 ,000 mg/kg group. These findings were considered to be no 

toxicological significance, since similar changes were not observed at the end of the 

dosing period; no related findings; histopathological changes of them might be 

spontaneous. The findings of testis were not considered related to administration of 

the test substance, since these changes were unilaterally and limited in one animal. 

The other statistically significant differences were not considered related to 

administration of the test substance, since there were no dose-relationship and with no 

related findings. 

Based on these results, the main effect of V -05H was increased relative kidney weights 

in female of the 1,000 mg/kg group. But the effect was very weak. Accordingly, the 

NOEL ofV -05H for rats was considered to be 200 mg/kg/day in this study. 

Historical control data 

Parameter (unit) 

Total Protein 

(g/dl) 

Mean±2S.D. 

At termination of dosing period 

Male 

5.9 ± 0.2 

n= 186 

- 14-

Female 
I 

_j 

I 

I ~ ! 

-1 

l 

I 

l 

I 

I 

/-·---""' 

~ 

y 

~; 

(g) 

450 

400 

350 

300 

250 

zoo 

150 

100 ~, () : Vehicle 

0 : 40 m

£::. : 200 m

<> : 1,000 m

Administration Recov

-1 1 3 5 8 10 12 15 17 19 22 24 26 28 1 

Figure 1 28-day repeated-dose oral toxicity study in rats 

Mean body weights : Male 

3 5 8 
~ ' l I 

N 

I 

1 

I 

I 

l 

~ i ~ ~ l ' ~ ~ 

-I 

i 

-I 

l 

(g) 

250 

200 

150 

100 

() : Vehicle 

0 : 40 

tJ. : 200 

<> : 1,000 m

Administration Recov

-1 1 3 5 8 10 12 15 17 19 22 24 26 28 1 3 5 8 

Figure 1 -Continued 

Mean body weights : Female 
; ~ I 

-1 

j 

I ~ I 

l 

! 

i 

• 

w 

/ 

~ 

y 

~ 

0 

40 

30 

>. 

o:! 20 

"0 

-

..., 

o:! 

.... 

-

bO 

10 

0 

/--~---- .... 

Administration 

1 4 8 11 15 18 22 

Figure 2 28-day repeated-dose oral toxicity study in rats Mean food consumption : Male 

25 28 4 

0 : Vehicle 

0 : 40 m

t:. : 200 m

<> : 1,000 m

Re

8 
~ I 

i ~ t 

1 ~ ! 

I 

r 

i 

I 

l 

l 

i 

I 

/--~ ~ ~ 

~ 

.-

40 

30 

>. 

201 <d "0 

-

...., 

<d 

.... 

-

'b.() ~ 10 

0 

,/ 

__.-A Ill &:::,..__ 

~ 

0 : Vehicle 

D : 40 m

6. : ZOO m

0 : 1,000 m

Administration Rec

1 4 8 11 15 18 22 25 28 4 8 

Figure Z -Continued 

Mean food consumption : Female 
Bll-0443 

Table 28-day repeated-dose oral toxicity study in rats 

Clinical signs 

Sex Signs Administration Period Recovery Period 

\ mg/kg/day vc vc 40 200 1,000 1,000 vc 1,000 

(R) (R) 

Male. ta ta ta ta ta ta ta ta 

6' 6 6 6 6 6 6 6 

No-abnormalities detected 6 6 6 6 6 6 6 6 

Female ta ta ta ta ta ta ta ta 

6' 6 6 6 6 6 6 6 

No abnormalities detected 6 6 6 6 6 6 5 6 ------------

Loss of hair 

* Num'ler of animals examined. 

VC, Vehicle control;(R),Recovery. 

ta,terminal autopsy. 

- 1 -
// /---, 

Table 2 28-day repeated-dose oral toxicity study in rats Bll-044

Mean body weights(g) 

Sex Exp.group Number of Administration 

(mg/kg/day) animals -1 1 3 5 8 10 12 (days) 

Vehicle 12 127.7 134.5 153.3 171.2 196.8 215.7 232.0 control ± 3.1 ± 2.8 ± 4.1 ± 5.2 ± 5.8 ± 7.0 ± 7.6 

40 6 127.9 134.8 150.9 167.7 191.1 208.0 222.6 Hale ± 3.0 ± 3.0 ± 3.7 ± 4.7 ± 7.1 ± 8.0 ± 8.5 

200 6 127.9 135.2 151.4 166.3 190.1 206.6 221.6 

± 3.3 ± 4.3 ± 6.8 ± 8.3 ± 11.5 ± 13.6 ± 20.1 

1,000 12 127.8 135.3 150.7 168.8 193.3 211.8 227.7 

± 3.1 ± 3.0 ± 3.7 ± 4.1 ± 5.1 ± 7.7 ± 9.5 

Vehicle 12 113.1 118.9 129.7 140.3 151.5 161.3 166.5 control ± 2.8 ± 2.7 ± 3.5 ± 4.4 ± 6.8 ± 8.6 ± 10.1 

40 6 113.2 118.2 128.9 140.7 150.4 161.2 163.9 Female ± 2.9 ± 2.9 ± 4.6 ± 2.4 ± 2.8 ± 5.2 ± 5.0 

200 6 113.2 118.7 131.0 139.7 155.4 161.6 170.3 

± 2.9 ± 4.5 ± 6.1 ± 8.2 ± 12.6 ± 11.6 ± 14. 1 

1,000 12 113.5 117.7 129.4 138.7 150.7 160.5 166.6 ~ ± 2.5 ± 3.2 ± 4.5 ± 6.4 ± 7.7 ± 9.2 ± 10.4 Hean ±S.D. 

t-v * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 

! 

l 

l 

I 

1 

~ l y ~ ~ 

I ~ 

I 

l 

l 

l 

I 
/-~ ,~-~~- 

Table 2 -Continued Bll-044

Mean body weights(g) 

Sex Exp.group Number of Administration 

(mg/kg/day) animals 15 17 19 22 24 26 28 (days) 

Vehicle 12 257.2 277.3 291.7 315.5 331.5 341.3 352.9 control ± 10.4 ± 10.5 ± 12.0 ± 14.8 ± 15.6 ± 15.8 ± 17.2 

40 6 244.0 260.8 274.0 295.3 307.6 318.4 327.4 Hale ± 10.1 ± 10.4 ± 12.1 ± 14.8 ± 14.2 ± 16.1 ± 16.1 

200 6 245.8 263.4 276.4 296.0 310.0 320.0 331.1 

± 20.3 ± 22.5 ± 26.4 ± 28.0 ± 29.5 ± 32.2 ± 31.3 

1,000 12 252.3 268.9 282.7 304.1 318.3 328.1 339.9 

± 11.5 ± 12.8 ± 15.2 ± 16.5 ± 19.1 ± 20.9 ± 20.9 

Vehicle 12 172.8 183.0 187.0 197.3 202.0 207.8 210.8 control ± 10.1 ± 10.8 ± 12.3 ± 11.9 ± 14.5 ± 12.6 ± 15.7 

40 6 173.5 179.1 187.2 199.0 201.0 206.1 208.8 Female ± 6.1 ± 8.4 ± 9.3 ± 7.1 ± 10.6 ± 11.1 ± 11.3 

200 6 178.0 187.3 193.4 202.0 211.3 213.2 218.1 

± 12.7 ± 10.4 ± 12.7 ± 13.7 ± 15.8 ± 15.5 ± 19.4 

1,000 12 172.9 181.6 187.3 196.4 202.0 205.8 211.3 

----1 ± 12.5 ± 13.2 ± 14.0 ± 18.6 ± 16.7 ± 17.3 ± 17.7 

I 

l 

w i 

' 

Hean ±S.D. 

* : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. i 

l 

! 

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l ~ 

I ~ 

I ~ 

I ~ 

1 ~ l 

I 
,_,--,, 

Table 2 -Continued Bll-044

Mean body weights(g) 

Sex Exp.group Number of Recovery 

(mg/kg/day) animals 1 3 5 8 10 12 14 (days) 

Vehicle 6 362.1 372.6 376.3 397.4 409.0 413.1 423.0 Male control ± 14.4 ± 15.9 ± 16. 4. ± 17.8 ± 18.9 ± 20.6 ± 21.6 

1,000 6 348.8 360.7 367.4 385.4 396.5 401.0 415.5 

± 20.3 ± 21.7 ± 25.4 ± 27.7 ± 28. 1 ± 29.3 ± 30.2 

Vehicle 6 216.4 220;9 224.2 229.7 237.5 238.8 246.2 Female control ± 17.2 ± 17.4 ± 17.1 ± 21.5 ± 21.2 ± 19.7 ± 22.1 

1,000 6 211.6 214.9 219.7 225.2 230.1 232.0 235.2 

± 17.4 ± 17.9 ± 18.6 ± 19.3 ± 22.5 ± 20.0 ± 21.3 

Mean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. ~ ' l 

...,. 

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V\ I 

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~ ~ ~ 

~ 

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/--

Table 3 28-day repeated-dose oral toxicity study in rats 

Mean food consumption(g/rat/day) 

Sex Exp.group Number of 

(mg/kg/day) animals 1 4 

Vehicle 12 16.2 20.2 

control ± 1.3 ± 1.2 

40 6 16.3 19.7 

Hale ± 1.4 ± 1.0 

200 6 15.5 19.6 

± 1.0 ± 1.3 

1,000 12 15.7 20.2 

± 1.0 ± 1.0 

Vehicle 12 13.9 16.3 

control ± 1.3 ± 0.9 

40 6 14.5 16.5 

Female ± 1.0 ± 0.7 

200 6 13.6 16.5 

± 2.2 ± 1.9 

1,000 12 12.3 16.2 

± 1.7 ± 1.1 

Mean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 

8 

-

23.1 

1. 3. 

22.1 

± 1.5 ± 

21.6 

1.8 

22.8 

1.2 

17.4 

± 1.4 ± 

17.4 

± 1.9 

17.9 

± 2.0 ± 

17.1 

± 1.1 ± 

Administration 

11 15 18 22 

25.1 26.1 27.4 28.0 

1.5 ± 1.8 ± 1.6 ± 1.9 

23.8 24.3 24.9 25.5 

1.7 ± 1.8 ± 1.7 ± 1.3 

23.3 25.0 25.4 26.1 

2.9 ± 3.1 ± 3.4 ± 2.9 

25.2 26.1 26.5 27.1 

2.0 ± 1.8 ± 1.8 ± 2.2 

17.7 17.7 16.6 17.9 

1.7 ± 1.8 ± 1.5 ± 1.2 

17.9 17.6 15.5 17.8 

1.9 ± 1.2 ± 1.8 ± 1.4 

18.1 18.0 16.8 18.5 

1.7 ± 1.3 ± 1.5 ± 1.5 

17.8 17.7 16.7 18.1 

1.1 ± 1.2 ± 1.1 ± 1.2 
_j ~ l 

I 

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Table 3 -Continued 

Mean food consumption(g/rat/day) 

Sex Exp.group Number of 

( mg/kg/ day) animals 4 8 

Vehicle 6 26.3 27.0 Hale control ± 1.3 ± 1.2 

1,000 6 25.7 27.1 

± 2.9 ± 2.5 

Vehicle 6 18.5 19.2 Female control ± 2.4 ± 2.6 

1,000 6 17.9 18.5 

± 1.2 ± 1.3 

Mean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 

Bll-0443 

Recovery 

11 14 (days) 

27.8 27.5 

± 1.5 ± 2.0 

27.3 27.9 

± 1.7 ± 2.2 

19.7 20.4 

± 2.2 ± 3.2 

18.1 18.8 

± 1.8 ± 1.7 
---: 

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"" ~ ( Table 4 28-day repeated-dose oral toxicity study in rats Hematology Sex Hale Female Mean ±S.D. Exp,group (mg/kg/day) Vehicle control 40 200 1,000 Recovery __ _ Vehicle control 1,000 Vehicle control 40 200 1,000 Number of animals 6 6 6 6 6 6 6 6 6 6 RBC WBC Hb (x10 4 /mm3 ) (x102 /mm3 ) (g/dl) 741 112 15.3 ± 41 ± 16 ± 0.6 765 95 15.3 ± 30 ± 22 ± 0.5 754 100 15.2 ± 21 ± 25 ± 0_4 762 100 15.4 ± 3L _____ LB__ ± 0.4 Ht (%) 44.1 ± 1. 7 44.7 ± 1.4 44.4 ± 1. 2 45.2 HCV (.um3) 59.6 ± 1. 6 58.4 ± 1. 8 58.9 ± 1. 0 59.4 __ LU ______ L2. 3 HCH (pg) 20.7 ± 0.6 19.9 ± 0.7 20.2 ± 0.2 20.2 HCHC (%) 34.8 ± 0.6 34.1 ± 0.3 34.3 ± 0.6 34.0 ± 0& __ ~ 0.3 825 93 15.8 45.9 55.7 19.1 34.3 ± 23 ± 11 ± 0 . 4 ± 1. 3 ± 2 . 5 ± 0 . 7 ± 0 • 7 816 107 15.7 45.6 55.9 19.2 34~4 ± 36 ± 25 ± 0.3 ± 1.0 ± 1.7 ± 0.6 ± 0.5 761 79 14.9 42.8 56.3 19.7 34.9 ± 37 ± 26 ± 0. 3 ± 1. 5 ± 1. 8 ± 0. 8 ± 0. 6 763 75 15.3 44.0 57.7 20.0 34.7 ± 27 ± 22 ± 0.5 ± 1.3 ± 1.4 ± 0.5 ± 0.3 769 79 15.4 43.7 56.8 20.0 35.2 ± 33 ± 22 ± 0. 4 ± 1. 3 ± 0. 9 ± 0. 5 ± 0. 6 772 90 15.5 44.1 57.2 20.1 35.2 ± 40 ± 29 ± 0.6 ± 1.1 ± 1.8 ± 0.5 ± 0.7 i\ecove"fY------------------------------------------------------- Vehicle 6 789 75 15.4 43.6 55.3 19.5 35.3 control ± 24 ± 15 ± 0. 6 ± 1. 7 ± 0. 9 ± 0. 2 ± 0. 5 1,000 6 813 77 15.6 44.9 55.2 19.2 34.7 ± 18 ± 25 ± 0.2 ± 0.7 ± 1.0 ± 0.4 ± 0.3 * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 

Platelet Reticulo 

(xl0 

4 /mm3 ) (%,) 

126.1 ± 14.6 124.6 ±_ 14.8 118.5 ± 12.8 117.6 ,± 4. 9 29 ± 9 25 ± 4 26 ± 5 30 ± 5 

120.8 24 

± 17.6 ± 7 

116.6 21 

± 7.3 ± 4 

111.3 22 

± 18. 2 ± 4 

126.7 17 

± 17.9 ± 5 

121.9 26 

± 10.5 ±10 

124.1 22 

± 10. 4 _ _!:_!.9 ___

113.7 ± 6.9 106.8 ± 10.7 26 ± 5 20* ± 3 
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Table 4 -Continued 

Hematology Bll-0443 

Sex 

Male 

Female 

Mean ±S.D. 

Exp.group 

(mg/kg/day) 

Vehicle 

control 

40 

200 

1,000 

Recovery 

Vehicle 

control 

1,000 

Vehicle 

control 

40 

200 

1,000 

Number of 

animals 

6 

6 

6 

6 

6 

6 

6 

6 

6 

6 

Differentiation of leukocyte (96) --

N-Band N-Seg Eosino Baso LYJllph Mono 

0.0 12.6 0.7 0.0 86.3 0.4 

±0.0 ± 4.0 ±0.8 ±0.0 ± 4.3 ±0.8 

0.2 8.6 0.4 0.0 90.7 0.2 

±0.3 ± 1. 7 ±0.5 ±0.0 ± 1. 9 ±0.3 

0.2 11.3 0.8 0.0 87.6 0.1 

±0.3 ± 3.3 ±0.4 ±0.0 ± 3.1 ±0.2 

0.3 10.2 0.7 o.o 88.8 0.1 

±o_. 3 ± 5.4 tQ.:..L_ ±o.o ___ ± 6.2 ____ ±o__,_? _____ 

0.0 5.4 0.9 0.0 93.4 0.3 

±0.0 ± 1.8 ±1.1 ±0.0 ± 2.0 ±0.3 

0.0 6.0 0.6 0.0 93.2 0.3 

±0.0 ± 2.6 ±0. 5 ±0.0 ± 3.0 ±0.3 

0.3 9.2 1.3 0.0 89.3 0.0 

±0.3 ± 2.5 ±1.0 ±0.0 ± 2.6 ±0.0 

0.2 8.6 0,8 0.0 90.4 0.1 

±0.3 ± 3.7 ±0.7 ±0.0 ± 4.1 ±0.2 

0.2 10.9 1.1 0.0 87.8 0.1 

±0.3 ± 7.6 ±1.0 ±0.0 ± 7.6 ±0.2 

0.1 9.1 1.1 0.0 89.4 0.3 

Re-c-overy-------------------------------------------------------------- ±0.2 ± 3.5 ±0.5 ±0.0 ± 3.4 ±0.4 

Vehicle 6 0.0 6.1 0.3 0.0 93.4 0.2 control ±0.0 ± 4.2 ±0.3 ±o.o ± 4.2 ±0.3 

1,000 6 0.0 6.8 1.1* 0.0 92.0 0.2 

±0.0 ± 2.2 ±0.8 ±0.0 ± 2.1 ±0.3 

* : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 
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Table 5 28-day repeated-dose oral toxicity study in rats Blood chemistry 

Exp.group Number of GOT GPT 

Sex (mg/kg/day) animals (IU/1) (I U/1) 

Vehicle 6 81 20 

control ±12 ± 1 

40 6 76 20 

±17 ± 3 

200 6 74 20 Hale ±11 ± 3 

1,000 6 81 21 

± 9 ± 2 

Recovery Vehicle 6 67 19 

control ± 8 ± 2 

1,000 6 66 24 

± 7 ± 8 

Vehicle 6 75 17 

control ± 5 ± 2 

40 6 62* 16 

± 6 ± 2 

200 6 71 18 Female ±12 ± 5 

1,000 6 78 17 

/~ 

ALP ChE y-{ITP T-Cho TG Glucose T-prote

(IU/1) (IU/1) (IU/1) (mg/dl) (mg/dl) (mg/dl) (g/dl) 

363 57 0.7 73 110 116.0 6.0 

±104 ± 7 ±0.2 ± 8 ± 32 ± 8.4 ±0. 2 

383 61 0.4* 69 96 106.9 5.8 

± 90 ± 23 ±0 .1 ± 8 ± 55 ± 10.6 ±0 .1 

350 53 0.4* 63 124 124.5 5.7* 

± 97 ± 18 ±0 .1 ± 9 ± 24 ± 15.2 ±0.1 

±_~ 

440 59 0.6 64 114 105.2 5.7** 

__ ± 17 ___ ±O.L ___ ±_s ___ ± 42_ ± 4.4 ±0.1 

328 54 0.6 64 81 138.8 6.0 

± 42 ± 22 ±0.1 ±12 ± 24 ± 22.8 ±0.1 

274 51 0.7 54 so 152.5 5.9 

± 67 ± 7 ±0. 2 ± 5 ± 20 ± 13.0 ±0. 2 

249 338 0.7 55 39 108.7 6.0 

± 69 ±102 ±o .1 ± 7 ± 9 ± 9.5 ±0.4 

253 414 0.7 54 37 125.2 6.1 

± 61 ± 82 ±0.3 ± 9 ± 10 ± 8.8 ±0.3 

232 359 0.9 64 38 119.3 6.0 

± 50 ±137 ±0.1 ±12 ± 6 ± 15.2 ±0. 2 

216 325 0.9 66 34 125.0 6.1 

Re-covery---------------------------------------------------------------------------------------

± 8 ± 2 ± 23 ± 73 ±0.3 ± 8 ± 5 ± 11.1 ±0.3 

Vehicle 6 70 19 181 control ± 5 ± 3 ± 42 

1,000 6 66 17 197 

± 2 ± 4 ± 47 

Mean ±S.D. * : Significantly different from vehicle control at P<0.05. 

** : Significantly different from vehicle control at P<O.Ol. 

385 0.9 

±144 ±0.3 

417 0.9 

±191 ±0.2 

69 43 140.8 6.5 

±15 ± 14 ± 28.9 ±0. 3 

72 38 133.7 6.4 

± 8 ± 6 ± 23.0 ±0 .2 
----1 

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Table 5 -Continued Bll-0

Blood chemistry 

Exp,group Number of BUN Creatinine T-Bil Ca IP Na K Cl 

Sex (mg/kg/day) animals (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mEq/l) (mEq/1) (mEq/l) 

Vehicle 6 13.9 0.37 0.27 10.2 7.7 144 4.2 107.1 control ± 1.8 ±0.11 ±0.05 ± 0.2 ±0.4 ± 1 ±0.2 ± 1.5 

40 6 12.0 0.32 0.28 10.6 7.7 144 4.3 106.4 

± 2.1 ±0.05 ±0.04 ± 0.2 ±0.4 ± 1 ±0.3 ± 1.6 

200 6 13.7 0.38 0.26 10.1 7.9 143 4.4 106.5 Hale ± 1. 5 ±0.05 ±0.05 ± 0.3 ±0.3 ± 1 ±0.3 ± 1.0 

1,000 6 13.1 0.38 0.25 10.2 7.4 144 4.4 107.1 

± 1.0 ±0.03 _±0. O,?__ ___ i_O. 4 ±0.5 __ _j_ __ l_ __ ____tQ.,L ____ L_Q_._?_ _

Recovery Vehicle 6 12.4 0.40 0.23 9.8 7.4 144 4.4 105.5 control ± 1.1 ±0.11 ±0.07 ± 0.2 ±0.3 ± 1 ±0.4 ± 1.1 

1,000 6 12.6 0.39 0.20 9.6 7.7 144 4.3 105.5 

± 1.0 ±0.04 ±0.02 ± 0.3 ±0.7 ± 2 ±0.4 ± 1.3 

Vehicle 6 15.1 0.47 0.20 10.5 7.6 142 4.2 107.8 control ± 0.8 ±0.04 ±0.02 ± 0.5 ±1. 2 ± 1 ±0.3 ± 0.5 

40 6 13.6 0.46 0.21 10.6 7.4 141 4.3 108.1 

± 0.8 ±0.03 ±0.02 ± 0.3 ±0.6 ± 1 ±0.1 ± 0.5 

200 6 14.3 0.44 0.21 10.5 7.5 142 4.3 108.0 Female ± 1. 6 ±0.05 ±0.04 ± 0.3 ±0.7 ± 1 ±0.3 ± 1.1 

1,000 6 14.4 0.51 0.18 10.8 7.6 142 4.2 108.0 

Rec-overT 

± --~~_7_ 

_______ i<? .. ! _ _o_1 ____ _lg_,_Q_? ________ j __ g_,_L _____ tQ_,_~ _________ t .... L ___ ±__g_,_3 _______ L_!_._I__ _

Vehicle 6 14.9 0.45 

control ± 2.2 ±0.06 

1,000 6 14.3 0.40 

± 1. 7 ±0.05 

Hean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 

0.21 9.7 

±0.02 ± 0.2 

0.24 9.6 

±0.05 ± 0.3 

6.8 142 4.2 106.8 

±0.6 ± 1 ±0.3 ± 0.4 

6.8 142 4.1 107.2 

±0.3 ± 1 ±0.3 ± 1.8 
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Table 6 28-day repeated-dose oral toxicity study in rats 

Urinalysis 

Sex 

Male 

Female 

Exp.group 

(mg/kg/day) 

Vehicle 

control 

40 

200 

1,000 

Recovery 

Vehicle 

control 

1,000 

Vehicle 

control 

40 

200 

1,000 

Recovery 

Vehicle 

control 

1,000 

a) Mean±S;D. 

Number of 

animals 

6 

6 

6 

6 

6 

6 

6 

6 

6 

6 

6 

6 

Volume•> 

(ml) 

25 

±11 

24 

±12 

18 

± 6 

18 

±10 

21 

± 8 

12 

± 6 

12 

± 5 

8 

±4 

14 

± 4 

15 

±13 

10 

± 7 

11 

± 4 

Color 

SY Y 

5 

4 2 

5 

4 2 

5 

2 4 

5 

5 

4 2 

3 3 

2 4 

3 3 

* Significantly different from vehicle control at P<0.05. ** Significantly different from vehicle control at P<O.Ol. SY Slightly yellow 

Y Yellow 

PH 

6.0 6.5 7.0 

0 3 3 

0 3 3 0 2 4 

0 3 3 

0 2 4 

0 4 2 

z 3 

2 4 0 

0 4 2 0 4 2 0 2 4 

4 

_ 

Protein Ketones Bilirubin 

± + ++ - ± + 

0 5 0 0 6 0 6 

0 4 2 0 0 6 0 6 

0 4 2 0 0 6 0 6 

0 2 3 0 6 0 6 

0 4 2 0 0 6 0 6 

0 5 0 0 5 6 

0 5 0 6 0 0 6 

0 3 z 6 0 0 6 0 6 0 0 6 0 0 6 

2 3 0 6 0 0 6 

3 2 0 6 0 0 6 

0 5 0 6 0 0 6 

Occult B

± 

6 

6 

6 

5 

6 

6 

5 

6 

6 6 6 6 
---1 

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Table 7 

Sex 

Male 

Female 

28-day repeated-dose oral toxicity study in rats 

Absolute organ weights 

Adrenal 

Exp.group Number of Spleen Liver Kidney Brain Testis gland Ovary 

(mg/kg/day) animals (g) (g) (g) (g) (g) (mg) (mg) 

Vehicle 6 0.60 9.58 2.26 1. 95 2.95 53.9 - 

control ± 0.09 ± 1.10 ± 0.19 ± 0.08 ± 0.19 ± 3.4 - 

40 6 0.56 8.86 2.31 1. 90 2.84 49.3 - 

0.07 ± 0.54 ± 0.14 ± 0.04 ± 0.10 ± 7.0 - 

200 6 0.59 8.89 2.22 1.85 2.77 47.4 - 

± 0.11 ± 1.45 ± 0.23 ± 0. 06 ± 0.21 ± 8.0 - 

1,000 6 0.59 8.48 2.33 1. 94 2.87 46.8 - 

± 0.11 ± 0.89 ± 0.18 ± 0.09 ± 0.14 Recovery ---------------------------------------------- 5.7 -----=------~___

Vehicle 6 0.66 11.13 2.51 2.00 3.20 51.5 - 

control ± 0.07 ± 1. 02 ± 0.13 ± 0.09 ± 0.16 ± 4.0 - 

1,000 6 0.67 11.12 2.65 1. 99 3.09 55.9 - 

± 0.15 ± 0.96 ± 0.21 ± 0. 06 ± 0.54 ± 10.6 - 

Vehicle 6 0.48 5.77 1. 53 1.81 60.4 99'. 0 

control ± 0.08 ± 0.32 ± 0.11 ± 0.10 ± 3.8 ± 28.6 

40 6 0.41 5.90 1. 61 1. 79 65.9 95.8 

0.04 ± 0.32 ± 0.12 ± 0. 09 ± 9.3 ± 12.2 

200 6 0.43 5.96 1.60 1.82 - 61.2 93.4 

0.06 ± 0.90 ± 0.23 ± 0.11 - ± 8.9 ± 14.7 

1,000 6 0.41 5.93 1.64 1.85 - 66.9 96.7 

Recovery-------------------------------------------------------------------------------------

± 0.06 ± 0.48 ± 0.14 ± 0.06 - ± 9.5 ± 22.9 

Vehicle 6 0.45 6.37 1.60 1.87 - 63.7 90.8 

control ± 0.04 ± 0.70 ± 0.08 ± 0.05 - ± 8.8 ± 8.9 

1,000 6 0.46 

± 0.06 

6.07 

± 0.55 

1. 71 

± 0.14 

1.81 

0.08 

66.3 

9.2 

89.1 

18.7 

Mean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. ~~ 
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Table 8 

Sex 

Hale 

Female 

/-

28-day repeated-dose oral toxicity study in rats Relative organ weights 

Exp.group Number of 

(mg/kg/day) animals 

Vehicle 

control 

40 

200 

1,000 

6 

6 

6 

6 

Spleen 

(g/lOOg) 

Liver 

(g/lOOg) 

0.19 2.98 

±0.02 ±0.22 

0.18 2.91 

±0.02 ±0.15 

0.19 2.88 

±0.02 ±0.25 

0.19 2.77 

±0.03 ±0.15 

Kidney 

(g/lOOg) 

Brain 

(g/lOOg) 

Testis 

(g/lOOg) 

Adrenal 

gland 

(mg/lOOg) Ovary 

(mg/lOOg) 

0.71 0.61 0.92 16.8 - 

±0.05 ±0.03 ±0.05 ± 0.8 - 

0.76 0.63 0.93 16.3 - 

±0.06 ±0.02 ±0.06 ± 2.5 - 

0.72 0.61 0.91 15.7 - 

±0.03 ±0.05 ±0.11 ± 4.0 - 

0.76 0.63 0.94 15.3 - 

ii"fico'Ver:-y----------------------- _j_~_Q..iL_ _____ i_Q_._9_1 ____ ±o :.9L.. _ _l__l~ ___ -___ 

vehicle 6 0.17 2.81 0.63 0.51 0.81 13.0 - 

control ±0.02 ±0.15 ±0.04 ±0.02 ±0.06 ± 1.2 - 

1,000 6 0.18 2.88 0.69 0.52 0.80 14.4 - 

Vehicle 

control 

40 

200 

1,000 

6 

6 

6 

6 

±0.03 ±0.12 ±0.05 ±0.03 ±0.13 ± 2.3 - 

0.24 2.90 0.77 0.91 - 30.5 49.2 

±o. 04 ±o. 07 ±o. 04 ±o. 07 - ± 3. 6 ± 11. 5 

0.21 3.02 0.82 0.92 - 33.8 49.1 

±o.01 ±0.13 ±0.02 ±0.06 - ± 5.7 ± 7.0 

0.21 2.93 0.79 0.90 - 30.3 46.0 

±0.03 ±0.26 ±0.06 ±0.02 - ± 4.9 ± 6.7 

0 . 21 3 . 0 6 0 . 8 5** 0 . 9 6 - 3 4 . 4 4 9 . 5 

iiecoveii---------------------------------------------------------------------

±0.02 ±0.06 ±0.04 ±0.07 - ± 3.3 ± 9.0 

Vehicle 6 0.20 2.83 0.72 0.84 - 28.5 40.4 

control ±0.03 ±0.16 ±0.05 ±0.05 - ± 5.2 ± 3.3 

1,000 6 0.21 2.78 0.78 0.83 - 30.4 40.7 

±0.01 ±0.18 ±0.07 ±0.07 - ± 3.8 ± 7.3 

Hean ±S.D. * : Significantly different from vehicle control at P<0.05. ** : Significantly different from vehicle control at P<O.Ol. 
~ l 

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Table 9 28-day repeated-dose oral toxicity study in rats 

Gross pathological findings 

Findings 

Vehicle 

control 

Vehicle 

control 

,/--

Male 

40 200 1,000 1,000 

Vehicle 

control 

Vehicle 

control 

F

40 

(Recovery) __ (Recovery) (Recovery) 

ta* ta ta ta ta ta ta ta ta 

6** 6 6 6 6 6 6 6 6 

Kidney 

Apparent spotty pattern of 

surface 0 0 0 0 0 1 0 0 0 

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

Pelvic dilatation 0 0 0 0 0 0 0 0 0 

Glandular stomach 

Blackish region of mucosa 1 0 0 0 0 0 0 0 0 

Testis 

Small 0 0 0 0 0 

Cerebrum 

Partial defect of cortex 0 0 0 0 0 0 0 0 

Skin 

..,. 

Loss of hair 0 0 0 0 0 0 0 0 

* ta,tern1inal autopsy. 

**Number of animals examined. 

::)? 

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:.n ~ '3i ~ //_. ___ Table 10 28-day repeated-dose oral toxicity study in rats Histopathological findings Findings Vehicle control Vehicle control .. ....--------... Male 40 200 1,000 1,000 Vehicle control Vehicle control F

40 

(Recovery) (_Recovery) {Recovery) 

ta* ta ta ta ta ta ta ta ta 

Grade 6** 6 6 6 6 6 6 6 6 

Liver .. :t:!:<:> .. ~.~-~<:>~.~l.\~~~-~ .. ~~~~-~~~~•••••••ou•ouoouoououuouo~/~.,~~•~•••uuuuu•u•=••••••••••••••••••ou•u••••=•••uuu•u•••••••uu•u•••=u••U''''"'''"''''''"'''''?.!.~••••••Um''"'"''''''''''"'='"''''"'''"'''"'''"''"'''''"'"''''''?./~

Microgranuloma + 2/6 l/6 l/6 

Spleen No abnormalities detected 6/6 6/6 6/6 

Kidney .,;t:!:<:>.,~.~-~.?.~.~-l.i~~~~.,~~~~-~~~~ . ., ... .,.,.,.,.,.,.,.,.,., . .,.,?./~_, . .,., ..... .,.,., .. .,.,.,.,::::., .. .,.,.,., .... .,.,.,.,.,.,.,::::.,.,.,,., • .,., .. .,.,.,., . .,.,:::::.,.,.,,.,.,., .. .,.,.,.,.,.,~!.~ . .,.,., .. .,., . .,., ..... .,.9.!.l.,.,.,.,., .. .,., . .,., . .,.,.,.,.,.,,.?.(,~.,.,.,., .. ., ..... ., ...... ?.!?. . ., ...... .,., ... ., .. ., .. .,.,::::., .... ., 

. 1.3..~-~<:>P!Y.F~ .. ~?.1:!.!~~ .......... ., .. ., ..... ., .. . ., .. ., .. .,.,., . ., .. .,.,l/~ .. ., ........ ., .. ., ... ., .. .,.::::: ..... ., . ., ..... .,., .... ., .. .,:::: .. ., .... ., .. ., ... ., .......... ::::: .. ., ........... ., .. ., ...... Q!.?. .................... ., .... 2!..1 ... ., .... .,., ..... ., ................ 2t.~ .......... .,., .. ., ....... 9.!.?. .......... ., ......... .,., . .,:::: ........

... ~.Y..~~.,f.?.~.~~~<:>~ .... ., .. .,,.., .. ., ..... ., ..... ., .. :: .. ., ..... ., ...... ., .. .,,9/?. ... .,.,., ................... :::::., ... ., .. ., .. .,.,., .. .,., . .,:::::., ....... .,., ....... ., ... ., .. :::: ........... ., ............... Q!..?. .. ., ... ., ......... ., ... .,.2!..! . .,.,.,.,., ........... ., ... ., .... .,.9/~_, .... ., ... ., .. ., ..... .,.9.!?. .. .,., ... .,.,,. .... .,., ..... ::::., .. .,, .

.!.)~r.<:>~.\.~_, ........... .,., ............. ., ...... ., ........... :: .... ., ..... ., .. ., .. ., .. .Y?.., .......... ., . ., ... ., .. ., .. :::: ............ .,., .............. :::: ............................. ::::: .......... .,.,.,,.., .... .,9.!.?.., .. ., ......... ., ... .,.,.,9.!.I .......... .,., .. ., .... ., ........ ., .. 9.(.~ ... ., .. ., ........... ., .. .2!.?. ........... ., ............... :::: .. ., . .,.

Increased eosinophilic bodies ++ 016 - - - 0/6 111 0/6 0/6 - 

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

Pelvic dilatation + 0/6 - - - 0/6 Oil 016 016 

Heart No abnormalities detected 6/6 - - - 6/6 - 6/6 

Forestomach No abnormalities detected 6/6 - - - 6/6 - 6/6 6/6 

Glandular stomach .. :t:!:<:> .. ~.\?.~<:>~~-~-~~i.~~ .. ~~~~-~-~~~ ............................... ?./~ ............................ ::::: ............................. :::: ............................. ::::: ........................... ?.!.?. ............................ :::: ..................................... ~t.~ ......................... §!.§ ............................ :::: ........

Necrosis of mucosa + 1/6 - - - 0/6 - 0/6 0/6 - 

* ta, terminal autopsy. **Number of animals autopsied. * * * Ntunber of animals affected I Ntunber of animals examined. -,Not examined. +,slight;++, moderate. 
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~ 

-=1 -~·· Table 10- Continued Histopathological fmdings Findings Grade Duodenum No abnonnalities detected Jejunum No abnonnalities detected ileum No abnonnalities detected Cecum No abnonnalities detected Colon No abnonnalities detected Rectum No abnonnalities detected Testis Vehicle control ta 6 6/6 6/6 6/6 6/6 6/6 6/6 Vehicle control 40 Male 200 I,OOO 1,000 (R.ecoveryl_ . ___ _ _ __ (Recovery) ta ta ta ta ta 6 6 6 6 6 6/6 6/6 6/6 6/6 6/6 6/6 No abnonnalities detected 6/6 6/6 - - 6/6 5/6 Vehicle control ta 6 6/6 6/6 6/6 6/6 6/6 6/6 Vehicle control 40 F

(Recovery) ___

ta ta 

6 6 

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

Focal atrophy of seminiferous tubules ++ 0/6 0/6 016 116 Adrenal No abnonnalities detected 6/6 6/6 6/6 

Cerebrum Partial defect of cortex + Ill Skin Atrophy of hair follicles + Ill 
( 

Photo. 1 

Photo. 2 

( 

I 

\ 

.l:Hl-U44..; 

Testis of a male rat given vehicle orally for 28 days (recovery group). 

Normal. 

' No. 7 animal. HE. x90. 

Testis of a male rat given test substance at a dose level of 1,000 mg/kglday 

orally for 28 days (recovery group). 

Focal atrophy of seminiferous tubules. 

No. 31 animal. HE. x90. 

- 1-

---~~~---
/ 

\ 

\ 

Addendum I 28-day repeated-dose oral toxicity study in rats 

Clinical signs 

Vehicle control 

Administration 

Signs Sex 2 3 

1,2,3,4,' 1,2,3,4, 1,2,3,4, 

5,6,7,8, 5,6,7,8, 5,6,7,8, 

9,10,1 1, 9,10,1 I, 9,10,1 I, No abnormalities detected Male 

12 12 12 

37,38,39, 37,38,39, 37,38,39, 

40,41,42, 40,41,42, 40,41,42, 

43,44,45, 43,44,45, 43,44,45, Female 

46 47 48 46 47 48 46 47 48 

Loss of hair (ventral neck) Male 

Female. 

- 1 -

BJ 1-0443 

Recovery 

4 2 (week) 

1,2,3,4, 

5,6,7,8, 7,8,9,10, 7,8,9,10, 

9,10,11, 11,12 I 1,12 

12 

37,38,39, 

40,41,42, 43,44,45, 44,45,46, 

43,44,45, 46,47,48 47,48 

. 46 47 48 

43 
( 

\ 

Addendum 1 -Continued 

Clinical signs 

40 mg/kg/day 

Signs 

No abnormalities detected 

* Animal number. 

Bll-0443 

Administration Recovery 

Sex 1 2 3 4 1 2 (week) 

Male 13,14,15, 13,14,15, 13,14,15, 13,14,15, 

................... J.§,J.?.,J.?. ...... !..?.!!..?.!!..?. .... J?.!!..?.!!..?. ...... ~.?.!n~~---····························································· 49,50,51, 49,50,51, 49,50,51, 49,50,51, Female 52,53,54 52,53,54 52,53,54 52,53,54 

- 2 -
( 

\ 

( 

Addendum 1 -Continued 

Clinical signs 

200 mg/kg/day 

Signs 

No abnormalities detected 

* Animal number. 

Bll-0443 

Administration Recovery 

Sex 2 3 4 1 2 (week) 

M I 19 20,21 • 19,20,21, 19,20,21, 19,20,21, a e t ' 

..........••...•.... &.'?.,&.:?.,&.4.. ..... ?..?.!t2.!?.:± ...... ?.?:!?.~.~?.± ..... .?.?.~?.~.'.?.~ ................................................................ . 55,56,57, 55,56,57, 55,56,57, 55,56,57, Female 58,59,60 58,59,60 58,59,60 58,59,60 

- 3 -
Bll-0443 

Addendum I -Continued 

Clinical signs 

1,000 mg/kg/day 

( 

,-

Administration Recovery 

Signs Sex 2 3 4 I 2 (week) 

25,26,27, . 25,26,27, 25,26,27, 25,26,27, 

No abnormalities detected Male 28,29,30, 28,29,30, 28,29,30, 28,29,30, 31,32,33, 31,32,33, 

31,32,33, 31,32,33, 31,32,33, 31 ,32,33, 34,35,36 34,35,36 

34,12 36 34,35,36 34,35,36 34,35,36 

61,62,63, 61,62,63, 61,62,63, 61,62,63, 

Female 64,65,66, 64,65,66, 64,65,66, 64,65,66, 67,68,69, 67,68,69, 

67,68,69, 67,68,69, 67,68,69, 67,68,69, 70,71,72 70,71,72 

70 71 72 70 71 72 70 71 72 70 71 72 

- 4 -
Addendum 2 28-day repeated-dose oral toxicity study in rats Bll-044

Body weights(g) 

Sex Exp.group Administration 

(mg/kg/day) Animal No. -1 1 3 5 8 10 12 (days) 

1 130.7 134.0 158.2 175.7 203.1 220.2 238.0 2 128.5 133.8 152.3 176.3 199.4 220.3 237.0 3 126.5 135.7 155.6 172.6 199.5 218.6 235.6 4 132.1 136.5 156.3 172.6 196.2 214.3 234.6 5 123.7 130.8 147.6 162.5 183.9 199.0 215.0 Vehicle 6 124.3 132.2 148.7 168.3 196.1 214.3 228.1 control 7 125.7 136.6 155.4 175.5 199.3 220.4 236.9 8 127.0 136.0 154.2 172.3 201.5 217.7 237.2 9 130.7 134.7 151.0 166.8 193.1 213.3 228.1 10 128.2 136.3 151.0 170.7 191.5 213.2 225.2 11 131.3 138.6 160.8 178.6 204.7 227.2 242.3 12 123.4 128.8 149.0 163.0 193.6 209.4 226.5 

13 124.2 130.5 147.2 161.6 182.4 198.2 214.6 14 130.3 136.9 151.8 168.9 196.9 213.3 224.7 40 15 126.6 133.6 149.2 167.6 192.2 210.1 225.4 16 125.2 132.7 147.8 164.6 185.7 202.6 214.7 17 131.8 136 .. 7 157.3 175.6 201.1 220.0 237.0 Hale 18 129.3 138.4 152.2 168.0 188.1 203.5 218.9 

19 126.1 135.6 150.9 168.3 198.2 216.0 234.3 20 128.3 132.3 147.1 162.9 190.0 206.4 216.9 200 21 133.5 142 .. 8 163.1 178.1 203.2 219.8 243.0 

C.l1 22 129.3 136.6 154.9 170.4 192.4 210.9 227.2 23 126.0 132.7 148.7 165.1 186.6 205.3 223.4 24 124.1 131.0 143.7 153.2 169.9 181.2 184.9 25 132.1 141.9 155.9 171.9 197.7 218.0 233.3 

l 26 126.3 133.7 147.8 164.5 184.9 200.2 210.4 27 130.6 135.7 152.7 168.7 192.1 211.2 225.4 ~ 28 124.0 131.8 147.4 162.8 187.8 204.1 219.5 29 128.3 135.2 149.8 166.9 190.1 205.1 218.5 1,000 30 125.4 132.9 145.4 167.4 193.4 210.0 226.9 31 130.8 135.8 149.0 168.2 191.6 207,0 221.9 1 32 123.2 132.5 146.9 165.5 190.4 208.8 225.8 33 128.7 138.8 154.9 172.9 198.4 220.0 233.7 

I 34 125.4 133.6 151.4 168.7 192.9 213.0 234.7 35 131.9 138.0 156.7 178.1 203.8 227.2 244.7 l 36 126.7 133.1 150.9 170.0 196.2 217.0 237.2 l 

~ ~ i ~ 

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1 

l 

I 

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i 
Addendum 2 -Continued Bll-044

Body weights(g) 

Sex Exp.group Administration 

(mg/kg/day) Animal No. 15 17 19 22 24 26 28 (days) 

1 263.6 282.5 297.6 321.1 337.0 344.7 357.1 

2 256.8 279.0 288.0 307.7 322.5 329.4 337.5 

3 264.7 286.5 297.9 332.2 346.6 354.1 369.1 

4 260.5 278.9 301.4 323.3 345.2 356.5 371.1 

5 234.1 253.1 264.5 285.5 301.8 311.4 321.0 

Vehicle 6 251.5 269.8 285.5 302.2 322.1 326.0 337.2 

control 7 257.2 277.8 292.7 317.0 328.6 342.0 354.1 

8 263.1 280.7 297.6 317.4 334.0 346.9 359.8 

9 253.8 274.6 287.4 310.7 321.9 333.9 341.6 

10 254.4 278.3 290.0 313.1 329.6 339.9 352.9 

11 277.2 296.6 314.0 344.7 363.6 372.4 383.6 

12 249.8 269.5 283.6 311.2 325.6 338.6 349.8 

13 239.7 255.1 269.8 292.3 304.8 318.2 326.7 

14 247.1 264.9 279.1 305.7 315.2 322.1 333.4 

40 15 245.1 265.6 277.5 297.9 314.7 327.2 333.8 

16 233.7 250.0 261.5 280.4 294.1 307.0 314.0 

17 261.8 277.4 293.7 316.9 327.2 340.9 350.9 

Hale 18 236.7 251.8 262.5 278.6 289.7 294.7 305.5 

_j 19 259.7 277.1 294.6 315.2 331.4 339.7 355.6 

20 245.2 260.9 271.0 291.2 306.4 316.4 322.9 

200 21 263.7 284.6 301.1 322.9 336.3 352.4 360.3 

O'l 22 250.4 269.7 280.2 298.9 310.2 320.5 330,6 23 248.7 267.2 284.1 304.3 320.8 331.0 342.9 

24 206.8 220.6 227.1 243.7 254.7 259.8 274.1 

25 263.7 282.2 298.5 322.7 339.3 354.7 368.4 

26 232.4 246.7 256.2 278.9 288.9 296.6 313.5 

27 247.0 261.7 275.2 295.1 305.9 320.2 329.7 28 248.4 262.8 274.6 296.7 307.1 314.3 327.7 29 240.5 258.1 267.2 285.3 301.6 308.7 317.2 1,000 30 250.8 268.2 283.8 302.7 315.5 329.2 334.7 31 245.1 260.6 273.4 293.0 307.4 312.4 326.9 32 248.8 265.7 279.6 304.9 314.5 325.6 334.0 33 251.1 266.1 280.1 298.2 313.1 318.6 333.7 34 265.5 284.2 299.7 324.3 345.3 356.6 372.5 l 

1 35 273.0 291.3 308.5 333.2 351.9 362.9 374.1 

I 36 261.3 279.4 295.1 313.8 329.4 337.6 346.1 

~ ~ ~ 

~. 

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1 

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Addendum 2 -Continued Bll-044

Body weights(g) 

Sex Exp.group Administration 

(mg/kg/day) Animal No. -1 1 3 5 8 10 12 (days) 

37 116.2 121.4 133.1 145.1 157.7 165.1 173.0 38 111.2 116.5 127.9 135.3 144.1 157.6 153.6 39 110.0 117.3 126.1 138.5 148.9 157.5 165.5 40 115.0 118.1 129.5 143.6 156.1 168.3 172.4 41 113.6 120.2 132.5 144.3 158.9 170.1 176.0 Vehicle 42 112.9 119.3 124.6 136.5 142.5 148.9 156.4 control 43 114.9 118.5 133.9 137.8 147.5 155.3 154.3 44 110.6 115.6 127.5 137.7 154.7 163.2 174.0 45 113.6 121.4 132.9 144.1 158.9 174.2 177.3 46 107 .,8 114.5 124.3 134.0 143.9 152.2 157.4 47 113.7 119.6 130.5 138.8 145.3 151.8 157.4 48 117.8 123.8 133.3 147.6 160.0 171.7 180.2 

49 113.6 120.9 128.7 141.1 149.2 156.6 159.1 50 114.5 li9.1 134.6 143.4 153.8 167.1 168.7 40 51 115.3 118.5 130.5 138.0 146.8 154.7 157.0 52 108.2 113.1 121.0 139.2 153.9 166.5 168.7 53 111.7 116.9 127.2 139.0 149.2 158.9 163.2 Female 54 115.9 120.9 131.5 143.7 149.6 163.5 166.4 ~ 55 109.1 114.3 126.5 130.8 145.3 147.7 161.0 56 112.1 116.3 130.7 139.6 154.8 160.0 169.9 200 57 115.0 122.9 137.4 148.3 168.4 175.3 183.7 

--.] 58 111.9 116.5 127.2 138.3 150.5 161.8 168.2 59 117.5 125.7 139.5 150.1 172.5 174.5 188.3 60 113.8 116.6 124.6 131.3 141.1 150.3 150.4 

61 112.3 117.7 131.0 139.2 152.7 160.2 170.5 

j 62 116.8 121.9 136.6 149.5 159.2 171.2 177.6 63 115.5 117.4 129.9 142.3 155.0 164.7 170.5 i 64 114.2 119.9 134.7 144.5 165.0 173.1 182.3 

---1 

65 110.5 116.6 126.9 138.6 144.7 156.8 159.6 1,000 66 111.4 111.1 120.6 126.2 138.8 147.8 149.4 I 67 114.7 119.1 126.8 131.4 140.2 143.8 152.4 ' ~ 68 110.8 113.5 125.7 134.0 148.4 159.8 167.3 69 115.4 122.2 135.0 143.6 156.5 169.8 178.2 70 112.7 116.6 128.9 137.7 151.8 166.9 171.0 71 117.2 119.2 129.9 142.2 150.5 159.5 160.5 l 72 110.2 117.4 126.6 135.0 146.0 152.6 159.5 

-I 

~ 

~· 

. 

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I 
Addendum 2 -Continued Bll-044

Body weights(g) 

Sex Exp,group Administration 

(mg/kg/day) Animal No. 15 17 19 22 24 26 28 (days) 

37 179.9 186.6 196.3 198.2 214.1 208.1 218.6 38 169.5 177.7 181.9. 194.2 189.0 203.3 197.4 39 164.8 182.5 177.3 197.6 201.5 207.9 212.7 40 181.7 195.9 198.5 212.1 216.5 227.1 228.5 41 177.8 192.9 200.1 207.1 216.1 214.0 228.9 Vehicle 42 156.5 168.3 168.4 178.5 185.3 189.7 190.4 control 43 165.2 173.1 175.7 184.4 183.5 194.9 191.2 44 175.5 184.6 191.0 198.3 208.6 214.6 214.1 45 190.5 197.9 207.4 217.2 219.1 227.8 231.2 46 159.5 169.1 173.4 182.3 186.3 194.9 196.9 47 171.3 173.1 180.7 192.2 188.2 195.6 195.6 48 181.6 194.8 192.9 206.0 216.3 216.0 224.5 49 172.3 183.8 187.9 197.5 204.9 199.6 213.9 50 177.7 184.7 187.7 202.6 197.6 209.5 204.2 40 51 163.7 163.1 173.9 187.6 186.0 188.2 195.6 52 181.5 184.6 202.1 207.2 218.3 220.9 228.5 53 171.0 176.5 182.1 195.0 201.1 207.0 206.2 Female 54 174.5 182.0 189.4 203.8 198.0 211.3 204.6 55 163.3 178.0 178.1 191.7 195.6 199.5 201.3 -1 56 177.3 190.0 194.4 200.6 210.5 218.2 211.4 200 57 191.9 194.1 206.0 219.4 224.1 228.9 234.0 ex:> 58 176.7 186.3 188.5 197.5 207.5 208.8 211.2 59 193.4 201.8 210.1 217.3 234.9 230.8 249.3 60 165.2 173.7 183.1 185.4 195.0 192.7 201.1 61 174.6 180.3 185.4 188.3 199.6 194.6 210.3 62 173.6 197.6 191.7 212.2 215.4 219.6 223.3 1 63 184.3 192.5 199.3 218.4 213.2 214.5 225.1 • 64 193.1 191.1 211.0 222.1 228.6 231.4 239.2 ~ 65 169.1 178.5 183.9 192.5 202.1 203.5 210.1 1,000 66 150.8 157.5 165.0 165.8 174.3 176.7 181.8 67 157.1 161.1 166.6 168,0 178.2 178.7 187.9 68 168.9 181.6 188.1 197.0 203.1 211:7 212.4 69 188.3 197.0 202.6 213.1 220.1 224.8 230.3 70 lSi. 8 191.9 197.2 206.8 209.0 216.5 217.9 l 71 168.0 174.4 180.2 185.0 184.8 194.3 189.4 72 H)5. 3 176.0 176.6 187.4 195.9 203.3 207.8 --------~ i 

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----~, 

Addendum 2 -Continued Bll-0443 Body weights(g) 

Sex Exp.group Recovery 

(mg/kg/day) Animal No. 1 3 5 8 10 12 14 (days) 

7 359.2 369.1 369.3 387.2 398.2 397.9 404.4 8 359.9 372.5 373.8 393.0 404.3 409.7 418.9 Vehicle 9 348.6 354.3 360.7 382.9 391.0 395.7 403.9 control 10 357.9 368.5 373.9 395.2 409.7 413.4 425.8 11 390.3 402.3 408.2 432.6 445.1 452.7 463.0 Male 12 356.4 368.7 371.6 393.5 405.7 409.1 422.1 31 331.1 341.7 342.3 353.0 366.5 368.6 380.2 32 335.4 345.4 351.0 370.9 377.0 382.1 398.9 1,000 33 333.7 347.5 353.0 369.6 380.8 385.0 399.0 34 371.3 384.0 395.3 410.3 421.8 430.9 448.0 35 377.3 392.3 403.1 426.7 438.7 442.3 456.6 36 343.8 353.4 359.5 381.9 394.1 396.9 410.0 43 201.0 207.4 208.9 208.5 217.9 220.6 224.0 44 220.3 229.0 232.7 232.6 243.3 241.6 258.2 Vehicle 45 242.7 247.9 248.7 259.1 269.5 266.9 275.0 control 46 201.3 203.2 207.7 214.4 219.8 219.8 224.3 47 204.4 208.5 211.4 212.4 221.4 227.3 232.4 Female 48 228.9 229.3 235.5 250.9 252.9 256.8 263.5 

j 67 191.4 189.0 196.7 202.2 198.1 209.3 208.4 I 68 219.0 219.8 229.3 235.3 236.9 244.8 241.1 

£) 

1,000 69 232.4 235.4 239.7 245.7 255.1 250.7 259.8 70 223.6 229.6 231.8 237.6 246.4 241.1 249.8 71 189.9 198.8 196.4 200.1 207.0 203.9 209.6 72 213.1 217.0 224.2 230.1 236.9 242.2 242.4 

l 

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Addendwn 3 28-day repeated-dose oral toxicity study in rats 

Food consumption(g/rat/day) 

Sex Exp,group Administration 

(mg/kg/day) Animal No. 1 4 8 11 15 .18 22 

1 16.7 21.0 24.6 26.1 27.0 27.7 29.3 

2 15.7 21.1 24.3 26.4 26.0 26.5 26.8 

3 15.9 21.2 24.5 26.4 28.2 29.2 30.4 

4 15.2 20.5 23.1 24.8 27.9 28.3 29.4 

Vehicle 

5 14.5 18.6 20.6 21.9 22.2 23.3 24.4 

6 16.6 18.5 22.2 23.5 25.6 27.1 28.0 

control 7 17.9 21.8 23.1 26.1 24.9 26.6 26.2 

8 19.1 21.2 24.3 26.2 27.9 27.9 27.7 

9 16.4 18.4 22.4 24.3 23.9 26.3 25.7 

10 15.8 19.6 22.3 24.4 26.1 28.8 28.8 

11 16.0 20.9 24.1 27.0 27.2 29.1 30.8 

12 14.3 19.5 21.9 24.4 26.0 27.5 28.9 

13 15.8 18.7 20.9 22.8 23.5 24.3 25.4 

14 17.3 20.4 23.4 23.2 23.4 24.9 25.3 

40 15 16.9 19.4 22.3 25.0 25.1 26.2 25.9 

16 13.8 18.5 19.7 21.6 21.9 22.0 23.7 

Hale 

17 16.6 20.7 23.6 26.5 27.0 26.9 27.6 

18 17.5 20.7 22.4 23.6 25.1 25 .. 0 25.3 

19 15.3 19.4 22.1 24.8 26. 1. 25.2 27.5 

---1 20 14.0 18.8 21.2 22.2 25.1 25.0 25.4 

I 

I-' 

200 21 17.0 21.3 23.9 26.7 27.6 28.8 29.2 

0 

22 15.7 20.5 22.6 24.7 26.4 27.6 26.1 

23 15.5 20.0 21.4 23.2 26.1 26.4 27.6 

24 15.7 17.6 18.5 18.3 18.9 19.1 20.9 

25 15.9 21.0 22.3 24.7 26.9 27.0 29.3 

26 13.6 18.8 20.5 22.5 23.0 23.5 23.4 

28 

27 15.5 20.6 22.8 24.1 25.3 25.9 25.4 

29 

15.4 18.9 21.7 24.2 25.4 25.8 26.5 

17.7 21.4 23.2 24.6 25.5 26.7 26.9 

1,000 

31 

30 14.1 19.3 22.2 23.3 25.2 25.2 26.0 

32 

16.1 19.7 22.1 23.4 24.3 25.1 25.3 

33 

15.8 19.6 23.0 25.5 26.0 27.6 27.5 

34 

16.3 

15.9 

20.5 23.9 25.7 25.9 24.6 25.6 

35 16.3 

21.0 23.0 28.2 29.7 29.2 30.7 

36 

22.1 25.4 29.1 28.4 29.5 30.5 

15.8 19.9 23.1 26.8 27.1 27.4 27.6 

-I S> %) 

-1 ~ 
/-- --~, Addendum 3 -Continued 

Food consumption(g/rat/day) 

Sex Exp.group Administration 

(mg/kg/day) Animal No. 1 4 8 11 15 18 22 

37 12.1 16.1 17.2 17.8 17.7 15.2 17.7 

38 12.6 14.8 15. 9. 17.2 17.1 16.0 17.5 

39 13.9 16.4 17.1 17.2 18.0 17.7 18.2 

40 13.9 16.2 17.2 17.3 17.5 17.0 17.5 

Vehicle 

41 14.1 l6.3 17.5 18.8 18.1 17.8 18.7 

42 12.5 15.4 16.1 15.8 15.8 16.2 16.9 

control 43 13.1 16.1 16.2 16.3 15.5 14.5 15.6 

44 12.7 15.5 18.9 18.4 18.3 16.3 19.1 

45 16.1 17.7 19.9 21.2 21.3 18.9 19.8 

46 14.3 16.4 16.6 17.8 15.9 15.0 16.2 

47 15.2 16.9 16.4 15.0 16.9 15.7 17.9 

48 15.7 17.9 20.0 19.4 20.6 19.0 19.2 

49 13.0 15.8 15.5 16.2 16.6 15.7 16.4 

50 14.2 17.2 17.7 18.6 17.5 15.1 17.4 

40 51 14.1 16.2 16.1 15.8 16.2 13.0 16.4 

52 15.2 15.8 19.4 19.6 19.1 17.3 19.6 

53 14.4 16.4 15.9 16.6 17.0 14.1 17.4 

Female 54 15.8 17.3 19.8 20.4 19.0 17.6 19.4 

55 14.3 15.2 16.4 16.0 16.9 16.0 17.7 

--: 56 11.9 16.2 17.2 17.6 17.7 15.5 18.3 

,_. 

200 57 15.0 18.8 20.7 20.6 19.4 18.9 20.0 

- 58 10.2 14.9 16.2 17.3 16.5 15.2 16.9 

59 16.3 18.8 20.2 19.9 19 .a 18.3 20.7 

60 13.7 15.0 16.6 17.4 17.6 16.9 17.4 

61 12.1 16.0 17.4 17.5 18.0 16.0 17.8 

l 62 14.0 17.3 17.6 19.4 18.3 17.8 19.3 

63 10.8 15.9 17.8 18.5 17.9 16.8 17.3 

64 13.8 17.8 18.8 18.4 19.6 16.6 20.3 

-~ 1,000 65 13.8 16.8 16.5 17.1 16.8 16.2 17.9 

66 9.7 15.8 17.2 17.2 17.7 16.6 17.9 

I 67 12.1 15.2 14.3 15.4 15.4 14.0 15.7 

~ 68 9.6 14.4 16.3 17.3 17.2 16.4 17.9 

69 14.4 17.7 18.3 19.1 19.5 17.9 19.8 

- 70 11.3 16.5 17.4 18.2 18.6 17.2 18.3 

l 71 12.4 15.1 17.1 17.8 16.6 17.9 17.2 

I 

72 13.5 15.4 16.7 18.1 16.8 16.6 17.9 

:y 

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Addendum 3 

Sex 

Hale 

Female 

-Continued 

Food consumption(g/rat/day) 

Exp.group 

(rog/kg/day) Animal No. 4 

7 25.4 . 

8 27.0 

Vehicle 9 25.5 

control 10 24.6 

11 27.9 

12 27. 3 .. 

31 22.2 

32 25.2 

1,000 33 24.4 

34 27.3 

35 30.5 

36 24.7 

43 16.5 

44 20.5 

Vehicle 45 :20.1 

control 46 16.4 

47 16.3 

48 2L3 

67 16.2 

68 18.1 

1,000 69 18.1 

70 t8.4 

71 16.8 

72 19.6 

-------- 

·---- -·- --

Bll-0443 

Recovery 

8 11 14 (days) 

25.6 26.6 24.4 

26.9 27.3 27.1 

26.1 26.6 27.1 

26.9 27.6 28.7 

28.9 30.7 30.5 

27.6 28.2 27.3 

23.4 . 25.5 24.4 

26.9 26.7 27.7 

26.5 26.3 26.7 

27.4 28.1 29.0 

31.2 30.3 30.4 

27.0 27.1 29.4 

16.3 17.6 16.9 

19.3 20.3 21.5 

21.8 22.2 21.9 

16.9 17.5 17.3 

18.1 18.2 19.6 

22.8 22.1 25.4 

16.8 15.3 18.6 

18.1 17.7 20.0 

19.5 19.3 19.4 

18.3 18.5 17.3 

17.8 17.4 16.5 

20.3 20.6 20.9 
,/--, 

Addendum 4 28-day repeated-dose oral toxicity study in rats 

Hematology 

Exp.group RBC WBC Hb Ht HCV HCH HCHC Platelet 

Sex (mg/kg/day) Animal No. (xl0 4 /mm3 ) (xl02 /mm3 ) (g/dl) (%) ( J.tm3 ) (pg) (%) (xl04 /mm

1 769 128 15.7 44.0 57.2 20.4 35.7 108.7 

2 768 109 16.2 46.3 60.3 21.1 35.0 107.2 

3 748 130 15.2 44.1 59.0 20.3 34.5 139.1 

4 686 116 14.7 42.2 61.5 21.4 34.8 140.1 

5 694 91 14.7 42.2 60.8 21.2 34.8 130.3 

Vehicle 6 779 95 15.5 45.8 58.8 19.9 33.8 131.1 

control iie-c-ove-ii-----------------------------------c----------------------------------------------------------------------------

7 832 98 16.3 46.3 55.6 19.6 35.2 137.4 

8 793 85 15.6 44.8 56.5 19.7 34.8 96.5 

9 860 91 15.2 43.9 51.0 17.7 34.6 127.3 

10 834 77 15.8 46.8 56.1 18.9 33.8 140.7 

11 810 94 15.8 47.3 58.4 19.5 33.4 105.3 

12 819 110 15.8 46.5 56.8 19.3 34.0 117.3 

13 727 134 14.4 42.7 58.7 19.8 33.7 145.9 

14 810 80 15.8 46.5 57.4 19.5 34.0 132.6 

40 15 778 71 14.8 43.6 56.0 19.0 33.9 121.6 

16 781 97 15.4 45.1 57.7 19.7 34.1 120.7 

17 746 91 15.4 45.0 60.3 20.6 34.2 125.5 

~ Hale 18 750 99 15.7 45.4 60.5 20.9 34.6 101.1 

19 738 131 14.8 42.8 58.0 20.1 34.6 100.0 

20 769 101 15.6 45.5 59.2 20.3 34.3 126.2 

..... 

w 

200 21 724 127 14.7 43.7 60.4 20.3 33.6 123.3 

22 761 72 15.3 45.5 59.8 20.1 33.6 126.4 

23 753 81 15.4 43.6 57.9 20.5 35.3 130.6 

24 780 86 15.5 45.4 58.2 19.9 34.1 104.7 

1 25 711 139 14.7 42.8 60.2 20.7 34.3 120.4 

26 804 93 15.8 46.4 57.7 19.7 34.1 117.4 

I 27 793 74 15.5 45.4 57.3 19.5 34.1 110.9 

~ 28 783 92 15.1 44.9 57.3 19.3 33.6 123.8 

29 738 102 15.5 45.4 61.5 21.0 34.1 113.0 

1,000 iiecoveri-------------------------------------------------------------------------------------------------------------------------

30 745 98 15.7 46.5 62.4 21.1 33.8 120.0 

31 8'26 143 15.6 45.8 55.4 18.9 34.1 104.4 

32 843 89 16.2 46.0 54.6 19.2 35.2 121.9 

J 33 861 98 15.9 47.2 54.8 18.5 33.7 115.9 

i 34 757 96 15.4 44.9 59.3 20.3 34.3 113.1 

35 807 134 15.5 45.2 56.0 19.2 34.3 124.8 

J 

36 803 82 15.5 44.5 55.4 19.3 34.8 119.4 

~ 

~ 

~ 

-..... 
/ 

____, 

...... 

.... 

l 

~ 

I 

~ • 1 

l ~ :i< 

I 

~ J ~ 

! 

~)i 

Addendum 4 -Continued 

Hematology 

Sex 

Hale 

Exp,group 

(rog/kg/day) 

Vehicle 

control 

40 

200 

1,000 

------·----

. 

---·-~----- ------

Bll-0443 

Differentiation of leukocyte (%) 

Animal No. N-Band N-Seg Eosino Baso Lymph Mono 

1 0.0 8.5 0.0 0.0 89.5 2.0 2 0.0 17.0 0.5 0.0 82.5 0.0 3 0.0 9.5 0.0 0.0 90.5 0.0 4 0.0 17.0 2.0 0.0 80.5 0.5 5 0.0 9.0 1.0 0.0 90.0 0.0 R.ec-ovii-ii------------------------------------------------------------------------------------------------------- 6 0.0 14.5 0.5 0.0 85.0 0.0 

7 0.0 5.5 1.0 0.0 93.0 0.5 8 0.0 6.5 1.0 0.0 92.0 0.5 9 0.0 8.5 0.0 0.0 91.0 0.5 10 0.0 4.0 3.0 0.0 93.0 0.0 11 0.0 4.0 0.0 0.0 96.0 0.0 12 0.0 4.0 0.5 0.0 95.5 0.0 13 0.0 10.5 0.0 0.0 89.5 0.0 14 0.5 9.5 0.0 0.0 90.0 0.0 15 0.0 8.5 0.0 0.0 91.0 0.5 16 0.0 6.5 1.0 0.0 92.5 0.0 17 0.0 6.5 0.5 0.0 93.0 0.0 18 0.5 10.0 1.0 0.0 88.0 0.5 19 0.5 15.5 0.5 o.o 83.5 0.0 20 0.0 6.5 1.0 0.0 92.5 0.0 21 0.0 12.0 1.0 0.0 86.5 0.5 22 0.0 11.0 0.5 0.0 88.5 0.0 23 0.0 14.0 0.5 0.0 85.5 0.0 24 0.5 9.0 1.5 0.0 89.0 0.0 25 0.0 5.0 0.0 0.0 95.0 0.0 26 0.0 9.5 1.5 0.0 89.0 0.0 27 0.5 8.0 0.5 0.0 91.0 0.0 28 0.5 6.5 0.0 0.0 93.0 0.0 29 0.5 20.0 2.0 0.0 77.5 0.0 

R.e-coviiri-------------------------------------------------------------------------------------------------------- 30 0.0 12.0 0.0 0.0 87.5 0.5 

31 0.0 6.0 0.5 0.0 93.0 0.5 32 0.0 5.5 1.0 0.0 93.5 0.0 33 0.0 6.0 0.0 0.0 94.0 0.0 34 0.0 2.5 0.0 0.0 97.5 0.0 35 0.0 5.5 1.0 0.0 93.0 0.5 36 o.o 10.5 1.0 0.0 88.0 0.5 
~ ' 

l ~ ~ i 

-

CJ1 

~ 

~ 

~ 

~ 

~ 

,/·~ 

Addendum 4 -Continued 

Sex 

Female 

Hematology 

Exp.group 

(mg/kg/day) 

Vehicle 

control 

40 

200 

1,000 

RBC WBC Hb Ht MCV HCH MCHC Platelet 

Animal No. (xl04 /mm3 ) (xl0 2 /mm3 ) (g/dl) (%) (,um 3 ) (pg) (%) (xl04 /mm

37 748 53 15.0 43.6 58.3 20.1 34.4 121.1 

38 763 127 14.7 41.8 54.8 19.3 35.2 79.2 

39 774 70 15.1 42.9 55.4 19.5 35.2 101.9 

40 825 62 15.3 45.1 54.7 18.5 33.9 118.9 

41 735 77 14.5 40.8 55.5 19.7 35.5 130.3 

Recover:Y---------------------------------------------------~--------------------------------------------------------------------

42 718 86 14.9 42.3 58.9 20.8 35.2 116.4 

43 792 77 15.5 43.4 54.8 19.6 35.7 105.6 

44 767 98 14.8 41.6 54.2 19.3 35.6 122.6 

45 774 60 14.9 42.2 54.5 19.3 35.3 115.7 

46 815 83 16.1 45.1 55.3 19.8 35.7 119.8 

47 764 59 15.0 43.1 56.4 19.6 34.8 107.0 

48 820 74 15.9 46.2 56.3 19.4 34.4 111.4 

49 746 40 14.7 43.0 57.6 19.7 34.2 129.1 

50 765 106 15.8 45.3 59.2 20.7 34.9 114.8 

51 749 62 15.2 43.9 58.6 20.3 34.6 158.1 

52 735 76 14.6 42.0 57.1 19.9 34.8 129.5 

53 810 82 15.6 44.8 55.3 19.3 34.8 123.1 

54 773 86 15.6 45.1 58.3 20.2 34.6 105.4 

55 759 62 15.7 43.6 57.4 20.7 36.0 118.9 

56 769 54 15.4 43.2 56.2 20.0 35.6 117.1 

57 746 109 14.9 43.2 57.9 20.0 34.5 128.8 

58 827 62 16.0 45.7 55.3 19.3 35.0 114.3 

59 734 87 14.9 41.8 56.9 20.3 35.6 112.4 

60 780 97 15.4 44.5 57.1 19.7 34.6 140.0 

61 821 70 16.2 45.3 55.2 19.7 35.8, 138.3 

62 727 90 14.8 42.8 58.9 20.4 34.6 121.9 

63 751 71 14.8 43.5 57.9 19.7 34.0 134.3 

64 766 143 15.9 45.0 58.7 20.8 35.3 115.4 

65 821 99 16.1 44.9 54.7 19.6 35.9 111.9 

Re-cove-r:Y-------------------------------------------------------------------------------------------------------------------------

66 744 65 15.2 43.0 57.8 20.4 35.3 122.8 

67 807 79 15.4 43.9 54.4 19.1 35.1 99.3 

68 811 89 15.8 45.5 56.1 19.5 34.7 98.1 

69 840 93 15.7 45.3 53.9 18.7 34.7 107.3 

70 807 48 15.5 44.4 55.0 19.2 34.9 125.6 

71 825 46 15.5 45.5 55.2 18.8 34.1 111.8 

72 787 107 15.4 44.5 56.5 19.6 34.6 98.9 
-1 

' 0 

l 

...... 

' O"l I 

i 

• 

l ~ ~ l 

1 

~ 

~ 

($"""-., ~ /-~, Addendum 4 -Continued Hematology Exp.group Bll-0443 Differentiation of leukocyte ( %) Sex (mg/kg/day) Animal No. N-Band N-Seg Eosino Baso Lymph Mono Female Vehicle control 40 zoo 1,000 37 0.5 13.0 1.5 0.0 85.0 0.0 38 0.5 9.5 1.0 0.0 89.0 0.0 39 0.0 8.0 0.0 0.0 92.0 0.0 40 0.0 5. 5 3.0 0.0 91.5 0.0 41 0.5 9.0 0.5 0.0 90.0 0.0 42 0.5 10.0 1.5 0.0 88.0 0.0 iiecaveir"Y------------------------------------------------------------------------------------------------------ 43 0.0 5.0 0.0 0.0 95.0 0.0 44 0.0 14.0 0.5 0.0 85.5 0.0 45 0.0 6.0 0.5 0.0 93.5 0.0 46 o.o 6.0 0.5 0.0 93.0 0.5 47 o.o 2.0 0.5 0.0 97.5 0.0 48 0.0 3.5 0.0 0.0 96.0 0.5 49 0.5 10.5 0.0 0.0 89.0 0.0 50 0.0 7.0 0.5 0.0 92.5 0.0 51 0.5 8.5 0.5 0.0 90.5 0.0 52 0.0 15.0 2.0 0.0 83.0 0.0 53 o.o 5.0 1.0 0.0 94.0 0.0 54 0.0 5.5 0.5 0.0 93.5 0.5 55 0.0 4.5 0.5 0.0 95.0 0.0 56 0.5 23.0 1.0 0.0 75.5 0.0 57 0.0 8.5 2.0 0.0 89.5 0.0 58 0.0 7.0 0.5 0.0 92.5 0.0 59 0.5 17.5 0.0 0.0 81.5 0.5 60 0.0 5.0 2.5 0.0 92.5 0.0 61 0.0 11.0 0.5 0.0 88.0 0.5 62 0.0 15.0 1.5 0.0 83.5 0.0 63 0.0 7.0 1.5 0.0 91.5 0.0 64 o.o 9.5 1.0 0.0 89.0 0.5 65 0.5 6.5 1.5 0.0 91.5 0.0 66 o.o 5.5 0.5 0.0 93.0 1.0 iiec-over_y __________________________________________________________________________________________________________ 67 0.0 5.5 1.0 0.0 93.5 0.0 68 0.0 4.0 1.0 0.0 95.0 0.0 69 ci.o 10.0 1.0 0.0 89.0 0.0 70 0.0 6.0 2.5 0.0 91.5 0.0 71 0.0 6.5 1.0 0.0 92.0 0.5 72 0.0 8.5 0.0 0.0 91.0 0.5 
Addendum 5 28-day repeated-dose oral toxicity study in rats 

Blood chemistry 

Exp.group GOT GPT ALP ChE y-GTP T-Cho TG Glucose 

Sex (mg/kg/day) Animal No. (IU/1) (IU/1) (IU/1) (IU/1) (IU/1) (mg/dl) (mg/dl) (mg/dl) 

1 84 20 283 57 0.4 79 116 114.8 

2 70 20 523 62 0.5 62 99 120.5 

3 77 21 294 60 0.8 85 157 122.8 

4 85 22 256 67 0.9 73 125 125.1 

Vehicle 

5 101 20 386 51 0.8 70 59 103.5 

6 71 19 436 47 0.5 67 102 109.0 

control Recovery------------------------------------------------------------------------------------------------------------------------

7 79 21 284 41 0.7 65 56 109.7 

8 62 17 382 49 0.6 80 77 128.5 

9 65 20 374 31 0.6 56 78 160.5 

10 60 20 293 46 0.5 47 74 148.0 

11 75 20 334 92 0.5 62 127 165.9 

12 63 17 303 64 0.8 72 75 119.9 

13 57 18 376 106 0.3 83 154 116.2 

14 73 22 445 56 0.5 60 58 97.9 

40 15 65 19 341 40 0.3 71 153 111.7 

16 77 16 284 51 0.6 67 128 119.9 

17 107 20 528 49 0.4 64 35 93.4 

Hale 18 75 23 321 61 0.2 68 47 102.2 

_, 19 72 17 395 47 0.3 69 122 117.2 

..... 

20 60 20 247 83 0.6 76 106 112.5 

200 21 68 17 397 42 0.2 59 144 147.1 

-.) 22 84 21 250 67 0.4 50 95 140.3 

23 71 23 313 41 0.4 57 159 118.3 

24 91 24 496 40 0.5 64 118 111.8 

I 25 78 21 527 45 0.6 51 167 107.3 

26 69 19 454 59 0.5 63 58 99.9 

27 87 23 496 84 0.7 72 124 100.2 

l 28 91 20 328 37 0.6 73 122 107.4 

l 29 88 24 484 68 0.4 64 71 110.9 

1 1,000 Recove-ry-------------------------------------------------------------------------------------------------------------------------

30 72 21 350 61 0.9 61 141 105.4 

- 31 65 25 193 44 0.6 59 65 171.0 

32 56 23 356 45 0.7 51 83 159.5 

33 74 40 288 49 0.6 54 82 143.1 

l 34 60 19 318 48 0.5 62 110 159.8 

i 35 67 18 193 62 1.0 52 88 136.2 

I 36 72 21 293 55 0.9 48 51 145.5 

I ~ ~ • 

I ~ ~ 

I 

1 \.f\ 

I 

I 
/~, 

Addendum 5 

Sex 

--j Hale 

' j ~ ' co I l I 

' j 

l 

J 

-

l 

i ~ ~ 

_J 

I 

~ 

~ I 

···--------- -----

__ I 

-Continued 

Blood chemistry 

Exp.group 

(mg/kg/day) 

Vehicle 

control 

40 

200 

1,000 

BUN 

Animal No. (mg/dl) 

1 15.0 

2 14.5 

3 12.4 

4 14.9 

5 15.7 

Creatinine 

(mg/dl) 

0.35 

0.23 

0.49 

0.46 

0.43 

T-Bil 

(mg/dl) 

0.21 

0.31 

0.26 

0.27 

0.23 

Ca 

(mg/dl) 

10.5 

10.3 

10.2 

10.0 

10.1 

IP 

(mg/dl) 

7.4 

7.9 

7.5 

7.4 

8.3 

Na 

(mEq/1) 

143 

146 

145 

144 

144 

K 

(mEq/1) 

4.0 

3.9 

4.0 

4.3 

4.5 

iiEicoverY:--------------------------------------------------------------------------------------------------------------------

6 11.0 0.23 0.34 10.3 7.7 143 4.4 

7 10.6 0.29 0.27 9.4 7.0 143 5.0 

8 11.6 0.47 0.20 9.8 7.2 145 4.1 

9 12.4 0.48 0.17 10.0 7.8 144 4.1 

10 12.7 0.42 0.20 9.8 7.3 145 4.2' 

11 13.9 0.48 0.17 9.7 7.4 143 4.2 

12 13.0 0.23 0.36 9.9 7.8 143 4.5 

13 12.5 0.32 0.27 10.4 7.1 144 3.9 

14 12.3 0 0 36, 0.24 10.8 7.5 145 4.3 

15 8.7 0.33 0.27 10.4 7.7 144 4.4 

16 11.9 0.37 0.25 10.6 7.9 143 4.1 

17 15.1 0.22 0.36 10.7 8.4 143 4.9 

18 11.3 0.31 0.28 10.6 7.6 143 4.4 

19 14.3 0.40 0.26 9.8 8.1 142 4.2 

20 15.8 0.36 0.33 10.2 7.5 144 4.4 

21 12.9 0.36 0.25 10.4 8.3 144 4.3 

22 13.0 0.38 0.19 9.9 7.6 143 4.1 

23 11.4 0.31 0.27 10.6 8.0 142 4.9 

24 14.5 0.47 0.23 9.8 7.6 143 4.2 

25 13.3 0.39 0.27 10.1 7.9 142 4.3 

26 11.5 0.35 0.23 10.8 6.8 143 4.3 

27 13.2 0.36 0.25 10.0 7.0 144 4.3 

28 14.3 0.43 0.23 9.9 8.0 144 4.6 

29 13.9 0.37 0.28 9.9 7.3 144 4.3 

Rec-overY:-----------------------------------------------------------------------------------------------------------------------

30 12.2 0.35 0.23 10.4 7.6 144 4.4 

31 11.7 0.46 0.17 10.2 8.9 146 4.7 

32 13.7 0.39 0,20 9.6 7.6 143 3.8 

33 11.2 0.35 0.21 9.6 7.2 145 4.0 

34 12.6 0.38 0.19 9.5 7.1 142 4. 1 

35 13.7 0.38 0.19 9.4 7.3 142 4.9 

36 12.7 0.38 0.23 9.4 7.8 143 4.2 
----1 

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l 

._. 

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I ~ .I 

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1 

/-'-..._ 

Addendum 5 -Continued 

Blood chemistry 

Sex 

Female 

Exp.group 

(mg/kg/day) 

GOT 

Animal No. (IU/1) 

GPT 

(IU/1) 

ALP 

(IU/1) 

ChE 

(IU/1) 

y-GTP 

(IU/1) 

T-Cho 

(mg/dl) 

TG 

(mg/dl) 

Glucose 

(mg/dl) 

37 69 15 169 287 0.7 64 45 119.2 

38 77 18 235 349 0.7 44 29 97.7 

39 73 20 281 365 0.6 55 36 105.4 

40 84 16 357 192 0.6 56 55 109.4 

Vehicle 

41 73 14 266 330 0.9 50 36 120.1 

control iiecoveri---------------------------------------------------------------------------------------------------------------------

42 74 18 183 504 0.9 62 35 100.1 

43 65 17 226 247 0.9 64 39 112.6 

44 69 20 201 336 1.2 73 31 124.8 

45 72 19 169 315 0.7 96 70 135.4 

46 77 16 168 486 1.0 60 37 141.9 

47 65 16 110 630 0.6 61 35 133.9 

48 73 24 211 297 1.2 57 47 195.9 

49 56 17 265 428 0.3 37 30 125.4 

50 64 16 347 397 0.8 57 52 113.6 

40 51 71 15 273 286 1.0 56 38 135.1 

52 56 13 264 423 0.5 60. 43 115.8 

53 59 15 183 544 0.9 52 28 131.6 

54 67 18 186 408 0.9 63 29 129.9 

55 61 16 207 244 1.0 55 29 103.6 

56 65 23 267 349 0.8 74 34 111.3 

200 57 62 12 195 296 0.9 74 42 141.5 

58 68 15 209 624 1.0 78 39 113.1 

59 94 23 196 363 0.8 54 44 135.2 

60 75 16 318 276 1.0 50 41 111.3 

61 82 16 216 245 0.5 53 40 119.1 

62 70 19 217 420 0.6 64 32 116.4 

63 72 16 230 404 0.9 6! 29 115.9 

64 83 16 210 277 0.8 71 36 145.6 

65 89 15 245 276 1.2 66 27 125.8 

1,000 iie-cover"Y--------------------------------------------------------------------------------------------------------------------------

66 72 18 175 329 1.3 77 38 126.9 

67 64 24 182 791 0.8 73 43 110.9 

68 67 11 155 

33~ 1.0 77 40 133.9 

69 64 18 290 303 1.3 78 33 132.0 

70 68 15 194 274 0.7 59 30 126.7 

71 66 18 181 433 0.6 80 46 121.3 

72 65 14 181 362 0.9. 67 36 177.5 
/.-

___, 

: I 

; 

N 

0 

I 

I 

i 

. ~ ~ ~ l ~ :5? 

! 

~ 

I ~ ~ 

I 

I 

Addendum 5 -Continued 

Blood chemistry 

Sex 

Female 

Exp.group 

(mg/kg/day) 

Vehicle 

control 

40 

200 

1,000 

BUN 

Animal No. (mg/dl) 

Creatinine 

(mg/dl) 

T-Bil 

(mg/dl) 

Ca 

(mg/dl) 

IP 

(mg/dl) 

Na 

(mEq/1) 

K 

(mEq/1) 

37 15.1 0.46 0.23 10.4 6.5 143 4.5 

38 15.4 0.48 0.23 10.2 7.0 142 3.9 

39 16.4 0.48 0.19 10.2 6.9 141 4.0 

40 13.8 0.47 0.20 10.2 7.5 140 4.5 

41 15,0 0.39 0.19 10.4 8.2 141 4.5 

Recover::y--------------------------------------------------------------------------------------------------------------

42 14.8 0.51 0.17 11.5 9.7 143 4.0 

43 13.5 0.42 0.22 9.4 6.5 142 4.3 

44 13.4 0.48 0.22 9.7 6.4 143 4.2 

45 15.1 0.46 0.21 9.7 6.1 143 3.9 

46 13.2 0.44 0.19 9.9 6.9 141 3.9 

47 15.0 0.35 0.24 9.7 7.6 142 4.6 

48 19.1 0.53 0.17 10.0 7.5 142 4.3 

49 13.2 0.41 0.21 10.1 7.4 141 4.5 

50 14.2 0.45 0.24 10.5 7.7 142 4.4 

51 14.2 0.49 0.23 10.4 6.2 141 4.1 

52 14.2 0.46 0.22 10.7 7.8 142 4.4 

53 13.6 0.43 0.18 11.0 7.3 141 4.3 

54 12.3 0.49 0.19 10.6 7.7 140 4.3 

55 15.1 0.38 0.23 10.3 7.0 141 4.5 

56 13.8 0.43 0.22 10.4 6.5 141 4.0 

57 12.8 0.42 0.21 10.5 7.6 142 4.2 

58 16.9 0.51 0.20 10.4 7.7 142 4.2 

59 14.6 0.47 0.13 10.6 7.9 142 4.0 

60 12.8 0.40 0.25 11.0 8.4 141 4.6 

61 11.8 0.39 0.22 10.4 7.4 142 4.3 

62 16.3 0.51 0.18 11.2 7.6 141 4.1 

63 16.0 o:54 0.20 10.9 6.7 143 4.1 

64 14.0 0.60 0.17 11.0 7.9 143 3.9 

65 13.6 0.51 0.16 10.7 8.3 140 4.6 

Rec-av-er::y----------------------------------------------------------------------------------------------------------------------

66 14.8 0.51 0.17 10.4 7.5 142 3.9 

67 14.0 0.36 0.22 9.8 7.0 141 3.7 

68 14.1 0.46 0.20 9.7 6.5 142 3.9 

69 14.0 0.36 0.31 9.5 6.9 141 4.5 

70 16.1 0.39 0.27 9.3 6.4 142 4.4 

71 15.9 0.38 0.24 10.1 6.7 143 4.0 

72 11.5 0.46 0.19 9.4 7.0 141 3.9 
_j 

I 

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l ~ ~ ! 

l ~ I 

i 

l 

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N 

y 

)...:> ~ ~ --~--~ ---~ 

Addendum 6 28-day repeated-dose oral toxicity study in rats Urinalysis 

Sex Male Exp.group (mg/kg/day) Vehicle control 40 200 1,000 SY : Slightly yellow Y : Yellow Animal No. VolUJDe (ml) Color pH Protein Ketones Bilirubin Occult Blood 

1 24 SY 6.5 ± ± - - 

2 7 y 6.5 + ± - - 

3 35 SY 6. 5 ± ± - - 

4 21 SY 7.0 ± ± - - 

5 39 SY 7. 0 ± ± - - 

6 23 SY 7. 0 ± ± - - 

-!lecciver'Y·---·----·-·---·--·-------·----------------------·--·-·----···---------------·----····--··----·--------------------------

7 14 SY 6.5 ± ± - - 

8 21 SY 6.5 ± ± - - 

9 10 y 7.0 + ± - - 

10 31 SY '7.0 ± ± - - 

11 18 SY 7.0 + ± - - 

12 29 SY 7. 0 ± ± - - 

13 35 SY 7.0 ± ± - - 

14 28 SY 6. 5 ± ± - - 

15 34 SY 7. 0 ± ± - - 

16 8 y 6.5 + ± - - 

17 25 SY 7. 0 ± ± - - 

18 11 y 6.5 + ± - - 

19 20 SY 7.0 ± ± - - 

20 25 SY 7. 0 ± ± - - 

21 14 SY 6.5 + ± - - 

22 21 SY 7.0. ± ± - - 

23 20 SY 7.0 ± ± - - 

24 8 y 6.5 + ± - - 

25 33 SY 7. 0 ± ± - ± 

26 26 SY 7. 0 ± ± - - 

27 16 SY 6.5 + ± - - 

28 7 y 6. 5 t+ ± - - 

29 9 y 6.5 + ± - - 

30 14 SY 7. 0 + ± - - 

·ife.ca'Ver'Y _____________________ ................................................................................................................................................

31 14 SY 6.5 + ± - - 

32 22 SY 7. 0 ± ± - - 

33 9 y 6. 5 + ± - - 

34 12 y 6.5 + + - - 

35 9 y 7.0 + ± - - 

36 5 y 6.5 + ± - - 
__, 

1 

l 

l ~ j -~ N N ~ ~ )-) 

...._j ~ Addendum 6 Sex Female -Continued Urinalysis Exp.group (mg/kg/day) Vehicle control 40 zoo 1,000 SY : sllgntly yellow Y : Yellow 

Animal No. Volume (rnl) Color pH Protein Ketones Bilirubin Occult Blood 

37 10 SY 6.0 ± - - - 

38 4 y 6.0 + - - - 

39 14 SY 6.5 ± - - - 

40 12 SY 6.5 ± - - - 

41 11 SY 6.5 ± - - - 

42 19 SY 7.0 ± - - ± 

-fecovei:;;·-··-----------------------------------------------------------------------------------------------------------------------

43 24 SY 7. 0 - - - - 

44 6 y 6. 5 + - - - 

45 12 SY 7.0 ± - - - 

46 4 y 6. 5 + - - - 

47 10 y 7.0 ± - - - 

48 5 y 7.0 ± - - - 

49 15 SY 6.5 ± - - - 

50 5 y 6.0 + - - - 

51 3 y 6.0 ++ - - - 

52 10 y 6.5 ± - - - 

53 5 y 6. 5 + - - - 

54 10 y 6.5 ± - - - 

55 13 SY 6. 5 ± - - - 

56 16 SY 6.5 ± - - - 

57 10 y 7.0 ± - - - 

58 ZO SY 7. 0 ± - - - 

59 13 SY 6.5 ± - - - 

60 9 y 6. 5 ± - - - 

61 39 SY 7. 0 - - - - 

62 18 SY 6. 5 ± - - - 

63 12 SY 7. 0 ± - - - 

64 7 y 6.5 + - - - 

65 6 y 6.5 + - - - 

66 5 y 6.5 + - - - 

'lfecover"Y······----······-·-·····----------------------·-------------------------------------------------------------------------------------------·----------·-----

67 1 Z SY 6. 5 ± - - - 

68 6 y 6.5 ± - - - 

69 15 SY 6.5 ± - - - 

70 10 y 7. 0 ± - - - 

71 15 SY 6.5 ± - - - 

72 5 y 6.0 + - - - 
-i 

-I ~ 9 ~:') w 

-1 iv ~ 

-1 ~ I 

l 

j 

I 

Addendum 7 28-day repeated-dose oral toxicity study in rats 

Absolute organ weights 

Bll-0

Sex 

Male 

Exp. group Spleen Liver 

(g) Kidney (g) Brain (g) Testis (g) 

Adrenal 

gland (mg) Ovary (mg) Body weig

(mg/kg/day) Animal No. (g) (g) 

Vehicle 

control 

40 

200 

1,000 

1 0.66 10.16 2.27 2.09 3,03 53.6 - 328.5 

2 0.55 8.63 2.46 1.93 2.63 56.5 - 311.4 

3 0.70 10.41 2.29 1.97 3.05 58.0 - 337.3 

4 0.68 10.52 2.44 1.98 3.17 55.5 - 342.4 

5 0.54 7.83 1.94 1.89 2.95 50.2 - 294.1 

6 0.49 9.93 2.17 1.86 2.87 49.7 - 309.7 iiec-overi-------------------------------------------------------------------------------------------------------

7 0.66 9.99 2.70 1.97 3.33 49.7 - 381.0 

8 0.70 11.51 2.46 1.90 2.95 46.0 - 396.8 

9 0.54 10.44 2.33 1.97 3.09 55.5 - 380.2 

10 0.65 10.52 2.42 1.94 3.30 56.6 - 396.7 

11 0.76 12.78 2.61 2.16 3.13 51.6 - 432.1 

12 0.62 11.55 2.51 2.04 3.37 49.4 - 389.8 

13 0.63 9.12 2.08 1.92 2.92 53.3 - 304.6 

14 0.47 8.39 2.32 1.96 2.81 40.9 - 308.6 

15 0.49 9.06 2.20 1.88 2.76 41.4 - 311.9 

16 0.57 9.34 2.40 1.90 2.99 58.3 - 295.2 

17 0.65 9.24 2.47 1.92 2.83 53.0 - 326.1 

18 0.53 7.99 2.37 1.83 2.71 49.0 - 280.2 

19 0.79 9.50 2.48 1.86 2.72 35.3 - 334.5 

20 0.54 9.35 2.14 1.86 3.11 46.7 - 298.5 

21 0.63 10.35 2.48 1.91 2.55 42.0 - 335.6 

22 0.56 8.60 2.04 1.83 2.69 56.5 - 306.5 

23 0.55 9.37 2.23 1.88 2.92 48.8 - 317.9 

24 0.48 6.16 1.93 1.74 2.61 55.0 - 252.3 

25 0,78 10.00 2.45 2.01 2.86 48.4 - 334.4 

26 0.50 7.29 2.19 1.83 2.93 47.4 - 283.9 

27 0.51 8.60 2.27 1.95 3.05 45.8 - 302.6 

28 0.49 8.58 2.64 1.81 2.81 41.9 - 303.9 

29 0.64 8.00 2.21 2.03 2.90 40.6 - 294.5 

30 0.61 8.40 2.21 1.98 2.64 56.5 - 317.4 iiecover-i--------------------------------------------------------------------------------------------------------------------------

31 0.63 10.09 2.33 1.90 2.14 46.8 - 354.6 

32 0.51 11.01 2.64 1.94 3.18 44.6 - 373.7 

33 0.54 10.51 2.88 2.02 3.60 62.6 - 368.7 

34 0.81 12.70 2.81 2.06 3.53 50.8 - 414.3 

35 0.88 11.77 2.75 2.02 3.24 72.5 - 428.5 

36 0.63 10.63 2.49 1.97 2.87 58.0 - 381.4 
---' 

l i -I ~ 1 

: l ~ N ..;::. ~ ~ ~ ...j ~ ---------···· ------------·--------- ---·-------- -- ----·-- Addendum 7 -Continued Bll-0

Sex Female Absolute organ weights Exp.group Spleen Liver (g) Kidney (g) Brain (g) Testis (g) Adrenal gland (mg) Ovary (mg) Body weig

(g) (mg/kg/day) Animal No. (g) Vehicle control 40 200 1,000 37 0.40 5.88 1.47 1.71 - 60.0 83.0 203.4 38 0.43 5.45 1.50 1.74 - 64.7 93.4 187.0 39 0.54 5.67 1.55 1.75 - 53.8 99.4 201.3 40 0.39 6.07 1.54 1.86 - 62.5 102.9 212.9 41 0.60 6.17 1.72 1.98 - 59.2 150.3 214.7 42 0.49 5.38 1.41 1.82 - 62.4 64.9 177.5 ii"Eic-over"Y---------------------------------------------------------------------------------------------------------------------

43 0.47 5.52 1.61 1.83 - 53.9 96.4 206.4 44 0.51 6.31 1.63 1.92 - 55.0 91.8 232.9 45 0.47 7.22 1.73 1.94 - 64.6 99.5 254.5 46 0.44 6.19 1.55 1.83 - 78.1 77.8 206.2 47 0.43 5.81 1.59 1.82 - 65.9 82.2 214.2 48 0.39 7.19 1.51 1.89 - 64.8 97.2 236.7 49 0.45 5.80 1.61 1.73 - 68.7 79.4 197.2 50 0.43 5.51 1.56 1.71 - 50.7 85.1 194.1 51 0.38 5.70 1.46 1.82 - 77.4 106.5 177.7 52 0.46 6.36 1.81 1.93 - 72.5 106.5 213.4 53 0.38 5.82 1.57 1.85 - 63.2 91.1 191.6 54 0.38 6.20 1.65 1.72 - 62.9 106.2 199.6 55 0.44 5.45 1.39 1.72 - 65.6 81.9 190.2 56 0.40 5.68 1.61 1.84 - 65.3 99.0 201.2 57 0.50 7.36 1.86 1.95 - 67.6 108.1 219.1 58 0.34 5.12 1.42 1.78 - 43.4 69.2 198.6 59 0.45 6.78 1.91 1.93 - 61.5 101.6 223.7 60 0.47 5.35 1.42 1.69 - 63.6 100.3 184.8 61 0.42 5.71 1.62 1.86 - 50.8 71.1 182.9 62 0.44 6.29 1.86 1.87 - 72.5 124;4 211.3 63 0.48 6.09 1.66 1.89 - 72.1 123.1 198.5 64 0.44 6.57 1.69 1.87 - 76.8 99.4 213.1 65 0.35 5.64 1.57 1.89 - 68.2 84.5 190.1 66 0.34 5.25 1.43 1.73 - 60.8 77.9 168.6 iiec-over-:Y---------------------------------------------------------------------------------------------------------------------------

67 0.38 5.46 1.61 1.77 - 63.4 79.7 194.0 68 0.50 6.71 1.96 1.92 - 72.8 122.3 225.7 69 0.55 6.23 1.71 1.73 - 55.6 88.9 239.3 70 0.45 5.86 1.59 1.84 - 72.5 77.6 232.1 71 0.41 5.48 1.60 1.71 - 56.2 69.7 192.0 72 0.47 6.65 1.77 1.87 - 77.1 96.5 228.7 
__J 

N 

CJl 

l I 

' 

l 

-1 ~ • 1 

i 

I 

• 

~ ~ i ~ 

_J 

I 

~ 

i 

' i 

I 

1 

l 

Addendum 8 28-day repeated-dose oral toxicity study in rats 

Relative organ weights 

Bll-0

Sex 

Hale 

Exp.group Spleen 

(mg/kg/day) Animal No. (g/lOOg) 

Liver 

(g/lOOg) 

Kidney 

(g/lOOg) 

Brain 

(g/lOOg) 

Testis 

(g/lOOg) 

Adrenal 

gland Ovary Body weig

(mg/lOOg) (mg/lOOg) (g) 

1 0.20 3.09 0.69 0.64 0.92 16.3 - 328.5 2 0.18 2.77 0.79 0.62 0.84 18.1 - 311.4 3 0.21 3.09 0.68 0.58 0.90 17.2 - 337.3 4 0.20 3.07 0.71 0.58 0.93 16.2 - 342.4 5 0.18 2.66 0.66 0.64 1.00 17.1 - 294.1 

control Vehicle Recover'Y----------------------------------------------------------------------------------------------------------------------

6 0.16 3.21 0.70 0.60 0.93 16.0 - 309.7 

7 0.17 2.62 0.71 0.52 0.87 13.0 - 381.0 8 0.18 2.90 0.62 0.48 0.74 11.6 - 396.8 9 0.14 2.75 0.61 0.52 0.81 14.6 - 380.2 10 0.16 2.65 0.61 0.49 0.83 14.3 - 396.7 11 0.18 2.96 0.60 0.50 0.72 11.9 - 432.1 12 0.16 2.96 0.64 0.52 0.86 12.7 - 389.8 

13 0.21 2.99 0.68 0.63 0.96 17.5 - 304.6 14 0.15 2.72 0.75 0.64 0.91 13.3 - 308.6 40 15 0.16 2.90 0.71 0.60 0.88 13.3 - 311.9 16 0.19 3.16 0.81 0.64 1.01 19.7 - 295.2 17 0.20 2.83 0.76 0.59 0.87 16.3 - 326.1 18 0.19 2.85 0.85 0.65 0.97 17.5 - 280.2 

19 0.24 2.84 0.74 0.56 0.81 10.6 - 334.5 20 0.18 3.13 0.72 0.62 1.04 15.6 - 298.5 200 21 0.19 3.08 0.74 0.57 0.76 12.5 - 335.6 22 0.18 2.81 0.67 0.60 0.88 18.4 - 306.5 23 0.17 2.95 0.70 0.59 0.92 15.4 - 317.9 24 0.19 2.44 0.76 0.69 1.03 21.8 - 252.3 

25 0.23 2.99 0.73 0.60 0.86 14.5 - 334.4 26 0.18 2.57 0.77 0.64 1.03 16.7 - 283.9 27 0.17 2.84 0.75 0.64 1.01 15.1 - 302.6 28 0.16 2.82 0.87 0.60 0.92 13.8 - 303.9 29 0.22 2.72 0.75 0.69 0.98 13.8 - 294.5 1,000 30 0.19 2.65 0.70 0.62 0.83 17.8 - 317.4 iiec-ove-r'Y---------------------------------------------------------------------------------------------------------------------------

31 0.18 2.85 0.66 0.54 0.60 13.2 - 354.6 32 0.14 2.95 0.71 0.52 0.85 11.9 - 373.7 33 0.15 2.85 0.78 0.55 0.98 17.0 - 368.7 34 0.20 3.07 0.68 0.50 0.85 12.3 - 414.3 35 0.21 2.75 0.64 0.47 0.76 16.9 - 428.5 36 0.17 2.79 0.65 0.52 0.75 15.2 - 381.4 
I 

l 

I 

----! 

I 

I 

N 

0> 

J 

I 

I 

i ~ ~ l 

n 

-I ~ 1---' ._,_j 

l 

I -c: 

! 

1 

l 

I 

I 

l 

' l 

I /--~ Addendum 8 -Continued Bll-0

Sex Female Relative organ weights Adrenal Exp.group Spleen Liver (g/lOOg) KidneY (g/lOOg) Brain (g/lOOg) Testis (g/lOOg) gland Ovary Body weig

(mg/kg/day) Animal No. (g/lOOg) (mg/lOOg) (mg/lOOg) (g) Vehicle control 40 200 1,000 37 0.20 2.89 0.72 0.84 - 29.5 40.8 203.4 38 0.23 2.91 0.80 0.93 - 34.6 49.9 187.0 39 0.27 2.82 0.77 0.87 - 26.7 49.4 201.3 40 0.18 2.85 0.72 0.87 - 29.4 48.3 212.9 41 0.28 2.87 0.80 0.92 - 27.6 70.0 214.7 Recover'Y---------------------------------------------------------------------------------------------------------------------

42 0.28 3.03 0.79 1.03 - 35.2 36.6 177.5 43 0.23 2.67 0.78 0.89 - 26.1 46.7 206.4 44 0.22 2.71 0.70 0.82 - 23.6 39.4 232.9 45 0.18 2.84 0.68 0.76 - 25.4 39.1 254. 5

46 0.21 3.00 0.75 0.89 - 37.9 37.7 206.2 47 0.20 2.71 0.74 0.85 - 30.8 38.4 214.2 48 0.16 3.04 0.64 0.80 - 27.4 41.1 236.7 49 0.23 2.94 0.82 0.88 - 34.8 40.3 197.2 50 0.22 2.84 0.80 0.88 - 26.1 43.8 194.1 51 0.21 3.21 0.82 1. 02 - 43.6 59.9 177.7 52 0.22 2.98 0.85 0.90 - 34.0 49.9 213.4 53 0.20 3.04 0.82 0.97 - 33.0 47.5 191.6 54 0.19 3.11 0.83 0.86 - 31.5 53.2 199.6 55 0.23 2.87 0.73 0.90 - 34.5 43.1 190.2 56 0.20 2.82 0.80 0.91 - 32.5 49.2 201.2 57 0.23 3.36 0.85 0.89 - 30.9 49.3 219.1 58 0.17 2.58 0.72 0.90 - 21.9 34.8 198.6 59 0.20 3.03 0.85 0.86 - 27.5 45.4 223.7 60 0.25 2.90 0.77 0.91 - 34.4 54.3 184.8 61 0.23 3.12 0.89 1.02 - 27.8 38.9 182.9 62 0.21 2.98 0.88 0.88 - 34.3 58.9 211.3 63 0.24 3.07 0.84 0.95 - 36.3 62.0 198.5 64 0.21 3.08 0.79 0.88 - 36.0 46.6 213.1 65 0.18 2.97 0.83 0.99 - 35.9 44.5 190.1 Rec-overY:--------------------------------------------------------------------------------------------------------------------------

66 0.20 3.11 0.85 1.03 - 36.1 46.2 168.6 67 0.20 2.81 0.83 0.91 - 32.7 41.1 194.0 68 0.22 2.97 0.87 0.85 - 32.3 54.2 225.7 69 0.23 2.60 0.71 0.72 - 23.2 37.2 239.3 70 0.19 2.52 0.69 0.79 - 31.2 33.4 232.1 71 0.21 2.85 0.83 0.89 - 29.3 36.3 192.0 72 0.21 2.91 0.77 0.82 - 33.7 42.2 228.7 
( 

( 

Addendum 9 

Sex 

28-day repeated-dose oral toxicity study in rats 

Pathological findings 

Bll-0443 

Exp.group Animal Fate Gross findings (mg/kglday) No. Histopathological findings 

1 ta Glandular stomach Glandular stomach 

Blackish region of mucosa Necrosis of mucosa+ 

................................... _ .......................... G. .. E~8.!.?..~~?. .. ~P.?..~!>..'l.. .................................................................................................................................................... . 2 ta NAD Liver 

·····-··---·····-····-········-· .. ·······················--·-··-····-··············-········ .. ·················-·························-·············~·!.~~?..W..~?.~~~.~~ ... ~ .......................................... . 3 ta NAD Liver 

.................................................................................................................................................... -.................... ~.!.~!..?..~~?.~!..9..~-~-~······························-·····-·-· 4 ta NAD NAD ·········-····-················-·--············-·····-·········-···················-························ .. ········· .. ·-·······················-··················-··--···················-·············-········-················-······-·········· 5 ta NAD NAD .............................................................................................................................................................................................................................................................................. 

Vehicle control 6 ta NAD Kidney 

Basophilic tubules + 

Fibrosis+ 

Recovery group 

7 ta NAD NAD 

8 ta NAD NAD 

9 ta NAD NAD 

Male 10 ta NAD NAD 

11 ta NAD NAD 

12 . ta NAD NAD 

13 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

14 ta NAD Not examined 

15 ta Cerebrum Cerebrum 

40 Partial defect of cortex Partial defect of cortex+ 

16 ta NAD Not examined 

17 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

18 ta NAD Not examined 

19 ta NAD Not examined 

20 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

200 

21 ta NAD Not examined .............................................................................................................................................................................................................................................................................. 

22 ta NAD Not examined 

23 ta NAD Not examined ....................................... -......... -...................................................................................................................................................................................................................... .. 

24 ta NAD Not examined 

ta, terminal autopsy. 

NAD, no abnormalities detected. 

+,slight; ++, moderate. 

- 27 - P1 )-75 

I 1' 
Addendum 9 

Sex 

Male 

( 

\ 

.l:Hl-U44J 

- Continued 

Pathological findings 

Exp.group Animal Fate Gross findings Histopathological findings 

(mglkg/day) No. 

1,000 

25 ta NAD Liver 

······-··································································································································································~·i·~~~W..~!:?:~.l.~.I?.~ ... :!:. ........................ _ ..._ .......... . 26 ta NAD NAD ··················-···-·········-··-·····--··-···········-·········-.. -··-······· .. ··················-······················-· .. ·····--·················--·······························-···-··-.. ········-···-·-··············-···-····-···· 27 ta NAD NAD ............................................................................................................................................................................................................................................................................... 

28 ta 

29 ta 

30 ta 

Recovery group 

31 ta 

NAD 

NAD 

NAD 

Testis 

Small (right) 

NAD 

NAD 

NAD 

Testis 

Focal atrophy of seminiferous 

tubules++ 

32 ta NAD NAD ............................... ·-···--··-············ .. ··---··----·-·--·· .. -· .. --.. --................................. _ ..... -.............................................. _ ... ,, ... _ ...................... _ ..... -....... -............ . 

33 ta Kidney Kidney 

Apparent spotty pattern of Increased eosinophilic 

-···-··-········-··-··-·····-······················-··~·~E!.~.~~ .. Q?.i!~.t.~~.~!2... .................... -···········-··--··········~.?..9.~~ .. 2:.±.······················-·-··········-·····-···-··-·-·-······ 

34 ta NAD NAD ....... ,_ ................................................................................ ,_ ........................................................................................................................................... -................................ . 

35 ta NAD NAD 

36 ta NAD NAD 

- 28 -
( 

\ 

/ 

Bll-0443 

Addendum 9 - Continued 

Pathological findings 

Sex 

Female 

Exp.group 

(mglkg/day) 

Animal 

No. 

37 

Fate 

ta NAD 

Gross findings Histopathological fmdings 

NAD 

38 ta NAD NAD ·····················-······-·································-························-·······························································································-································································-··············· 39 ta NAD NAD 

40 ta NAD NAD .............................................................................................................................................................................................................................................................................. 

41 ta NAD NAD 

42 ta NAD Liver 

··---·-------·--·--·····-··---------------·-······-·······-···········---····--·-·····--··-···········-·················-·····-················--------~!-~~?.~~~~-!.?..~~-::!:. __________ : _________________________ _ Vehicle control Recovery group 

43 ta Skin Skin 

······--------·-············--····-·--······-----------!:.?.~~--~-f..~~i~ .. {~~!Y~.~~Q ________________________________ ~!!.~P.-~Y. .. ?..f..?..~~~--f.?.!!~~-!-~-~---:!::······-·····-·-· 44 ta NAD NAD 

45 ta NAD NAD ··-···-·-····-·-········-·--·--···-··················-·-·-········-······--······-·-··· .. ········-·····-······-·········· .. ·····-···-···-·---········ .................................................. _.,, .. , ____ .................... . 

46 ta NAD NAD 

47 ta NAD NAD .......................................................................................... ,_, ........................... _ .................................................... _ ....................................... ,_,,, ............. ,_, _______________ .. 

48 ta NAD NAD 

49 ta NAD Not examined 

50 ta NAD Not examined ····················-·····-············-·······································································-·································--·--.. ··-··--..................................................................................................... . 

40 

51 ta NAD Not examined 

52 ta NAD Not examined 

53 ta NAD Not examined 

54 ta NAD Not examined 

55 ta NAD Kidney 

··········--········--··--···········-·················--··········--··---·--------------······--···--····------·-······--···············--···--················--··----··gy~! .. f.?.~.~~i?.~ .. ::!: .................................... _ ......... . 56 ta NAD NAD 

57 ta NAD NAD .............................................................................................................................................................................................................................................................................. 

200 58 ta NAD Kidney 

Basophilic tubules + 

Fibrosis+ 

59 ta NAD NAD 

60 ta NAD NAD 

- 29 -
(_ 

Bl1-0443 

Addendum 9 - Continued 

Pathological findings 

Sex Histopathological fmdings Exp.group Animal 

(mglkg/day) No. 

Fate Gross findings 

61 ta Kidney Kidney 

..... ·-·-······-··-············-·····-·····-···········}~~~Y.~~--~~~-~!~!.!~~ .. J~!.!~!.~~-~!!. ..................... -.... ~~-~Y.!.~ .. ~~~-~-~£!.~~---~·····-·········-···-····-········-·- 62 ta NAD NAD 

63 ta NAD NAD ···········-··-··-··-·-····--· .. ···-···········--··-.. ······-······--·--·-·······--···········-········-································-·······--·······-····-········--·······--···········-···-·····-········--···-···--···· 

64 ta NAD NAD 

65 ta Glandular stomach Glandular stomach 

Blackish region of mucosa Necrosis of mucosa + 

······-··--·--·····--·······-···-···--·······-····(9_::?..~ .. ~ ... ~>...-.. ·····-··-·······---··-········-·············-··--··-·····-···-·-····-·-···-·······-··--···-·----·--··-··-··-·-··-· 

Female 1,000 66 ta NAD NAD 

Recovery group 

67 ta NAD NAD .... ,_ ................................. _ ......... __ .,_ ...... , _____ ,,, ................................................. _,, ....................... ; __ ,,, .......... -... ---···-··-·······-··-·····-···-·-· .. ·-·--·-...... ... 

68 ta NAD NAD ................. ---·------............................................................................. _ ................................................ _ .. _____ ., .......... --------·-· .. ---·-·-·----............... . 

69 ta NAD NAD 

70 ta NAD NAD .................................... ---···· ......................................................................... _ ................................................ -. .......................................... __ , .......... _ .................. _._,. 

71 ta NAD NAD 

72 ta NAD Kidney 

Cyst formation + 

- 30 -

·-~~~-- •. '--~-·-~·· .. _ _, -"'""'-2< -- -~-~-~- -~----~~-·· 

' 
Bll-0443 

( 

APPENDIX 

PHYSICOCHEMICAL REPORT 

Person in charge of Chemical Analysis: 

Signed in original May 29, 1998 

Takeharu Wada, M.S. 

-I~ 
( 

( 

Bll-0443 

Sponsor: NISSHINBO INDUSTORIES, INC. R & D CENTER 

Testing Facilities: Hita Research Laboratories, Chemical Biotesting Center, 

Chemicals Inspection & Testing Institute, Japan 

Test substance: V-OSH (Lot No. 70616) 

Tests: 1) Stability Test of Test Substance 

2) Stability and Uniformity Tests of Test Substance in Formulation 

Date ofTest: 1) December 24, 1997 and March 3, 1998 

2) January 22, 26, 29, 1998 

Methods: 1) Stability Test of Test Substance 

The test sample was taken (n=l) before and after the dosing period, 

and analyzed with infra-red spectrophotometer (IR-470, Shimadzu). 

2) Stability and Uniformity Tests of Test Substance in Formulation 

Formulations were prepared before dosing and following tests were 

performed. The formulations were stored in the cold and dark 

place during the test period. 

(1) Stability Test 

Middle layers of the formulation were taken (n=3) just after 

preparation, at interval, and 7 days after. After pre-treatment, 

the test substance in the formulations was quantitatively 

analyzed by the high performance liquid chromatography 

(HPLC). 

(2) Uniformity Test 

Upper, middle and lower layer of formulations were taken 

(n=3) after preparation. After pre-treatment, the test substance 

in formations was analyzed by HPLC to estimate the 

uniformity of test substance. Stability test and uniformity test 

were performed at the same time, and the mean value (n=3) of 

results of the uniformity test was served as the data of the 

stability test just after preparation. 

-2-
Results: 

( 

Bll-0443 

1) Stability Test of Test Substance (Fig. 1, 2, 3) 

IR spectrum of the test substance was identical with IR spectrum 

provided from the Sponsor. There were no differences of IR 

spectrum pattern between before and after dosing periods. 

Therefore, the test substance was stable during the test period. 

2) Stability and Uniformity Tests of Test Substance in Formulations 

(Table 1, 2) 

The test substance in formulations was stable (within ±10%) for 7 

days. The uniformity of the test substance among the three layers 

was virtually good with less than 5% relative standard deviation 

(R.S.D.%). 

-3-

~~--·-~-
( 

\, 

Bll-0443 

1. Pre-treatment ofF ormulation 

1.1 1 0 w/v% formulation 

Accurate 1 ml of formulation was added to tetrahydrofuran (THF) to make 50 ml, 

and treated in an ultrasonic bath for 2 minuites. Accurate 0.5 ml of this solution 

was added to THF to make 50 ml and served for HPLC. 

The dilution rate of the sample was 5,000. 

1.2 2 w/v% formulation 

Accurate 1 ml of formulation was added to THF to make 25 ml. Accurate 0.5 ml 

of this solution was added to THF to make 20 ml, and served for HPLC. 

The dilution rate of the sample was 1 ,000. 

2. Preparation of Standard Solution 

Approximately 0.5 g (0.5500 g) of the test substance was accurately weighted, and 

dissolved in THF to make 100 ml (undiluted standard solution; 5,500 J.tg/ml). 

Accurate 1 ml of this solution was added to THF to make 25 ml. Accurate 2 ml of 

this solution was added to THF to make 20 ml (standard solution; 22.0 J.tg/ml). 

3. Analytical Conditions 

Instrument: High performance liquid chromatography 

(Model L-6200, Hitachi Ltd.) 

Column: Shodex Asahipak GF-310 HQ 8.0 mm l.D. X 300 mm 

T t 4ooc empera ure: 

Eluent: 1 0 mM sodium perchlorate tetrahydrofuran solution 

Flow rate: 1.0 mllmin 

Detector: 

Wave length: 

Recorder: 

Injected amount: 

UV-VIS detector (Model L-4250, Hitachi Ltd.) 

260nm 

HPLC system manager (Model D-7000, Hitachi Ltd.) 

100 J.tl 

Under the analytical conditions mentioned above, the. waking curve was a straight 

lines which passed through the origin of the coordinates, and obtained linearity 

with a correlation coefficient of 0.999 (Fig. 4). 

The typical chromatogram is shown in Fig. 5. 

-4-
( 

( 

Bll-0443 

4. Calculation 

The total peak area of the test substance in each HPLC sample was measured to 

calculate concentration of the test substance in each dosing solution (C: w/v%) using 

the following equation: 

-C= CsxAxD 

Asx 10,000 

where: 

Cs: Concentration of the test substance in the standard solution (J.tg/ml) 

As: Total peak area of the test substance in the standard solution 

A: Total peak area of the test substance in each HPLC sample 

D: Dilution rate in each HPLC sample 

-5-
I:S) 13:1 IS.! t:=;.J 

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4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 

Fig.3 IR spectrum provided by the sponsor 
( 

180,000 

150,000 

120,000 

y = 3247x 

~ R= 0.999 Cl) 

.... 

~ 90,000 

~ Cl) 

~ 

60,000 

30,000 

0 

0 10 20 30 40 50 

Concentration (!lg/ml) 

Fig. 4 Calibration curve 

Concentration (!lg/ml) Peak area 

11.0 34,495 

22.0 71,176 

55.0 178,935 

-9-
( 

\ 

\ 

2 3 4 5 6 7 

Fig. 5 Typical chromatogram 

-10-

8 9 10 11 12 

f1 J_ 9/l 

! i 
Table 1 Chemical stability of the test substance in dose formulations 

I 

I 

\ Time point of Nominal Found cone. Dilution Actual cone. Mean cone. R.P. 

measurement conc.(w/v%) (~g/ml) rate (w/v%) (w/v%) (%) 

Immediately 10.0 9.61 

after 

preparation 

2.0 1.99 

19.407 5,000 9.70 

10.0 18.855 5,000 9.43 9.63 100 

4 days after 19.534 5,000 9.77 

preparation 19.939 1,000 1.99 

2.0 19.055 1,000 1.91 1.94 97.3 

19.089 1,000 1.91 

19.782 5,000 9.89 

10.0 19.473 5,000 9.74 9.88 103 

7 days after 20.074 5,000 10.0 

preparation 20.391 1,000 2.04 

2.0 20.267 1,000 2.03 2.04 102 

20.376 1,000 2.04 

R.P.(%): Rate to the concentration measured immediately after preparation 

Table 2 

( 

Physical stability of the test substance in dose formulations 

\ 

Nominal .Layer of Found cone. Dilution Actual cone . Mean cone. R.S.D. 

conc.(w/v%) measurement (~g/ml) rate (w/v%). (w/v%) (%) 

18.845 5,000 9.42 

top 18.523 5,000 9.26 9.41 

19.118 5,000 9.56 

19.496 5,000 9.75 

10.0 middle 19.409 5,000 9.70 9.74 9.61 2.4 

19.559 5,000 9.78 

19.497 5,000 9.75 

bottom 19.294 5,000 9.65 9.67 

19.190 5,000 9.60 

20.191 1,000 2.02 

top 20.059 1,000 2.01 2.01 

20.002 1,000 2.00 

20.507 1,000 2.05 

2.0 middle 20.416 1,000 2.04 2.05 1.99 3.3 

20.601 1,000 2.06 

18.359 1,000 1.84 

bottom 19.845 1,000 1.98 1.92 

19.459 1,000 1.95 

R.S.D.(%) :Relative standard deviation 

/ 

\ 

-11- ~( ~~1 
( 

( 

( 

\ 

Accepted number 

Study number 

P03-2314 

82314 

STUDY ·REPORT - I 

Measurement of molecular weight distribution of V -05H 

September 9, 2003 

Kurume Laboratory 

Chemicals Evaluation and ~esearch Institute, Japan 

f1 ~1D 
( 

\ 

Sponsor 

Title 

Study number 

STATEMENT 

NISSHINBO INDUS1RIES, INC. 

82314 

Kurume Laboratory 

Chemicals Evaluation and 

Research Institute, Japan 

Measurement of molecular weight distribution of V -05H 

82314 

I, the undersigned, hereby declare that this report provides a correct English translation 

of the Study Report (Study No.82314, issued on September 9, 2003). 

Date 

Study Director 

Takanori Matsumoto 
( 

SUMMARY 

Title 

Measurement of molecular weight distribution ofV-05H 

Test conditions 

(1) Test method GPCmethod 

2000mg!L 

82314 

(2) Concentration of test item 

(3)Data treatment system Waters Corporation MILLENNIUM 201 O(GPCN) 

Results 

The molecular weight of the test item was calculated from the calibration curve of the 

polystyrene standards. The average content of the component of the molecular weight less 

than 1000 for the test item was 5.53 %. 

Results of molecular weight distribution of test item 

Number average molecular weight (Mn) 4.}2 X 103 

Weight average molecular weight (Mw) 1.65 X 104 

Degree of dispersion (Mw/Mn) 4.01 

Average content of component of molecular 

weight less than 1 000 (%) · 5.53 
( 

> e 

( 

:E .. 

,g 

:;:, "'

3: 

"' 

Molecular weight distribution by polystyrene standards 

Figure of molecular weight distribution 

Number average molecular weight (Mn) 

Weight average molecular weight (Mw) 

Z average molecular weight ( Mz ) 

Degree of dispersion ( Mw I Mn ) 

See figures 

4.12 X 103 

1.65 X 104 

3.39 X 104 

4.01 

Average content of component of molecular 

weight less than 1000 (%) 

5.53 

40.00 

30.00 

20.00 

10.00 

0.00 

-10.00 

-20.00 

(The values in the table are the average of duplicate measurement.) 

GPO chromatogram 

.~, '' i \ ; I , I I ' 

g 

0 

.-1 

II 

i \ ~ i \ 'i::l 

I \ ,.,:; 

i I \ \ ~ i \ 

I ' I "'---..}\ ........................................................................... ______ , ............. L--............ . 

0.00 5.00 10.00 15.00' 20.00 25.00 30.00 35.00 40.00 

0.80 

0.60 

0.40 

0.20 

0.00 

min 

Sample Test item-1 Vial 6 Date 2003/07/16 15:24:12 

. gl _ ....... -1 00 00 ~ /IT--...__·'- oL__ .•. -··"_... . . 

~ I ~ ·"-. ~-----·····"! ('I') I '::J:ij \ I" .,~' ,_: 

1!J I u?F \ ~__.. ti -80.00 

~ -::£ ; ....-:-~ v~ -c 'I 0 1/ /\ c: «< ~ y / '-::£ E 

~+' / ,/ . \~ ~. 1-oo.oo tP. 

/ \ I f 

~.1 /. '\ 1 ... f I I ' ~MO 

I .// ~'- I p, -20.00 

/ .. ··· '-......j !\ -

.. I I / r--·--·'. I \ ,_... I '-'\_ 

......... -~::<------··-./ j \ -o.oo 

5.00 ' 4.50 4.o'o 3.50 ~.oo 2~- LDg~Mw 

.............. Molecular weight distribution 

.............. Total area 

82314 
82314 

( 

CONTENTS 

page 

Title 1 

Sponsor 1 

Test facility 1 

Objective 1 

Dates 1 

Personnel · 1 

( Approval of study report 1 

1. Test item 2 

2. Performance of test 3 

3. Results 4 

4. Remarks 4 
Figures 

82314 

Fig. 1 Chromatogram of GPC analysis and calibration curve for standard 

polystyrene 

Fig. 2 Chromatogram and results of GPC analysis for test item -1 

Fig. 3 Chromatogram and results of GPC analysis for test item -2 

Fig. 4 Chromatogram of GPC analysis for solvent blank 
( 

( 

( 

82314 

Title 

Measurement of the molecular weight distribution of V -05H 

Sponsor 

NISSHINBO INDUSTRIES, INC. 

1-2-3, Onodai, Midori-Ku, Chiba, 267-0056, Japan 

Test facility 

Objective 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

19-14 Chuomachi, Kurume, Fukuoka 830-0023, Japan 

This study was performed to evaluate the molecular weight and the molecular 

weight distribution ofV-05H. 

Dates 

Study initiation date July 15,2003 

Study completion date September 9, 2003 

Personnel 

Study Director Takanori Matsumoto 

Study personnel Tamaki Hata 

Approval of study report 

Study Director Date September 9, 2003 . 

Signature Signed in original 

Takanori Matsumoto 

1 
( 

"'--
( 

( 

82314 

2. Performance of test 

2.1 Test method 

The molecular weight distribution of the test item was measured by using 

high-peiformance liquid chromatography (gel permeation chromatography). The 

measurement was conducted in duplicate. The number average molecular weight and 

the weight average molecular weight were calculated from the calibration curve of the 

standard items of molecular weight. 

2.2 Preparation of test item solution 

20 mg of the test item was dissolved and filled up to 10 mL with the eluent; The 

mixture was allowed to stand at 25± 1 oc for about 24 hours to prepare 2000 mg/L 

solution of the test item. Then the solution was filtrated with a membrane filter (pore 

size 0.45 11m) and the filtrate was analyzed. 

2.3 Preparation of standard solution 

The polystyrene (PS) standards were used as the standard items of molecular 

weight. The PS standards, whose average molecular weights are described bello~, 

were dissolved in the eluent to prepare the standard solution. The concentration of 

each PS standard in the solution was 1000 mg/L. Then the standard solution was 

filtrated with a membrane filter (pore size 0.45. 11m) and the filtrate was analyzed. 

PS Standard 

(TSK Standard) 

F-700 

F-128 

F-40 

F-10 

F-4 

F-1 

A-2500 

A-500 

2.4 Measurement conditions 

Instrument 

GPC system 

Data treatment system 

Column 

Column temperature 

Eluent 

Flow rate 

Sample size 

Average molecular weight 

6770000 

1260000 

355000 

J02000 

43900 

9500 

'2800 

526 

High-performance liquid chromatograph 

Showa Denco K.K. GPC SYSTEM 21 

Waters Corporation MILLENNIUM 2010(GPCN) 

Shodex GPC KF-806L x 2 

30 em x 8.0 mm I.D. stainless steel 

Shodex GPC KF-800D x 1 

10 em x 8.0 mm I.D. stainless steel 

40°C 

Tetrahydrofuran 

0.8mL/min 

100 JlL 

3 
/ 

\ 

( 

\ 

3. Results 

Results of molecular weight distribution of test item 

No.1 

Number average molecular weight (Mn) 4.13 X 103 

Weight average molecular weight (Mw) 1.65 X 104 

Degree of dispersion ( Mw I Mn ) 4.00 

Content of component of molecular 5.50 weight less than 1000 (%) 

4.Remarks 

Instruments and apparatuses 

High-performance liquid chromatograph : 

see page 3 

Data treatment system : see page 3 

No.2 

4.10 X 103 

1.65 X 104 

4.02 

5.55 

Electronic analytical balance: Metler Toledo type AT201 

82314 

Average 

4.12 X 103 

1.65 X 104 

4.01 

5.53 

Special apparatus 

Membrane filter : ADV ANTEC DISMIC-25HP pore size 0.45 J..lffi 

Reagent 

Tetnihydrofuran: W ako Pure Chemical Industries, Ltd. 

4 
( 

Study No. 82314 

Date 2003/07/17 

Page 1 I 1 

> 

E 

GPO chromatogram 

30.00-.----------,---------0

:=--;-o"o--------------, 

0 g8~ 0 oiONMo 

0 10 o.._,. 10 g g (") ~! 0) g 

~ ~ i\f~\ 1\ A ~ t\ : \ i \ I 1 \ I\ I \ .t 1 1 1 • J if l \ : I : \ it'-. 

f \ i \! \i i \ i \ I \ i l 

10.00 

20.00 

;'-JYT!\.jvv· \ , .......... -.--------····-·•••····-L ...... J •••••••••••••••• I. •••• u•••·•··••·•••·•·••··"--··\ (\ !r-···-~·-···-···••·· 

\, \ f'.! 

0.00 

-10.00 

-20.00 

~ .. 

0 

~ 

I ' 0.00 

8.00 

7.00 

6.00 

5.00 

4.00 

3.00 

2.00 I 

14.00 

• 

1 

2 

3 

4 

5 

6 

7 

8 

Fig.1 

·1. i \f 

I v 

i I 

\i 

'/ 

v I I I I I I I I I ' I I ,, I I I I I I I I I I I I I I I ,, I I I I 

5.00 10.00 15.00 20.00 25.00 30.00 35.00 Min. .. 

Sample PS STD Vial 5 Date 2003/07/16 11:29:58 

I 16.00 

.. ,__. 

GPO calibration curve 

I 18.00 I 20.00 

Elution volume 

I 

22.00 

GPO calibration table 

Molecular weight Retention time(min.) Calculated M.W. 

6670000 19.079 6649582 

1260000 21.356 1323919 

355000 23.017 320041 

102000 24.313 98435 

43900 25.074 48828 

9500 26.841 9948 

2800 28.476 2588 

526 30.907 536 

I 24.00 

Chromatogram of GPC analysis and calibration curve 

for standard polystyrene. 

I ' 40.00 

I 26.00 
c '· 

( 

I 

\ 

> 

E 

Study No. 82314 

Date 2003/07/17 

40.00 

30.00 

20.00 

10.00 

GPO chromatogram 

~~. j \ i \ ; I ' I I ' 

i \ j \ 

i \ 

f \ J \ 

! \ I \ ; \ 

i ~ 

Page 1 I 2 

0.00 

I --./\ 

························································-·················-"-..,-·············-·-L--·············---'--\ (\ . r-"-··················-

\1 I 1\ I 

-10.00 

I i i! 

It . 

-20.00 

p 

.f . ij 

. 0.00 5.00 10.00 15.00 20.00 25.00 

Min. 

30.00 35.00 40.00 

Sample Test item-1 Vial 6 Date 2003/07/16 15:24:12 

Results 

• Ret. time(time) Mn Mw Mw1Mn r Mz Mz/Mw <MW1000(%) 

1 26.380 4127 16498 3.998004 33889 2.054170 5.499 

0.80-

csi ....... -1 oo oo 

g /iT--.._·,, _ ....... ---·-·*·· ·-···-····-··-- - . ~ I ~ \ --· (') I ~ ' r- ~" -: 

~ I ~ \ /~/ ~i -80.00 

0.60- !?,LO 1/ II 10! -~ y ' ~ Ei 

~ 

g -;F.! ,...:r:-1 ;X··~ -r· 

.!-+' I \, 3:! 

~I // \\ ::EI 

-60.00 ~ 

~ ~ 0.40 3: ., 

0.20 

0.00 

I / ·. I 

I / '""' 1 

l -40.00 .... 

/ "" I 

II

/ _. ....__ I f\ -2o.oo 

/ ,,_j t\ 

I // '1-1 ·--·-·---.. t \ - / / V\ ..... --~-.-::-~ ... -·-·--· I '--::.O.oo I I 

5.00 4.50 I I 

4.00 3.50 3.00 

Log Mw 

.............. Molecular weight distribution 

.............. Totalarea 

2.50 

Fig. 2-1 Chromatowam and results of GPO analysis for test item -1. 
( 

( 

Study No. 82314 

Date 2003/07/17 

GPC slice table 

Page 2 I 2 

M lp: 14924216522N . arne: P ea k1 

r. Retention time(min.) Peak area M.W. Log M.W. 

-~" dwt/d(logM) 

1 23.627 19 184513 5.266026 0.002 0.000531 

2 23.904 171 14~261 5.156129 0.026 0.004699 

3 24.181 837 111098 5.045704 0.164 0.022898 

4 24.459 2722 86089 .f.934949 0.680 0.074346 

5 24.736 6550 66690 4.824058 2.063 0.178826 

6 25.o13 12017 51669 4.713226 4.904 0.328553 

7 25.290 17152 40054 4.602650 9.395 0.470476 

8 25.567 . 21513 31083 4.492525 15.288 0.593036 

9 25.844 25930 241.57 4.383046 22.516 . 0.719654 

10 26.121 29078 .·18811. 4.274409 30.923 0.814061 

11 26.399 29997 14683 4.166810 39.926 0.848687 

12 26.676 . 29085 11493 4.060443 48.896 0.833247 

13 26.953 26894 .9026 3.955505 57.375 0.781769 

14 27230 23800 7115 3.852191 65.038 0.703450 

15 27.507 20151 5632 3.750697 71.665 0.606968 -. 

16 27.784 16369 4479 3.651218 77.155 0.503640 

17 28.061 12945 3581 3.553949 81.543 0.407858 

18 28.339 10274 2878 3.459087 85.013 .0.332348 

19 28.616 8223 2327 3.366827 87.783 0.273925 

20 28.893 6483 1894 3.277363 89.985 0.223070 

21 29.170 5077 1552 3.190893 91.713 . 0.181038 

22 29.447 4044 1281 3.107611 93.075 0.149996 

23 29.724 3333 1066 3.027712 94.179 0.129162 

24 30.001 2825 894 2.951394 95.088 0.114852 

25 30279 2488 757 2.878850 95.892 0.106731 

26 30.556 2298 646 2.810276 96.625 0.104573 

27 30.833 2223 557 2.745869 97.290 0.108123 

28 31.110 1403 485 2.685823 97.862 0.073514 

29 31.387 1731 427 2.630334 98.268 0.098592 

30 31.664 3072 380 2.579598 99.068 0.192574 

31 31.941 1286 342 2.533810 99.734 0.090007 

32 32.219 286 311 2.493166 99.931 0.022808 

33 32.496 90 287 2.457861 99.977 0.008382 

34 32.773 33 268 2.428091 99.995 0.003703 

35 33.050 0 254 2.404051 100.000 0.000000 

Fig. 2-2 Chromatogram and results of GPC analysis for test item -1. 
~-. 

i 

"-

! 

Study No. 82314 

Date 2003/07/17 

40.00 ~ 

30.00 

20.00 

GPC chromatogram 

J, 

i \ : \ 

! \ I \ 

I ' i i 

I \ 

i \ : \ 

Page 1 I 2 

10.00 

> I \ 

E i \ 

I \ 

0.00 

i ., __ i' 

••••••••••ooooooouuoo•oooonouoo•••••••••n•••••••u••o•o•••••••••••••••••----dt ......... --••••::'••:\..... ••• , f'\ --·•••••••••••••••••••" 

\' i ( .J \ l1! 

-10.00 

1 IV 

\! ;I 

II 

-20.00 v 

I I I I· I I I I I I I I I I I I I I I I I I I I ' I I I ' I ' ' I ' 0.00 5.00 10.00 15.00 . . 20.00 25.00 30.00 35.00 40.00 

Min. 

Sample Test item-2 Vial 7 Date 2003/07/16 16:09:10 

Results 

• Ret. time(time) Mn Mw Mw/Mn Mz Mz/Mw <MW1 000(%) 

1 26.384 4100 16472 4.017202 33914 2.058943 5.548 

~~ - ..,.. ;JT-, 8!_ _______ ...... ---··- ::-loo.oo 

0. 80 ,... "'-.. --'::1 ~ I ~ \ /.~···· ~~ <'>j ~ o __ / t! ~ I (i!F \ 9-- -R• -80.00 

0 60 N ij/ \ II f>'l 

N :::E I .-":':'j \ /~ ,, 

. ~ ; .· \ :§ ~~~ % ~ / /... \{ --£0.00 ~ ..2 t.,/ / \ i 1! 1 .0.40 :::EI · I ~ 

• 0.20 I // ~ Ill . -40.00 

I .• --...., 1\ -20.00 

/ /// ~1-··-----, I \ 

/ \.. . / ,/~-- I . 0.00- ········-""··-·····-· 1 \. __ ~.oo 

I I 

5.00 4.50 I 

4.00 

Log Mw 

I 

3.50 3.00 

Molecular weight distribution 

--- Total area 

I 

2.50 

Fig. 3-1 Chromatogram and results of GPO analysis for test item -2. 
' ! 

\ 

( 

Study No. 82314 

Date 2003/07/17 

GPC slice table 

Page 2 I 2 

Mp· . 14875 . 818326 Name: Peak1 

• Retention. time(min.) Peak area M.W. Log M.W. 

-~" dwt/ d(logM) 

1 23.581 15 192432 5.284277 0.003 0.000398 

2 23.862 130 148927 5.172973 0.021 0.003482 

3 24.143 703 115106 5.061097 0.132 0.018804 

4 . 24.424 2438 88890 4.948852 0.579 0.065097 

5 24.705 6143 68619 4.836443 1.843 0.163954 

6 24.986 11711 52975 4.724073 4.542 0.312922 

7 25.267 17124 40921 4.611945 8.943 0.458985 

8 25.548 21639 31642 4.500264 14.802 0.582858 

9 25.829 26211 24504 4.389233 22.023 0.710821 

10 26.110 29589 19013• 4.279056 30.474 0.809412 

11 26.390 30634 14789 4.169936 39.571 0.846950 

12 26.671 29736 11537 4.062078· 48.656 0.832564 

13 26.952 27499 9030 3.955684 57.247 0.781355 

14 27233 24306 7095 3.850959 65.006 0.702413 

15 27.514 20534 5599. 3.748107 71.707 0.604917 

16 27.795 16622 4439 3.647331 77.241 0.500373 

17 28.076 13104 3539 3.548834 81.651 0.404123 

18 28.357 10381 2837 3.452821 85.129 0.328900 

19 28.638 8285 2288 3.359495 87.900 0.270455 

20 28.919 6508 1858 3.269061 90.095 0.219603 

21 29.200 5082 1520 3.181720 91.811 0.177861 

22 29.481 4054 1252 3.097679 93.164 0.147767 

23 29.762 3337 1040 3.017139 94.262 0.127204 

24 30.043 2876 872 2.940305 95.171 0.115177 

25 30.324 2527 737 2.867380 95.972 0.106930 

26 30.605 2282 629 2.798569 96.703 0.102691 

27 30.886 2253 542 2.734074 97.378 0.108546 

28 31.167 1287 472 2.674101 97.907 0.066970 

29 31.448 2189 416 2.618851 98.366 0.124334 

30 31.729 2901 370 2.568529 99242 0.182103 

31 32.010 982 334 2.523340 99.794 0.069261 

32 32290 216 304 2.483485 99.940 0.017431 

33 32.571 83 281 2.449170 99.978 0.007904 

34 32.852 32 263 2.420598 99.995 0.003702 

35 33.133 3 250 2.397972 100.000 0.000518 

Fig. 3-2 Chromatogram and results of GPC analysis for test item -2. 

il ··-~~---
Study No. 82314 

Date 2003/07/17 

Page 1 I 1 

> E 

GPO chromatogram 

40.00.,-----------,.------------'-----------, 

30.00 

20.00 

10.00 

0.00 

-10.00 

-20.00 

I 

0.00 

··························································-···-·······\ f\ r-········-·····~·-·· 

II I n I I !\I \ i v 

\ i .I 

\1 

V· I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 

5.00 10.00 15.00 20.00 25.00 30.00 35.00 

Min. 

··············· Sample BL THF Vial 8 Date 2003/07/16 16:54:12 

I I I I I '. 

40.00 

Fig.4 Chromatogram of GPO analysis for solvent blank. 
( 

'\__ 

Accepted number P03-2315 

Study number 82315 

STUDY REPORT - IT 

Study on "Safety Evaluation Flow Scheme for Polymer" ofV-OSH 

September 9, 2003 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 
( 

( 

Sponsor 

Title 

Study number 

STATEMENT 

NISSHINBO INDUSTRIES, INC. 

Kunnne Laboratory 

Chemicals Evaluation and 

Research Institute;-Japan 

Study on "Safety Evaluation Flow Scheme for Polymer" ofV-05H 

82315 

823l5 

I, the undersigned, hereby declare that this report provides a correct English translation 

of the Study Report (StudyNo.82315, issued on September 9, 2003). 

Date Octohw 23 r ) 003 

Study Director 

Takanori Matsumoto 

P1 Jo 7 
j 

J 

I 

I 

l ~ l 

] ~ I 

/~' 

~', 

Suqunruy of study results of flow scheme for polymer 

I. Number average molecular weight (by polystyrene standard) 

Mn=4.J2 X 103 

2. Stability test ~ ~ 'rJ ~ Test solutions pH 1.2 (2000mg/L) pH4 (2000mg/L) pH7 (2000mg/L) pH9 (2000mg/L) Before 403.8 404.5 411.1 405.4 402.7 407.5 405.3 400.9 Weight change Weight (m~:) Rate of weight Average After b..W change (%) (%) 404.1 -0.3 -0.1 0.0 404.2 0.3 0.1 409.3 1.8 0.4 0.3 405.1' 0.3 0.1 401.6 1.1 0.3 0.4 405.5 2.0 0.5 404.5 0.8 0.2 oA 398.7 2.2 0.5 \ TOCchange Concentration ofTOC (m C/L) After Control b.TOC Average 6.1 0.7 5.4 6.0 7.2 6.5 / !/ I/ v 8.8 8.2 ' 0.6 8.7 9.8 9.2 6.3 0.9 5.4 6.9 9.3 8.4 

Views by IR spectrum 

- No difference was observed in 

IR spectra before and after the te

. . 

I Befo

b. W = weight befo

Rate of weight chan

A

= weight 
( 

( 

\ 

( 

82315 

3. Molecular weight distribution by polystyrene standards 

> E 

% 

~ ~ 3: .., 

40.00 

30.00 

20.00 

10.00 

0.00 

-10.00 

-20.00 

Figure of molecular weight distribution 

Number average molecular weight (Mn) 

Weight average molecular weight (Mw) 

Z average molecular weight ( Mz) 

Degree of dispersion ( Mw I Mn) 

Average content of component of molecular 

weight less than 1000 (%) 

See figures 

4.12 X 103 

1.65 X 104 

3.39 X 104 

'4.01 

5.53 

(The values in the table are the average of duplicate measurement.) 

GPC chromatogram 

0 J, 8 i \ ~ i \ · II 

! I ~ I I ~ i \ ~ 

I \ ~ I ·\_~f· ........................................................................... _ ..................... _l__ ............. _· -'--, r· ,_ .................. - '.] \ -d 

I l\1 

i 1 v 

\I 

\1 ll 

v 

0.00 I 

5.00 10.00 15.00 20~00 25~00 35~00 40~00 

0.80 

0.60 

0.40 

0.20 

0.00 

min 

.............. Sample Test item-1 Vial 6 Date 2003/07/16 15:24:12 

T

-.... .. ~0· . . _ ....... ::.1 00 00 en /J " o -·-·---· · CD ., • __ ... • 

CD f cx7=t ·\ __ __..... II -

~ I ~ ' r- // ..-I - 0) \!,// ,.z- \ 2" ; II -80.00 o ::E ·lii v· if ~ -

~ v ~ /\~ ~, - ~ ; / 'i ~~ -so.oo 1;1! 

N' / \ • 'i 

::E' . ' I .... 

,

,; / \ l I / ·, I -40.00 ~ 

I ./ ""' I I / ' I I .· " ...... I /\ t-20.00 

___ .. / ---- //,... r--------,,j \ = 

......... -.--~~:::. ______ •• --· 1 \_ ~.oo 

I 

5.00 4.50 I 4.00 3.50 3.00 

Log Mw 

Molecular weight distribution 

Total area 

I 

2.50 

----· ·-----.~-~ 
82315 

CONTENTS 

( 

page 

Title 1 

Sponsor 1 

Test facility 1 

Objective 1 

Test method 1 

Dates 2 

( 

Personnel 2 

Approval of study report 2 

1. Test item 3 

2. Stability test 4 

3. Results 9 

4. Summary 12 

5. Remarks 12 

--- -~---~-, 
82315 

Figlires 

Fig. 1 IR spectrum of test item before stability test 

Fig. 2 IR spectrum of test item (pH 1.2) after stability test 

Fig.3 IR spectrum of test item (pH 4) after stability test 

Fig;4 IR spectrum of test item (pH 7) after stability test 

Fig. 5 IR spectrum of test item (pH 9) after stability test 

Fig. 6 · GPC chromatogram of polystyrene standards and calibration curve 

Fig. 7 GPC chromatogram and results of test item before stability test 

Fig. 8 GPC chromatogram and results of test item after stability test (pH 1.2) 

Fig. 9 GPG chromatogram and results of test item after stability test (pH 4) 

Fig. 10 GPC chromatogram and results of test item after stability test (pH 7) 

( Fig. 11 GPC chromatogram and results of test item after stability test (pH 9) 

\ 

Fig. 12 GPC chromatogram of solvent blank 

~f }!( 
( 

' 

( 

( 

82315 

Title 

Study on "Safety Evaluation Flow Scheme for Polymer" ofV-05H 

Sponsor 

· NISSHINBO INDUSTRIES, INC. 

1-2-3, Onodai, Midori-Ku, Ch;iba, 267-0056, Japan 

Test facility 

Kurume Laboratory 

Chemicals Evaluation and Research Institute, Japan 

19-14 Chuo-machi,Kurume-shi, Fukuoka 830-0023, Japan 

Objective 

This study was performed to evaluate the information on safety evaluation for 

polymer. 

Test method 

This study was conducted according to "Stability Test and Solubility Test in Water . 

and Organic Solvents" based on "Safety Evaluation Flow Scheme for Polymer (The 

Report Compiled by Polymer Commission, November, 1985)". 

P1 )IV 
( 

( 

( 

\ 

Dates 

Study initiation date 

Study completion date 

Personnel 

Study Director 

Study personnel 

Approval of study report 

Study Director 

82315 

July 28, 2003 

September 9, 2003 

Tak:anori Matsmnoto 

Tamak:i Hata 

Date September 9, 2003 

Signature Signed in original. 

Tak:anori Matsmnoto 

2 rfJ( '3/3 
( 

----,-=--
( 

' 

82315 

2. Stability test 

2.1 Test method 

2.2 

2.3 

About 400 mg of the test item was accurately weighed and 200 mL of the buffer 

solution described in 2.3 was added to obtain test solutions. After the test sol~tions 

were stirred in a constant temperature bath at the test temperature ( 40 ± 2 °C) for two 

weeks (24 hours for the test solution of pH 1.2), they were equilibrated at 25 ± 2 °C, and 

then analyzed for the stability test of the test item. 

Test conditions 

(1) Test concentration 2000 mg!L ( 400 mg I 200 mL) 

(2) Test temperature 40±2°C 

(3) Test period 2 weeks (24 hours for the test solution of pH 1.2.) 

( 4) Equilibrium temperature 25 ±2 oc 

(5) Equilibrium time 24 hours 

(6) Light Room~light 

~ 

(7) Number of test repetition 2 

Preparation of buffer solution 

··A buffer solution of pH 1.2 

16.1 mL of 2.0 mol/L hydrochloric acid and 12.5 mL of 2.0 mol/L potassium 

chloride solution were mixed and filled up to 500 mL with purified water. 

• A buffer solution of pH 4 

2.0 mL of 0.1 mol/L sodium hydroxide solution and 100 mL of 0.25 mol/L 

potassium hydrogen phthalate solution were mixed and filled up to 500 mL with 

purified water. 

· A buffer solution ofpH 7 

14.8 mL of 1.0 mol/L sodium hydroxide solution and 50.0 mL of 0.5 mol/L 

potassium phosphate solution were mixed and filled up to 500 mL with purified 

water. 

· A buffer solution of pH 9 

10.7 mL of 1.0 mol/L sodium hydroxide solution and 50.0 mL of 0.5 mol/L 

potassium chloride and 0.5 mol/L boric acid solution were mixed and filled up to 500 

mL with purified water. 

4 

tf}( 715" 
( 

\ 

/ 

( 

82315 

2.4 Analysis of test solution 

The test solutions were pretreated for each analysis according to the following 

flow scheme. 

Flow scheme (pH1.2) 

Test solution 

• Decantation 

Supernatant Residue. 

·Washing (purified water) 

• Drying(under reduced pressure, 40 °C) 

Sample for TOC analysis Sample for gravimetric analysis 

Sample for IR analysis 

• Weighing 20 mg 

(electronic analytical balance) 

~Tetrahydrofuran 10 mL 

(measuring pipet) 

• Standing (25 °C, about 24 hours) 

• Filtration (DISMIC-25HP) 

Sample for HPLC analysis 

5 
( 

Flow scheme (pH4, pH7 and pH9) 

·Taking out 

(komagome pipet) 

82315 

Supernatant Residue 

• Taking out 10 mL 

(measuring pipet) 

• Centrifugation 

(1000 X g, 10 min) 

• Washing (purified water) 

• Drying (under reduced pressUre, 40 °C) 

Sample for gravimetric analysis 

Sample for TOC analysis 

(Except for the test 

soluion of pH 4) 

Sample for IR analysis 

• Weighing 20 mg 

(electronic analytical balance) 

~Tetrahydrofuran 10 mL 

(measuring pipet) 

• Standing (25 °C, about 24 hours) 

• Filtration (DISMIC-25i-IP) 

Sample for HPLC analysis 

6 

!){ 117 
( 

( 

82315 

(1) Analysis oftotal organic carbon (TOC) 

The samples for TOC analysis were analyzed under the following conditions. 

The test solutions of pH 4 were not analyzed because these solutions contained 

potassium hydrogen phthalate. 

Analytical conditions 

Instrument 

Temperature of furnace 

Flow rate 

Injection volume 

·sensitivity 

(2) Gravimetric analysis 

Total organic carbon analyzer 

Shimadzu Corporation type TOC-5000 

680°C 

150mL/min 

33 J.tL 

Range 5 

· The ·samples for·· gravimetric analysis were weighed after the constant weight 

were obtained. 

(3) Measurement of infrared absorption spectrum (IR) 

The samples for IR analysis were measured under the following conditions. 

Measurement conditions 

Instrument 

Sample preparation method 

7 

Fourier transform infrared spectrophotometer 

Shimadzu Corporation type FTIR-8200PC 

Neat 
\ 

( 

\ 

( 

82315 

( 4) Measurement of molecular weight distribution 

The samples for HPLC analysis were applied to measurement of the molecular 

. weight distribution under the following conditions. The molecular weight 

disutribution: of the test item was also mesured. 

Measurement conditoions 

Instrument 

_GPC system 

High.,-performance liquid chromatograph 

Showa Denco K.K. GPC SYSTEM 21 

Data treatment system Waters Corporation MILLENIUM 2010 (GPCN) 

Column Shodex GPC KF-806L x 2 

Coltnnil temperature 

Eluent 

Flow rate 

Sample size 

30 em x 8.0 mm J.D. stainless steel 

Shodex GPC KF-800D x 1 

10 em x 8.0 mm J.D. stainless steel 

40°C 

Tetrahydro:furan 

0.8rnUmin 

100 J!L 

· Standard items for measurement of molecular weight 

Polystyrene standards Average molecular weight 

6770000, 1260000,3§5000, 102000,43900,9500,2800,526 

8 
( 

\ 

82315 

3. Results 

(1) Analytical results ofTOC 

The concentrations ofTOC in the test solutions are as follows. 

*3 

*2 Concentration ofTOC after test Theoretical Solubilization 

Test (mgC/L) concentration rate of 

solution ofTOC component 

(mgC/L) carbon(%) 

After Control ATOC Average Average test 

pH 1.2 6.1 0.7 5.4 6.0 1574.8 0.3 OA 

7.2 6.5 1577.6 0.4 

pH7 

8.8 0.6 8.2 8.7 1570.5 0.5 0.6 9~8 9.2 1589.3 0.6 

pH9 6.3 0.9 5.4 6.9 1580.7 0.3 0.4 

9.3 8.4 1563.5 0.5 

*2 Theoretical concentration ofTOC: 

The theoretical concentration of TOC means the concentration of TOC in the -

case that the test iteni is completely dissolved in the test solution, and was calculated 

by the following equation. 

Theoretical concentration ofTOC (mgC/L) 

_ Weight of test item before test (mg) [ Weight of carbon in segment J - Volume of test solution (L) x Morecular weight of segment in test itemj 

[ 

Weight of carbon in segment . J 

where M 1 .gh. t f 1. t t .t - was calculated from the orecu ar wet o segmen m es 1 em 

structural formula described in 1.2, and the results was 0.78. 

*3 Solubilization rate of component carbon : · 

Percentage of the measured concentration of TOC in the test solution to the 

theoretical concentration ofTOC, expressed by the following formula. 

• • • · 0 _ Concentration ofTOC 

Solubthzation rate of component carbon (Yo) - Theoretical concentration ofTOC x 100 

9 
I 

\ 

82315 

(2) Weight change and rate of weight change (%) in gravimetric analysis 

Test Weight (mg) Weight change (%) *

5 

solution Before test After test A.w*4 Average 

pH 1.2 403.8 404.1 -0.3 -0:1 0.0 

404.5 404.2 0.3 0.1 

pH4 

411.1 409.3 1.8 0.4 0.3 

405.4 405.1 0,3 0.1 

pH7 

402.7 401.6 1.1 0.3 0.4 

407.5 405.5 2.0 0.5 

pH9 

405.3 404.5 0.8 0.2 0.4 

400.9 398.7 2.2 0.5 

*4 f). W =weight before test -· weight after test 

*5 Rate of weight change(%)= W 'gh f . bee. · x 100 e1 t o test Item 10re test 

10 

I I I i I I I 
( 

82315 

(3) Results of measurement of infrared spectra (IR) (see Figs. 1 - 6) 

No difference was observed between the IR spectra of the test item before the 

test and those after the test. The characteristic fundamental vibrations of the test 

item are as follows. 

Wave number (cm-1

) 

3000-2800 

Around2270 

Around2140 

1600-1400 

Reversion 

C - H Stretching vibration 

- N = C = 0 Stretching vibration 

· - N = C = N- Stretching vibration 

C = C Ring vibration 

(4) Results of measurement of molecular weight distribution (see Figs. 7- 11) 

The number average molecular weight· and the weight avarage molecul~ 

weight of the test item before and after the test ate as follows. 

Results of measurement of molecular weight distribution 

Average molecular weight · 

by polystyrene standards 

Sample Number average Weight average Degree of dispersion molecular weight molecular weight - (Mw) (Mw/Mn) (Mn) 

Before 4.38 X 103 1.83 X 104 4.18 

pH 1.2 4.31 X 103 1.83 X 104 4.24 

pH4 4.22 X 103 1.80 X 104 4.26 

pH7 4.37 X 103 1.85 X 104 4.24 

pH9 4.32 X 103 1.83 X 104 4.24 

11 
\ 

( 

( 

82315 

4. Summary 

The weight change of the test item before and after the test was less than 2.2 mg 

(the rate of weight change was less than 0.5 %) and the concentration of the dissolved 

organic carbo.n was less than 9.2 mgCIL (solubilization rate of component organic 

carbon was less than 0.6 %). In the measurement of infrared absorption spectra and 

molecular weight of the test item, no difference was observed between those measured 

before the test and those measured after the test. 

5.Remarks 

Instruments and apparatuses 

High-performance liquid chromatograph : 

see page 8 

Fourier transform infrared : see page 7 

· Total organic carbon analyzer : see page 7 

Constant-temperature incubator :Thomas Scientific Instrument Co., Ltd.· 

typeAT-24R 

Electronic analytical balance : 

Data treatment system : 

Special apparatus 

Membrane filter : 

Reagents 

Purified water: 

Tetrahydrofuran: 

Hydrochloric acid : 

Potassium chloride : 

Sodium hydroxide : 

Potassium hydrogen phthalate : 

Potassium phosphate : 

Boric acid: 

Metler type AT201 

see page 8 

Advantec INC. 

DISMIC-25HP pore size 0.45Jlm 

Takasugi Seiyaku Co., Ltd. 

Wako Pure Chemical Industries, Ltd. 

Kanto C)J.emical Co., Inc. 

Wako Pure Chemical Industries, Ltd. 

Kanto Chemical Co., Inc. 

W ako Pure Chemical Industries, Ltd. 

Wako Pure Chemical Industries, Ltd. 

Wako Pure Chemical Industries, Ltd. 

12 
( 

( 

100.0 --;-, ---------.-----------------

" 1 I 

i 

"i 

20.0 ····· ....... -- -----

0.0 +-,--r-,-.---+-.---.---.-.-t----,,--,---r-,--+---r-.--.--..-i--,-.,.-.,--,-+---l 

4000.0 3000.0 2000.0 1500.0 1000.0 500.0 

1/cm 

Fig. 1 IR spectrum of test item before stability test. 

Study No. : 

Method 

Date 

Name 

82315 

Neat 

September 1, 2003 

·r A M fl fC 1. H A 1 A 
( 

\ 

100:0 -:---~------,---------------

j 

%T ~ 

t.J .. "'· 

:: ~~-~ I 

! 

40.0 --······· ··-·-···-····-···-·····-·····-·-·-! ···----·--·--------·----- . - ----·-······--·-····-·- ····--·-· 

I 

--·-··· ·····--·· .. ····---······ --·-···1 

I i 

20.0 - - .... . ....... ----- -·r I --·----·- ···- ... -·-·-· .. .. . ---- ........ . 

l· 

i . ...... ; ..... . 

! 

I 

0.0 --+--.--i---.--.--+-.-.--.----.--!----J-.~-.-+--,---r'--r-.---+----.--.--,---,---+---l 

4000.0 3000.0 2000.0 1500.0 1000.0 

Fig.2 IR spectrum of test item (pH 1.2) after 

stab i I i ty test. 

Study No. : 

Method.· 

Date 

Name 

82315 

Neat 

September 1, 2003 

"TAHA!<=-L HAI.A 

500.0 

1/cm 
( 

100.0 -,-----:-----~--r-------------"------, 

%T 

I 

20.0 ········· -- - ---- . ---- ---l ---- ---- --- --- -- --·-- -··-- -- -- :--- 1 ' 

i 

··--· ··-····· ... 

! 

I 

0.0 -j--,----,---.---,---j-,--,--,-,-----;!---J----.---.---.-+1 ---.--.--.----,---+---.----.--.--.----+---l 

4000.0 3000.0 2000.0 1500.0 1000.0 500.0 

1/cm 

Fig.3 IR spectrum of test item (pH 4) after 

stab i I i ty test. 

Study No. : 

Method · 

Date 

Name 

82315 

Neat 

September 1, 2003 

\AMA!<:1- HATA 

f1 ))b 

T·-.---1 -, ..-~~·---· ·~~ 
I 

\ 

100.0 ,,------------,-----------.,..-------. 

l 

%T l 

80.0 J 

40.0 

t 

I 

l 

i 

I 

········-IJY_····· 

·-·- -------·-··-· -·-·t ... ··-············-·· -·-·---····· ·······-········- ! 

! I . . t-·· - ---- ... 

! 

: l 

' 

0.0 -j--,--,--,--,--i---,--,--,--,--j-.,-.,-.,-,-+1 ---,---,-,--;r,-+---,--,--,--,--;--j 

4000.0 3000.0 2000.0 1500.0 1000.0 

Fig.4 IR spectrum of test item (pH 7) after 

stab i I i ty test. 

Study No. : 82315 

Method · Neat 

Date September 

Name T A t-1 A ~ r 

1' 2003 

HATA 

soo:o 

1/cm 
( 100.0 ~ 

%T l 

80.0 

~'v~ (\ i / . ~~ 1'( 

60.0 

.... ¥tJtwtvJ:~v 

40.0 _ ................ ____ - -·---- --.-,ll -........... --.-

i ! 

i ......... , ____ , ____ ....... --- -..... ·"'t--... i 

'.i 

f 

i 

20.0 ---- -- ________ l .. --..... ---- ------------------- --- ---- r· I --------

0.0 --l------.--,---,--.--+--.----.--.--,------cl----r--r--r----r--+--.-.---,--'--r--+----,---,--,---,--+--1 

4000.0 3000.0 2000.0 1500.0 1000.0 

Fig.5 IR spectrum of test item (pH 9) after 

stab i I i ty test. 

Study No. : 

Method 

Date 

Name 

82315 

Neat 

September 1, 2003 

IAMA!c-1 HATA 

-... ----T~- ~ ----·---- ·~· i I I I 

500.0 

1/cm 
Study No. 82315 

Date 2003/08/27 

Page 1 I 1 

GPC chromatogram -so.oo,.J _______ ___,_ ________________ ......._,...------, 

4o.ooJ 

0 00 

o 8889o 

g g:g~o . 0 . co ' <? ~""" :g g 

> ::.::: ~ .1 ! A I 0) . 

E . co I ,, ''" ~ C\1 co 10.00 . ~ ~ I: it:: ! i I C\1 · i~ : \ J \: \ :. •1\ LO 

!I :\ I: i Y' ! I P 1 I \ ! \ ; i I l \ I \ ; i ,.1', 

30.00 

20.00 

0.00 I '- .f ·-{ V i \. ; '} \/ "". -························n·-----··---------------------..l~-----~------l---~----X ..... ~ ........ : ........... ._ ___ "\ r·, ,.--···---------------···· 

\I \ ., I 

-10.00 ,; t' 1\ l 

: 1 \} 

\I 

-20.00 v 

~ .. 

0 

-' 

I I I I ! I I I I I I I I I I I I I I I I I I I I I I I I I I I·' ' I I I I I I I I 

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 

8.00 

7.00 -- 6.00 

5.00 

4.00 

3.00 

Min. 

Sample PS STD Vial1 Date 2003/08/26 11:51:05 

GPC calibration curve 

' ' . 

--s-~.· 

: .. ·~ 

.... ~ .··-. 

t ... 

"'-.....~, . . .. ;-....__............ ,., 

~~---------~ ... , ---- 2.00+--,--.---r-r-...----r---r--r---o-T-.---,I,--.,--,--,---,---,.-,---~....--,---r---r--f 

14~0 16~0 18~0 20~0 22~0 24~0 . 26~0 

Elution volume 

GPC calibration table 

• Molecular weight Retention time(min.) Calculat~d M.W. 

1 6770000 19.174 6726918 

2 1260000 21.463 1335798 

3 355000 23.153 319889 

4 102000 24.469 98087 

5 . 43900 25.243 48520 

6 9500 27.024 9953 

7 2800 28.669 2605 

8 526 31.118 535 

Fig.6 Chromatogram of GPC analysis and calibration curve 

for standard polystyrene. 

I ' 
I ' 

\ 

( 

Study No. 82315 

Date 2003/08/27 

50.00 

40.00 

30.00 

20.00 

> E 

10.00 

GPO chromatogram 

co 

0 

,_ 

co 

.,.... 

,+\ 

! \ I \ I I 

i \ I ' i \ 

i \ I \ 

Page 1 I 2 

0.00 . ! \ ___ J\ ·································································································-zsL·~·-··········--·······7S"\ (\ . ~-------················ 

. . v \ {\ t 

-10.00 

.-20.00 

0.00 

• 1 

0.80 

0.60 

5.00 10.00 15.00 20.00 25.00 

Min. 

30~00 

\IV 

\ i 

11 

i/ 

.\1 

~ 

35~00 

Sample Test item Via19 Date 2003/08/26 18:01:35 

Results 

40.00 

Ret. time(min.) Mn Mw Mw/Mn Mz Mz/Mw 

26.430 4383 18311 4.177234 37216 2:032453 

-80.00 

51 -

~ 1/W'"' ____ .. --·-···-~ _ ....---------·---·-··-- ~1 oo.oo 

r- / jg \. ---- ~ ~I ~ \ //·-·· N I ~ ~ 

~ ;:Et I' , //<0 ....r I I \ C'? M :/, )( ~ 

~I .,/\\!: II / /.. \ ;::E .,.... I ' . -60.00 ~ 

-40.00 

+ / ,, 

~/ / ' y // \ 

: 

I / .!/ \"--·-~ I

I ./ . __ .. / '·-... - _(\, -20.00 

I ' I .. / --------- I \ 

0.40 

0.20 

•, /// \.)\ . \ 

0.00 .......... -.,c::., ... --··-·// '-·- -D.OO 

5.00 4.50 4.00 3.50. 

Log Mw 

Molecular weight distribution 

--------- Total area 

3.00 2.50 

Fig. 7-1 Chromatogram and results of GPC analysis for test item 

before stability test. 

J 
Study No. 82315 Page 2 I 2 

Date 2003/08/27 

G PC slice table 

( Mp: 16706.362227 Name: Peak 1 

( 

\ 

-Retention time(min.) Peak area M,W. log M.W. 

-~" dwt/d(logM) 

1 23.549 2 224996 5.352174 0.000 0.000066 

2 23.831 60 174627 5.242112 0.008 0.001671 

3 24.114 427 135328 5.131387 0.072 0.011800 

4 24.396 1718 104759 5.020191 0.379 0.047345 

5 24.679 4738 81044 4.908721 1.342 0.130379 

'6 24.961 9942 62686 4.797170 3.592 0.273622 

7 25.243 15927 48499 4.685732 7.631 0.439178 

8 25.526 20570 37549 4.574603 13.310 0.569274 

9 25.808 24589 29106 4.463975 20.271 0.684204 

10 26.090 28207 22597 4.354045 28.436 0.790586 

11 26.373 29833 17579 4.245006 37.416 0.843775 

12 26.655 29304 13710 4.137053 46.530 0.837946 

13 26.938 27317 10725 4.030379 55.227 0.791261 

14 27.220 24391 8417 3.925180 63.153 0.717165 

' 

15 27.502 20878 6632 3.821650 70.077 0.624413 

16 27.785 17131 5248 3.719984 75.876 0.522334 

17 28.067 13585 ·. 4172 3.620375 80.545 0.423284 

18 28.350 1066~ 3334 3.523018 84.219 0.340556 

19 28.632 8488 2680 3.428107 87.123 0.278279 

20 28.914 6739 2167 3.335838 89.436 0.227607 

21 29.197 5295 1764 3.246403 91.260 0.184795 

22 29.479 4200 1445 3.159999 92.697 0.151972 

23 29.761 3428 1193 3.076819 93.852 0.129084 

24 30.044 2826 993 2.997057 94.807 0.111218 

25 30.326 2545 834 2.920908 95.634 0.105160 

26 30.609 2312 706 2.848566 96.365 0.100834 

27 30.891 2163 60,3 2.780226 97.039 0.100172 

28 31.173 1780 520 2.716083 97.687 0.088172 

29 31.456 1300 453 2.656330 98.095 0.069397 

30 31.738 3005 399 2.601162 98.757 0.174643 

31 32.020 1909 355 2.550773 99.591 0.122239 

32 32.303 478 320 2.505358 99.909 0.034224 

33 32.585 100 292 2.465111 99.977 0.008143 

34 32.868 37 269 2.430227 99.995 0.003569 

35 33.150 0 252 2.400900 100.000 0.000000 

Fig. 7-2 Chromatogram and results of GPO analysis for test item 

before stability test. 
' I 

'·· 

( 

( 

Study No. 82315 

Date 2003/08/27 

Page 1 I 2 

> 

GPO chromatogram 50.00.------------~----------------~--. 

40.00 

30.00 

20.00 

10 

'<t 

10 

<0 

,t, 

I' f ·\ ; \ I \ I \ 

! \ 

E 10.00 

I \ 

! \ 

% 

..9 

';:! 

'-... 

l: 

., 

I \ I \ 

. I '\__ 

0.00 --·······················----····················•··:··-··-···-·-·r;·)·:······--· -~~~~\. .... 1\'"l ;"o r·-··-················· , ___ \ '--~ \ 1 \ I 

-10.00 

-20.00 

0.00 

• 1 

0.80 

0.60 

0.40 

0.20 

5.00 10.00 15.00 20.00 25.00 30.00 

Min. 

'J .I i\ ! 

: i \} 1! I: 

\ i 

ii 

•I l! 

35.00 

Sample pH1.2 Vial 10 Date 2003/08/26 18:46:35 

Resu:lts 

40.00 

Ret. time(mln.) Mn Mw Mw/Mn Mz Mz/Mw 

26.441 4307 18276 4.243566 37437 2.048449 

~ 

..... /1r'"" g ........ -··_ ....... -1 oo.oo . ~ I ~ " /----------:§ .......... - - WI~\_../ ~ 

m Nf ..-':':'~ \/r< 1-o ~~ \···. g e ~y \'<t ll: 

10' /,•'\ 1t :s II I ·~ ~ I / \: t., I ,, ~! / \ 

i 1/ \"'· I /./ ,~- , /•' ' _,1\, / . . '·,,,, --·-...... ./\/ \ 

-80.00 

--60.00 ~ 

i 

-40.00 •,!! 

-20.00 

./ _./ 

0.00 ------·~~::. ...... ----/ \ __ -o.oo 

' I ' 

5.00 

I I I I I I I I I I I I I I I I I I 

. 4.50 4.00 . 3.50 3.00 

Log Mw 

Molecular weight distribution ......... ,..... Total area 

I I ,-

2.50 

Fig. 8-1 Chromatogram of GPC analysis and results for residue after 

stability test ( pH 1.2 ). 
( 

\ 

Study No. 82315 

Date 2003/08/27 

GPO slice table 

Page 2/ 2 

Mp: 16544.618685Name: Peak1 

18 Retention time(min.) Peak area M.W. Log M.W. -~~ dwt/d(logM) 

1 23.549 5 224996 5.352174 0.001 0.000155 

2 23.831 64 174627 5.242112 0.010 0.001831 

3 24.114 439 135328 5.131387 0.078 0.012400 

4 24.396 1732 104759 5.020191 0.396 0.048774 

5 24.679 4690 81044 4.908721 1.378 0.131852 

6 24.961 9733 62·686 4.797170 3.637 0.273700 

7 25.243 . 15520 48499 4.685732 7.664 0.437257 

8 25.526 19982 37549 4.574603 13.310 0.565022 

9 25.808 23821 29106 4.463975 20.207 0.677257 

10 26.090 27355 22597 4.354045 28.291 0.783368 

11 26.373 29013 17579 4.245006 37.202 0.838396 

12 26.655 28555 13710 4.137053 46.268 0.8342'49 

13 26.938 26683 10725 4.030379 54.938 0.789707 

14 27.220 23872 8417 3.925180 62.856 0.717132 

15 27.502 20478 6632 3.821650 69.788 0.625754 

16 27.785 16834 5248 3.719984 75.604 0.524443 

17 28.067 13361 4172 3.620375 80.295 0.425340 

1.8 28.350 10481 3334 3.523018 83.985 0.341818 

19 28.632 8335 2680 3.428107 86.898 0.279219 .· 

20 28.914 6624 2167 3.335838 89.219 0.228565 

21 29.197 5201 1764 3.246403 91.052 0.185450 

22 29.479 4127 1445 3.159999 92.494 0.152586 

23 29.761 3404 1193 3.076819 93.660 0.130967 

24 30.044 2792 993 2.997057 94.629 0.112278 

25 30.326 2520 834 2.920908 95.460 0.106388 

26 30.609 2285 706 2.848566 96.198 0.101834 

27 30.891 2147 603 ·2.780226 96.882 0.101585 

28 31.173 2066 520 2.716083 97.581 0.104533 

29 31.456 1370 453 2.656330 98.071 0.074713 

30 31.738 2837 399 2.601162 98.712 0.168506 

31 32.020 1971 355 2.550773 99.557 0.128927 

32 32.303 521 320 2.505358 99.900 0.038119 

33 32.585 109 292 2.465111 99.976 0.009082 

34 32.868 37 269 2.430227 99.995 0.003623 

35 33.150 0 252 2.400900 100.000 0.000000 

Fig. 8-2 Chromatogram of GPO analysis and results for residue after 

stability test ( pH 1.2 ). 
> E 

Study No. 82315 

Date 2003/08/27 

50.00 

40.00 

30.00 

20.00 

10.00 

GPC chromatogram 

It) 

It) 

<? 

<0 

~ 

J, 

I \ 

i \ ! \ I \ 

I \ 

! \ 

f \ : \ 

Page 1 I 2 

0.00 

J \ 

··---------···········--·-································: ................ _. _______ i.'S):............. . · . ....._ .. J\ w.'. (\ r-················· 

\t I ("I 

-10.00 

-20.00' 

0.00 

• 1 

0.80 

0.60 

0.40 

0.20-

5.00 

v \ !V 

I! ii L 

ij 

10~0 15~0 20~0 25~0 30~0 35~0 

Min. 

Sample pH4 Via,111 Date 2003/08/26 19:31:35 

Results 

40.00 

Ret. time(min.) Mn Mw Mw/Mn Mz Mz/Mw 

26.455 4224 17986 4.258285 37013 2.057809 

~ ~· /rf"'·"" 81 ...................... -- -100.00 

~ / i -,\ / / __ _,_ .. ----·-rr .... ----

v ~~ ~ X\ ··~ ~i Q) v ~ ~· ~· ~ I ·· \ II ""'l, It) I _. \ s:: ""' II I I \::i! i 

~/ ./ ~ li N, / \ 

~I / ' 

1/ I ' ' f I \ ~~ , I \ 

I I ' .1 I / \ ... "' 

:-80.00 

H>o.oo ~ 

i 

.s 40.00 

II

' // .. '- ,i ,, ..... i·, -20.00 ............ ! f \ 

/ ..... 't--···-"""\ j \ I ./ I \.i \ 

. .// _/__./ I \ .. 0.00-....... ,.--~e:._ ........ -·- ! ''--o.oo 

5.00 4.50 4.00 3.50 

Log Mw· 

Molecular weight distribution 

----------- Total area 

3.00 2.50 

Fig. 9-1 Chromatogram of GPC analysis and results for residue after 

stabi lity test ( pH4 ) . 
Study No. 82315 Page 2 I 2 

Date 2003/08/27 

GPO slice table 

C Mp: 16354.943394 Name: Peak1 

~ Retention time(min.) Peak area M.W. log M.W. 

-~" dwt/d(logM) 

1 23.550 ·o 224804 5.351804 0.001 0.000012 

2 23.833 63 174328 5.241367 0.009 0.001870 

3 24.117 400 134978 5.130264 0.075 0.011890 

4 24.400 1585 104397 5.018689 0.382 0.046991 

5 24.683 4338 80694 4.906840 1.337 0.128422 

6 24.967 9061 62361· 4.794913 3.553 0.268297 

7 25.250 14500 48206 4.683104 7.521 0.430182 

8 25.533 . 18744 37292 4.571610 13.104 0.558157 

9 25.817 22459 . 28882 4.460627 19.960 0.672466 

10 26.100 25830 22405 4.350352 28.021 0.779044 

11 26,383 27400 17417 4.240980 36.911 0.834008 

12 £6.667 26994 13574 4.132709 45.961 0.830801 

13 26.950 25251 .1 0610 4.025735 54.625 0.787361 

14 27.233 22618 8322 3.920253 62.547 0.715986 

15 27.517 19421 6553 3.816461 69.489 0.625469 

16 27.800 15978 5183 3.714554 75.31.9 0.524708 

17 28.083 12698 4118 3.614730 80.026 0.426211 

1.8 28.367 10008 3290 3.517184 83.740 0.344181 

19 28.650 7993 ' 2643 3.422113 86.685 0.282428 

20 28.933 6364 2137 3.329714 89.040 0.231715 

21 29.217 5012 1739 3.240182 90.902 0.188639 

22 29.500 3989 1425 3.153714 92.372 0.155735-

23 29.783 3288 1176 3.070506 93.561 0.133662 

24 30.067 2718 979 2.990756 94.550 0.115536 

25 30.350 2474 822 2.914658 95.413 0.110488 

26 30.633 2290 696 2.842411 96.193 0.107977 

27 30.917 2133 595 2.774209 96.909 0.106883 

28 31.200 1744 513 2.710249 97.595 0.093535 

29 31.483 1366 447 2.650728 98.043 ' 0.079075 

30 31.767 2931 394 2.595843 98.767 0.184976 

31 32.050 1721 351 2.545789 99.607 0.119879 

32 32.333 418 317 2.500762 99.913 0.032655 

33 32.617 83 289 2.460960 99.978 0.007449 

34 32.900 33 267 2.426578 99.995 0.003416 

35 33.183 2 250 2.397814 100.000 0.000219 

Fig. 9-2 Chromatogram of GPC analysis and results for residue after 

stability test ( pH4 ). 

"""t I 
( 

> E 

'i 

.. 

~ ....... 

.... 

,. . .., 

Study No. 82315 

Date 2003/08/27 

Page 1 I 2 

GPO chromatogram 

50.00 

40.00 

30.00 ~ 

/1\ 

! \ 

20.00 ! \ ! \ 

f \ 

10.00 

f \ 

! \ 

I \ : \. 

0.00 / "'--·,..1\ ••••••••••·-·-••••-•••••-••·-·---·-·--··•--•-••-o•mo-oom•ooooooom•.l<.•-'••-••-""""mmoou I' ' .r\ J'_ .................. .. 

'--~ W \It .,., ! 

-10.00 

-20.00 

0.00 

• 1 

5.00 10.00 15.00 20.00 25.00 30.00 

Min. 

\I\ fl.! v ·: j v 

I: l/ \i 

:I 

v 

35.00 40.00 

Sample pH7 Vial 12 Date 2003/08/26 20:16:30 

Results 

Ret. time(min.) Mn Mw Mw/Mn Mz Mz/Mw 

26.428 4367 18535 4.244455 37847 2.041903 

g .--·-······-1 00.00 ;1r'·"' o -··- o:8o ~ / ~ \ /__.--··---·-····J· ·-·-····-·· - II 1 g;; \ /'_.. § '"' ....

0.60 

0.40 

0.20 

:E / ~ X\ . ·t; -80.00 ~ ,; ~ (') e ~ ! .. \ ~ £ ~ / / \ :::;; -60.00 ~ 

~~ // \ -~F ':ii./ . \ 6h ,, ,/ \ ! / \ -40.00 .s 

I / / "' , 

I / ""· r, -2o.oo I ___ ... / ................... /\ 

/

1 / .. t-·---·~-/"'\/ \ // ' - o.oo ···········-··.,< ........ ---· "· -o.oo 

5.00 4.50 4.00 3.50 

Log Mw 

Molecular weight distribution 

··············· Total area 

3.00 2.50 

Fig. 1 0~1 Chromatogram of GPC analysis and res1,.1lts for residue .after 

stability test ( pH7 ) . 
( 

( 

~-

( 

Study No. 82315 

Date 2003/08/27 

GPO slice table 

Page 2 I 2 

Mp: 16739.626799 Name· . Peak 1 

•• Retention time(min.) Peak area M.W. Log M.W. -~% dwt/d(logM) 

1 23.549 1 224996 5.352174 0.001 0.000020 

2 23.831 72 174627 5.242112 0.009 0.002002 

3 24.114 480 135328 5.131387 0.082 0.013249 

4 24.396 1868 104759 5.020191 0.418 0.051361 

5 24.679 5006 81044 4.908721 1.445 0.137408 

6 24.961 10280 62686 4.797170 3.785 0.282229 

7 25.243 16250 48499 4.685732 7.916 0.446994 

8 25.526 20783 37549 4.574603 13.664 0.573753 

9 25.808 24670 29106 4.463975 20.651 0.684765 

10 26.090 28215 22597 4.354045 28.807 0.788864 

11 26.373 29796 17579 4.245006 37.760 0.840639 

12 '26.655 29226 13710 4.137053 46.833 0~833643 

13 26.938 27220 10725 4.030379 55.482 0.786504 

14 27.220 24289 8417 3.925180 63.357 0.712406 

15 27.502 20786 6632 3.821650 70.234 0.620135 .. 

16 27.785 17049 5248 3.719984 75.992 0.518562 

17 28.067 13504 4172 3.620375 80.626 '0.419721 

18 28.350 10587 3334 3.523018 84.266 0.337092 

19 28.632 8412 2680 3.428107 87.137 0.275109 

20 28.914 6678 2167 3.335838 89.424 0.224987 

21 29:197 5245 1764 3.246403 91.227 0.182584 

22. 29.479 4160 1445 3.159999 92.647 0.150152 

23 29.761 3439 1193 3.076819 93.795 0.129213 

24 30.044 2814 993 2.997057 94.751 0.110496 

25 30.326 2536 834 2.920908 95.568 0.104546 .. 

26 30.609 2310 706 2.848566 96.295 0.100518 

27 30.891 2147 603 2.780226 96.966 0.099193 

28 31.173 1915 520 2.716083 97.628 0.094628 

29 31.456 1297 453 2.656330 98.063 0.069091 

30 31.738 2932 399 2.601162 98.698 0.169999 

31 32.020 2034 355 2.550773 99.548 0.129936 

32 32.303 540 320 2.505358 99.896 0.038602 

33 32.585 111 292 2.465111 99.974 0.009035 

34 32.868 42 269 2.430227 99.994 0.004046 

35 33.150 0 252 2.400900 100.000 0.000000 

Fig. 10-2 Chromatogram of GPC analysis and results for residue after 

stability test ( pH7 ). 

fJ'i 317 
( 

( 

\. 

I 

! 

Study No. 82315 

Date 2003/08/27 

Page 1 I 2 

> E 

50.00 

40.00 

30.00 

20.00 

10.00 

0.00 

GPO chromatogram 

"' 

-co 

co 

-

A, 

j \ 

! \. 1 \ I I I \ 

i \ 

j \ I ' ! \ i \.,_ I -.. ,-'\. ···································································-,-----·---------·zs~······--··--. -··········'-~\ /\ n r-··-----·············· 

\} l 1\ ! 

\ i ') -10.00 

-20.00 

\! 

'I \; 

if 

v I I I I I I I I I I I I 1. I I I I I I I I I I I I I I I I I I I I I I I I I I 

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 

• 1 

0.80-

0.60-

0.40 

0.20 

Min. 

-- Sample pH9 Vial 13 Date 2003/08/26 21:01:32 

Results 

Ret. time(minJ Mn Mw Mw/Mn Mz Mz/Mw 

26.437 4322 18329 4.240298 37474 2.044569 

I 

5.00 4.50 

g 

----------!- .... -····- ~/··- ] 

. 4.00 3.50 

Log Mw 

Molecular weight distribution 

·············· Total area 

.. 

. ~ 

3.00 

~---······· . 

I 

2.50 

100.00 

80.00 

f--60.00 

40.00 

20.00 

~ 

i 

... = 

Fig. 11-1 Chromatogram of GPC analysis and results for residue after 

stability test ( pH9 ). 
( 

\ 

' \ 

Study No. 82315 

Date 2003/08/27 

Page 2 I 2 

GPC slice table 

- Retention time(min.) Peak area M.W. log M.W. 

-~" dwt/d(logM) 

Mp: 16611.60547QName: Peak1 

1 23.550 3 224804 5.351804 0.001 0.000094 

2 23.833 62 174328 5.241367 0.008 0.001812 

3 24.117 441 134978 5.130264 0.079 0.012728 

4 24.400 1732 104397 5.018689 0.405 0.049859 

5 24.683 4681 80694 4.906840 1.410 0.134556 

6 24.967 9688 1 62361 4.794913 3.719 0.278539 

7 25.250 15392 48206 4.683104 7.823 0.443394 

8 25.533 19739 37292 4.571610 13.553 0.570701 

9 25.817 23494 28882 4.460627 20.535 0.683015 

10 26,100 26908 22405 4.350352 28.703 0.788010 

11 26.383 28426 17417 4.240980 37.676 0.840135 

12 26.667 27881 13574 4.132709 46.770 0.833206 

13 26.950 25969 10610 4.025735 55.440 0.786231 

14 27.233 23166 8322 3.920253 63.333 0.712035 

15 27.517 19817 6553 3.816461 70.224 0.619688 

16 27.800 16238 5183 3.714554 . 75.988 0.517762 

17 28;083 12846 4118 3.614730 80.621 . 0.418660 

18 28.367 10069 3290 3.517184 84:260 0.336227 

19 28.650 8015 2643 3.422113 87.132 0.274988 

20 28.933 6357 2137 3.329714 89.420 0.224747 

21 29.217 4986 1739 3.240182 91.222 0.182222 

22 29.500 3955 .1425 3.153714. 92.641 0.149937 

23 29.783 . 3276 1176 3.070506 93.788 0.129284 

24 30.067 2676 979 ·2.990756 94.741 0.110427 

25 30.350 2408 822 2.914658 95.561 0.104399 

26 30.633 2213 696 2.842411 96.292 0.101345 

27 30.917 2097 595 2.774209 96.970 0.102063 

28 31200 1812 513 2.710249 97.642 0.094349 

29 31.483 1352 447 2.650728 98.089 0.075983 

30 31.767 2953 394 2.595843 98.787 0.180949 

31 32.050 1754 351 2.545789 99.618 0.118630 

32 32.333 419 317 2.500762 99.918 0.031788 

33 32.617 83 289 2.460960 99.979 0.007158 

34 32.900 26 267 2.426578 99.995 0.002626 

35 33.183 0 250 2.397814 100.000 0.000000 

Fig. 11-2 Chromatogram of GPC analysis and results for residue after 

stability test ( pH9 ). 

I, ,T 
I 

> E 

( 

___ j 

Study No. 82315 

Date 2003/08/27 

50.00 

40.00 

30.00 

20.00 

10.00 

PageL/ 1 

GPO chromatogram 

0.00 

---············································--------··············--------------, f\ r···· ~-

v l {'I 

' j \; I! -10.00 

-20.00 

I I I I I I I I I I I I I I I I I . I· I I I I I I I I 

0.00 5.00 10~0 15~0 20~0 25~0 30~0 

Min. 

i/ 

I• q 

~ 

I I I I I I 

35.00 40.00 

-- Sample BL THF Vial14 Date 2003/08/26 21:46:30 

Fig.12 Chromatogram of GPC analysis for solvent blank.