PMN Page 1 Page 1 of 1,1 0' 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

PREMANUFACTURE 1:"& .. [: 15 '.\1'"""':">. 

I"" : ~ ~.i-_ "_, 

t; 1111111111111111 mIII~ IIIIIIIIIIIII~ 11111 ~IIIIIIIIIIIIIIII~IIII ~II :S 

5 1 1 000 0 0 1 3 6 I S 

Company Sanitized 

D.C. 20460 

GENERAL INSTRUCTIONS 

You must provide all information requested in this form to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates if you do 

not have actual data. 

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

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

If a user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remember, 

your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, Washington Financial Management Center (3303), P.O. 

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

Part I - GENERAL INFORMATION 

You must provide the currently COlTecl Chemical Abstracts (CA) Name of the new 

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

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

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

in support ofyour submission should reference your TS user fee identification number. 

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

claimed any information as confidential, a single sanitized copy must also be submitted. 

Part 11-HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

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

sections A and B of this notice, reproduce the sections as needed. 

Part 111-LIST OF ATTACHMENTS 

Attach additional sheets ifthere is not enough space to answer a question fully. Label each 

continuation sheet with the corresponding section heading. In Part Ill. list these 

attachments, any test data or other data and any optional information included in the notice. 

OPTIONAL INFORMATION 

You may include any information that you want EPA to consider in evaluating the new 

substance. On page 11 of this form, space has been provided for you to describe 

pollution prevention and recycling information you may have regarding the new substance. 

So-<:alled "binding" boxes are included throughout this form for you to indicate your 

willingness to be bound to certain statements you make in this section. such as use, 

production volume, protective equipment ... This option is intended to reduce delays that 

routinely accompany the development of consent orders or Significant New Use Rules. 

Except in the case of exemption applications (such as TMEA, LVE, LOREX) where 

certain infotrnation provided in such notification is binding on the submitter when the 

Agency approves the exemption application, checking a binding box in this notice ~ 

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 assen 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 attachmen~ circle or bracket the infonnation you claim as 

confidential. If you claim infonnation in the notices as confidential you must also provide 

a sanitized version of the notice (including attachments). For additional instructions on 

claiming information as confidential, read the Instructions ManuaL 

Mark (x) ifany information in this notice is claimed as confidential 

TEST DATA AND OTHER DATA 

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


provide a description of all other data known to or reasonably ascertainable by 


you, if these data are related to the health and environmental effects on the 


manufacture, processing, distribution in commerce, use, or disposal ofthe new 


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


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


of data. must be submitted ifthey do not appear in the open literature. You should 


clearly identify whether test data is on the substance or on art analog. Also, the 


chemical composition ofthe 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). 


r--.: 


Test Data (Check Below any included in this notice) ~r 

('".:;:) 

Environmental fate data Yes Other data (,,"::,,­

",... 

Health effects data Yes Risk assessments ;Q 

n·,:'fes 

Environmental effects data 0 Yes Structure/activity relia.ticmslup',r:l 

~ 

PhysicallChemlcai Properties' 0 Yes Test data not in ~tssesslo~ or control of the' itter B 

A phYSIcal and chemIcal properties woTksheet IS located on the last page~iS form. 

TYPE OF NOTICE (Check Only One) - o PMN (Premanufacture Notice) 

o lNTERMEDlATE PMN (submitted in sequence with final product PMN) 

o SNUN (Significant New Use Notice) 

D TMEA (Test Marketing Exemption Application) 

o LVE (Low Volume Exemption) @40 CFR 723.50(0)(1) 

D LOREX (Low Release/Low Exposure Exemption) @ 40 CFR 723.50(c)(2) 

o 	 LVE Modification o LOREX Modification

IS THIS A CONSOLIDATED PMN? o 
 Yes

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

181 
PMN Page 2 ; Page 2 of 1,101 

Public reporting burden for this collection ofinformation 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 

information. Send comments regarding the burden estimate or any other aspect ofthis collection of information, including suggestions for 

reducing this burden, to Director, Collection Strategies Division (2822), U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., N.W., 

Washington, D.C. 20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

CERTIFICATION -- A Printed copy of this signature page, with original Signature, must be submitted 

I certifY that to the best ofmy knowledge and belief: 

1. The company named in Part I, section A, subsection 1 a ofthis notice fonn 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 infonnation provided in this notice is complete and truthful as of the date ofsubmission. 

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 ofthe Premanufacture Notification Rule. 

Additional Certification Statements: 

Ifyou are submitting a PMN, Intennediate PMN, Consolidated PMN, or SNUN, check the following user fee certification 

statement that applies: 

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

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

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

o The Company named in Part J Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of $1 00 

in accordance with 40 CFR 700.45(b). 

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

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

statements: 

o 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 tenns of40 CFR 723.50. 

o The manufacturer is familiar with the tenns ofthis section and will comply with those tenns; and 

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

o Ifthis application is for an LVE in accordance with 40 CFR 723.50(c)(J), the manufacturer intends to commence 

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

review period. 

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

described herein. Any knowinl!ud willful misinterpretation is subject to criminal penalty pursuant to 18 USC 1001. 

Signatun: and title ofAuthorized Official (Original Signature Required) Date 

Signatun: of agent • (if applicable) Date 

Confidential 

D 

EPA FORM 7710·25 (Rev. 5·95) Page 2 
; Page 3 of 1.10' 

la. 	Person 

Submitting 

Notice (in U.S.) 

Company 

Mailing address (number and street) 

City, State Postal Code 

b. Agent(if Name of authorized official 

applicable) 

Mailing address (number and street) 

City, State 	 Postal Code Telephone (include area code) 

c. IfYOu~eSubmi~;·n~g~~Krti~;;P;tQr;~~~~~~~~~~~bo~X:.::::::::::::::::::::::::::::::::::::::::::=i~~ 

Joint Submitter (if Position 

applicable) 

Company 

Postal Telephone (include country or area code) 

2. Technical 

Contact (in 

U.S.) 

Name of authorized official 

Dr. Richard Jourdenais 

Company 

Technology Sciences Group Inc. 

Position 

Director, Chemicals Division 

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

EPA assigned a PC Number 10 Ihe notice. enlerth :::::e.::n=um::be=r.~___-+~ 

4. 	 If you previously submined an exemption application for the chemical 

substance covered by this notice. enter the exemption number assigned by 

EPA. If you previously submitted a PMN for this substance enler the PMN 

number EPA withdrawn or ~ 

5. 	 If you have submined a notice of Bona fide inlenllo manufacture or import 

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

assigned by EPA. ~ 

D Manufacture I. 6. Type of Notice - M~k (X) Only 

D Binding Option 

Mark 

Mailinl! address (number and street) 

1150 18th Street NW Suite 1000 

Postal Code Telephone (include area code) 

Washington, DC 20036 202-828-8992 

12. ~ Import 

Only 

M~k(X) 

ifnone 

Mark (X) 

if none 

Mark (X) 

if none 

1 D 


13. Both 

D Binding Option ,

I 

M~k 

EPA FORM 7710-25 (Rev. 5-95) 	 Page 3 
PMN Page 4 ; Page 4 of 1,101 

Part I -- GENERAL INFORMATION -- Continued 

Section B -- CHEMICAL IDENTITY INFORMATION: I

You must provide a currently correct Chemical Abstracts (CA) Mme ofthe substance based on the nintb 

Collective Index (9CI) ofCA nomenclature rules and conventions. 

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

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

If another person will submit chemical identity infonnation for you (for either Item 1 or 2), mark (X) the box at the right. 0 

Identify the name, company, and address of that person in a continuation sheet. .. 

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

a. Class of substance - Mark (X) I 0 Class I or 2 0 Class 2 

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

Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name or CA Preferred Name must be provided, 

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

c. 

d. 

e. 

Please identify which method you used to develop or obtain the specified chemical identity infonnation reported in this notice: (check one). 

r-;l Method I (CAS Inventory Expert Service a copy of the Identification D Method 2 (Other Source) 

L.:.J report obtained from the CAS Inventory Expert Services must be 

submitted as an attachment to this notice) 

Molecular fonnula CBI CAS Registry Number 

(ifa number already 

exists for the substance) 

For a class 1 substance, provide a complete and correct chemical structure diagram. For a class 2 substance, provide a correct representative or 

partial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained. Please see the E-PMN Instruction Manual for 

discussion of "native fonnat" diagram software which can be helpful in reviewing your substance. 

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

Confidential 

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

PMN Page 4a ; Page 5 of 1,101 

For a class 2 substance· (I) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction or process. 

(3) Indicate the range ofcomposition and the typical composition (where appropriate). 

e. (1) List the immediate precursor substances with their respective CAS Registry Numbers. ConfiName (CAS #) dential 

D 

e. (2) Describe the nature of the reaction or process. D 

e. (3) Indicate the range ofcomposition and the typical comJlOsition (where appropriate). D 

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

EPA FORM 7710·25 (Rev. 5·95) Page 4a 
PMN Page 5 ; Page 6 of 1,10 

Part I -- G.ENERAL INFORMA nON -- Continued 

Section B - CHEMICAL IDEJ't.TITY INFORMATION - Continued 

2. Polymers (For a definition of polymer, see the Instructions Manual.) Confidential 

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

Indicate 

below I 000 

maximum 

absolute 

weight 

molecular 

percent 

weight 

of low 

of

molecular 

that composition. 

weight species (not including residual monomers, reactants, or solvents) below 500 and D

Describe the methods of measurement or the basis for your estimates: GPCU Other U: (SpecifY below) 

(i) lowest number average molecular weight: 

I I 

(ii) maximum weight % below 500 molecular weight: 

(iii) maximum weight % below 1000 molecular weight: 

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

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

the polymer. 

(2) Mark (X) this column ifentry in column (I) is confidential. 

(3) - Indicate the typical weight percent of each monomer or other reactant in the polymer. 

(4) - Choose "yes" from drop down menu if you want a monomer or other reactant used at two weight percent or less to be listed as part ofthe polymer 

description on the TSCA Chemical Substance Inventory. 

(5) - Mark (X) this column if entries in columns (3) and (4) are confidentiaL 

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

commercial purposes. 

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

Monomer or other reactant and CAS Registry Number Confi­ ~lncludein Confi­ M"dm=: Confidential . identity dential residual dential 

(I) (2) 3 (4) (5) (6) (7) 

D % D % 

D % 0 D % D 

D % D . % D 

D % D D % 

% D % 

---­ D % 0 D % 

.... D

D % 0 D % 

CJ c:=J % 

CJ c:=J % 

c:=J % , c:=J % I I : c:=J % c:=J % I I 

c:=J %R~ % '. 

CJ % %

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

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

PMN Page Sa; Page 7 of 1.10 

c, 	 D Please identifY which method you used to develop or obtain the specified chemical identityC:rnnation reported in this notice (check one), COl

Method I (CAS Inventory Expert Service· a copy of the identification report Method 2 (other source) 


obtained from CAS Inventory Expert Service must be submitted as an attachment to this notice) 
 D 

d, The currently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCA Inventory listings for similar polymers, 

D 


CAS Registry Number (if a number already exists for the substance) 

e, 	 Provide a correct representative or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertained" Please 

see the E·PMN Instruction Manual for discussion of "native format" diagram software which can be helpful in reviewing your substance, 

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

EPA FORM 7710·25 (Rev 5·95) 	 Page Sa 
PMN Page 6; Page 8 of 1,10 

Impurities 

(a) 	 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. Ifthere are unidentified impurities, enter "unidentlfied.rt 

Estimate the maximum % of . . . . estimate their total %. 

Impurity and CAS Registxy Number Maximum Confipercent dential 

% 0 

% 

0 

% 0 

% D 

% D 

% D 

% D 

Confidential 

0 


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

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

KarenzMr®PEl,PEl-VM,PEMB,PEMB and PE3MB, KarenzMT(PEMB and PE3MBl 

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

6. 	 you you must name your substance that reveals 

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

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

Ppll.ythiol 

manufacture, processing, use, or disposal of the new chemical substance. Provide the CAS Registxy 

CAS 

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

Number Confidential 
PMN Page 7 ; Page 9 of 1,101 

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. 

1. Production volume ­ Estimate the maximum production volume during the first 12 months of production. Also estimate the maximum 

production volume for any consecutive 12-month period during the first three years of production. Estimates should be on 100% new chemical 

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

10,000 kg/yr, specify the volume and mark (x) in the binding box. If granted, you are bound to this volume 

Maximum fi,,, 12-month prod","on (kg/y<) Maximum 12-month production (kg/y<) :Ij::''''''' 

.[ (100% new chemical substance basis) (100% new chemical substance basis) dentlal Ma!;)

2. Use Infonnation - 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. (J) --Describe each intended category of use ofthe new chemical substance by function and application. (2) --Mark (X) this column if entry column (1) is 

~onfidential business information (CBI). (3) --Indicate your willingness to have the information provided in column (I) 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)-­

~stimate the percent of the new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under 

your control associated with each category of use. (7) --Mark (X) this column if entry in column (6) is confidential business information (CBI). (8) -Indicate % of 

product volume expected for the listed "use" sectors, Mark more than one box if appropriate. Mark (X) to indicate your willingness to have the use type provided in (8) 

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

Category of use (1) (by function and application i.e. a CBI Binding Prod- CBI °/0 in CBI % of substance expected per use CBI 

dispersive dye for finishing polyester fibers) Option uction Fonnu (8) MiIrk % latian (x) 

(2) (3) (4) (5) (6) (7) Cons- Indus- Com- Binding (9) .mer trial mercial OptiM 

D % ~ % ~ ~ ; :' 

'" 

D % % - ',)7 0 " " " ,'.; . . 

. 

'l'o 'l'o .. 

',' 

.i!. D

DD % D % r--­ 0 ,..-­

.. - % % 

- ~ - D

D % - % r--­ .. ' 

'-----­

.. .. .. D

DD % D % \:; 

'--­

• If you have identified a "consumer» use, please provide on a continuation sheet a detailed description of the use(s) of this chemical substance in consumer products. 

In addition include estimates of the concentration of the new chemical substance as expected in consumer products and describe the chemical reactions by which this Id bstance loses its identity in the consumer product. 

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

b. Generic use If you claim any category of use description in subsection 2a as confidential, enter a generic description of that category. Read the Instruction 

description Manual for examples of generic use descriptions. 

Monome:r:for industrial paints, coatings, ink and adhesives 

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

3. Hazard Information - Include in the notice a copy of reasonable facsimile of any hazard warning statement, label, material safety data sheet, or other Binding 

information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or practices for Option 

the safe handing, transport, use, or disposal of the new substance. List in part III hazard information you include. Mark (x) 

~ Mark (X) this box if you attach hazard information. I I 

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

PMN Page 8 ; Page 10 of 1, 1C 

Section A -- INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER box next to any item you 

Complete section A for each type of manufacture, processing, or operation involving the new chemical substance at industrial sites you 

control. Importers do not have to complete this section for operali0l1s outside the U.S.; however, you may still have reporting requirements if 

there are further industrial or use' must describe these See instructions manual 

l. 

of the site at which the will occur. 

Site address (number and street) 

If the same operation will occur at more than one site, enter the number of sites. IdentifY the 

additional sites on a continuation sheet, and if any of the sites have significantly different 

production rates or operations, include all the infonnation requested in this section for those 

sites as attachments. 

J, Batch 

2. Continuous 

Mil.X,mIUm kg/day (100% new chemical 

substance) 

Use 

BatchesJyear 

Days/year 

Confidential 

D 

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

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

drum, rail car, tank truck, etc,). 

(2) Provide the identity, the approximate weight (by kg/day or kglbatch 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 intem1ittent releases, to the environment of the new chemical substance, If releasing to two media 

at the same step, assign a second release number for the second medium, 

attach a continuation sheet 

EPA FORM 7710-25 (Rev, 5-95) Page 8 

PMN Page 8a ; Page 11 of 1,10 

Confidential 

D 


EPA FORM 7710-25 (Rev. 5-95) Page Sa 
PMN Page 9 ; Page 12 of 1 ,1 C 

D 

D 

D 

Part II-w HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE wW 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, number of 

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

(I) -- Describe the activities (i.e. bag dumping, tote filling, unloading drums, sampling, cleaning, etc.) in which \MJrkers 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 formes) of the new chemical substance (e.g., solid: crystal, granule, powder, or dust) and % new chemical substance (if part ofa mixture) at the 

time ofexposure. 

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

(8) -- Estimate the maximum number of\MJrkers involved in each activity for all sites combmed. 

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

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

(12) -- Mark (X) this column ifentries in columns (10) and (II) are confidential business information (CBI). 

Worker activity 

(i.e., bag dumping, filling drums) 

(I) 

CBI 

(2) 

Protective Equipmenti 

Engineering Controls 

(3) 

Binding 

Option 

Mark (x) 

(4) 

Physical 

forms(s) 

and % new 

substance 

(5) 

Bindmg 

Option 

Mark (x) 

(6) 

CBI 

(7) 

# of 

Workers 

Exposed 

(8) 

CBI 

(9) 

Maximum duration CBI 

(12) 

Hrs/day 

(10) 

Days/yr 

(ll) 

'. 

.'. 

....i» ... 

'. 

. ··c. 

•• < 

'.' . . 

.. 

.. - " 

. ..' 

. '... 

D - r-­

- .. -

-

--­ - '--­ .''''r 

D D- '--­

D D .' 

D D -

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

EPA FORM 7710-25 (Rev. 5-95) 9 
PMN Page 9a; Page 13 of 1,10 

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

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

(I) -- Enter the number of each release point identified in the process description, part II, section A, subsection Id(3). 

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

(3) -- Mark (X) this column ifentries in columns (1) and (2) are confidential business information (CBI). 

(4) -- Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, P01W, or other (specify» to 

which the new substance will be released from that release point. 

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

disposed ofon land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet, for 

each site describe any additional disposal methods that will be used and whether the waste is subject to secondary or tertiary on-site treatment. b. Estimate the 

amount released to the environment after control technology (in kg/day). 

(6) -- Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CBI). 

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

NPDES numbers of the P01W (Publicly Owned Treatment Works). Mark (X) if the P01W name or NPDES # is confidential business information (CBI). 

Release Amount of new CBI Medium of Control technology and efficiency (you may wish to optionally attach CBI 

Number substance released release efficiency data) 

(1 ) (2a) (2b) e.g. sLack.if Binding 

(4) Mark (3) (5a) (Xl (5b) (6) 

D -

D " ',' 

: ", 

) D D D

D r--­

", 

' ! 

I '--­ D

D -

--­ I .. 

... 

I,· 

. [ D D

'" 

•••• 

.' 

'", 

.. 

. Q D

:' 

. 

I 

D D D

I,' D D ..·..

y 

··.i " ". D " 

D - (7) Mark (X) the destination(s) ofreleases to water. NPDES# CRI 

D P01W--provide 

name(s) D

0 Navigable waterway-­

provide name(s) D

D Other--Specify 

D 

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

EPA FORM 7710-25 (Rev. 5-95) Page 9a 
PMN Page 10; Page 14 of 1,10 

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 chemical substance at sites you do not control. Importers do not have to complete this section for 

operations outside the U.s.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate section B for each type af 

pracessing, ar lISe aperatian irrvalving the new chemical substance. If the same operation is performed at more than one site describe the typical operation common to these 

sites. IdentitY additional sites on a continuation sheet. 

1(a). Operation Description -- To claim infonnation in this section as confidential, circle or bracket the specific infonnation 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, identify by letter and briefly describe each worker activity. (2) -- Either in the diagram or in the text field l(b) below, provide the . 

identity, the approximate weight (by kg/day or kglbatch, on an 100"10 new chemical substance basis), and entry point ofall 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) -- Either in the diagram or in the 

text field I(b) below, 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 ofthese sites on a continuation sheet); eBI # ofsites 

I(b). (Optional) This space is for a text description to clarify the diagram above. 

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

EPA FORM 7710-25 {Rev. 5-95) Page 10 
PMN Page 10a ; Page 15 of 1,10 

2. Worker [xposure/Environmental 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 ofexposure 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 ofthe new substance as formulated when packaged or used as a final product 

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

(10) -- Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kglbatch), 

(12) -- Describe media of release i.e, stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, or other (specifY) 

and control technology, if any, that will be used to limit the release of the new substance to the environment 

(14) -- IdentifY byproducts which may result from the operation, 

_(3). (5). (8 , (11), 13) and (15) -- Mark (X) this column ifany of the proceeding entries are confidential business information (CB!), 

Lett." #of CBI Duration Of CBt Protective Equip.lEngineering ControlslPhysical Form and! % new substance 0/0 in CBt 

of Workers Exposure Fonn­ (Cti. Exposed ulation 

Vlly 

• (I) (2) (3) (4a) (4b) (5) _(6) (7) (8) 

D 10 Pleas$see the attachment .... 0 r--I 0 0 , . ," --II·· 

I D 0 10 . ,. D , ..... 0 ,,' 0 

D . -D '. 0 

Release Amounlof CBl Media of Release & Control Technology CBl 

Number New 

Subs1ance ; 

Released I 

(9) lOa) lOb (II) (12) (13) 

" ; 0 ·i 0 ,-­ 0 ". ". L....­ . 

0 I 

- 0 '. ; 

i . .'. 0

". Oi .... . " 01 

14) -- Byproducts: (15) 

. 0 

I!l Mark (X) this box if you attach a continuation sheet, 

OPTIONAL POLLUTION PREVENTION INFORMATION 

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

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

associated with activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new information 

pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due to the new 

chemical substance. Source reduction includes the reduction in the amount or toxicity 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 ofpollution 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 nonenvironmental 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 PMN Instructions 

Manual and Pollution Prevention Guidance manual for I'tuidance and examples. 

EPA FORM 7710-25 {Rev, 5-95) Page lOa 
PMN Page 11 : Page 16 of 1.10' 

Optional Pollution Prevention Information (Continued) Describe the expected net benefits, such as (I) an overall reduction in risk to 

human health or the environment; (2) a reduction in the volume manufactured; (3) a reduction in the generation ofwaste materials 

through recycling, source reduction or other means; (4) a reduction in potential toxicity or human exposure andlor environmental 

release; (5) an increase in product performance, a decrease in the cost of production andlor improved operation efficiency ofthe new 

chemical substance in comparison to existing chemical substances used in similar application; or (6) the extent to which the new 

chemical substance rna be a substitute for an existin substance that oses a reater overall risk to human health or the environment. 

COl 

D 

ou attach a continuation sheet. 

EPA FORM 7710-25 (Rev. 5-95) Page 11 
PMN Page 12 ; Page 17 of 1,10 

J 

Part III -- LIST OF ATTACHMENTS 

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

structure/activity infonnation), and optional infonnation after this page. Clearly identify the attachment and the section of the fonn to 

which it relates, if appropriate. Number consecutively the pages of any paper attachments. In the column below, enter the inclusive page 

numbers of each attachment. Electronic attachments can be identified by filename. 

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 infonnation in an attachment as confidential. You must include with the sanitized copy of the notice fonn a sanitized 

version of any attachment in which you claim infonnation as confidential. 

# Attachment name Attachment Filename Attachment 

page number(s) 

Confidential 

~ 

~ 

- ~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

[RJ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

~ 

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

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

I 
PMN Page 13 ; Page 18 of 1,10 

PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

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

include it in the notice, IdentifY the property measured, the page of the notice on which the property appears, the value ofthe property, 

the units in which the property is measured (as necessary), and whether or not the property is claimed as confidentiaL If the attachments 

are electronic, give the attachment number (found on page 12) at (b), The physical state of the neat substance should be provided, 

These measured properties should be for the neat (100"10 pure) chemical substance, Properties that are measured fur mixtures or 

formulations should be so noted (% PMN substance in --->, You are not required to submit this worksheet; however, EPA strongly 

recommends that you do so, as it will simplifY review and ensure that confidential information is properly protected, You should submit 

this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission oftest data, 

Property Mark (X Page Value Measured 0 Conl\­

iC number Estimate dentisl 

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

(M or E) (d) 

Physical state of neat substance 0 D (s) 0(1) D(g) [Xl 

Vapor pressure lZJ [Xl @ Temperature °C Torr 

Density/relative density i[Z] glcm3 [Xl 

Solubility 

@ Temperature °C 0 [Xl 

Solvent giL 

Solubility in water@ Temperature °C lZ] 0% gIL [Xl 

Melting temperature lZJ °C [Xl 

Boiling / sublimation temperature@ torr pressure lZJ °C [Xl 

Spectra lZJ [Xl 

Dissociation constant 0 0 

Particle size distribution 0 Oi 

Octanol/ water partition coefficient lZJ [Xl 

H~'s Law constant 0 0 

Volatilization from water 0 D 

Volatilization from soil 0 D 

0 . 

[Xl pH . @ concentration 

Flammability lZJ [Xl 

Explodability [ZJ [Xl 

Adsorption I coefficient lZJ [Xli 

Other - SpecifY [Zl [K] 

Other ­ Specify [ZJ !Xl 

D Mark (X) this be. if you atlach a continuation sheet Enter Iha atlachment nama and number, 

ATTACHMENT 


PAGE 13 

.t.............. "' ....................................................... . ...... . ........ _- .. ~ .......
~ ~ 

14 
Inventory Expe'!§ervice 


A division of the Ameritan Chemical Society Phone: 800·631·1884, 614447-3870 

Fax: 6t4-441·3747 

E-mal: answers@cas.org 

Web: www.cas.orglproducts~dientl 

INVENTORY EXPERT SERVICE REPORT 

..... -_ ................... -..... --...... -......... -."' ..... -....... _.......... -........ -......................... _.. -_ ... _........ _.......... -_ .................... -.... -............ --: 


Please print the above CA Index Nama on the appropriate page of your PHN. 

o If this box is checked, CAS haa made correction(a) marked in red to 

your :rES order. Please make the same corraction(s) to your PMR before 

submitting it to the &PA. 

CAS 2510 Olellt.1ngy River Road P.O,8ox 3343 Columbus. Ohio ;3210-0334 USA 

15 
Inventory Expert Service' 


A division of the American Chemical Society Phone: 800·631-1 BB4. 611·447-3870 

Faldl4-447-3747 

E.mail: answers@cas.org 

Web: WoNW.cas.orgfproduasTdienti 

INVF~TORY EXPERT SERVICE REPORT 

Plea.e print the above CA Index Name on the appropriate page of your PUN. 

o If this box i. checked., CAS has maCle correction(s) marked. il1 red. to 

your IES order. Please make the same correction(s) to your PMH before 

submitting it to the EPA. 

CAS 254Q Oleotan8Y Ri....., Road P.O, Box 33-43 Columbus. Ohio 43210-033~ USA 

16 
....................................................................................................... j 


. . 


. . 
 ~~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::~ ...................................... 


. . 

17 
[•..••..•.......••..•...•..•...........•.......•...••.........••...•.••...•...•..•...•...•............. 


18 
Industrial Sites Controlled by Others Worker ExposLI'e/Environmental Release 

Amount of 

lin New Media of Letter #of DI.ntion of Protective Equipment! Release Substance Release & of worIters COl Engineering ControV Physical FonrIJIa COl Released into COl Control COl ExposLI'e Number Activity Exposed Form and I new substance lion control Technology 

technology ....•.......•...•••......••..•.•......•1-- .....................................;--

· 

~ X · :X 

I-- · :-

· X IX :X 

· I-- I-- iX X ·

· · ~ I-- ~ 

X X . :X 

· E 1 1 J ·····························]0······· 1 1 

F 1 1 I 10 I I . . .. 

X X X 

~ f-- I--

X X X 

I-- ~ I--

X X 

I-- ~ ~ 

X X X 

'-~':J() ..."'.J" ............r···T···........ ,.---

K I I I 

L 1 1 J 10 I 1 1 

19 

: .•...................•..........................•....................•...........••..••...-•...••........•.·······························1 


,

, 

20 . . t.. ___ ........ __ .. __ ....... __ .. "" .. __ ..... _________ ....... __ .... __ .... '" __ "' ........ _4 

., 21 ••••• __ • __ •• __ ••• __ • ___ •• * _________ • __ • _____ .~ ______ • ________ a_ •• ___ ._~~. __ • __ • ___ • ___ • __ •• __ • ______________ _____ ••• _____ •••• _ •• ______ .: 
..........................· ................................................................................................................ . 
 j 

. 

22 


.....-..--.. -~.--- ..--.--..-----.... ----~----- .. 
l............................................................................................................................................ 


23 
............................................................................................................ ································1 


" 

.: 

;: 

, 

'., 

",'.

· 

" ·, 

.. 

, , 

" 

,

, 

, · 


t ........................................................................................................................................... 


24 
..................................................................................................................... 


25 
r....·•....·•··..•·•····•·····························........:...:•.........................••.....•....•.....••....••....•••... ~ 

I .................................................................................................................... ~~......._ 

PAGE 1 OF 90 PAGES 

Mixture of PEMB and PE3MB: 


DETERMINATION OF PHYSICO-CHEMICAL 


PROPERTIES 


PROJECT NUMBER: 014510193 


AUTHORS: 	 R E Butler 

AJ Woolley 

D FWhite 

STUDY SPONSOR: TEST FACILITY: 

Showa Denko K.K. Harlan Laboratories Ltd 

13-9, Shiba Daimon 1-Chome Shardlow Business Park 

Minato-ku Shard low 

TOKYO 105-8518 Derbyshire 

JAPAN DE722GD 

UK 

Telephone: +44 (0) 1332 792896 

Facsimile: +44 (0) 1332799018 

27 
PROJECT NUMBER: 014510193 PAGE 2 

QUALITY ASSURANCE REPORT 


This study type is classed as short-term. Inspection of the routine and repetitive 

procedures that constitute the study is carried out as a continuous process designed to 

encompass the major phases at or about the time this study was in progress. In 

addition, inspection of general facilities not specifically related to this study are done 

monthly or annually in accordance with QA Standard Procedure. 

This report has been audited by the Quality Assurance Unit, and is considered to be an 

accurate account of the data generated and of the procedures followed. 

In each case, the outcome of QA evaluation is reported to the Study Director and 

Management on the day of evaluation. Audits of study documentation, and process 

inspections appropriate to the type and schedule of this study were as follows: 

§ 19 November 2008 Protocol Compliance Audit 

09 March 2009 Freezing Temperature 

07,28 January 2009 Boiling Temperature 

07 January 2009 Relative Density 

06 April 2009 Vapour Pressure 

07 April 2009 Surface Tension 

02,03 March 2009 Water Solubility 

16 January 2009 Partition Coefficient 

15 January 2009 Flash Point 

05 January 2009 Auto-Ignition Temperature (Liquids and Gases) 

09 January 2009 Adsorption Coefficient 

03 April 2009 Fat Solubility 

§ 18 May 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

. 05 JUN 2009 ...~:)j..~~~.................... 
DATE ................................................ . 


For the Quality Assurance Unit* 

'"Authorised QA Signatures: 

Manager, Quality Assurance: J G Riley sSe (Hons) MRQA 

Deputy Head of Department JM Crowther MISeT MRQA 

Senior Audit Staff: G Wren ONC MRQA 

28 

PROJECT NUMBER: 0145/0193 PAGE 3 

GLP COMPLIANCE STATEMENT 


With the exception of the explosive properties and oxidising properties calculations, the 

work described was performed in compliance with UK GLP standards (Schedule 1, Good 

Laboratory Practice Regulations 1999 (51 1999/3106 as amended by 512004/0994»). 

These Regulations are in accordance with GLP standards published as OECD Principles 

on Good Laboratory Practice (revised 1997. ENV/MC/CHEM(98)17); and are in 

accordance with, and implement. the reqUirements of Directives 2004/9/EC and 

2004/10/EC. 

This report fully and accurately reflects the procedures used and data generated . 

. - ~ JUN 2009 ............. ................................. DATE ............................................... .. 

R E Butler GRSC 

STUDY DIRECTOR 

This report may be presented in finai form as a digital (pelf) doClKllent. Such documents are prepared by scanning the paper original. 

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However. in all cases the 

hand-signed paper Original, held In secure archlves, Is the definitive document. 

29 
PROJECT NUMBER: 0145/0193 PAGE 4 

CONTENTS 


QUALITY ASSURANCE REPORT 2 


GLP COMPLIANCE STATEMENT 3 


CONTENTS 4 


SUMMARY 7 


1. INTRODUCTION 9 


2. TEST MATERIAL 9 


2.1 Test Material Description, Identification and Storage Conditions 9 


2.2 Chemical Structure 9 


3. ARCHIVES 10 


4. MELTING/FREEZING TEMPERATURE 11 


4.1 Method 11 


4.2 Procedure 11 


4.3 Calculation 11 


4.4 Results 12 


4.5 Conclusion 15 


5. BOILING TEMPERATURE 16 


5.1 Method 16 


5.2 Procedure 16 


5.3 Calculation 17 


5.4 Results 18 


5.5 Discussion 20 


5.6 Conclusion 20 


6. RELATIVE DENSITY 21 


6.1 Method 21 


6.2 Procedure 21 


6.3 Calculations 21 


6.4 Results 22 


6.5 Conclusion 22 


7. VAPOUR PRESSURE 23 


7.1 Method 23 


7.2 Procedure 23 


7.3 Calculation 25 


7.4 Results 26 


7.5 Conclusion 40 


8. SURFACE TENSION 41 


8.1 Method 41 

8.2 Procedure 41 

8.3 Calculation 46 

8.4 Results 47 

8.5 Validation 49 

8.6 Discussion 49 


8.7 Conclusion 49 

30 

PROJECT NUMBER: 014510193 PAGE 5 

9. WATER SOLUBILITY 60 

9.1 Method 50 

9.2 Procedure 50 

9.3 Calculation 54 

9.4 Results 55 

9.5 Validation 57 

9.6 Discussion 57 

9.7 Conclusion 58 

10. PARTITION COEFFICIENT 69 

10.1 Method 59 

10.2 Procedure 59 

10.3 Calculation 63 

10.4 Results 64 

10.5 Discussion 66 

10.6 Conclusion 66 

11. FLASH POINT 67 

11.1 Method 67 

11.2 Procedure 67 

11.3 Calculation 67 

11.4 Results 68 

11.5 Conclusion 70 

11.6 Reference 70 

12. EXPLOSIVE PROPERTIES 71 

12.1 Method 71 

12.2 Procedure 71 

12.3 Results 72 

12.4 Conclusion 72 

13. AUTO-IGNITION TEMPERATURE (LIQUIDS AND GASES) 73 

13.1 Method 73 

13.2 Procedure 73 

13.3 Results 73 

13.4 Conclusion 75 

13.5 Reference 75 

14. OXIDISING PROPERTIES (LIQUIDS) 76 

14.1 Method 76 

14.2 Procedure 76 

14.3 Results 76 

14.4 Conclusion 76 

31 
PROJECT NUMBER: 014510193 PAGES 

15. ADSORPTION COEFFICIENT 77 


15.1 Method 77 


15.2 Procedure 77 


15.3 Calculation 81 


15.4 Results 82 


15.5 Discussion 84 


15.6 Conclusion 85 


16. FAT SOLUBILITY 86 


16.1 Method 86 


16.2 Procedure 86 


16.3 Results 87 


16.4 Discussion 88 


16.5 Conclusion 88 


Appendix 1 Coconut Oil Certificate of Analysis 89 


Appendix 2 Statement of GLP Compliance in accordance with Directive 


2D0419IEC 90 


32 

PROJECT NUMBER: 014510193 PAGE 7 

Mixture of PEMB and PE3MB: 


DETERMINATION OF PHYSICO-CHEMICAL 


PROPERTIES 


SUMMARY 
 ..-.-...-.--..-.....--..--. . . 
 Melting/Freezing Temperature. l 

~ 

l using 854633: Method for the 

Determination of Crystallizing pofriC·MethOd··Af·Melting/Freezing Temperature of 

Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 102 of the 

OECD Guidelines for Testing of Chemicals, 27 July 1995 . 

...~~iJ!!!fl..T.~PP.'!~~~!8 The test material was found to boil with decomposition C~~~~~l 

: :by differential scanning calorimetry, using ASTM E537-86, Method 
 . . ···JX2··Boliiiig·Teimperature of Commission Regulation (EC) No 440/2008 of 30 May 2008 

and Method 103 of the OECD Guidelines for Testing of Chemicals, 27 July 1995 . 

........ 00_ ........ _ .... __ ....... - .... --- .. - .......... -.


. . Relative Denslty~ lusing the pycnometer method, Method A3 

Relative Density of"Corrimrssion'~eguiafion (EC) No 440/2008 of 30 May 2008 and 

Method 109 of the OECD Guidelines for Testing of Chemicals, 27 July 1995 . ......... _........ -.. -.... -..................... ,., ........... ,
 . . Vapour Pressure. 1 ~sing a vapour pressure balance, Method A4 

Vapour Pressure orCoiiimlssioii··Regulati~n (EC) No 440/2008 of 30 May 2008 and 

Method 104 of the OECD Guidelines for Testing of Chemicals, 23 March 2006. 

,....... _......................................................................... -_ ............................................ ", ........... -................ --................. -: 

Surface Tension. : : ,........................... "' ......................... -.............................,... -.................................. -........................................................................................... 
 : using a ring method based on ISO 304, Method AS ..... __ ....... ~ ........................... _~ .... _ ............. __ .... _.J 


Surface Tension of Commission Regulation (EC) No 440/2008 of 30 May 2008 and 

Method 115 of the OECD Guidelines for Testing of Chemicals, 27 July 1995. The test 

material is considered not to be a surface-active material. 

Water Solubility. The water solubility the test material has been determined using the 

flask method, Method A6 Water Solubility of Commission Regulation (EC) No 44012008 

of 30 May 2008 and Method 105 of the OECD Guidelines for Testing of Chemicals, 27 

....~.~~¥.~.~.~~: ..~~~ .~~~~~!~. ~~~ .~~C?~.~.!ry. !~~.!~I~~~ine .~~~;................................................. 


33 

PROJECT NUMBER: 014510193 PAGES 

Partition Coefficient The partition coefficient of the test material has been determined 

using the HPLC method, Method A8 Partition Coefficient of Commission Regulation (EC) 

No 440/2008 of 30 May 2008 and Method 117 of the OECD Guidelines for Testing of 

Chemicals, 13 April 2004. The results are shown in the followi~.Q.~?'!~.:..............................,
 r-------====""""""',..."..,.........--~-.................. ,
 ,.L..............................__ ...'" .e ••• •i.' 


. 

, 

, · . 
 · . 


, 

· , . : ................ _ .............................................................................. _ ................................ _ ......................................................"'.. : .........:::"'......:_..: .. ,. .... .1 


'··i=iiis-';·PCiiiit.··The test material has been determined to have a flash point of: : 1 ...... _ .... _ ...... _ ........ ~ 

using a closed cup equilibrium method, Method A9 of Commission Regulation (EC) 

No 440/2008 of 30 May 2008 

Explosive Properties. Based on the chemical structures of the components of the test 

material, the test material has been predicted to be····················] using Method A 14 

Explosive Properties of Commission Regulation (EC) No44oiioos'C;i30 May 2008. 

Auto-Ignition Temperature (Liquids and Gases). The test material has been ............ -..................., 


determined to have an auto-ignition temperature of: : using Method A 15 of ~ .. "'- .......... - ... - ..... .1 


Commission Regulation (EC) No 440/2008 of 30 May 2008. 

Oxidising Properties. Based on the chemical structures of the components of the test 

material, the test material has been predicted to be:···················1using Method A21 

! .. -_ ........ - ... - .......... -_.1 


Oxidising Properties (Liquids) of Commission Regulation (EC) No 44012008 of 30 May 

2008. 

Adsorption Coefficient The adsorption coefficient was determined using the HPLC 

screening method, Method C19 Adsorption Coefficient of Council Regulation (EC) No 

440/2008 of 30 May 2008 and Method 121 of the OECD Guidelines for Testing of 

Chemicals, 22 January 2001. The results are shown in the following table: 

...................-------.. -................................... _.............. _.. -........ -............................ _.............. "' .............. -_ .. "' ................ -...... -_ ........................... .. 


· 


· 


· 

.... " ... : .............................................................. -.................................................... -............ .-_ .... .., ............... 


······Fi;i·s~i~biiiiY:··;:h~.~~~~.~~~~!~~'--~~~ut~~~rmined to be soluble in the range 0(..........1 


f·························· ...................... _ ...~._:. using a procedure based on Method 116 of 
 ............................... _..................... .. 


the OECD Guidelines for Testing of Chemicals, 12 May 1981. 

34 
PROJECT NUMBER: 0145/0193 PAGE 9 

Mixture of PEMB and PE3MB: 


DETERMINATION OF PHYSICO-CHEMICAl 


PROPERTIES 


1. INTRODUCTION 

The physico~chemical properties of the test material have been determined. 

Methods employed complied with those specified in Commission Regulation (EC) 

No 440/2008 of 30 May 2008, Part A: Methods for the determination of physico-chemical 

properties and Part C: Methods for the determination of ecotoxicity and the current 

OECD Guidelines for Testing of Chemicals. 

Testing was conducted between 07 January 2009 and 17 April 2009. 

2. TEST MATERIAL 

2.1 Test Material Description, Identification and Storage Conditions 

Sponsor's identification Mixture of PEMB and PE3MB , 

, 

-....--...-.-.. ~--.---.-- ....-.., Description , , , ·, , 

· 

, , 

, Purity , , 

· 

·, 

,

, 

, . 

.

,

, 

, 

Batch number : . 1...... -_ .. "'._ .... - ................ - .......... -_ ........ .. 


Date received 15 December 2008 

Storage conditions room temperature in the dark 

The integrity of supplied data relating to the identity, purity and stability of the test 

material is the responsibility of the Sponsor. 

2.2 Chemical Structure 


The Sponsor provided the following chemical structure for the test material. 


Component A Component B 

35 

PROJECT NUMBER: 014510193 PAGE 10 

3. ARCHIVES 

Unless instructed otherwise by the Sponsor, all original data and the final report will be 

retained in the Harlan Laboratories Ltd, Shard low, UK archives for five years, after which 

instructions will be sought as to further retention or disposal. 

36 

PROJECT NUMBER: 0145/0193 PAGE 11 

4. MELTlNGIFREEZING TEMPERATURE 

4.1 Method 

The determination was carried out using B84633: Method for the Determination of 

Crystallizing Point, Method A1 MeltinglFreezing Temperature of Commission Regulation 

(EC) No 440/2008 of 30 May 2008 and in accordance with Method 102 of the OECD 

Guidelines for Testing of Chemicals, 27 July 1995. 

4.2 Procedure 

An aliquot of test material was placed into a test tube containing a thermometer. A wire 

loop with which the sample could be agitated was placed inside the tube to ensure 

sample homogeneity during cooling. The test tube was surrounded by a glass jacket and 

then placed into a large beaker containing a dry ice/acetone mixture. The temperature 

and observations of the test material were recorded approximately every 30 seconds. 

The dry ice/acetone bath was maintained until the temperature had reached 

apprOximately -20°C or until the test material solidified. 

The determination was carried out in duplicate. 

Graphs of temperature (K) versus time (seconds) were constructed to obtain the freezing 

temperature (Figure 4.1 and Figure 4.2). 

4.3 Calculation 

Measured temperatures were converted from °C to K using Equation 4.1. 

Equation 4.1 

T =t+ 273.15 

where: 

T = temperature (K) 

t = temperature (0C) 

37 

PROJECT NUMBER: 0145/0193 PAGE 12 

4.4 Results 

Cooling data for Determinations 1 and 2 are shown in the following tables: •...•....•• __ .••.........•. _..••...•....•....•....••....•..........•.............••...••.. __ ..-.........--... ~ ....----.---..--;


, 

38 

PROJECT NUMBER: 0145/0193 PAGE 13 

....... _-- '*' .. - ................ __ .. "' ......... _.... __ .......... _ ................ _ ...... __ ............................................... - •• -_ ...... - ........ - .......................... "' ........ .., .. - ................ - ................


. . 


39 

PROJECT NUMBER: 014510193 PAGE 14 

Graphs of temperature (K) versus time (seconds) for Determinations 1 and 2 are shown 

in Figure 4.1 and Figure 4.2 respectively. 

40 

PROJECT NUMBER: 014510193 PAGE 15 

~ ...-...-.-.. ~ ........-.-....---...--......... -.-.~--- ...-....-.-............--..--....-....-...... -~.-- ..-....-..-...... ~. 

, ' , ' . 

~. __ •• _ •••• ___ •• _ •••• _ •• _. __ •• __ ••• __ •• __ ••• _ •••• _ •• __ ••• __ ••• __ •• __ •• _______ •• _. __ ••• __ •• ___ •• __ ._ •• _ ••• __ •• _----_._. ___ 1 

41 

PROJECT NUMBER: 014510193 PAGE 16 

5. BOILING TEMPERATURE 

5.1 Method 

The determination was carried out by differential scanning calorimetry (DSC) using the 

procedure specified in ASTM E537-86. Method A2 Boiling Temperature of Commission 

Regulation (EC) No 44012008 of 30 May 2008 and in accordance with Method 103 of the 

OECD Guidelines for Testing of Chemicals, 27 July 1995. 

5.2 Procedure 

5.2.1 Calibration 

The temperature accuracy of the differential scanning calorimeter was assessed using 

an indium reference standard (purity*99.999%). The melting temperature was 

determined to be 156.4°C and within the defined tolerance (156.6 ± 0.5°C). The 

instrument was therefore considered acceptable for use. 

5.2.2 Sample 

Aliquots (see following table) of test material were placed in pierced aluminium crucibles. 

Table 5.1 

Determination Mass Taken (g) 

1 O.OOSO 

2 0.0053 

* Value quoted by supplier 

42 
PROJECT NUMBER: 0145/0193 	 PAGE 17 

5.2.3 Analysis 

The instrument parameters were as follows: 

Calorimeter 	 Mettler Toledo DSC822e 

ThermoHaake Cooler EK45/MT 

Temperature program 	 initial: 25.0oC 

rate: 20.00C/min 

final: 400.0oC 

Atmosphere 	 Determination 1 - air (static) 

Determination 2 - nitrogen 

5.3 Calculation 

Measured temperatures were converted from °C to K using Equation 5.1. 

Equation 5.1 

T", t+273.15 

where: 

T = temperature (K) 

t = temperature (OC) 

43 

PROJECT NUMBER: 0145/0193 PAGE 18 

5.4 Results 

Thermograms and thermographic data for Determinations 1 and 2 are shown in 

Figures 5.1 and Figure 5.2 and in the following tables respectively. ___ ••• w __ •• ___ • ______ •• _________ .~. __________ .~ __ • _________ •• _________________ •• ___ ••• ___________________ w_ . .. ________________ , 

,

, 

· 

· , 

,

, 

,

,

,

,

, 

, 

· 

, · , 

· · 

· · 

· 

· 

·,

,

, 

44 

PROJECT NUMBER: 0145/0193 PAGE 19 

I.~ ___ •• - ••••• ~ ••• ---~ •••••-.---_ ••••••• _ •• _ ••• ______ • ••• _ ••••••• ___ ._ ••• __ •• __ •••••••• ___ • __ ••• ___ • ______ •• _ •• ~ __ 6 __ ._._. ____ •• _1 . 

45 

PROJECT NUMBER: 0145/0193 PAGE 20 

5.5 Discussion 

, , ••.• _. __ _____ ~ ___ .••... _~ ____ ••••. _.•. ________ •••••.•. ____ •• _...•.... ____ •••. _•... _______ •..•• ______ ••.. _... ____ .t... _* •••••• J 

46 

PROJECT NUMBER: 014510193 PAGE 21 

6. RELATIVE DENSITY 

6.1 Method 

The determination was carried out using the pycnometer method, Method A3 Relative 

Density of Commission Regulation (EC) No 44012008 of 30 May 2008 and in accordance 

with Method 109 of the OECD Guidelines for Testing of Chemicals, 27 July 1995. 

6.2 Procedure 

6.2.1 Sample 

A glass pycnometer of 25 ml nominal capacity was cleaned and dried to a constant 

weight. 

Distilled water and test material were equilibrated at 20.0 ± 0.5°C. 

A calibration was carried out by determining the mass of distilled water required to fill the 

pycnometer. The pycnometer was then emptied and dried to constant weight. The mass 

of test material required to fill the pycnometer was then determined. 

6.3 Calculations 

The relative density was calculated using Equation 6.1 and Equation 6.2. 

Equation 6.1 

Density(kg/m3 ) =-...:d(,--c-_a-:...) 

(b-a) 

where: 

a = mass of pycnometer (g) 


b = mass of pycnometer filled with water (g) 


c = mass of pycnometer filled with test material (g) 


d = density of water at test temperature (kg/m1 


47 

PROJECT NUMBER: 014510183 PAGE 22 

Equation 6.2 

Relative density = P 

PH:P. 4·C 

where: 

P = density of test material (kg/m3

) 

density of water at 4°C (999.97 kg/m3

) 

6.4 Results 

I-~·-----~-·~~-~-------···-----------~------------------------------.--------------------.-.------------ .. -----------.-~-.------:

· .
 · · .. · .
 · .
 · .
 · .
 · . 


· · 

· 

· 

· · · 

48 

PROJECT NUMBER: 014510193 PAGE 23 

7. VAPOUR PRESSURE 

7.1 Method 

The vapour pressure was detennined using a vapour pressure balance with 

measurements being made at several temperatures and linear regression analysis used 

to calculate the vapour pressure at 25°C. Testing was conducted using Method A4 

Vapour Pressure of Commission Regulation (EC) No 440/2008 of 30 May 3008 and in 

accordance with Method 104 specified in the OECD Guidelines for Testing of Chemicals, 

23 March 2006. 

7.2 Procedure 

The vapour pressure was detennined using a vapour pressure balance. The 

temperature of the sample was controlled electronically. The mass and temperature 

readings were recorded automatically into a computer file. 

A diagram of the cross~section of the vapour pressure balance is represented in Figure 

7.1. After evacuating the system, opening the shutter above the sample oven causes the 

escaping vapour jet to be directed at the scale pan. The difference in mass readings 

with the orifice covered and uncovered is proportional to the vapour pressure at the given 

oven temperature. 

A sequence of runs was started after a sample of test material had been under vacuum 

for approximately 44 '!4 hours. Temperature and pressure readings were taken between 

160 and 170°C with a one hour dwell at 1600C between runs. 

49 

PROJECT NUMBER: 014510193 PAGE 24 

Figure 7.1 Schematic Diagram ofthe Apparatus Used 

2 

4 5 

1 Microbalance 

2 Oven 

3 Glass viewing panel 

4 Balance pan 

.5 Shutter with orifice 

6 Test sample 

50 

PROJECT NUMBER: 014510193 PAGE 25 

7.3 Calculation 

The vapour pressure is related to the observed mass difference by Equation 7.1. 

Equation 7.1 

Vp = om.g 

A 

where: 

Vp = vapour pressure (Pa) 

om = mass difference (kg) 

g = acceleration due to gravity (9.813 m S-2) 

A = area of the orifice (7.06858x10-6m2) 

Vapour pressure is related to temperature by Equation 7.2. 

Equation 7.2 

slope . Log1o [Vp (Pa)] = +Intercept temperature (K) 

A plot of LOgl0 Vp (Pa) versus reciprocal temperature (11T(K» therefore gives a straight 

line graph. 

The vapour pressure of the sample was measured over a range of temperatures to 

enable extrapolation to 298.15 K. 

51 

PROJECT NUMBER: 014510193 PAGE2S 

7.4 Results 

52 

PROJECT NUMBER: 014510193 PAGE 27 

~---~ ...........---.--..-.......----.--.--...-.-.... ~. ---_._-- ............_---- .......... _---- .......... _---._ .. _._._----.
 . . 


53 

PROJECT NUMBER: 014510183 PAGE 28 

;--.-.........-...---..-..-.--.....-.. --.~--- ......--.. _ ................. _- .........................-----_..----..----.. --.--.----.~

. 

: . ...---------....-----------...-------..----------....-_.----------_.------_._._-....---------------_.------------------------._---, 

54 

____ 

PROJECT NUMBER: 014510193 PAGE 29 

...--.-------.......-..-..-.----------....... ------~-- ---.-_._ ... __ .... _-_ .......••••. _-_ ......••••-_ ..._---..-.-..-...
------~

. 

,

M __ •••• _. ______ • ___ ••• ___ ._ •••••••••• ___ •• ____________ ._._._.~ _______ ••• ____ ._ •• ___ •••• ________ • ___ •••• __ ••• __ .* ._. ___ . __ , 

55 

PROJECT NUMBER: 014510193 PAGE 30 

,-_. __ .__ ......._---_ ....................-..-.............-........... _---......--------...... _- .........-----.....-..--.----.. -.~

. .
 : . 

' ••• __ •• -----••••••••--•• __ ••• _-------_ •••_-_ •• _-----._ ••• a ______ ••••••• _ ••• __ ._. __ ._._. _____ • __ • __ • ___ • ____ ._ •• _ •• __ .~ ___________ • 

56 

PROJECT NUMBER: 014510193 PAGE 31 

~- ..-..--....-.-------.--....... -.~ .. ~-.-----.------.-- ...-.... ------.-----.-~-- ..-------.--.-..------------......----.
 . : 

57 

PROJECT NUMBER: 0145/0193 PAGE 32 

~.- ... ~.-~ ....--.---------.--.-....----.-..-.--.-.--.-..._----_._._------_ .. -------------------------------- -----.--------------~

.. 

58 

PROJECT NUMBER: 014510193 PAGE 33 

59 

PROJECT NUMBER: 014510193 PAGE 34 

60 

PROJECT NUMBER: 014510193 PAGE 35 

,... __ ....•.......... _--_ .. ---------_.--------_ .. _---- -------.~ .. -------------.---------------------------~.-~.-------------:

. . 


. 

---------------------------.----------.-~-.----------- ------------------------------~----.-.--.----.-------------.--- ... -~ 

61 
PROJECT NUMBER: 0145/0193 PAGE 36 

62 

PROJECT NUMBER: 014510193 PAGE 37 

63 

PROJECT NUMBER: 014510193 PAGE 38 


__________ ._. ____ ••. _______ • _________________ ._. ______ • ______ ~_k __________________ w _________ ._~ ___ • __ •••• ____________________ w ___ ~ ___ 1 


. .
 : . . 

64 

PROJECT NUMBER: 014510193 PAGE 39 

.. ---~ .....-.-.-.••...••...••.. ~ ...-....-....----.......---...-... -.~ ... --~ .......-..-..-........-........--............ ~ 

, , 

..___ .. ____ ... _ ..__ ... ~ __ ..... _~_._.* ____ .__ .____ .________ .__ ._. __ ~ ____ ... ____________ .___ .~ _______ ._______ .____ .____ w __ ._~ 

65 

PROJECT NUMBER: 014510193 PAGE 40 

A summary of the results is shown in the following table: :-........... __ ......... _-_ ... _....... _...... _.. __ ...---_ .. _--._._--.._.---.._---._.---_._---...-.._._--_._.---.---.--.-..-.. 
--.~ 

,

, 

~--.-.---- ...--.--...----..-.. ----.-----.-.-.--------.-----_._----------..--.. _-------------..-------..._----------_._---------.: 

66 

PROJECT NUMBER: 014510193 	 PAGE 41 

8. SURFACE TENSION 

8.1 Method 

The determination was carried out using a White Electrical Institute interfacial tension 

balance and a procedure based on the ISO 304 ring method. With the exception of the 

following deviation, the experimental procedure used complied with that specified in 

Method AS Surface Tension of Commission Regulation (EC) No 440/2008 of 30 May 

2008 and in accordance with Method 115 of the OECD Guidelines for the Testing of 

Chemicals, 27 July 1995. 

1) 	 The surface tension result was not corrected using the Harkins-Jordan correction 

table as the correction is not applicable to the apparatus used. Once calibrated, 

the balance and ring assembly used in this test give a direct reading for surface 

tension that is within the required accuracy (± 0.5 mN/m); this is as a result of the 

reduced ring dimensions. 

This deviation has been considered not to have affected the integrity of the study. 

8.2 Procedure 

8.2.1 Cleaning of apparatus 

The platinum ring and all glassware were cleaned with acetone, tap water, chromic acid, 

tap water and finally glass double-distilled water. The platinum ring was dried over a 

methanol flame and cleaned between each surface tension measurement. 

8.2.2 	 Preparation of sample solution 

Table 8.1 

Sample Preparation Time (mins) 

An aliquot (0.1007 g) of test material was diluted to 100 ml with glass double-distilled oto 10 water. 


The sample solution was shaken at approximately 30°C for 96 hours at approximately 
 15 to 5775 200 rpm. 


The sample solution was equilibrated at 20°C for 24 hours. 
 5775 to 7215 

The sample solution was centrifuged at 13500 rpm for 20 minutes. 7220 to 7240 

The sample was sampled using a pipette. 7240 to 7245 

The supernatant was diluted to 90% of its former concentration with glass double­ 7245 to 7250 distilled water. 


The sample solution was transferred to the measuring vessel. 
 7255 

67 

PROJECT NUMBER: 0145/0193 PAGE 42 

8.2.3 Surface tension readings 

The surface tension of the sample solution was measured at intervals until a constant 

reading was obtained. The elapsed time from transferral to the measuring vessel to 

obtaining each surface tension reading was recorded. 

A calibration reading of glass double-distilled water was taken with each sample reading. 

Readings were taken of the minimum force required to detach the ring from the surface 

of the liquid. Temperature readings were taken directly after each sample and calibration 

reading. 

8.2.4 Analysis of sample solution 

The concentration of test material in the sample solution was determined by high 

performance liquid chromatography (HPLC). 

Samples 

Duplicate aliquots (A and B) of the sample solution were diluted by a factor of 2 

using acetonitrile. 

Standards 

Duplicate standard solutions of test material were prepared in acetonitrile:water (1:1 v/v) 

at two nominal concentrations of 1 mg/l and 100 mg/l. 

Matrix blank 

Acetonitrile:water (1:1 v/v). 

68 

PROJECT NUMBER: 014510193 PAGE 43 

Analysis 

The standard and sample solutions were analysed by HPLC using the following 

conditions: 

HPLC System Agilent Technologies 1100, incorporating 

autosampler and workstation 

Column Prodigy 00S3 100A 51J (250 x 4.6 mm id) 

Column temperature 40°C 

Mobile phase acetonitrile:water containing 0.1 % phosphoric 

acid (75:25 vlv) 

Flow-rate 1.0 mllmin 

UV detector wavelength 200nm 

Injection volume 100 IJI 

Retention time Component A: - 4.7 mins 

Component B: - 8.7 mins 

69 

PROJECT NUMBER: 014510193 PAGE 44 

Typical Chromatography 


Standard 108 mgn 


VW01 A. WavelenlJlh=2OO nm (PFT853101+o1401,D) 

mAU 

600 


500 


400 


300 


200 


100 


o ~ - \, 

Standard 1.01 mgn 

VW01 A. Wawlengt/l=aog nm (PFTIl531013-1301.D) 

mAU 

7.5 


5 
 ! I ~ 2.5 "!

... 

0 ~L lJv

-2.5 


-6 


-7.5 


-10 0 2 4 10 12 14 

70 

PROJECT NUMBER: 014510193 PAGE 45 

~ ___ . ___ R_~ __ ~.~_. _______________ ... _____ ~. _____ R_._~ _____ ... ______ .. _____ ._ .. ___________ .. ______________ ~~ .. ____ . ______ . _____ .

71 

PROJECT NUMBER: 014510193 PAGE 46 

8.3 Calculation 

8.3.1 Sample solution concentration 

The mean peak area of both standards for each concentration was corrected to the 

nominal concentration of 1 mg/l and 100 mgll and the mean values taken. 

The sample solution concentration (gil) was calculated using Equation 8.1. 

Equation 8.1 

P 1 P C ~~xC xDx---x--

IIpI P ltd 1000 100 

$til 

where: 

Cspl = sample concentration (g/l) 

Pspl = mean peak area of sample solution 

Pstd = mean peak area of standard solution I corrected to nominal standard 

concentration 

Cstd = nominal standard concentration (1 mg/l or 100 mg/l) 

D = sample dilution factor (2) 

P = purity of component (A = 14.3 %, B =83.0 %) 

8.3.2 Calibration factor (;) 

The calibration factor (tJ). by which the measured surface tension of the sample solution 

is multiplied, was determined using Equation 8.2. 

Equation 8.2 

where: 


¢ = calibration factor 


VL = literature value for surface tension of water at test temperature (mN/m) 


VM = measured value for surface tension of water at test temperature (mN/m) 


72 

PROJECT NUMBER: 0145/0193 PAGE 47 

8.4 Results 

8.4.1 Sample solution concentration 

The mean peak areas relating to the standard and sample solutions are shown in the 

following table: 

i--········· __ ··-_·-_·································..............--.----.-------...•......-..----.... 


.....-_ ....__ ........... _--_ ..--_ ........------_ .......... __ .........•••...... _--_ ...._--_ ..._---_ .. _-­

73 

PROJECT NUMBER: 014510193 PAGE 48 

8.4.2 Calibration factor (f6) 

The readings, temperatures and the corresponding calibration factors for glass doubledistilled water are shown in the following table: I--·······---·--~····-·-·-·-······--····-·---··--····· ...-.---..........---.....---------.....---.---.......-----------------._ ..-:
 . .
 . : 

· 

· · · 

74 

PROJECT NUMBER: 0145/0193 _________________________________________________________________________________________________________________________ PAGE 49 .w __

75 

PROJECT NUMBER: 014510193 PAGE 50 

9. WATER SOLUBILITY 

9.1 Method 

The determination was carried out using the flask method, Method A6 Water Solubility of 

Commission Regulation (EC) No 44012008 of 30 May 2008 and in accordance with 

Method 105 of the OECD Guidelines for Testing of Chemicals, 27 July 1995. 

9.2 Procedure 

9.2.1 Preliminary test 

An aliquot (1.01 g) of test material was diluted to 1000 ml with glass double-distilled 

water. After shaking at 30°C for 6~ hours and standing at 20°C for 19 hours, the 

solution was centrifuged at 13500 rpm for 20 minutes and analysed. 

9.2.2 Definitive test 

Based on the preliminary result. mixtures (see following table) of test material and glass 

double-distilled water were added to six separate flasks. 

Table 9.1 

Sample Number Mass of Test Material (g) Volume of Water (ml) 

1 1.00 1000 

2 1.00 1000 

3 1.00 1000 

4 1.01 1000 

5 1.01 1000 

6 1.01 1000 

After addition of glass double-distilled water to the flasks, they were shaken at 

approximately 30°C and, after standing at 20°C for a period of 24 hours, the contents of 

the 'flasks were centrifuged at 13500 rpm for 20 minutes. 

The pH of each solution was measured. 

9.2.3 Analysis of sample solution 

The concentration of test material in the sample solutions was determined by high 

performance liquid chromatography (HPLC). 

76 

PROJECT NUMBER: 014510193 PAGE 51 

Samples 

Duplicate aliquots (A and B) of the sample solution were diluted by a factor of 2 

using acetonitrile. 

Standards 

Duplicate standard solutions of test material were prepared in acetonitrile:water (1:1 v/v) 

at two nominal concentrations of 5 mgll and 50 mgt!. 

Matrix blank 

Acetonitrile:water (1:1 v/v). 

Analysis 

The standard and sample solutions were analysed by HPLC using the following 

conditions: 

HPLC System Agilent Technologies 1100. incorporating 

autosampler and workstation 

Column Prodigy SJ,J 00S3 (250 x 4.6 mm id) 

Column temperature 40°C 

Mobile phase acetonitrile:water containing 0.1 % phosphoric 

acid (75:25 vtv) 

Flow-rate 1.0 mllmin 

UV detector wavelength 200nm 

Injection volume 100 J,JI 

Retention time Component A: -4.6mins 

Component B: -8.5 mins 

77 

--

PROJECT NUMBER: 014510193 PAGE 52 

Typical Chromatography 


Standard 65.5 mg/l 


VWD1 A. Wal/lllength=200 nm (pm42IOO7..Q701.D) 

N mAU 

~o 

300 


250 


20D 


150 


lDO 


50 


0 


:2 4 ;, -r .~ .'., 14 -, 

Standard 6.55 mg/I 

~ 

\. 

mAU 

4D 

30 

2D 

10 

0 

VWD1 A. WavelenI!Jl.h"200 nm (pm421OO9..Q901.D) 

~ 

rU~ 

"! 

? ~ 

fJ 

;, i 

\.. 

10 1~ 

78 


14 
I 

PROJECT NUMBER: 014510193 PAGE 53 

-.-~ ...--.....---.. ~-- .. ~~-----------.-- .. ---.---.--------.~.---------- ... --------------~---------------------~.----.~----------~-~, 

Matrix Blank 

VWD1 A, WIMIIeng1h-200 nm (PFT7421011-1101.D) 

mAU 


10 


7.5 


5 


2.5 


0 
 - ~~ -2.5 


-5 


2 4 R it <? ." 14 

79 


-7.5 
PROJECT NUMBER: 014510193 PAGE 54 

9.3 Calculation 

The mean peak area of both standards for each concentration was corrected to the 

nominal concentrations of 5 mg/l and 50 mgll and the mean values taken. 

The sample solution concentration (gil) was calculated using Equation 9.1. 

Equation 9.1 

PaPI C D 1 P C =-x x x--xspl PSId sid 1000 1 00 

where: 

Cspl = sample concentration (gil) 

Pspl = mean peak area of sample solution 

Pstd = mean peak area of standard solution, corrected to nominal standard 

concentration 

Cstd = nominal standard concentration (5 mgll or 50 mg/l) 

0 = sample dilution factor (2) 

P = purity of component (A =14.3 %, B =83.0 %) 

80 

PROJECT NUMBER: 014510193 PAGE 55 

9.4 Results 

,.... :~:1......~~!!~.i.t:J~!Y. .~~t ....................................................................................--: 
 , . 

81 

PROJECT NUMBER: 014510193 PAGE 56 

--_ .....-_ ......---_ ..--_ ..-......_---_._---_ .. -------.---.---.-.~.- ..... -----------~ .~.- .....--------------.-..---.. --~----.--------- : 

* Standard and sample mean peak areas have been corrected for the mean peak area of the matrix blank. 

t Data not used included in the mean concentration 

82 
PROJECT NUMBER: 014510193 PAGE 57 

The mean concentrations (g/l) of Components A and B in the 96, 120 and 144 hour 

sample solutions are shown in the following table: 

...................................,......................riIble.9,6.. ....................... " ............................... . 


....'9:5"""" Vaifiicition .....··.......................................................................................... 

.--.-...-.-.--.--.-.. ---.-.-.-.-.-.~--.--.----. ..-...••...••.. __ .... -_ .•.....••. __ ...... _-_ ......_---_ ..._-------_._------_._ .. _.

· · · · ,

· 

·,

· 

· 


· 


· · · · 


· 


· , 

· 

· 

83 

PROJECT NUMBER: 014510193 PAGE 58 

.~~. __ • __ • __ ~ •• __ •• _M •••• __ • ___ •• _____ • ________ W •• ____ ----•• ----•• ---.---~---------.--.---.---.-~-.---.-~--.--.--.------- •• -----~

. 

~ _____ ~ _______ ~ _______ ~. _______ • _______ • _________ • ____ _ w _______ • ______ M ___________________ • _______ • _________ ----•• -----.--------~ 

. 

84 

PROJECT NUMBER: 014510193 PAGE 59 

10. PARTITION COEFFICIENT 

10.1 Method 

The determination was carried out using the HPLC Method, Method A8 Partition 

Coefficient of Commission Regulation (EC) No 440/2008 of 30 May 2008 and in 

accordance with Method 117 of the OECD Guidelines for Testing of Chemicals, 13 April 

2004. 

10.2 Procedure 

10.2.1 Preliminary estimate 

A preliminary assessment of the partition coefficient was made based on the 

approximate solubilities of the test material in n-octanol and water. This was carried out 

by visual assessment. 

10.2.2 Definitive test 

Preparation ofsample solution 

Test material (0.1093 g) was diluted to 100 ml with acetonitrile. 

Preparation ofdead time solution 

The dead time was determined by measuring the retention time of thiourea (purity* 

>99%,21 mg/l solution in mobile phase). 

Preparation ofreference standard solution 

A solution of reference standards (see following table) was prepared in acetonitrile. 

Table 10.1 

Standard Purity (%)* Concentration (mgll) 

2-Butanone 99+ 5.13 x 103 

Benzene 99.98 5.02 x 103 

Toluene 99 3.33 x 103 

Naphthalene ;;:99 424 

Phenanthrene ;;:97 27.0 

Triphenylamine 98 219 

.. Value quoted by supplier 

85 
PROJECT NUMBER: 014810193 PAGE 60 

Determination ofretention time 

The sample, dead time and reference standard solutions were injected in duplicate using 

the following HPLC parameters: 

HPLC System 

Column 

Column temperature 

Mobile phase 

pH of mobile phase 

Flow-rate 

Injection volume 

UV detector wavelength 

Construction ofcalibration curve 

Agilent Technologies 1100, incorporating 


workstation 


Prodigy ODS 3 100A 5jJm (250 x 4.6 mm id) 


400(; 


acetonitrile:water (75:25 v/v) 


6.B 

1.0 mllmin 

20 JJI 

dead time and reference standards: 240 nm 

sample: 220 nm 

A calibration curve was constructed from the retention time data of the dead time and 

reference standard solutions (Figure 10.1). The capacity factors (k) for the reference 

standards were calculated using Equation 10.2. Log1o Pow values of the reference 

standards are those quoted in Method 117 of the OECD Guidelines for Testing of 

Chemicals, 13 April 2004. 

Partition coefficient ofsample 

The capacity factor was calculated using Equation 10.2 and the 10910 PC1W value 

determined using Equation 10.3 with reference to the calibration curve (Figure 10.1). 

86 

PROJECT NUMBER: 014510193 PAGE 61 

Typical Chromatography 


Dead Time 


\/wDi A, WaveJengtlr-240 nm (PFT188\O01"()102.D) 

mAU 

~ 1000 

800 

600 

400 

200 

I 

0~~~~~==~~~=r~==~~~~~~==~~~~~1 4 sJ 

mAU 

GOD 

500 

400 

31)0 

200 

100 

0 

Reference Standards 

\/w01 A, wavetangth,.240 nm (PFT18S\004..()401.D) ... i - '" 

I 


.. 


:d 

! 


~ 

i 

, 

i1i 

'" I ~ 

\. 

10 1'5 20 

87 

PROJECT NUMBER: 014510193 PAGE 62 

88 

PROJECT NUMBER: 014510193 PAGE 63 

10.3 Calculation 

10.3.1 Capacity factor 


The capacity factor was determined using Equation 10.2. 


Equation 10.2 

where: 

= capacity factor 


= retention time (min) 


= dead time (min) 


10.3.2 Partition Coefficient 

The partition coefficient was calculated using Equation 10.3. 

Equation 10.3 

P Log10 k-A l Og'0 ow = B 

where: 

Pow = partition coefficient 


k = capacity factor 


A = intercept of the calibration curve (Figure 10.1) 


B = slope of the calibration curve (Figure 10.1) 


89 

PROJECT NUMBER: 014510193 PAGE 64 

10.4 Results 

10.4.1 Preliminary estimate .----.--..... -----~ ....----...---.-.....-.-......-.----_. __ ........ -_ ..----_ .......-----_ ...........-.....................: 


. 

. 

10.4.2 Definitive test 

Calibration 

The retention times of the dead-time and the retention times, capacity factors (k) and 

10910 Pow values for the reference standards are shown in the following tables: 

Table 10.2 

Dead Time 

Retention Time (mins) 

Injection 1 Injection 2 

Mean Retention Time 

(mins) 

Thiourea 2.323 2.324 2.324 

Table 10.3 

Standard 

Retention Time (mins) Mean 

Retention 

Time (mins) 

Capacity 

Factor 

(k) 

LOQICl k Log10 Pow 

Injection 1 Injection 2 

2-Butanone 3.051 3.052 3.052 0.313 -0.504 0.3 

Benzene 4.661 4.663 4.662 1.006 2.80 x 10-3 2.1 

Toluene 5.657 5.660 5.659 1.435 0.157 2.7 

Naphthalene 6.381 6.385 6.383 1.747 0.242 3.6 

Phenanthrene 9.394 9.400 9.397 3.044 0.483 4.5 

Triphenylamine 16.854 16.854 16.854 6.254 0.796 5.7 

90 

PROJECT NUMBER: 014510193 PAGE 65 

Figure 10.1 Calibration Curve 

HPLC Pow Call1lmlan CW'YI _ k ~ UIII _ 

o 2.1. v.a i 2.!IlO _ lUG 11.157 

... OAII 

1.!IlO 0:''11 A-41III .-a,:m ,.­

0.81 

0.67. 

DZI 

~i~~--~--~~--~--~----~--~-- -Q.24 0.78 1.76 2.78 3.•78 4.71 5.78 

1og10Paw 

Partition coefficient ofsample 

The retention times, capacity factors and log10 Pow values determined for the sample are 

shown in the following tables: 

91 

PROJECT NUMBER: 014510193 PAGE 66 

10.5 Discussion 

Substances having a log10 Pow greater than 3 are regarded as having the potential to 

bioaccumulate in the environment. 

.-------~~--------~.-------.---.------------ .. -----------------------*.-----.-~------.-------~---------------------------_., 

· 

· 

· · 

·· · :------------------.--------*------~---------.-------.--------- ..._---... _-----------------------------------.-------------~ 

10.6 Conclusion 

The partition coefficient of the test material has been determined and is shown in the 

following table: 

..-------.-.--.----.---...--.--... -~------------ ... ---.--.---~--- ...--.-.--.-...-----....---....------...--------_.. _----------_ ...: 

. 

92 

PROJECT NUMBER: 014510193 PAGE 67 

11. FLASH POINT 

11.1 Method 

The flash point was determined using a closed cup equilibrium method, Method A9 Flash 

Point of Commission Regulation (EC) No 44012008 of 30 May 2008. 

11.2 Procedure 

An aliquot (2 ml for temperatures below 70c C and 4ml for temperatures over 70°C) of the 

test material was injected into the sample cup of the Setaflash 30000-0 tester. The 

sample was heated and allowed to equilibrate at the set temperature. The test flame 

was introduced into the sample cup for approximately 2 seconds by sliding the cup 

shutter open. Observations were made for ignition of the vapour. If no ignition occurred, 

the temperature was increased and the test flame re-introduced. This was repeated until 

the lowest reproducible temperature at which a flash occurred, using a fresh sample, was 

determined, or until the sample boiled. 

11.3 Calculation 

The flash point temperature was corrected to a pressure of 101.325 kPa using Equation 

11.1. 

Equation 11.1 

FP ==C +0.23(101.325-8) 

Where: 

FP = corrected flash point (0C) 

C = observed flash point (0C) 

B = ambient atmospheric pressure (kPa) 

93 

PROJECT NUMBER: 0145/0193 PAGE 68 

11.4 Results 


The results are shown in the following table: 
 ................ _ ......................... ____ 00_ .............. ____ ............................................ _ .. - ............. _ .............. _ ..................... _ .......... - ....................... "' .......... - ............................. "': 


94 

PROJECT NUMBER: 0145/0193 PAGE 69 

95 

PROJECT NUMBER: 014510193 PAGE 70 

11.5 Conclusion 

.....-.--.--...---­ The flash point of the test material has been determined to b~ .

'''' .... -~ ...... -...... --.......... --.. 


11.6 Reference 

Based on. Anon (1983) Paints, Varnishes. Petroleum and Related Products ­

Determination of Flash Point - Rapid Equilibrium Method. International Standard ISO 

3679-1983 (EJ. Second Edition - 1983-12-01, P1-7 and Anon (1989) Flash Point of 

Liquids by Setaflash Closed-Cup Apparatus. ASTM 0-3278-89, P1-7. 

96 

PROJECT NUMBER: 0145/0193 PAGE 71 

12. EXPLOSIVE PROPERTIES 

12.1 Method 

The explosive properties were predicted using Method A 14 Explosive Properties of 

Commission Regulation (EC) No 44012008 of 30 May 2008. 

12.2 Procedure 

The structure of the test material was assessed for chemical groups that imply explosive 

properties. Examples of such groups are C-C unsaturated, C-metal. N-metal, contiguous 

oxygen atoms, contiguous nitrogen atoms, N-halogens. O-halogens. N-O. Examples of 

these groups are given below. 

StnJctur.aIFeatu~s Examples 

C-C unsaturated Acetylenes, acetylides, 1,2 dienes 

C-metal, N-metal Grignard reagents, organo-lithium compounds 

Contiguous oxygen atoms Peroxides, ozonides 

Contiguous nitrogen atoms Azides, aliphatic azo compounds, diazonium 

salts, hydrazines, sulphonyl hydrazides 

N-halogens Chloramines, fluoroamines 

O-halogens Chlorates, perchlorates, iodosyl compounds 

N-O Hydroxylamines, nitrates, nitro compounds, 

N-oxides, 1 ,2-oxazoles 

Full structural details are given in Bretherick's Handbook of Reactive Chemical Hazards, 

4th Edition, Butterworths, London. 1990. 

12.3 Results 


The test material has the following chemical structures: 


97 

PROJECT NUMBER: 014510193 PAGE 72 

12.4 Conclusion 

Based on the chemical structure of both components of the test material the result for the 

explosive properties has been predicted;················: . . .... --_ .................... ' 


98 

PROJECT NUMBER: 014510193 PAGE 73 

13. AUTO·IGNrnON TEMPERATURE (LIQUIDS AND GASES) 

13.1 Method 

The auto-ignition temperature was determined by heating aliquots of the test material in 

a flask and observing for any ignition. Testing was conducted using Method A15 AutoIgnition Temperature (Liquids and Gases) of Commission Regulation (EC) No 44012008 

of 30 May 2008. 

13.2 Procedure 

The test flask was heated in a flask heater. Aliquots of test material (tested as received) 

were injected into the flask using a syringe and the flask was observed for signs of 

ignition over a 300 second period. This procedure was repeated, varying the sample 

size, as necessary, until the lowest temperature at which ignition, if any, occurred within 

300 seconds of insertion, was determined. 

13.3 Results 

........ .T.~~. ~~.~~I~ .~~ .~h~~I). iI)J.~~ f~~I~W!~.9. ~~Q!~.:............................................................... . 


99 

PROJECT NUMBER: 014510193 PAGE 74 

..

~ ______ .. _.. ~ .. __ .____ .. ________ ...__ .... ~_. ____ ...__ .______ .________ .__ .__ ._._*._.... _.~~ .. _. _________ ..._.._. ______________ ~ .. J .

100 

PROJECT NUMBER: 014510193 PAGE 75 

13.5 Reference 

Anon (1987). Electrical Apparatus for Explosive Gas Atmospheres. Part 4: Method of 

Test for Ignition Temperature. IEC Publications 79-4, P1-19. 

101 

PROJECT NUMBER: 014510193 PAGE 76 

14. OXIDISING PROPERTIES (LIQUIDS) 

14.1 Method 

The oxidising properties were predicted using Method A21 Oxidising Properties (Liquids) 

of Commission Regulation (EC) No 44012008 of 30 May 2008. 

14.2 Procedure 

The structure of the test material was assessed for chemical groups that would imply 

oxidising properties. Examples of such groups are nitrates, metal oxides, hypofluorites, 

difluoroaminopolynitroaryls, perchlorates, bromates and iodites. 

Full structural details are given in Bretherick's Handbook of Reactive Chemical Hazards. 

4th Edition, Butterworths, London, 1990. 

14.3 Results 

The test material has the following chemical structures: 

.-..... --------~-----.- ...--....-.---...--.-----.--.. -...... -----.----------~--.-- ..--------------.-.-------_._._, ,

,

· 

· 

· · 

14.4 Conclusion 

Based on the chemical structures of each component, the result for the oxidising 

properties has been predicted f·····..····..1 .

.-.. ----........ --..... 
~ 

102 
PROJECT NUMBER: 014510193 PAGE 77 

15. ADSORPTION COEFFICIENT 

15.1 Method 

The determination was carried out using the HPLC screening method. Method C19 

Adsorption Coefficient of Commission Regulation (EC) No 440/2008 of 30 May 2008 and 

in accordance with Method 121 of the DECO Guidelines for Testing of Chemicals, 22 

January 2001. 

15.2 Procedure 

Preparation of sample solution 

Test material (0.1014 g) was diluted to 100 ml with acetonitrile. 

Preparation of dead time solution 

The dead time was determined by measuring the retention time of formam ide (purity* 

:i!99.5%, 1.43 x 103 mgll solution in mobile phase). 

Preparation of reference standard solutions 

Solutions of reference standards (see following table) were prepared in acetonitrile. 

Table 15.1 

Standard Purity (%)* Concentration (mgll) 

Acetanilide 99+ 104 

Phenol 99+ 114 

Atrazine 97.4 100 

Isoproturon 99.8 100 

TriadimenoJ 98.4 110 

Linuron 99.5 109 

Naphthalene ~9.0 139 

Endosulfan-diol 99,8 106 

Fenthion 97.8 101 

a-Endosulfan 99.6 103 

Phenanthrene 0::97.0 105 

Diclofop-methyl 99.5 104 

DDT 98 120 

.. value quoted by supplier 

103 
PROJECT NUMBER: 014510193 PAGE 78 

Determination of retention time 

The sample, dead time and reference standard solutions were injected in duplicate using 

the following HPLC parameters: 

HPLC System Agilent Technologies 1100, incorporating 

autosampler and workstation 

Column Zorbax 58 CN 5~m (250 x 4.6 mm id) 

Column temperature 40°C 

Mobile phase acetonitrile:water (55:45 v/v) 

Typical pH of mobile phase 7.2 

Flow-rate 1.0 ml/min 

Injection volume 20 ~I 

UV detector wavelength dead time and reference standards: 210 nm 

sample: 220 nm 

Construction of calibration curve 

A calibration curve was constructed from the retention time data of the dead-time and 

reference standard solutions (Figure 15.1). The capacity factors (k) for the reference 

standards were calculated using Equation 15.1. 

Adsorption coefficient of sample 

The capacity factor was calculated using Equation 15.1 and the log1oKoc value 

detennined using Equation 15.2 with reference to the calibration curve (Figure 15.1). 

104 

PROJECT NUMBER: 0145/0193 PAGE 79 

Typical Chromatography 

Dead Time 

VWD1 A, WIMllength=210 nm (PFT235\OO1·0101.D) 

mAU 

1000 

600 

400 

Naphthalene Reference Standard 

VWD1 A. Wave!eng\h"210 nm (PFT235\010-1001.D) 

mAU 0

2000 

1500 

1000 "' 

. 

; 

500 

0 

) 

[) 2:5 ;; 7'!i 1'~ 1~ ~ ,'.. 17.& -~ 

105 
PROJECT NUMBER: 014510193 PAGE 80 

Typical Chromatography 

. .
 :_-_. __ ......... __ .......-_ ......... __ ...........••....-.--.........-..... ~.------- ...-........ -.-.~- ...--.--_... _--..........' 


106 

8 

PROJECT NUMBER: 0145/0193 	 PAGE 81 

15.3 Calculation 

15.3.1 Capacity factor 

The capacity factor was determined using Equation 15.1. 

Equation 15.1 

where: 

= capacity factor 


= retention time (min) 


= dead time (min) 


15.3.2 Adsorption Coefficient 

The adsorption coefficient was calculated using Equation 15.2. 

Equation 15.2 

v _ LogfO k - A LOg10 ''OC - ---:;:"":'::""8-­

where: 

Koc 	 = adsorption coefficient 

k 	 = capacity factor 

A 	 = intercept of the calibration curve (Figure 15.1) 

= slope of the calibration curve (Figure 15.1) 

107 

PROJECT NUMBER: 014510193 PAGE 82 

15.4 Results 

Calibration 

The retention times of formam ide and the retention times, capacity factors (k) and 

10910 Koc values for the reference standards are shown in the two following tables: 

Table 15.2 

Dead Time Retention Time (mins) Mean Retention Time 

(mins) Injection 1 Injection 2 

Fonnamide 2.589 2.588 2.589 

Table 15.3 

Standard 

Retention Time (mins) Mean 

Retention 

Time (mins) 

Capacity 

Factor (k) Log,o k Log10 Koc: 

Injection 1 Injection 2 

Acetanilide 3.362 3.362 3.362 0.299 -0.525 1.25 

I Phenol 3.462 3.461 3.462 0.337 -0.472 1.32 

Atrazine 4.163 4.162 4.163 0.608 . -0.216 1.81 

Isoproturon 4.250 4.249 4.250 I 0.642, -0.193 ' 1.86 

Tliadimenol 4.678 4.678 4.678 0.807 ' -9.30 x10.2 2.40 

Llnuron 5.053 5.056 5.065 0.953 -2.11)( 10-2 2.59 

Naphthalene 5.440 5.440 5.440 1.10 ' 4.20 X 10.2 2.75 

Endosulfan-dlol 5.113 5.113 5.113 0.975 -1.09 x 10.2 3.02 

Fenthion 6.821 6.818 6.820 1.64 0.213 3.31 

a-Endosulfan 8.693 8.695 8.694 2.36 0.373 4.09 

Phenanthrene 7.056 7.055 7.056 1.73 0.237 4.09 

Diclofop-methyl 8.378 8.376 8.377 2.24 0.350 4.20 

DDT 12.842 12.838 12.840 3.96 0.598 5.63 

108 
PROJECT NUMBER: 0145/0193 PAGE 83 

Figure 15.1 Calibration Curve 

HPLC KOC ClUbl'ltlon CUrve 

~. 

j ,.. 4S25 

1__ -0.472 

Q 

1.810 -o~e 1._ 0(1.1. 

~ .~!-02 

2.8110 ~~'iillE,QI 

2.no .Q03E,QI 

3.Q20 -1.tl17E,02 

3310 Q.213 

...oao o.m ...., om 5_ <l2OO OJIIII o.a

"'-..0.713 a-C1.248 

,"&.17.\ 

O. 

Q 

• 

~.&'~--~--T-----~~----~--~--~-----T- 0.81 3.S1 5.81 

log10t(oc 

109 

PROJECT NUMBER: 014510193 PAGE 84 

Adsorption coefficient of sample 

The retention times, capacity factors and log10 Koc values determined for the sample are 

shown in the following table: 

_w __________________ ·~~ __________ • __ •• ________________ ---------~---------.------------------------~------------.-----------~.--: 

· 


· 
 · 


·
· 
 · 
 · 


· 


· 
 · 


· 
 · 
 · 


110 

PROJECT NUMBER: 014510193 PAGE8S 

16.8 Conclusion 

The adsorption coefficient (Koc) of the test material has been determined and is shown in 

the following table: 

.--...----.. -.~-.-- ... -~.- ..-...-.--..-.....-.....-.................. --_ .....---.--_...----..-------... ---_.--~----.----.-.-.--~

.. 

........ _-_ ....---•..••.•.... _-_ ..-------------...--_ .......---_ ......._-.. _------_ .. __ .--_ ...........------._------..-_._.-.--­

111 

PROJECT NUMBER: 014510193 PAGE 86 

16. FAT SOLUBILITY 

16.1 Method 

The determination was carried out using a procedure based on Method 116 of the OECD 

Guidelines for Testing of Chemicals, 12 May 1981. 

16.2 Procedure 

16.2.1 Preliminary test 

A mixture of test material and liquefied coconut oil (see Appendix 1) was prepared at 

48.5% test material and shaken at 3rC for at least 12 hours. After this period the 

mixture was visually assessed for excess test material. 

16.2.2 Definitive test 

Mixtures (see following table) of test material and liquefied coconut oil (see Appendix 1 

for Certificate of Analysis) were added to seven separate vessels. 

Table 16.1 

Sample Number Mass of Test Material (g) Mass of Coconut Oil (9)

1 2.0450 18.0098

2 4.4703 16.0682 

3 6.0083 14.0114

4 2.4006 17.6046 

5 2.8092 17.2110 

6 3.2709 16.8199 

7 3.6348 16.4363 

After the addition of the liquefied coconut oil, the samples were shaken at 37.0 ± 0.5°C 

for 3 hours and equilibrated at 37.0 ± 0.5°C for at least one hour, then shaken for a 

further 18% hours at 37.0 ± 0.5°C and equilibrated at 37.0 ± O.soC for one hour and 

visually assessed for dissolution of the test material. 

112 

PROJECT NUMBER: 014510193 PAGE 87 

16.3 Results 

16.3.1 Preliminary test . . .................................................. s


.--.. -~---.--.~-.- ... --.-------.~------- ....-------.-.-.-----_._._----- .... .. 

113 

PROJECT NUMBER: 014510193 PAGE 88 

16.4 Discussion .---.-------.. --.. ~---~- ..... -------.~*.~--.------.¥- .. -----------~------------.~-.--------- .. -----------.-.---~--------.---.-----: 

· · 

· · 

· · · · 

....... __ ..................... __ ...... __ ...... _ .................... _ ........................ _ .................... _ ........... ___ .... _ ...................................... __ .................. _ ............... _ ............... _......· · c 


The composition of the coconut oil is shown in Appendix 1. It was necessary to use 


coconut oil for the determination as the standard fat produced by the NATEC Company 


detailed in the OECD Guideline is no longer in production. Coconut oil has been 


identified as the most appropriate SUbstitute as attempts to locate an alternative 


manufacturer of standard fat, including communication directly with the OEeD, have 


been unsuccessful. 


16.5 Conclusion ......... , ...-_.--------------....-.... _--_ ....... _-_ ....--_ ..... _- .......... _-_ ...............-----------.... -_ ...---... _.....-......-....---_.------.. --_ .. ­

114 

PROJECT NUMBER: 014510193 PAGE 89 

Appendix 1 Coconut Oil Certificate of Analysis 

Cert[ficate of~naly'sis .
 1fiIiiMA-"'. r:tFE:H' 

Product Number 

Product Brand 

CAS Numb.r 

TIST 

APPEARANCE (COLOR) 

APPeARANCE (FORM) 

REMARKS ON OC 

MELl1NG POINT 

NMR SPECTRUM 1H 

IODINE VALUE 

ACID VALUE 

SAPONIFICATION VALUE 

DATE OF QC-RELEASE 

Edellraud SthWlrzler. Manager 

Quail" Control 

Buchs,SWltzeriand 

COCOnut au from Cocos nuc/fllm. 

mp 20-28 'C(Rl) 

60893 

FLUKA 

8001-31-8 

LOT 'UQn ReSULTI 

FAINTLY YEU.OW 

HONEY LIKE LIQUID 

C12:0 46.8%. Ct4:0 19.0%. 016:0 9.9%. C18:1 7.5%. C18:2 7.9% 

250 

CORRESPONDS 

11 

0.09 

250 

08/0CTI04 

115 
PROJECT NUMBER: 0145(0193 PAGE 90 

Appendix 2 Statement of GLP Compliance in accordance with Directive 2004l9/EC 

,DH) DeP.8rtment ~ ofHealth 

TIlE DEPARTMENT OF HEALTH OFTBE GOVERNMENT 


OF TBEIJNrI'ED KINGDOM 


GOOD LABORATORY PRACTICE 

STA:I'EMENr O}i'COMPLIANCE 

If(ACCORDANCE WrmDnutCfiVE lOlW9IEC 

TESTTYPE 

RarlnnLaboratorlos Ltd. Anall'HcaJIClillical Chemistry 

Sbardlow BuSiness Park Envinmmental Fate 

.LondonRosd Envil"ODme.ntal Tox.1cology 

SbardlOw Mutagellic:ity 

Derby . PhysJChem 

DE712GD Toxicology 

1.9tbAulUst Z008 

A general inspection for compliance witb the Principles of <J1)od Labor.atory Praotice 


was carried out ~tthe aboVe test facllit)' as pattQf the t1l{ GLPCompUancc PrQgl'lUllme. 


At the time of inspection no ~eviations were foun~ of ~ufficienl magnitude to affect 


the. ~ratidity of non-elinklil stu.dies performed at these faci:\iues. 


~~ 11"'°"( 

})t.Andl-ew I.Gra,. ..... 

Head, UK GLPMomtorhlg Authority . . . ... • 

116 

STATEMENT 


TITLE OF SruDY 

Mutagenicity Test of Karenz MT (Mixture of PEMB and PE3MB) Using 

Microorganisms 

(Study Code KOl-4229) 

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

the fina] report of the above-mentioned study issued on March 5,2009. 

Study Director: 

Ard 24-, 2009 

Shozo Ogura Date 

CERIHita 

Chemicals Evaluation and Research Institute, Japan 

117 

cERi !Receipt No. 836-08-T -5965\ 

STUDY CODE: KOl-4229 

FINAL REPORT 

MUTAGENICITY TEST OF 


KARENZ MT (MIXTURE OF PEMB AND PE3MB) 


USING MICROORGANISMS 


March 2009 


CERI Hita 


Chemicals Evaluation and Research Institute. Japan 


11 B 
GLP STATEMENT 

CERlHita 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Denko K.K. 

Title: Mutagenicity Test of Karenz MT (Mixture of PEMB and PE3MB) Using 

Microorganisms 

Study Code: KOl-4229 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of 

Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000) and 

"Concerning Standard of the Testing Facilities Conducting the Test Relating to the New 

Chemical Substances" (Notification No. 1121003 of the Phannaceutical and Food Safety 

Bureau, MHLW, No.3 (2003.11.17) of the Manufacturing Industries Bureau, METI & No. 

031121004 of the Environmental Health Department, MOE (November 21,2003)). 

Management: Signed in original March 5, 2009 


Takaharu Hara 


11 119 
GLPSTATEMENT 

CERI Hita 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Denko K.K. 

Title: Mutagenicity Test of Kmnz MI (Mixture of PEMB and PE3MB) Using 

Microorganisms 

Study Code: KOl-4229 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of 

Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000) and 

"Concerning Standard of the Testing Facilities Conducting the Test Relating to the New 

Chemical Substances" (Notification No. 1121003 of the Phrumaceutical and Food Safety 

Bureau, MHL W, No.3 (2003.11.17) of the Manufacturing Industries Bureau, MEn & No. 

031121004 of the Environmental Health Department, MOE (November 21,2003». 

I also confinued that this report accurately reflected the raw data and the test data were 

valid. 

Study Director: Signed in original March 5, 2009 


Shozo Ogura 


iii 120 
KOl-4229 

1/2 

QUALITY ASSURANCE STATEMENT 

CERI Hita 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Denko K.K. 

Title: Mutagenicity Test of Karenz MT (Mixture of PEMB and PE3MB) Using 

Microorganisms 

Study Code: KOl-4229 

This study was inspected by Quality Assurance Unit of CERI Hita, Chemicals 

Evaluation and Research Institute, Japan. The dates inspected and the dates reported 

these results to the study director and management are as follows. 

Phase Date of Inspection 

Date Reported to Study 

Director and Management 

Protocol January 13,2009 January 13, 2009 

Confinnation of the Answer from 

Study Director (Protocol) January 14,2009 January 14,2009 

Approval of Protocol January 15,2009 January 15,2009 

Protocol Amendment January 16, 2009 January 16,2009 

COllfinnation of the Answer from 

Study Director (Protocol 

Amendment) 

January 16,2009 January 16, 2009 

Preparation of Test Substance January 22, 2009 January 22, 2009 

Treatment of Test Strains January 22, 2009 January 22, 2009 

Approval of Protocol Amendment January 23, 2009 January 23, 2009 

Culture Condition and 

Observation and Colony Count January 26,2009 January 26,2009 

Raw Data and Draft Final Report March 4, 2009 March 4, 2009 

Re-inspection of Draft Final 

Report 

March 5, 2009 March 5, 2009 

Final Report March 5, 2009 March 5, 2009 

The inspection result of following phase was reported to the study director and 

management based on the report of process-based inspection relevant to this study type 

and timeframe. 

iv 121 
KOl-4229 

2/2 

Phase Date of Inspection 

Date Reported to Study 

Director and Management 

Examination of Solubility of 

the Test Substance 

October 10, 2008 March 5,2009 

Preparation and Management of 

Positive Control Substances September 4, 2008 March 5, 2009 

Preparation of Culture Medium 

and Rea.gents 

December 8, 2008 March 5, 2009 

Management of Test Strains April 21, 22, 29, 30 and 

May 1,2008 

March 5, 2009 

Pre-cultures of Test Strains December 11 and 12,2008 March 5, 2009 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry 

of Labor, Notification No.76, September 1, 1988; partially revised on March 29, 2000), 

"Concerning Standard of the Testing Facilities Conducting the Test Relating to the New 

Chemical Substances" (Notification No. 1121003 of the Phannaceutical and Food 

Safety Bureau, MHLW, No. 3 (2003.1] .17) of the Manufacturing Industries Bureau, 

METI & No. 031121004 of the Environmental Health Department, MOE (November 21, 

2003)). 

This study was also conducted in compliance with "Standards for Toxicity 

Investigations" (Ministry of Labor, Notification No.77, September 1, 1988 and 

Notification No.67, June 2, 1997), "Procedures of Mutagenicity Test Using 

Microorganisms and Evaluati.on of Test Results" (Ministry of Labor, Official 

Notification, February 8, 1999), "Ill Mutagenicity test" of "Reverse-Mutation Assay in 

Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances" 

(Notification No. 1121002 of the Phannaceutical and Food Safety Bureau, MHLW, No. 

2 (2003.11.13) of the Manufacturing Industries Bureau, METI & No.031121002 of the 

Environmental Health Department, MOE (November 21,2003)). 

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. 

Head, Quality Assurance Unit: ___S:.tig..,n..:::;e.::::.d~in.!-o:::.!n::J'gi;>!i:.!!n=al'--__March 5, 2009 

Ryuichiro MizUb'llchi 

v 122 
KOI-4229 


TITLE 

SPONSOR 

TESTING FACILITY 

PURPOSE OF STUDY 

TESTING METIIOD 

GLP COMPLIANCE 

PERlOD OF STUDY 

T ABLE OF CONTENTS 

Page 


............................. , ........... "......................... 2 


................................................................... 2 


................................................................... 2 


................................................................... 2 


................................................................... 2 


................................................................... 2 


................................................................... 3 


LOCATION AND PERlOD FOR RETENTION OF DATA ............. 3 


RETENTION OF ORlGINAL DOCUMENTS .................... 3 


STUDY DIRECTOR AND NAMES, ASSIGNED SECTIONS AND 


JOB ASSIGNMENT OF PERSONNEL .................... 4 


AUTHOR OF FINAL REPORT .................... 4 


SUMMARY 	 5 


MA TERIALS AND METHODS 

1. 	 TEST SUBSTANCE AND POSITIVE CONTROL SUBSTA};CES 6 


2. 	 BACTERIAL STRAINS .................................................... 8 


3. 	 MEDIUM AND S9 MIX .................................................... 8 


4. 	 PRE-CULTURES OF THE TEST STRAINS ............................. 9 


5. 	 PREPARATION OF TEST SUBSTANCE AND POSITIVE 


CONTROL SUBSTANCES ................................................... 10 


6. 	 METHODS .................................................... 11 


7. 	 OBSERVATION AND COLONY COUNTING .................... 12 


8. 	 JUDGEMENT CRITERIA OF TEST RESULTS .................... 12 


FACTORS AFFECTED RELIABILITY OF TEST .................... 12 


TEST RESULTS 

1. 	 DOSE FINDING TEST 12 


2. 	 MAINTEST 13 


DISCUSSION AND CONCLUSION 13 


TABLE AND FIGURE 

TABLES .................................................... 14 


FIGURES (DOSE-RESPONSE CURVES) ..................................... 
 16 


HISTORlCAL DATA 	 ]8 

1­ 123 

KOI-4229 


Study Code: KO] -4229 

Test Substance Code: HR7645 

Sponsor Code: S-0170 

TITLE 

Mutagenicity Test of Karenz MT (Mixture of PEMB and PE3MB) Using 

Microorgani sms 

SPONSOR 

Showa Denko K.K. 

13-9, Shiba Daimon I-Chorne, Minato-ku, Tokyo 105-85] 8, Japan 

TESTING FACILITY 

CERI Hita 

Chemicals Evaluation and Research Institute, Japan 

822, 3-chome, Ishii-machi, Hila, Oita 877-0061, Japan 

PURPOSE OF STUDY 

The ability of the test substance to induce mutations was investigated by using 

Salmonella typhimurium and Escherichia coli. 

TESTING METHOD 

This study was conducted in accordance with "Standards for Toxicity Investigations" 

(Ministry of Labor, Notification No.77, September 1,1988 and Notification No.67, June 

2, 1997), "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of 

Test Results" (Ministry of Labor, Official Notification, February 8, 1999), "III 

Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing 

Methods Relating to the New Chemical Substances" (Notification No. 1121002 of the 

Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (2003.11.13) of the 

Manufacturing Industries Bureau, METI & No.031I21002 of the Enviromnental Health 

Department, MOE (November 21, 2003)). 

GLP COMPLIANCE 

This study was conducted in compliance with "Standards to be observed by Testing 

Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, 

September 1, 1988; partially revised on March 29, 2000), "Concerning Standard of the 

Testing Facilities Conducting the Test Relating to the New Chemical Substances" 

(Notification No. 1121003 of the Pharnlaceutical and Food Safety Bureau, MHLW, No. 

- 2 124 
KOI-4229 

3 (2003.11.17) of the Manufacturing Industries Bureau, METI & No. 031121004 of the 

Environmental Health Department, MOE (November 2],2003)). 

PERIOD OF STUDY 

Commencement of Study: January 8, 2009 

Initiation of Experiment 

(Initiation of Treatment of Dose Finding Test): January 22, 2009 

Completion ofExperiment 

(Completion of Colony Count): February 2, 2009 

Completion of Study: March 5, 2009 

LOCATION AND PERIOD FOR RETENTION OF DATA 

Raw data, protocol, letter of test request, questiormaire, final report and other 

documentation records will be retained in the archives and the test substance will be 

retained at the test substance storage room of the testing racility for 10 years after the 

date of the notification specified under Item 1 of Article 57-3 of Industrial Safety & 

Health Law. Date of the notification will be communicated from the sponsor to the 

testing facility. They also will be retained for 10 years after the date of the notification 

specified under Article 4, Paragraph 1 or Paragraph 2, Article 4-2, Paragraph 2, 

Paragraph 3 or Paragraph 8, Article 5-4, Paragraph Article 24, Paragraph 2 or Article 

25-3, Paragraph 2 of Notification on Testing Methods Relating to the New Chemical 

Substances. The sponsor \vill communicate the date of the notification to the testing 

facility. Treatment of data after tennination of the retention period will be carried out 

with the approval ofthe sponsor. 

RETENTION OF ORIGINAL DOCUMENTS 

An original and a duplicate of protocol and protocol amendment were drawn up. The 

fonner will be retained at the testing facility. The latter was sent to the sponsor. 

An original final report is drawn up and will be retained at the testing facility. A copy 

of the'original final report that was recognized to be an accurate copy by the study 

director will be sent to the sponsor. 

- 3 ­ 125 
KOl-4229 

STUDY DIRECTOR AND NAMES, ASSIGNED SECTIONS AND JOB ASSIGNMENT 

OF PERSONNEL 

Study Director: 

Study Staff: 

Management: 

Person in Charge of Storage 

ofArchives: 

Person in Charge of Test 

Substance Management: 

AUTHOR OF FINAL REPORT 

Study Director: 

Shozo Ogura 

Section 3, CERI Hita 

Shozo Ogura, Shinya Wakamatsu, Erika Mizuguchi and 

Mari Yoshimoto 

Section 3, CERl Hita 

(Preparation of the test substance, observation and . count 

of number ofrevertant colonies) 

Takaharu Hara 

CERlHita 

Shizuka Kouda 

General Affairs Section, CERl Hita 

Haruhiko Tajima 

Section 1, CERl Hita 

Signed in original March 5, 2009 

Shozo Ogura 

- 4 --­ 126 
KOl-4229 

SUMMARY 

The ability of Karenz MT (Mixture of PEMB and PE3MB) to induce mutations was 

investigated using Salmonella typhimurium strains TAIOO, TA1535, TA98 and TA1S37 

and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and 

absence of a metabolic activation system (89 mix). 

As a result, the mutagenicity of the test substance was judged to be negative because the 

numbers of revertant colonies in the test substance treatment groups were less than two 

times that in each negative control in all test strains with and without 89 mix. 

Therefore, it is concluded that Karenz MT (Mixture of PEMB and PE3MB) has no ability 

to induce mutations under the present test conditions. 

5 127 
KOl-4229 

MATERIALS AND METHODS 

1. TEST SUBSTANCE AND POSITIVE CONTROL SUBSTANCES 

1.1 Test Substance (Information Provided by the Sponsor) l--··········---·····~~--·····-·----··-··----·-·-····- ..-.---.-...-- ..---.---.. -.---~.--.- ...-...-----.-.---..... --...... . · 

· 

· · · · · 

· · · · · 

· ·, 

· · ·

· · 

· 


· 
 · ·· 

· · 

· · · · 


· 


· 

- 6 ­ 128 
KOl-4229 

7) Physicochemical properties 

8) 	 Storage conditions 

Stored at room temperature (cabinet No. 1 in the test substance storage room, 

pennissible range: 1 0-30°C) and to avoid contact with oxide. 

9) 	 Precautions 

Gloves, a mask, a head cap, safety glasses and a lab coat were worn. The test 

substance was used in the draft since peculiar stink. 

1 2 	 Positive Control Substances 

Name Manufacturer Lot No. Appearance Purity Grade 

AF-2·) 

Wako 

Pure Chemical 

Industries Ltd. 

WAP0369 Red-yellow 

crystalline powder 

100.2% Special 

grade 

NaN3•

2 

Wako 

Pure Chemical 

Industries Ltd. 

KLN3948 

White crystalline 

powder 

99.8% Special 

grade 

ICR-19t'3 Polysciences, blC. 534652 Yellow crystalline 

powder - -

2AA*4 

Wako 

Pure Chemical 

Industries, Ltd. 

ASMII01 Yellow-greenbrown powder 

97.4% -

*1: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide 


*2: Sodium azide 


*3: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl) aminopropylamino]acridine·2HCI 


*4: 2-Aminoanthraccne 


1) 	 Storage conditions 

Stored in a cool and dark place (refrigerator No. 005 in the test substance storage 

room ofthe main building, pennissible range: I-lO°C). 

- 7 

129 
KOl·4229 

2) 	 Precautions 


Gloves, a mask, a head cap, safety glasses and a lab coat were worn. 


2. BACTERlAf, STRAINS 

2.1 	 Strains and Reason for Selection 

Salmonella typhimurium (8. typhimurium) strains TAlOO, TAIS3S, TA98 and 

TA1S37 and Escherichia coli (E. coli) strain WP2uvrA were used. S. typhimul'ium 

strains and E. coli strain were supplied from Dr. Taijiro Matsushima, Japan Bioassay 

Research Center, on March 13, 2003 and on September 20, 2003, respectively. 

These test strains have been recommended to use for the mutagenicity test using 

microorganisms in "Standards for Toxicity Investigations" and "Testing Methods 

Relating to the New Chcmical Substances". 

2.2 	 Storage 

Amino acid requirement, sensitivity to ultraviolet-rays, lfa membrane mutation, 

presence or absence of plasmid pKMIOI and negative and positive control values of 

test strains were examined in the testing facility. Dimethyl sulfoxide (DMSO, Lot 

No. VV035, 100.0% in purity, spectrophotometric grade, DOJrNDO Laboratories) 

was added to fresh overnight cultures of the test strains which had been confinned to 

have these properties at a volume ratio of 0.9: 10. The mixture was frozen as a stock 

culture in an ultra-deep freezer (the Ames test room No. 1 of the main building, 

MDF-293AT, SANYO Electric Biomedical Co., Ltd.) below ·80°C. The mixture 

was thawed just before use. 

3. MEDIUMANDS9MIX 

3.1 	 Medium 

1) 	 Minimal glucose agar plate 


Tesmedia AN (Oriental Yeast Co., Ltd.) was used. 


Lot No.: ANI760KX (manufactured on November 13,2008) 


2) 	 Soft agar 

A solution containing 0.5 mM histidine and 0.5 mM biotin for S. typhimurium 

strains or 0.5 mM tryptophan for E. coli strain was added to a soft agar solution 

containi.ng 0.6 w/v% agar (Bacto Agar, Lot No. 8014026, Dlfco Laboratories) and 

0.5 w/v% NaC] at a volume ratio of 1 to 10. 

- 8 	-. 130 
KOl-4229 

3.2 S9 Mix 

1) Rat liver S9 

S9 (Lot No. 08100307, manufactured on October 3, 2008, Oriental Yeast Co., Ltd.) 

prepared from the liver of 7 week-old male SD rats (body weight: 206.0±S.8 g) 

administered with phenobarbital and 5,6-benzoflavone was used. The S9 was 

cryopreserved in an ultra-deep freezer (MDF-293AT in the Ames test room No. I 

of the main building, SANYO Electric Biomedical Co., Ltd.) below -80°C until 

use. S9 was thawed just before use. 

2) 	 Composition ofS9 mix 

S9 mix was prepared using Cofactor J® (Lot No. 999801, Oriental Yeast Co., Ltd.) 

for S9 mix immediately before use. One milliliter of S9 mix consisted of 8 limol 

MgCh, 33 limo] KCl, 5 limol Glucose-6-phosphate, 4 limol NADPH, 4 limol 

NADH, 100 limo I sodium-phosphate buffer (pH 7.4) and 0.1 mL ofS9. 

4. 	 PRE·CULTURES OF THE TEST STRAINS 

Thirty microliters (for TAl 00), 5-IiL (for WP2uvrA), and 20-IiL(for TA98, TA1535 

and TA1S37) aliquots of frozen stock culture of bacterial strains were respectively 

inoculated to 11 mL of Nutrient broth No.2 (Lot No. 464616, OXOID Ltd.) in each Ltube (volume: 27 mL). The culture was incubated at 37±0.5°C for 9 hours by shaking 

at about 50 times/minute in a seesaw type of shaker (MONOSIN-IIA, Taitec 

Corporation). The number of viable cells was calculated from O.D. value at 660 nm 

measured by a photo meter (Mini photo 518R, Taitec Corporation) at the end of 

incubation to calculate the number of viable cells. It was confinned that the numbers 

of viable cell s were more than 2.3 x 1 09 cells/mL in Salmonella typhimurium and more 

than 3.8xJ09 cells/mL in Escherichia coli strain. When the numbers of viable cells 

were more than 2.7xI09 cells/mL in Salmonella typhimurium and more than 4.2xl09 

celIs/mL in Escherichia coli strain, the O.D. value was measured at 660 nm by a photo 

meter (the same above) and were adjusted with Nutrient broth No. 2 at 2.3-2. 7x 1 09 

cells/mL and at 3 .8-4.2x 109 cells/mL, respectively. The culture was used without 

adjusting when the Salmonella typhimurium strains were in the range of 2.3-2.7x 109 

cells/mL. The final numbers of prepared viable cells are shown below: 

Test TAIOO TA1535 WP2uvrA TA98 TAt537 

Dose finding test 2.7 2.6 4.1 2.5 2.6 

Main test 2.7 2.6 4.1 2.7 2.6 

(x 109cells/mL) 

-9­

131 
KOI-4229 


5. 	 PREPARATION OF TEST SUBSTANCE AND POSITIVE CONTROL 

SUBSTANCES 

5.1 	 Preparation of Test Substance 

1) 	 Solvent 

DMSO (Lot No. WF032, 100.0% in purity, spectrophotometric grade, DOJfNDO 

Laboratories) was used. 

2) 	 Reason for selection of solvent 

The test substance was insoluble in distilled water (infonnation provided by the 

sponsor) and was soluble in DMSO at 500 mg/mL. The test substance solution of 

500 mg/mL prepared with DMSO was no change in color nor heat generation at 

room temperature within 2 hours after preparation. Therefore, DMSO was 

preferably selected a'i a solvent. The test substance solution of 50.0 mg/mL 

prepared with DMSO was no change in color nor heat generation at room 

temperature within 2 hours after preparation. 

3) 	 Preparation method 

DMSO was added to the test substance and mixed by a tube mixer to make a 50.0 

mg/mL solution. The test substance solution was diluted with the same solvent to 

give each required concentration. 

4) 	 Preparation time 

The test substance solutions were prepared immediately before use, kept under 

yellow lamps at room temperature and used within 2 hours. 

5.2 	 Preparation. of Positive Control Substances 

1) 	 Preparation method 

NaN3 was dissolved in distilled water (distilled water for injection, Lot No. K8B81, 

Otsuka Pharmaceutical Factory Inc.). AF-2, ICR-191 and 2AA were dissolved in 

DMSO (Lot No. VV035). 

2) 	 Storage conditions 

Positive control solutions were stored in an ultra-deep freezer (the Ames test room 

No.1 of the main building, MDF-293AT, SANYO Electric Biomedical Co., Ltd.) 

below -80°C. The solutions were thawed before use. 

-	 10 ­ 132 
KOI-4229 


6. 	 METHODS 

The test was perfonned by the pre-incubation method with and without S9 mlX. 

Triplicate plates were used for the negative control group, and duplicate plates were 

used for the test substance treatment group at each dose and the positive control group 

in the test. The test code number, name of test strain, presence or absence of S9 mix 

and dose level were noted on each plate. 

6.1 	 Procedures 

After 0.1 mL of the test substance solution, solvent or the positive control solution, 

0.5 mL of 0.1 M sodium phosphate buffer (PH 7.4) or S9 mix and 0.1 mL of the 

bacterial culture were added to a test tube, the mixture was shaken at 37±0.5"C for 20 

minutes. Two milliliters of the soft agar were then added to each tube and the 

mixture was poured onto a minimal glucose agar plate. The number of revertant 

colonies was counted after incubation at 37±O.5°C for 48 hours. 

6.2 	 Sterility 

The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 

mL) were respectively mixed with 2 rnL of the soft agar and were poured onto each 

minimal glucose agar plate in order to examine bacterial contamination. The plates 

were incubated at 37±0.5°C for 48 hours. 

6.3 	 Negative Control and Positive Controls 

The solvent used in the tests was employed as a negative control and the following 

positive controls were used for each bacterial strain. 

TA100 1'A1535 WP2uvrA TA98 TA1537 

AF-2 NaN3 AF-2 AF-2 ICR-191 

-S9 mix 

0.01 0.5 0.0] 0.1 0.5 

2AA 2AA 2AA 2AA 2AA 

+S9 mix 

1 2 10 0.5 2 

(Unit: Jlg/plate) 

6.4 Dose Selection 

1) Dose finding test 

A total of six doses consisting of 5000 flg/plate as the highest dose and five lower 

doses diluted with a geometric progression of 4 were set. 

2) Main test 

The results of the dose finding test showed that the number of revertant colonies in 

the test substance treatment groups was less than twice that in the solvent control. 

The bacterial growth inhibition was not observed at any doses in the groups of 

treatment in all test strains with and without 89 mix. The precipitation of the test 

substance was observed at 1250 t-tg/plate or more in all test strains without 89 mix. 

- 11 ­ 133 
KOI-4229 


Therefore, in the main test without S9 mix, the highest dose was selected at 5000 

Ilg/plate as observed the precipitation and lower 5 doses of 2500, 1250, 625 and 

313 flgiplate diluted with a geometric progression of 2, were employed. In the 

main test with S9 mix, the highest dose was selected at 5000 Jlg/plate and lower 5 

doses of 2500, 1250, 625 and 313 Ilg/plate diluted with a geometric progression of 

2, were employed. 

7. 	 OBSERVATION AND COLONY COUNTING 

7.1 	 Observation 

The precipitation of the test substance was observed by macroscopically and the 

bacterial growth inhibition was observed by using a stereomicroscope, at the end of 

the incubation period. 

7.2 	 Colony Counting 

For the plates in which the precipitation of the test substance was observed, the 

number of colonies was counted with a manual counter, and the other plates were 

counted by using a colony analyzer (CA-I1D, System Science Ltd.). Square 

correction and miss counting correction were performed in colony analyzer. 

8. 	 JUDGEMENT CRITERIA OF TEST RESULTS 

The test substance was judged to be positive when the number of revertant colonies 

increased to twice or more than that in the negative control in a concentration-dependent 

manner and also the reproducibility of the test results was obtained. 111 all other cases, 

it was judged to be negative. Any statistical methods were not used. 

FACTORS AFFECTED RELIABILITY OF TEST 

There were no factors that might have affected the reliability ofthe test. 

TEST RESULTS 

1. 	 DOSE FINDING TEST 

The test result is shown in Table 1 and the dose-response curves are shown in Figs. 1 

and 2. 

No increases in the number of the revertant colonies of any test strain were observed in 


the test substance lrealment groups with or without S9 mix. 


Growth inhibition was not observed at any dose with or without S9 mix. 


The precipitation of the test substance was observed at 1250 Jlg/plate or more in nll test 


-	 12 ­ 134 
KOl-4229 

strains without S9 mix. 

2. 	 MAIN TEST 

The test result is shown in Table 2 and the dose-response curves are shown in Figs. 3 

and 4. 

No increases in the number of the revertant colonies of any test strain were observed in 


the test substance treatment groups with or without S9 mix. 


Growth inhibition was not observed at any dose with or without 89 mix. 


The precipitation of the test substance was observed at 1250 flg/plate or more in all test 


strains without S9 mix. 


DISCUSSION AND CONCLUSION 

The mutagenicity of the test substance was judged to be negative because the number of the 

revertant colonies at all dose levels of the test substance was less than twice that of the 

negative control for each test strain in the test. 

The numbers oftbe revertant colonies in the negative and positive controls were within the 

range of historical data in the testing facility. The numbers of the revertant colonies in the 

positive controls were two or more times higher than those of the negative control. 

Microbial contamination was not observed in the dose finding test or the main test. This 

indicates that the present test data are valid. 

Therefore, it was concluded that Karenz MT (Mixture of PEMB and PE3MB) did not 

ability to induce mutations under the present test conditions. 

-	 13 ­ 135 
KOl-4229 

Table 1 Results of the dose finding test 

Test substanec' Karcnz MT (mixture ofPEMB - and PE3MB)

Test Period From January 22, 2009 to January 26, 2009 

Wlth(+)or Test substance Number of revenant colOnies per plate 

witbout(-} dose Base-pair substitution type Framcshift type 

S9mix (!lglpJate) TAJOO TA1535 WP2/JvrA TA98 TAlS37 

Negative 95 91 16 8 1314 27 20 19 15 20 

control ](14 ( 97 15 ( ( 29 26 ( 22 10 ( 15 

112 7 27 23 13 

4.88 120 ( 116 6 ( 7 42 ( 35 19 ( 21 13 { 13 

105 10 30 IS 15 

195 95 ( 100 8 ( 9 27 ( 29 18 ( 17 13 I 14 

-59 mix 102 II 31 19 12 

18.1 95 ( 99 l! ( II 20 ( 26 22 ( 21 26 ( 19 

98 18 25 19 22 

313 136 f 117 7 ( 13 27 ( 26 23 ( 21 II ( 17 

!!6 8 33 22 B 

1250 t 116 ( 101 7 { 8 30 ( 32 18 ( 20 7 ( 8 

115 S 36 17 7 

5000 t 94 ( 105 7 ( 6 39 ( 3ij 26 ( 22 7 ( 7 

Negative 104 116 14 12 34 28 38 45 35 34 

control 116 ( 112 12 ( 13 38 ( 33 34 ( 39 22 ( 30 

107 14 30 28 15 

4.88 96 ( 102 7 ( II 36 ( 33 18 ( 23 20 ( 18 

105 II 34 21 16 

19.5 94 ( 100 8 ( 10 43 ( 39 28 ( 25 25 ( 21 

+59 mix lOS 5 30 27 20 

78.l 99 ( 104 10 ( 8 35 ( 33 29 ( 2S 20 ( 20 

128 10 33 27 29 

313 126 { 127 13 ( !~ 26 ( 30 34 I 31 20 ( 25 

128 12 44 30 8 

1250 108 ( 118 13 ( 13 41 ( 43 28 ( 29 14 ( 11 

J 12 5 36 30 7 

5000 96 ( 104 7 ( 6 31 ( 34 31 ( 31 7 ( 7 

Chemical AF·2 NaN.. Af-:2 AF-2 ICK·l9! 

Positive Dose(!,g/plale) 001 OJ 0.01 O.J 0,5 

control Numbf:rof 614 530 314 521 1659 -S9 mix revenant 

.",)Qrueslpl<llf 592 ( 603 405 ( 468 308 ( 311 512 ( 517 1501 ( 1580 

Chemica 2M 2AA 2AA 2M 2M 

Positive Dose(JAg/plale) I :2 10 0.5 2 

control Number of 1015 249 674 268 247 +S9 mix teift.rtant 

colrJfli¢!lplatt 909 ( 962 242 ( 246 594 ( 1i34 261 ( 278 221 ! 234 

(Notes] 

• ( ): The mean number of colonies per plate, 


, t: Precipitate of tcst substance was observed at all plates. 


• AF-2: 2-(2-Furyl)·3-(5-nitro-2-furyl}acrylllmide 

• NaN): Sodium azide 

• ICR·191: 2-Methoxy·6·ch]oro-9-!3-{2-chloroethyl)aminopropylamillo]a"ridinc' 2HCl 

• 2M: 2-Aminoanthraccne 

- 14 

136 
KOl-4229 

Table 2 Results of the main test 


Test substance: Kllrenz MT (ml:((urc of PEMB and PE3MB) 


TeSI Period From january 29, 2009 10 February 2, 2009 

Wlth(+)or Test substance Number of revertant COlOnies per plate 

wilhoUl(-) dose rf substitution type F rarnesbift tYJl{': 

S9mix (lLg!platc) TAIOO TA 1535 WP2ul'rA TA98 TA1537 

Negative ll5 102 13 14 28 22 22 25 3 13 

control n ( J03 3 ( 10 33 ( 28 2~ ( 25 13 ( 10 

115 7 28 21 to 

313 118 ( 117 7 ( 7 23 ( 26 21 ( 21 ) 8 ( 

111 II 31 25 4 " 

-S9 mix 625 105 ( 108 6 { 9 30 { 31 20 [ 23 6 { 5 

98 7 34 25 6 

1250 t 109 ( 104 8 ( H 29 { 32 26 ( 26 6 { 6 

116 12 35 15 5 

2500 t 103 ( 110 12 ( 12 28 ( 32 15 { 15 6 ( 6 

94 7 23 25 7 

I 5000 t 122 ( 108 8 ( g 28 ( 26 22 [ 24 4 ( 6 

Negative 117 108 10 8 3; 31 36 34 27 22 

control 116 ( 114 3 ( 7 36 ( 33 27 ( 32 22 ( 24 

103 6 37 26 15 

313 107 ( 105 R ( 7 22 ( 30 30 { 28 16 ( 16 

147 8 2S 16 14 

+S9mix 625 132 ( 140 5 ( 7 33 ( 29 28 I 22 8 ( I) 

115 10 28 35 II 

1250 III ( 113 5 ( 8 28 ( :;l8 25 ( 30 16 ( 14 

III 5 31 47 6 

2500 112 t 112 1 ( 6 28 ( 30 21 I 37 8 ( 7 

126 13 35 35 R 

5000 108 ( 117 8 ( II 34 ( 35 27 ( 31 6 ( 7 

Chemical M-2 NaN, AF-2 AF-2 JCR-191 

Positive Dose(J.lg!plate) 0.01 0.5 0.01 0,1 0.5 

control Numbel of 692 347 394 541 1561 -S9mix fe~rtant 

colonies/plate 655 ( 674 340 ( 344 360 ( 377 490 ( 516 1320 ( 1441 

Chemical 2AA 2AA 2AA .AA 2AA 

Positive Dose(vg!plate) 1 2 10 05 2 

conlml Numbetof 889 191 649 375 293 +S9mix JlIl:vm~nt 

CilhmKe$/plate 1188 ( 1039 213 ( 202 665 ( 651 367 ( 371 274 ( 284 

[NQles] 

• (): Til.: meannurnber of colonies per plate. 


· t: Precipitate of lest substance was observed at all plates. 


• AF-2: 2-(2-Furyl)-3-(S-nitro-2-furyl)acrylamide 

• NaN): Sodium azide 

• ICR-191: 2-Methoxy-6-chlQro-9-p-(2-chlorocthyJ)aminopropylamino]acridine' 2HCI 

• 2AA: 2-Arninoanthracene 

15 

137 
KOl-4229 

140 

., 120 

'iii 

-a. 

... 100 ., 

Po 

Ji 

d 

0 

'0 80 u 

i 

1:: 60 , ., 

-+-TAlOO 

-----TAlS35 

--tr-- WP2uvrA 

~TA98 

~ -----TAl 537 .... 

0 r ... 

.8 

E 

:z

::> 

40 

o 4.88 19.5 7S.1 313 1250 5000

Test substance dose (~glplate) 

Fig. 1 Dose-response curve without 89 mix in the dose finding test 

140 r 

120 ~ 

-a. 

8. 100 

.~ 

§ -+-TAlOO 

"8 80 

I -----T A 1535 

--tr-- WP2uvrA 

60 ~TA98 

~ -+-TAlS37 'c> 

:

.8 

§

:z 

o ' -'--.--,.-'--------........--­

o 4.88 19.5 78.1 313 12S0 SOOO 

Test substance dose (~glplatc) 

Fig. 2 Dose-response curve with S9 mix in the dose finding test 

- 16 ­ 138 
KO]-4229 


140 

j 

120 

Q. 

.... 

~ 100 

-+-TAIOO 

so ___ TAJ535 

-o-WP2uvrA 

60 --*-TA98 

---TAL 537 

40 

2Of~:=:;~~~ o c, ____~_ 

o 313 625 1250 2500 5000 

Test substance dose ()lglplate) 

Fig. 3 Dose-response curve without S9 mix in tbe main test 

160 c 

140 

120 • 

100 -+-TAIOO 

---TA1535 

80 -o-WP2uvrA 

--*-TA98 

60 ---TA1537 

o 	 313 625 1250 2500 5000 

Test substance dose (}4g1pJate) 

Fig. 4 Dose-response curve with S9 mix in the main test 

- 17 ­ 139 
KOI-4229 


HISTORICAL DATA 

(Tesmedia AN) 


July 2008 - December 2008 


Negative Control (Mean±3S.D.) 

-S9 mix +S9mix 

TAl 00 TAI535 WP2u1.'rA TA98 TAl537 TAIOO TAl53S WP2uvrA TA98 TA 1 537 

Mean 

S.D. ...._ ........ __ ............ w ..... _ 

Upper Limit 

105 

15 

150 

10 

4 .......... -... 

22 

24 

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

45 

22 

6 .... -....... 

40 

9 

4 .. ............ 

21 

112 

17 .. ......... 

163 

9 

3 ............... ­ 18 

29 

7 .. ...............­...-­ 50 

31 

7 .....-... -.... 52 

20 

6 -...... _..... 

38 

Lower Limit 60 1 3 4 1 61 J 8 10 2 

Positive Control (Mean±3S.D.) 

-S9mix +89 mix 

TAtoO TA1535 WP2uvrA TA98 TAI537 TAIOO TAl535 WP2uvrA TA98 TAI537 

Chemical ....................... "".......... AF-2 NaN)

.. ............ AF-2 ........... ..... AF-2 .............. JCR·19J 

.. ............ 2M _.............,. 2M .... .......... ­ 2M .. ................... 2AA .., ...... _,., ... ­ -_ 2M....' ...... ­ Dose 

(Ilglplate) 0.01 0.5 0.01 0.1 0.5 I 2 10 0.5 2 

Mean 662 380 329 510 1533 943 229 617 279 234 

S.D. ....... ............................ ,. 96 ................ 53 ............. 72 .. ----­....... 78 ............ ­ 192 110 -......... __ .. 28 ............... 10.3 "' ... '"' .........­..... 39 .. .......... 34 ............. .. 

Upper Limit 950 539 545 744 2109 1273 313 926 396 336 

Lower Limit 374 221 113 276 957 613 145 308 162 J32 

[Notes] 

• AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 

• NaN): Sodium azide 

• ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino ]acridine' 2HCI 

• 2AA: 2-Aminoanthracene 

18 ­ 140 
STATEMENT 

TITLE OF STUDY 

Chromosomal Aberration Test of Karenz MT (Mixture of PEMB and 

PE3MB) Using Cultured Mammalian Cells (Study Code: K06~1279) 

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

of the final report (Study Code: K06~1279, issued on Apri115, 2009) audited by Quality 

Assurance Unit of eERI Hita, Chemicals Evaluation and Research Institute, Japan. 

Renge Suzuki Date 

CERI Hita 

Chemicals Evaluation and Research Institute, Japan 

141 
cERi IReceipt No. 837-08-T-5966 

STUDY CODE: K06-1279 

FINAL REPORT 

CHROMOSOMAL ABERRATION TEST OF 

KARENZ MT (MIXTURE OF PEMB AND PE3MB) 

USING CULTURED MAMMALIAN CELLS 

April,2009 

CERI Hita 

Chemicals Evaluation and Research Institute, Japan 

142 
K06-1279 


GLP STATEMENT 

CERI Hita 


Chemicals Evaluation and Research Institute, Japan 


Sponsor: Showa Denko K.K. 

Title: Chromosomal Aberration Test of Karenz MT (Mixture of PEMB and 

PE3MB) Using Cultured Mammalian Cells 

Study Code: K06-1279 

I, the undersigned, hereby declare that this study was conducted in compliance with 

"Concerning Standard of the Testing Facilities Conducting the Test Relating to the New 

Chemical Substances" on Japanese GLP (Notification No. 1121003 of the 

Pharmaceutical and Food Safety Bureau, MHLW, No.3 (November 17, 2003) of the 

Manufacturing Industries Bureau, MET! & No. 031121004 of the Environmental Health 

Department, MOE (November 21, 2003). 

I also confirnled that this report accurately reflected the raw data and the test data were 

valid. 

Study Director: Signed in original April 15,2009 

Shozo Ogura 

11 

143 
K06-1279 

112 

QUALITY ASSURANCE STATEMENT 

CERI Hita 

Chemicals Evaluation and Research Institute, Japan 

Sponsor: Showa Denko K.K. 

Title: Chromosomal Aberration Test of Karenz MT (Mixture of PEMB and 

PE3MB) Using Culturcd Mammalian Cells 

Study Code: K06-1279 

This study was inspected by Quality Assurance Unit of CERI Hita, Chemicals 

Evaluation and Research lnstitute, Japan. The datcs inspected and the dates reported 

these results to the study director and the management are as follows. 

Phase Date of Inspection 

Date Reported to Study 

Director and Management 

Protocol November 28, 2008 November 28,2008 

Cell Pre-culture November 28, 2008 November 28, 2001{ 

Preparation of Test Substance December 1,2008 December 1, 2008 

Treatment of Cells December 1,2008 December 1, 2008 

Confirmation of the Answer from Study 

Director (Protocol) 

December 2, 2008 December 2, 2008 

Protocol Amendment December 16, 2008 December 16.2008 

Confirmation of the Answer from Study 

Director (Protocol Amendment) 

December 17,2008 December 17,2008 

Protocol Amendment (No.2) January 7, 2009 January 7,2009 

Protocol Amendment (No.3) January 15,2009 January 15,2009 

Confirmation of the Answer from Study 

Director (Protocol Amendment (No.3) January 16, 2009 January 16,2009 

Observation of Specimens March I 3, 2009 March 13, 2009 

Protocol Amendment (No.4) March 23, 2009 March 23, 2009 

Raw Data and Draft Final Report April 9, 2009 April 9, 2009 

Re-inspection of Raw Data and Draft 

Final Report April 1 0, 2009 April 10, 2009 

Draft. Final Report (Second) April 14, 2009 April 14, 2009 

Re-inspection of Drall Final Report 

(Second) April 15,2009 April 15,2009 

Final Report April 15, 2009 April 15, 2009 

III ­

144