November 5, 2010 

Via Federal Express 

New Chemicals Program 

Off. of Pollution Prevention and Toxics 

Confidential Business Information Center [Rm. 6428] 

EPA East Building 

Ronald Reagan Complex 

1201 Constitution Ave., NW 

Washington, DC 20004-3302 

Company Sanitized 

P-11-083 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

Re: Premanufacture Notice Submission under TSCA Section 5 by [ 

] 

Submission Report Number: CMTP101105088166808 

Dear Sir/Madam: 

Enclosed you "vill fwd a Prcmanufacture Notice (PMN) submitted under Section S(a)(1) of 

the Toxic Substances Control Act. This PMN is being submitted to the {J.S. Environmental 

Protection Agency (EPA or the Agency) on behalf off 

] . 

This PMN is being submitted for the chemical substance identified below: 
U.S. Environmental Protection :\gency 

November 5, 2010 

Page 2 

CA Index Name: 

CAS Registry Number: 

Trade Name: 

TS Number: TS-CMTP01 

Submission Report Number: CMTP101105088166808 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

User Fee Payment Numbers: Pay.gov Tracking ID No.: 251QRS7P 

Agency Tracking ID No.: 74149916922 

This PMN submission is made in accordance with the regulations given at 40 CFR 720 et 

seq. We request that the Agency begin review upon receipt. Enclosed with this letter are the 

confidential and sanitized versions of the PMN. Both of these versions have been created 

using the EPA's new e-PMN software. 

] has also electronically remitted the PMN user fee of $2,500.00. The User Fee 

Payment Identity Numbers are given above. 

Finally, f ) is claiming certain information in the PMN, including the chemical identity 

and company identity, as Confidential Business Information (CBI) in accordance with the 

provisions at 40 CFR 720.80. 

* 

For all questions regarding the submission of this PMN, the technical contact is [ 

] of the Firm. He is reachable at [ J. 

Sincerely, 
U.S. Env-ironmental Protection Agency 

November 5, 2010 

Page 3 

cc: 

Enclosures: 

• Conjidential version of the PMN 

• Sanitized version of the PMN 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 
1111111111111111111 SANITIZED SUBMISSION 

PMN2010P1 PMN Page 1 

Form Approved. O.M.B. Nos. 2070-0012 and2070-0038 

U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY 

When 

completed 

send th1s 

form to !> 

tact Numoers: 202-564-8930/8940 

:s 

',--- Date of receipt: • ') "1 

51110000063 P-11-063 

Submission Re ort Number 

CMTP101105088166808 

TS Number 

196 Pay.gov 10: 251QRS7P; Agency 10: 74149916922 CMTP01 

GENERAL INSTRUCTIONS 

• You must proVIde all information requested in this form to the extent that it 1s known to or reasonably ascertainable by you Make reasonable estimales if you do nol have actual data. 

Before you complete this form. you should read the "Instructions Manual for Premanutacture 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. For mailing address information see the Help instructions 1n the e-PMN tool 

Part 1- GENERAL INFORMATION 

You must provide the currently correct Chemical Abstracts (CA) 

Name of the new chemical substance, even if you claim the 

identity as confidential. You may authorize another person to 

submit chemical identity information for you, but your submission 

will not be complete and the review will not begin until EPA 

receives this information. A letter in support of your submission 

should reference your TS user fee identification number. For all 

Section 5 Notice submissions (paper or electronic) you must 

submit an original notice including all test data; if you claimed any 

information as confidential, an original sanitized copy must also be 

submitted. 

Part II- HUMAN EXPOSURE AND ENVIRONMENTAL 

RELEASE 

If there are several manufacture, processing, or use operations to 

be described in Part II, sections A and B of this notice, reproduce 

the sections as needed. 

Part Ill- LIST OF ATTACHMENTS 

For paper submissions, attach additional sheets if there is not 

enough space to answer a question fully. Label each continuation 

sheet with the corresponding section heading. In Part 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. 

"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 ... The intention is to reduce delays that routinely 

accompany the development of consent orders or Significant New 

Use Rules. Checking a "binding" box in a PMN does not by itself 

prohibit the submitter from later deviating from the information 

(except chemical identity) reported in the form; however, in the 

case of exemption applications (such as TMEA, LVE, LOREX) 

certain information provided in such notifications is binding on the 

submitter when the Agency approves the exemption application, 

especially if the production volume "binding" box is chosen in a 

LVE. 

CONFIDENTIALITY CLAIMS 

You may claim any information in this notice as confidential. To 

assert a claim on the form, mark (X) the confidential box next to 

the information that you claim as confidential. To assert a claim in 

an attachment, circle or bracket the information you claim as 

confidential. If you claim information in the notices as confidential 

you must also provide a sanitized version of the notice (including 

attachments). For additional instructions on claiming information 

as confidential, read the Instructions Manual. 

EPA FORM 7710-25 (Rev. 6-09) 

TEST DATA AND OTHER DATA 

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

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

related to the health and environmental effects on the manufacture, processing, distribution in 

commerce, use, or disposal of the new chemical substance. Standard literature citations may 

be submitted for data in the open scientific literature. Complete test data (written in English), 

not summaries of data must be submitted if they do not appear in the open literature. You 

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

chemical composition of the tested material should be characterized. Following are examples 

of test data and other data. Data should be submitted according to the requirements of 

§720.50 of the Premanufacture Notification Rule (40 CFR Part 720). 

D 

0 

D 

0 

D 

0 

D 

D 

D 

D 

D 

D 

D 

D 

Test Data (Check Below any included in this notice) 

Environmental fate data D OtherData 

Health effects data D Risk Assessments 

Environmental effects data 0 Structure/activity relationships 

Physical/Chemical Properties (A physical and chemical properties worksheet is 

located on the last page of this form.) 

Test data not in the possession or control of the submitter 

TYPE OF NOTICE (Check Only One) 

PMN (Premanufacture Notice) 

SNUN (Significant New Use Notice) 

TMEA (Test Marketing Exemption Application) 

LVE (Low Volume Exemption)@ 40 CFR 723.50(c)(1) 

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

LVE Modification 

LOREX Modification 

Mock Submission 

Mark (X) if pending Letter of Support 

IS THIS A CONSOLIDATED PMN (Y/N)? 

#of chemicals or polymers (Prenotice Communication# required, enter# on 

p. 3). 

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

Replaces previous editions of EPA Form 7710-25 

Page 1 
SANITIZED SUBMISSION 

PMN Page 2 

The public reporting and recordkeeping burden for this collection of information is estimated to average 93 hours per response. Send 

comments on the Agency's need for this information, the accuracy of the provided burden estimates, and any suggested methods for 

minimizing respondent burden, including through the use of automated collection techniques to the Director, Collection Strategies 

Division, U.S. Environmental Protection Agency (2822T), 1200 Pennsylvania Ave., NW, Washington, D.C. 20460. Include the OMB 

control number in any correspondence. Do not send the completed EPA Form 7710-25 to this address. 

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

with CD or paper submission. 

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

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

manufacture, import or process for a commercial purpose, other than in small quantities solely for 

research and development, the substance identified in Part I, Section B. 

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

3. I am submitting with this notice all test data in my possession or control and a description of all 

other data known to or reasonably ascertainable by me as required by §720.50 of the 

Premanufacture Notification Rule. 

Additional Certification Statements: 

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

certification statement that applies: 

D 

D 

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

The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined @ 40 CFR 700.43) in 

accordance with 40 CFR 700.45(b), or 

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

accordance with 40 CFR 700.45(b). 

If you are submitting a Low Volume Exemption (LVE) application in accordance with 40 CFR 723.50(c)(1) or a 

Low Release and Low Exposure Exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check 

the following certification statements: 

D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, 

other than in small quantities solely for research and development, under the terms of 40 CFR 723.50. 

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

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

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

the exempted substance for commercial purposes within 1 year of the date of the expiration of the 30 day review period. 

The accuracy of the statements you make in this notice should reflect your best prediction of the 

anticipated facts regarding the chemical substance described herein. Any knowing and willful 

misrepresentation is subject to criminal penalty pursuant to 18 USC 1 001. 

Signature and title of 

Authorized Official (Original 

Signature Required) 

Date 

Confidential 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 2 
111111111111111111 SANITIZED SUBMISSION 

PMNP 3 

liTTER 

Mark (X) the ··1... ' box next to any '" ' you claim as ...u1111ut:lltial 

1a. Pan•u•· Sul.uuuu• Notice (in U.S.) I.JO!)IIUCIIUdl 

Name of Authorized Official (first) XXX (last) XXX 

Position XXX 

Company XXX 00 Mailing Address (number & street) XXX 

City XXX I State I Postal Code XXX 

email XXX 

b. Agent (if A ~) l,j!J,,..,.,,, ....... 

Name of Authorized Official (first>xxx (last) XXX 

Position XXX 

Company XXX 

Mailing 00 Address (number & street) XXX 

City XXX I State I Postal Code XXX 

e-mail XXX Telephone XXX (include area code) 

c. Joint Subr.nmn' (If 1.,\.lrlm.I.,II\!CII 

If you are submitting this notice as part of a joint submission, mark (X) I [ J 

Name of Authorized Official (first) (last) 

Position 

Company 

Mailing Address (number & street) 

City I State I Postal Code 

e-mail Telephone 

(include area code) 

2. Technical (In U.S.) C• •VIIIIUCIIUCII ·"· ·"· 

Name of Authorized Official (first>xxx (last) XXX 

Position XXX 

Company XXX lli] Mailing Address (number & street) XXX 

City XXX I State I Postal Code XXX 

e-mail XXX c' ~~~~d;·~~ea code) XXX 

If you have had a prenotice communication (PC) concerning XXX Mark (X} if none \...Uiiuu"'"'"" 

3. this notice and EPA assigned a PC Number to the notice, D enter the number. lli] 

If you. pr~vio_usly submitted an .... ~,.,, '"""v' u"""""."'u"v' 1 for the Mark (X) if none V\Jomu.,ntial XXX chemical substance covered by this notice, enter the 

4. exemption number assigned by EPA. If you previously D submitted a PMN for this substance enter the PMN number lli] 

o.aajnnorl by EPA (i.e. withdrawn or 

If you have submitted a notice of Bona fide intent to XXX Mark (X) if none vUr!UUCIIUCII 

5. manufacture or import for the chemical substance covered 

by this notice, enter the notice number assigned by EPA. D 

6. Type of Notice - Mark (X) 

Manufacture Only D Import Only [X] D 1. 2. 3. Both 

Binding Option D Binding Option D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 771D-25 

Page3 
1111111111111111111 

1. Class 1 or 2 chemical substances (for definitions of class 1 and class 

2 substances, see the Instructions Manual) 

a. Class of substance - Mark 

Class 1 

D 

SANITIZED SUBMISSION 

Class 2 CBI 

D D 

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

substances. For Class 1 substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name orCA 

Preferred Name must be which ever is based on current CA index nomenclature rules and 

Method 1 (CAS Inventory Expert Service - a copy of the 

Identification report obtained from the CAS Inventory Expert 

Enter Attachment filename for Part I, Section B, 1. c. 

d. 

e. 

Enter Attachment filename for Part I, Section B, 1. e. 

EPA FORM 7710-25 (Rev. 6-09) 

provide a correct D 

Replaces previous editions of EPA Form 7710-25 

Page 4 
111111111111111111111111111111111111111111111111111111111111 SANITIZED SUBMISSION 

PMN2010P4A PMN Page 4a 

For a class 2 substance- (1) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe 

the nature of the reaction or process. (3) Indicate the range of composition and the typical composition (where appropriate). 

e. (1) List the immediate precursor substance names with their respective CAS Registry Numbers. 

Enter Attachment filename for Part I, Section B, 1. e. (1) I e. (2) Describe the nature of the reaction or process. 

Enter Attachment filename for Part I, Section B, 1. e. (2) I 

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

Enter Attachment filename for Part I, Section B, 1. e. (3) I 

Confidential 

D 

D 

D 

D 

D 

D 

EPA FORM 7710-25 (Rev. 6-09) 

Page 5 

Repl~ces previous editions of EPA Form 7710-25 

-: ~ ,.; ~- ,:: J ,:_::1 -~ "·. 
1111111111111111111111111111111111111111111111111111111 SANITIZED SUBMISSION 

PMN2010P5 PMN P age 5 

Part I -- GENERAL INFORMATION - Continued 

Section B -- CHEMICAL IDENTITY 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 maximum weight percent of low molecular weight species (not including residual monomers, reactants, or solvents) [X] 

below 500 and below 1 ,000 absolute molecular weight of that composition. 

Describe the methods of measurement or the basis for your estimates: 

GPC D I Other (Specify Below) D I 

Specify Other: 

(i) lowest number average molecular (ii) maximum weight % below 500 molecular (iii) maximum weight % below 1000 molecular 

weight: weight: weight: 

XXX XXX XXX 

Enter Attachment filename for Part I, Section B, 2. a. I D 

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) of each monomer or other reactant used in the 

manufacture of the polymer. 

(2) - Mark (X) this column if entry in column (1) 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 of 

the 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 specific chemical name 

(1) 

XXX 

CAS Registry Number (1) XXX 

XXX 

CAS Registry Number (1) XXX 

XXX 

CAS Registry Number (1) XXX 

XXX 

CAS Registry Number (1) XXX 

XXX 

CAS Registry Number (1) I XXX 

Mark (X) this box if the data continues on the next page. 

EPA FORM 7710-25 (Rev. 6-09) 

Page 6 

Typical Include in Max 

CBI composition identity CBI residual CBI 

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

X XXX X XXX X 

X XXX X XXX X 

X XXX X XXX X 

X XXX X XXX X 

X XXX X XXX X 

IX] 

Replaces previous editions of EPA Form 7710-25 

:;t ... ,.; '....., ·,:_; ::)r· 
SANITIZED SUBMISSION 

PMN2010P5-1 PMN Page 5 (1) 

Part I --GENERAL INFORMATION - Continued 

Section B -- CHEMICAL IDENTITY 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. 

D Indicate maximum weight percent of low molecular weight species (not including residual monomers, reactants, or solvents) 

below 500 and below 1,000 absolute molecular weight of that composition. 

Describe the methods of measurement or the basis for your estimates: 

GPC D I Other (Specify Below) D I 

Specify Other: 

(i) lowest number average molecular (ii) maximum weight % below 500 molecular (iii) maximum weight % below 1000 molecular 

weight: weight: weight: 

Enter Attachment filename for Part I, Section B. 2. a. I D 

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) of each monomer or other reactant used in the 

manufacture of the polymer. 

(2) - Mark (X) this column if entry in column (1) 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 of 

the 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 specific chemical name Typical Include in Max 

CBI composition identity CBI residual CBI (1) (2) (3) (4) (5) (6) (7) 

XXX X XXX X XXX X 

CAS Registry Number (1) XXX 

XXX X XXX X XXX X 

CAS Registry Number (1) XXX 

XXX X XXX X XXX X 

CAS Registry Number (1) XXX 

CAS Registry Number (1) 

CAS Registry Number (1) 

Mark (X) this box if the data continues on the next page. D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 7 
1111111111111111111111111111111111111 1111111111111 SANITIZED SUBMISSION 

PMN2010P5A PMN Page Sa c. Please identify which method you used to develop or obtain the specified chemical identity information reported in this notice 

(check one). 

Method 1 (CAS Inventory Expert Service 

- a copy of the identification report obtained 

from CAS Inventory Expert Service must be 

submitted as an attachment to this notice) 

Enter Attachment filename for Part I, Section B, 2. c. 

IES Order 

Number 

152304 

Method 2 

(other source) D 

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

polymers. 

XXX 

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

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

ascertained. 

Enter Attachment filename for Part I, Section B, 2. e. I 

CBI 

D 

D 

D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 8 
SANITIZED SUBMISSION 

PMN P age 6 

Part I -- GENERAL INFORMATION -- Continued 

Section B -- CHEMICAL IDENTITY INFORMATION -- Continued 

3. Impurities 

(a) - Identify each impurity that may be reasonably anticipated to be present in the chemical substance as manufactured for commercial 

purpose. Provide the CAS Registry Number if available. If there are unidentified impurities, enter "unidentified." 

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

CAS Registry Maximum Conti- Impurity (a) Number Percent% 

(a) (b) dential 

Mark (X) this box if the data continues on the next page. D 

Enter Attachment filename for Part I, Section B, 3. I D 

4. Synonyms- Enter any chemical synonyms for the new chemical identified in subsection 1 or 2. 

XXX [Z] 

Enter Attachment filename for Part I, Section B, 4. I D 

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

XXX 

00 

Enter Attachment filename for Part I, Section B, 5. I D 

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

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

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

Perfluoroalkyl acrylate copolymer 

Enter Attachment filename for Part I, Section B, 6. I 

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

CAS Registry Number if available. 

Byproduct (1) CAS Registry Number Conti- . (2)- dential 

XXX XXX X 

Mark (X) this box if the data continues on the next page. D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 9 
11111111111111111111111111111111111111111 SANITIZED SUBMISSION 

PMN2010P7 PMN P age 7 

Part I -- GENERAL INFORMATION -- Continued 

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

The information on this page refers to consolidated chemical number(s): 01 02 03 04 Os Os 

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, if you 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 first 12-month production (kg/yr) Maximum 12-month production (kg/yr) Confidential Binding Option 

(100% new chemical substance basis) (100% new chemical substance basis) Mark (X) 

XXX XXX [X] 0 

Enter Attachment filename for Part I, Section C, 1. I CBI 0 

2. Use Information --You must make separate confidentiality claims for the description of the category of use, the percent of production volume devoted 

to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box nex1 to any item you claim as 

confidential. 

a. (1) --Describe each intended category of use of the new chemical substance by function and application. 

(2) --Mark (X) this column if entry column (1) is confidential business information (CBI). 

(3) --Indicate your willingness to have the information provided in column (1) binding. 

(4) --Estimate the percent of total production for the first three years devoted to each category of use. 

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

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

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

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

(8) --Indicate% of product volume expected for the listed "use" sectors. Mark more than one box if appropriate. Mark (X) to indicate your 

willingness to have the use type provided in (8) binding. 

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

Category of use ( 1) Binding Prod %in % of substance expected per use 

(by function and application i.e. a dispersive dye for CBI Option uction CBI Form- CBI (8) CBI 

finishing polyester fibers) Mark (X) % ulation Site- Con- Industrial Com- Binding 

(2) (3) (4) (5) (6) (7) limited sumer• mercial Option (9) 

Coating material for use in textiles and/or paper. XXX X XXX X XXX XXX XXX XXX X 

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

consumer products. In addition include estimates of the concentration of the new chemical substance as expected in consumer products and describe 

the chemical reactions by which this substance loses its identity in the consumer product. 

Mark (X) this box if the data continues on the nex1 page. 0 

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

description Read the Instruction Manual for examples of generic use descriptions. 

Not applicable. 

Enter Attachment filename for Part I, Section C, 2. b. I CBI 0 

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

data sheet, or other information which will be provided to any person who is reasonably likely to be exposed to this substance Binding Option 

regarding protective equipment or practices for the safe handing, transport, use, or disposal of the new substance. List in part Ill Mark (X) 

hazard information you include. 

Mark (X) this box if you attach hazard information. [XI 0 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 10 
SANITIZED SUBMISSION 

PMN2010P8 PMNP age 8 

Part II- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE 

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

any item you claim as confidential 

The information on pages 8 and Sa refer to consolidated chemical number(s): [ J 1 02 03 04 5 Os 

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

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

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

instructions manual 

1. Operation description Contia. Identity -- Enter the identity of the site at which the operation will occur. dential 

Name 

Site address (number and 

street) 0 

City County 

State ZIP code 

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

0 sites on a continuation sheet, and if any of the sites have significantly different production rates or 0 

operations, include all the information reguested in this section for those sites as attachments. ~ 

Mark (X) this box ifthe data continues on the next page. 0 

b. Type-- Manufacturing 0 Processing 0 Mark (X) Use 0 0 

c. Amount and Duration - Complete 1 or 2 as appropriate Contidential 

Maximum kg/batch 

(100% new chemical Hours/batch Batches/year 0 1. Batch substance) 

Maximum kg/day Hours/day Days/year ( 1 00% new chemical substance l 0 2. Continuous 

Mark (X) to indicate your willingness to 

d. Process description have your process description binding. 

~ .. (1) Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5 gallon 

pails, 55 gallon drum, rail car, tank truck, etc.). 

(2) Provide the identity, the approximate weight (by kg/day or kg/batch on a 100% new chemical substance basis), and entry point of all starting 

materials and feedstocks (including reactants. solvents, catalysts, etc.), and of all products, recycle streams, and wastes. Include cleaning 

chemicals (note frequency if not used daily or per batch.). 

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

releasing to two media at the same step, assign a second release number for the second medium. 

D 

See Attachments #5 and #6. 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 11 
1~111~ Ill~ 1111111111~11111111111111111 SANITIZED SUBMISSION 

PMN2010P8A PMNP age 8 a 

Confidential 

Diagram of the major unit operation steps. 

D 

Enter Attachment filename for Part II, Section A, 1. d. I [ J 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 12 

,., 
SANITIZED SUBMISSION 

PMN2010P9 PMNP age 9 

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

Section A - INDUSTRIAL SITES CONTROLLED BY THE SUBMITTER --Continued 

The Information on pages 9 and 9a refer to consolidated chemical number(s): [ J 1 l 2 03 [ ]4 Ds Ds 

2. Occupational Exposure-- You must make separate confidentiality claims for the description of worker activity, physical form of the new chemical 

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

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

substance. 

(2) Mark (X) this column if entry in column (1) is confidential business information (CBI). 

(3) -- Describe any protective equipment and engineering controls used to protect workers. 

(4) and (6) -- Indicate your willingness to have the information provided in column (3) or (5) binding. 

(5) ·- Indicate the physical form(s) of the new chemical substance (e.g., solid: crystal. granule, powder, or dust) and % new chemical substance (if 

part of a mixture) at the time of exposure. 

(7) -- Mark (X) this column if entries in columns (3) and (5) are confidential business information (CBI). 

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

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

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

(12) -Mark (X) this column if entries in columns (10) and (11) are confidential business information CBI). 

Worker activity Protective Equipment/ Binding Physical Binding ##of Maximum Duration (I.e., bag dumping, fiiUng CBI Option form( e) Option CBI Workers CBI CBI 

drums) Engineering Controls Mark (X) &%new Mark (X) Exposed eubstance Hrs!Day Days/Yr (12) (1) (2) (3) (4) (51 (6) (7) (8) {9) (10) (11) 

Mark (X) this box if the data continues on the next page. f I 

Enter Attachment filename for Part II, Section A on the bottom of page 9a. D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 13 
SANITIZED SUBMISSION 

PMN 20 10P9A PMNP age 9 a 

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

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

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

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

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

(4) -- Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, PO'TW, or 

other (specify)) to which the new substance will be released from that release point. 

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

For releases disposed of on land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. 

On a continuation sheet. for each site describe any additional disposal methods that will be used and whether the waste is subject to 

secondary or tertiary on-site treatment. b. Estimate the amount released to the environment after control technology (in kg/day). 

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

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

discharges or NPDES numbers of the PO'TW (Publicly Owned Treatment Works). Mark (X) if the PO'TW name or NPDES #is confidential 

business information (CBI). 

Release Amount of New Medium of release Control technology and efficiency (you may wish to 

Number Substance Released CBI e.g. Stack air optionally attach efficiency data) CBI 

Binding (1) (2a) (2b) (3) (4) (5a) Mark (5b) (6) 

(X} 

Mark (X) this box if the data continues on the next page. D 

(7) Marl< (X) the destination(s of releases to water. NPDES# CBI 

D POTW--provide 

name(s) D 

D Navigable waterway-

-provide name(s) D 

D Other--Specify D 

Enter Attachment filename for Part II, Section A. 1 D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 14 
11111~111~ 1111 SANITIZED SUBMISSION 

PMN2o1oP1o PMN Page 10 

Part II- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE- Continued 

Section B -- INDUSTRIAL SITES CONTROLLED BY OTHERS 

The information on pages 10 and 10a refer to consolidated chemical number(s): [ ] 1 [ ] 2 [ ] 3 Ds Ds 

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 of processing, or use operation involving the new chemical substance. If the same operation is performed at 

more than one site describe the typical operation common to these sites. Identify additional sites on a continuation sheet. 

1(a). Operation Description-- To claim information in this section as confidential, bracket (e.g.{}) the specific information that you claim as 

confidential. 

(1) Diagram the major unit operation steps and chemical conversions, including interim storage and transport containers (specify- e.g. 5 gallon 

pails, 55 gallon drums, rail cars, tank trucks, etc). On the diagram. identify by fetter and briefly describe each worker activity. 

(2) •• Either in the diagram or in the text field 1 (b) below, provide the identity, the approximate weight (by kg/day or kg/batch, on an 100% new 

chemical substance basis), and entry point of all feedstocks (including reactants. solvents and catalysts, etc) and all products. recycle 

streams, and wastes. Include cleaning chemicals (note frequency if not used daily or per batch). 

(3) -- Either in the diagram or in the text field 1 (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 of these sites on a continuation sheet): ...... I Number of Sites I I Confidential 

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

See Attachment #9. 

Enter Attachment filename for Part II, Section Bon the bottom of page 10a. I D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 15 -~ 

•.. 
Ill~ I~ Ill~ 11~1 ~~I IIIII SANITIZED SUBMISSION 

PMN2010P10-1 

Continuation Sheet 

ID I P10SB1 (a)(4)1 I Field I Part II, Section B, 1(a)(4). Operation Site Locations 

XXX 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 16 
~1111~ 111111111 1111 SANITIZED SUBMISSION 

PMN201 OP10 A PMNP age 10 a 

2. Worker Exposure/Environmental Release 

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

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

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

(6) -- Describe physical form of exposure and % new chemical substance (if in mixture), and any protective equipment and engineering controls, if 

any, used to protect workers. 

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

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

(1 0) -- Estimate the amount of the new substance released (a) directly to the environment or (b) into control technology to the environment (in 

kg/day or kg/batch). 

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

POTW, or other (specify) and control technology, if any, that will be used to limit the release of the new substance to the environment. 

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

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

Letter #of Duration of Protective Equlp./Englneering Controls/Physical %new %in of Workers CBI CBI CBI 

Activity Exposed Exposure Form substance Formulation 

(1) (2) (3) (4a) (4b) (5) (6) (6) (7) (8) 

XXX XXX X XXX XXX X XXX XXX XXX X 

XXX XXX X XXX XXX X XXX XXX XXX X 

XXX XXX X XXX XXX X XXX XXX XXX X 

Release Amount of New Substance Released CBI Media of Release & Control Technology CBI Number 

(9) (108) (10b) (11) (12) (13) 

XXX XXX XXX X XXX X 

XXX XXX XXX X XXX X 

XXX XXX XXX X XXX X 

Mark (X) this box if the data continues on the next page. [ J <<' 

' 

(14) Byproducts: I XXX I (15) CBI 00 

Enter Attachment filename for Part II, Section B. D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 17 l~ k) 

•,.J _:; 
111111111 SANITIZED SUBMISSION 

PMN2010P11 PMN Page 11 

OPTIONAL POLLUTION PREVENTION INFORMATION 

To claim information in the following section as confidential, bracket (e.g. 0) the specific information that you claim as confidentiaL 

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

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

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

substance. Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological modification. process and 

procedure modification, product reformulation, and/or raw materials substitution. 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. Quantitative or qualitative 

descriptions of pollution prevention, source reduction and recycling should emphasize potential risk reduction in addition to compliance with existing 

regulatory requirements. 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 media (e.g .• air to water) or nonenvironmental areas (e.g., occupational or consumer exposure). To the 

extent known. information about the technology being replaced will assist EPA in its relative risk determination. In addition. information on the 

relative cost or performance characteristics of the PMN substance to potential alternatives may be provided. 

Describe the expected net benefits, such as 

(1} an overall reduction in risk to human health or the environment; 

(2) a reduction in the generation of waste materials through recycling, source reduction or other means; 

(3) a reduction in the use of hazardous starting materials, reagents, or feedstocks; 

(4) a reduction in potential toxicity. human exposure and/or environmental release; or 

(5) the extent to which the new chemical substance may be a substitute for an existing substance that poses a greater overall risk to human 

health or the environment. 

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 guidance and examples. 

The new chemical substance is expected to offer the following Pollution Prevention benefits: 

{XXX} 

Enter Attachment filename for Pollution Prevention Page 11 I D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 18 
1111111111111111 SANITIZED SUBMISSION 

PMN2010P12 PMNP age 12 

Part Ill -- LIST OF ATTACHMENTS 

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

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

to which it relates, if appropriate. Number consecutively the pages of any paper attachments. In the Number of Pages column 

below, enter the inclusive page numbers of each attachment for paper submissions or enter the total number of pages for each 

attachment for electronic submissions. Electronic attachments can be identified by filename. 

Mark (X) the "Confidential" box next to any attachment name or filename you claim as confidential. Read the Instructions Manual 

for guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy of the 

notice form a sanitized version of any attachment in which you claim information as confidential. 

Nu Associated 

# Attachment Name Attachment Filename ofPag PMN Section CBI 

Number 

001 Material Safety Data Sheet 7 

002 Summary of Information on the New Chemical 1 

Substance 

003 CAS Inventory Expert Service Report and 4 PU, Sec. B. 2c. I Pt.l, Sec. B. 2e. 

Chemical Structure of the New Chemical 

004 Impurities Analysis on the New Chemical 2 Pt.l, Sec.B, 3. 

Substance 

005 Manufacturing and Processing Information on 2 

the New Chemical Substance at Industrial Sites 

006 Manufacturing and Processing Information on 1 

the New Chemical Substance at Industrial Sites 

007 Worker/Occupational Exposure Information on 1 

the New Chemical Substance at Industrial Sites 

008 Environmental Release and Disposal Information 1 

on the New Chemical Substance at Industrial 

009 Processing Information on the New Chemical 2 Pt.2, Sec.B, 1a. 

Substance at Industrial Sites Controlled by 

010 Worker/Occupational Exposure and 1 Pt.2, Sec.B, End 

Environmental Release Information on the New 

011 Relevant Data on Analogous Chemical 5 

Substances 

012 Physical and Chemical Properties Data on the 15 PU, Sec.B, 2a. 

013 Health Effects Data on the New Chemical 19 

Substance- Acute Oral Toxicity Test 

014 Health Effects Data on the New Chemical 33 

Substance- Skin Irritation Test 

015 Health Effects Data on the New Chemical 32 

016 Health Effects Data on the New Chemical 34 

Substance- Ames (Bacterial Reverse Mutation) 

Mark (X) this box if the data continues on the next page. D 

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Form 7710-25 

Page 19 
lllllmllllllll SANITIZED SUBMISSION 

PMN2010P13 PMNP age 13 

PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

The information on this page refers to chemical number(s): 01 [ J 2 l J 3 04 l J 5 06 

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 

I notice. Identify the property measured, the value of the property, the units in which the property is measured (as necessary), and whether or not the 

property is claimed as confidential. Give the attachment number (found on page 12) in column (b). The physical state of the neat substance should be 

provided. These measured properties should be for the neat (100% pure) chemical substance. Properties that are measured for mixtures or 

formulations should be so noted(% PMN substance in_). You are not required to submit this worksheet; however, EPA strongly recommends that 

you do so, as it will simplify the review and ensure that confidential information is properly protected. You should submit this worksheet as a 

supplement to your submission of lest data. This worksheet is not a substitute for submission of test data. 

Property Unit I Mark X if Attachment Value Measured CBI 

Number or Estimate Mark (X) (a) Provided (b) (C) (MorE) (d) 

0 (solid) I (liquid) (gas) 

Physical state of neat substance 0 [X] 0 Measured 

Vapor Pressure @ oc 0 Torr Temperature 

Density/relative density 0 gfcm3 

Solubility 

@ Temperature ·c 0 g/L 

Solvent 

Solubility in Water @ ·c D giL Temperature 

Melting Temperature D •c 

Boiling I Sublimation Torr 0 ·c 

temperature @ 

Spectra 0 

Dissociation constant 0 

Octanol/ water partition coefficient 0 

Henry's Law constant 0 

Volatilization from water D 

Volatilization from soil 0 

pH@ concentration 0 

Flammability 0 

Explodability 0 

Adsorption I Coefficient 0 

Particle Size Distribution D 

' 

Other - Specify D 

Page 20 
ATTACHMENT HEADER SHEET 

Attachment Number 001 

Attachment Name 

Material Safety Data Sheet 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP1 011 05088166808 
TS-CMTP01 

Attachment #1 

Material Safety Data Sheet 

The following page provides a representative material safety data sheet (MSDS) on the flnal 

formulated product containing the new chemical substance. There are expected to be various 

formulations containing the new chemical substance. However, the MSDSs are all expected 

to be similar. 
[ J MATERIAL SAFETY DATA SHEET 

[ J Page 1 

[ J 

I Section 01 - Product Information 

Revision Date: 09/1 0/2010 

Date of printing :09/10/2010 

Identification of the 

company: [ J 

[ Information of the substance/pjparation: 

Emergency tel. number: +1 800-424-9300 CHEMTREC 

Trade name: 

Material number: [ 

Chemical family: 

Primary product use: 

J 

Dispersion of 

perfluoroalkyl acrylic copolymerisate 

-preparation 

Intermediate product 

I Section 02 - Composition information on hazardous ingredients 

Hazardous ingredients: 

Component CAS-no. (Trade secret 

no.} 

Dipropylene Glycol 25265-71-8 

Ethoxylated Alcohols 

I Section 03 - Hazards identification 

Expected Route of entry: 

Inhalation: 

Skin contact: 

Eye contact: 

Ingestion: 

Skin absorption: 

Health effects of exposure: 

Propanol, oxybis- ( 25265-71-8) 

no 

yes 

yes 

yes 

no 

Concentration 

< 10% 

<5% 

Dipropylene Glycol: LD50 (oral)(rat): 14.85 g/kg; LD50 (dermal){rat) : > 20 mllkg. Has a low 

order of toxicity following acute ingestion, but little data are available. Based on animal data, it 

is estimated that the acute lethal dose in adults is greater than one pint. Liver and kidney 

damage were observed in animals. Minor corneal irritation was reported in animals. May 

cause mild skin irritation. Central nervous system depression was reported in dogs given 6 

mllkg intravenously. No toxicity was noted when 5 mllkg was given orally. Extremely large 

amounts may produce liver and kidney injury. No untoward effects have been reported from 

the use of dipropylene glycol nor would any be expected based on human experience. 
[ J 

MATERIAL SAFETY DATA SHEET 

[ J Page 2 

[ J 

Ethoxylated Alcohol 

Revision Date: 09/10/2010 

Date of printing :09/10/2010 

Some ethoxylated alcohols have been known to cause eye and skin irritation. Ingestion may 

cause gastrointestinal irritation, diarrhea, nausea and vomiting. 

Known effects on other illnesses: No known effects on other illnesses for this 

product. 

listed carcinogen: lARC: NO NTP: NO OSHA: NO 

Other: No 

HMIS: 

Health: 1* Flammability: 1 Reactivity: 0 Personal protection: C 

I Section 04 - First aid measures 

After inhalation: 

Get victim to fresh air. Give artificial respiration or oxygen if breathing has stopped. Get 

prompt medical attention. Do not give fluids if victim is unconscious. 

After contact with skin: 

Wash thoroughly with soap and water for 15 minutes. If skin irritation occurs, seek medical 

attention. 

After contact with eyes: 

Immediately flush eyes with running water for at least 15 minutes, occasionally lifting the 

upper and lower eyelids. Seek prompt medical attention if redness or irritation occurs. 

After ingestion: 

Seek medical attention immediately. 

Advice to doctor I Treatment: 

None known. 

I Section 05 - Fire fighting measures 

Flashpoint: ca. 212 oF 

Hazardous combustion products: 

In case of fires, hazardous combustion gases are formed: 

carbon oxides 

nitrogen oxides 

Hydrogen fluoride (HF) 

Extinguishing media: 

Special fire fighting procedure: 

water spray jet 

foam 

dry powder 

carbon dioxide 

Use NIOSH/MSHA approved self-contained breathing apparatus and other proper protective 

equipment where this chemical is involved in a fire. Use water spray to cool containers. 

Unusual fire and explosion 

hazards: 

Emits toxic fumes under fire conditions. 
MATERIAL SAFETY DATA SHEET 

[ J 

[ J Page 3 

[ J 

I Section 06 - Accidental release measures 

Steps to be taken in case of spill or leak: 

Revision Date: 09/10/2010 

Date of printing :09/10/2010 

Contain spill. Ensure adequate ventilation and wear appropriate personal protective 

equipment. Collect onto inert absorbent. Place in sealable container. Do not allow to 

contaminate water sources or sewers. 

I Section 07 - Handling and storage 

Advice on safe handling: 

Avoid formation of aerosols. 

Avoid breathing vapors and contact with skin, eyes. and clothing. 

Further info on storage conditions: 

Store in original container. 

Store in a cool, dry, well-ventilated area. Keep container sealed when not in use. 

Keep from freezing. 

I Section 08 - Exposure controls I personal protection 

Respiratory protection: If airborne concentrations pose a health hazard, become 

irritating or exceed recommended limits, use a NIOSH approved 

respirator in accordance with OSHA respiratory protection 

requirements under 29 CFR 1910.134 

Hand protection: Butyl Rubber, PVC Or Neoprene. 

Eye protection: Safety glasses or chemical splash goggles. 

Other protective equipment: Clothing suitable to prevent skin contact. 

Advice on system design: Local ventilation recommended - mechanical ventilation may 

be used. 

IDLH: 

Not Determined 

I Section 09 - Physical and chemical properties 

Form: 

Color: 

Odor: 

pH: 

Solubility in water: 

Density: 

Boiling point : 

Dispersion 

milky white to yellowish 

characteristic 

ca.4 

miscible 

1.1 g/cm3 

ca. 212 °F 
MATERIAL SAFETY DATA SHEET 

[ 

[ J Page 4 

[ J 

I Section 10 ·Stability and reactivitY 

Chemical stability: Stable. 

Hazardous Polymerization: Will not occur. 

Conditions to avoid: None known. 

Conditions to avoid: Strong oxidizing agents. 

I Section 11 -Toxicological information 

Product information: 

LD50 > 2,000 mg/kg (rat) 

Revision Date: 09/10/2010 

Date of printing :09/10/201 0 

II Acute oral toxic~ Directive 1999/45/EC, Information based on the main 

component. 

Skin irritation: 

Eye irritation: 

non-irritant (rabbit) 

Method: 1999/45/EC 

non-irritant (rabbit eye) 

Method: 1999/45/EC 

I Section 12 - Ecological information 

Product information: 

Fish toxicity: 

Bacteria toxicity: 

Chemical oxygen demand 

(COD): 

Remarks: 

LC50 > 100 mg/1 (96 h, zebra fish (brachydanio rerio)) 

Method: evaluated 

IC50 > 100 mg/1 (activated sludge) 

Method: evaluated 

550 - 600 mg/1 

Method: estimated from the components 

No ecological testing was carried out on the preparation 

Ecological data are determined by analogy 

I Section 13 - Disposal considerations 

Waste disposal information: 

Consult local, state, and federal regulations. 

RCRA hazardous waste: 

No -- Not as sold. 

RCRA number: NONE 

I Section 14- Transport information 

DOT 

lATA 

not restricted 

not restricted 

J 
MATERIAL SAFETY DATA SHEET 

[ J 

[ J Page 5 

J 

IMDG not restricted 

I Section 15 - Regulatory information 

TSCA Status: 

Revision Date: 09/10/2010 

Date of printing :09/10/2010 

A component of this product is not listed on the TSCA Inventory. The component meets the 

definition of polymer as found in 40 CFR 723.250 and thereby is exempt from certain 

Premanufacture Notification requirements of TSCA. 

SARA (section 311/312): 

Reactive hazard: no 

Pressure hazard: no 

Fire hazard: no 

Immediate/acute: yes 

Delayed/chronic: yes 

SARA 313 information: 

This product does not contain any toxic chemical listed under Section 313 of the Emergency 

Planning and Community Right-To-Know Act of 1986. 

Clean Water Act: 

Contains no known priority pollutants at concentrations greater than 0.1%. 

Volatile organic compounds VOC: 

Remarks: Not tested 

FDA: 

Permitted for Use per 

Section: 

I Section 16 - Other information 

Other precautions: 

NONE 

This product is not registered with the FDA. 

Avoid inhalation of this product in mist or aerosol forms. 

Label information: 

CAUTION! 

MAY CAUSE GASTROINTESTINAL DISTURBANCES IF INGESTED. REPEATED 

EXPOSURES MAY RESULT IN LIVER OR KIDNEY DAMAGE. 

Avoid breathing fumes, vapors, mists or spray. Avoid contact with skin and eyes. Use 

with adequate ventilation and/or respiratory protection. Wash thoroughly after handling. 

Keep container closed when not in use. Wear proper protective equipment when 

handling. 

Skin contact: wash thoroughly with soap and water for 15 minutes. If skin irritation 

occurs, seek medical attention. Wash contaminated clothing before reuse. Eye contact: 

flush with water for at least 15 minutes while holding eyelids open. Seek immediate 

medical attention. Ingestion: seek medical attention immediately. Inhalation: remove to 
MATERIAL SAFETY DATA SHEET 

[ 

[ J Page 6 

l J Revision Date: 09110/2010 

Date of printing :09/10/2010 

fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. 

Seek medical attention if respiratory irritation continues. 

NFPA: 

Health: 1 Flammability: 1 Reactivity: 0 

This information is supplied under the OSHA Hazard Communication Standard, 29 CFR 

1910.1200, and is offered in good faith based on data available to us that we believe to be 

true and accurate. The recommended industrial hygiene and safe handling procedures are 

believed to be generally applicable to the material. However, each user should review 

these recommendations in the specific context of the intended use and determine whether 

they are appropriate for that use. No warranty, express or implied, is made regarding the 

accuracy of this data, the hazards connected with the use of the material, or the results to 

be obtained from the use thereof. We assume no responsibility for damage or injury from 

the use of the product described herein. Data provided here are typical and not intended 

for use as product specifications. 

J 
ATTACHMENT HEADER SHEET 

Attachment Number 002 

Attachment Name 

Summary of Information on the New Chemical Substance 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #2 

Summary of Infonnation on the New Chemical Substance 

I 

'------------
ATIACHMENT HEADER SHEET 

Attachment Number 003 

Attachment Name 

CAS Inventory Expert Service Report and Chemical Structure of the New Chemical Substance 

Associated PMN Section Number 

Pt.l, Sec.B, 2c. I Pt.l, Sec.B, 2e. 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #3 

Pt. 1, Sec. B, 2 

CAS Invent01y Expert Service Report and Chemical Structure of the New Chemical 

Substance 
Inventory Expert Service 

A division of the American Chemical Society Phone: 800-631-1884, 614-447-3870 

Fax: 614-447-3747 

E-mail: answers@cas.org 

Web: www.cas.org/products/dientl 

[ 

INVENTORY EXPERT SERVICE REPORT 

IES-ORDER NUMBER: 152304 

REGISTRY NUMBER:[ J 

CA INDEX NAME 

Please print the above CA Index Name on the appropriate page of your PMN. 

CAS 

If this box is checked, CAS has made correction(s) marked in red to 

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

submitting it to the EPA. 

2540 Olentangy River Road P.O. Box 3343 Columbus, Ohio 43210-0334 USA 

J 


ATTACHMENT HEADER SHEET 

Attachment Number 004 

Attachment Name 

Impurities Analysis on the New Chemical Substance 

Associated PMN Section Number 

Pt.l, Sec.B, 3. 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #4 

Pt. 1, Sec. B, 3 

Impurities Analysis on the New Chemical Substance 

Impurity CASRN %(max.) Method of Comments 

Detection 
TS-CMTPOl 
ATTACHMENT HEADER SHEET 

Attachment Number 005 

Attachment Name 

Manufacturing and Processing Information on the New Chemical Substance at Industrial Sites Controlled by the 

Submitter- Manufacture 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTP01 

Attachment #5 

Pt. 2, Sec. A, td 

Manufacturing and Processing Information on the New Chemical Substance at 

Industrial Sites Controlled by the Submitter- Manufacture 
~S-CMTP01 

L 

Attachment #5 

Manufacturing and Processing Information on the New Chemical 

Substance at Industrial Sites Controlled bv the Submitter- Manufacture 
ATTACHMENT HEADER SHEET 

Attachment Number 006 

Attachment Name 

Manufacturing and Processing Information on the New Chemical Substance at Industrial Sites Controlled by the 

Submitter - Processing 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #6 

Pt. 2, Sec. A, ld 

Manufacturing and Processing Information on the New Chemical Substance at 

Industn'al Sites ControUed by the Submitter- Processing 
ATTACHMENT HEADER SHEET 

Attachment Number 007 

Attachment Name 

Worker/Occupational Exposure Information on the New Chemical Substance at Industrial Sites Controlled by the 

Submitter- Processing 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTP01 

Attachment #7 

Pt. 2, Sec. A, 2 

Worker/Occupational Exposure Information on the New Chemical Substance at 

Industrial Sites ControUed by the Submitter- Processing 
ATTACHMENT HEADER SHEET 

Attachment Number 008 

Attachment Name 

Environmental Release and Disposal Information on the New Chemical Substance at Industrial Sites Controlled by 

the Submitter- Processing 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOt 

Attachment #8 

Pt. 2, Sec. A, 3 

Environmental Release and Disposal Information on the New Chemical Substance 

at Industrial Sites ControUed by the Submitter- Processing 
ATTACHMENT HEADER SHEET 

Attachment Number 009 

Attachment Name 

Processing Information on the New Chemical Substance at Industrial Sites Controlled by Others 

Associated PMN Section Number 

Pt.2, Sec.B, 1a. 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTP01 

Attachment #9 

Pt. 2, Sec. B, la 

Processing Infonnation on the New Chemical Substance at Industrial Sites 

ControUed by Others 

: <f\ _; 
TS-CMTP01 Attachment 9 

Processing Information on the New Chemical 

Substance at Industrial Sites Controlled bv Others 
ATTACHMENT HEADER SHEET 

Attachment Number 010 

Attachment Name 

Worker/Occupational Exposure and Environmental Release Information on the New Chemical Substance at 

Industrial Sites Controlled by Others 

Associated PMN Section Number 

Pt.2, Sec.B, End 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #10 

Pt. 2, Sec. B, End 

Worker/Occupational Exposure and Environmental Release Information on the 

New Chemical Substance at Industrial Sites Controlled by Others 
ATTACHMENT HEADER SHEET 

Attachment Number 011 

Attachment Name 

Relevant Data on Analogous Chemical Substances 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOl 

Attachment #11 

Relevant Data on Analogous Chemical Substances 

2 

I 
TS-CMTP01 

4 

6 

6 
TS-CMTPOl 

B 

9 

10 

11 
TS-CMTPOl 

12 

14 

15 

16 

12 

13 

14 

15 

16 
TS-CMTPOl 

17 

18 

19 

20 

17 

18 

19 

20 
ATTACHMENT HEADER SHEET 

Attachment Number 012 

Attachment Name 

Physical and Chemical Properties Data on the New Chemical Substance - Molecular Weight Data 

Associated PMN Section Number 

Pt.l, Sec.B, 2a. 

Does not contain CBI 

Report Number 

CMTP101105088166808 
TS-CMTPOt 

Attachment #12 

Physical and Chemical Properties Data on the New Chemical Substance 

Molecular Weight Data 





1-








ATTACHMENT HEADER SHEET 

Attachment Number 013 

Attachment Name 

Health Effects Data on the New Chemical Substance- Acute Oral Toxicity Test 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP1 01105088166808 
TS-CMTPOl 

Attachment #13 

Health Effects Data on the New Chemical Substance 

Acute Oral Toxicity Test 
L 

REPORT 

Acute Oral Toxicity Study in Rats 

Stutly Director: 

Test Facility: 

Dr. C. Simon 

RCC Ltd 

Wolferstrasse 4 

J 

4414 Fiillinsdorf I Switzerland 

[ 

Study Identification: RCC Study Number B46462 

Version: Final 

Study Cmnplt-tion Date: 05-NOV-2007 

Page I of 18 

J 
[ RCC Study ~tun!K'r 1Ht•46l Report Page 2 

CONTENTS 

C()N'IFN'i'S .,.,, .. , ........................................................................................................................... 2 

TABLr: LIS·1· ... " ............................................................................................................................. 2 

SICiN AI'UH ES ................................................................................................................................ 3 

STATEMI.NI' OF ('0MPLIANCE ................................................................................................ 4 

QUALITY ASSURANCE .............................................................................................................. 5 

PREFACE ....................................................................................................................................... 6 

(ienerallnfonnation ............................................................................................................... 6 

Responsibilities ...................................................................................................................... 6 

Schedule ................................................................................................................................. 6 

Ciuichdincs .............................................................................................................................. 7 

Ani1nal Welt(lrc ...................................................................................................................... 7 

Archiving ............................................................................................................................... 7 

SUI\!Iiv1ARY ........................................................................................................................... 8 

2 l-·( JNl.'l.LJSION, ................. ~~························ .. ··~·~~···············~·····~·~········~~··~······~······················· 9 

3 PURP()SF ............................................................................................................................ 10 

4 !VIi\ I'ERIALS AND METHODS ......................................................................................... 11 

4. I Test Systen1 ............................................................................................................ II 

4.2 llusbanury .............................................................................................................. II 

4 . .1 Test lten1 ................................................................................................................ 12 

4.3.1 Test ltenl .................................................................................................. 12 

4 .. ~.2 Vehicle ..................................................................................................... 12 

4.4 T>ose Fonnulation .................................................................................................. 13 

4.5 Tn:at1nent ............................................................................................................... 13 

4.6 ()bservations .......................................................................................................... 14 

4.7 Pathology ............................................................................................................... 14 

4.7.1 Necropsy .................................................................................................. 14 

'l.X Statistical Analysis ................................................................................................ 14 

4.() [)ata Con1pilation ................................................................................................... 14 

5 RESULTS ............................................................................................................................ 15 

5.1 Mortality ................................................................................................................ 1 S 

5.2 Clinical Signs ......................................................................................................... IS 

5.3 Body Weights ........................................................................................................ 15 

SA Macroscopic Findings ............................................................................................ 15 

'> ') Median Lethal Dose ............................................................................................... 15 

6 INDIVIDUAl. FINDINGS .................................................................................................. 16 

7 CILP-CERTIFlCA TE ........................................................................................................... 18 

TABLE LIST 

Table I Mortality I Clinical Signs ............................................................................................ 16 

Table 2 Body Weights .............................................................................................................. 16 

Table 3 Macroscopic Findings ................................................................................................. 17 
[ 

RCC Study Number 1346462 

1. Report Page 3 

SIGNATURES 

Study Director: Dr. C. Simon 

Management: (f'} Dr. H. Fankhauser 

~ ·! ' 'i ' 

............. ..~ ... \1-::,.'fr!.l, Date: .. \ lt 

~ \.," 1 I . . \ ) 1 . J v lt~,, V\.• ~.v-~ . l L. .\...... ·} 
[ RCC Study Number 846462 Report Page4 

GOOD LAHORA TORY PRACTICE 

STATEMENT OF COMPLIANCE 

RCC Study Number: B46462 

Test Item: [ J 

Study Director: Dr. C. Simon 

Study Title: Acute Oral Toxicity Study in Rats 

The stability of the test item dilutions under the test conditions is unknown. The formulation 

trials were performed before the study initiation date. Therefore, they are excluded from this 

statement. 

This study has been performed in compliance with the Swiss Ordinance relating to Good 

Laboratory Practice, adopted May 18th, 2005 [RS 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

1997 by decision of the OECD Council [C(97) 186/Final]. 

There were no circumstances that may have affected the quality or integrity of the data. 

Study Director: Dr. C. Simon 

' 1/ 
[ RCC Study Number B46462 Report Page 5 

QUALITY ASSURANCE 

R CC Ltd, W olferstrasse 4, 4414 Fiillinsdorf I Switzerland 

STATEMENT 

RCC Study Number: B46462 

Test ltem: [ ;J 

Study Director: Dr. C. Simon 

Study Title: Acute Oral Toxicity Study in Rats 

The general facilities and activities are inspected periodically and the results are reported to the 

responsible person and the management. 

Study procedures, with exception of the formulation trials, were periodically audited. The study 

plan and this report were audited by the Quality Assurance. The dates are given below. 

Dates of Reports to 

Dates and Types of QA Inspections the Study Director 

and Test Facility 

Management 

30-AUG-2007 Study Plan 30-AUG-2007 

22-AUG-2007 Process Based 22-AUG-2007 

(Test System, Test Item, Raw Data, Dose 

Preparation, Treatment) 

25-0CT-2007 Report 25-0CT -2007 

This statement also confirms that this final report reflects the raw data. 

Quality Assurance: S. van Dongen 

·~ '\ ' ' 

...... :' ...... ~ .. >: .:·.~ ... :. /~.:~~:~.~~. ~.~.~ .... Dall': . . _ , .. > 1 ·, . • • . • 

\,_,' '-· ~. \ » •• 1._'-i.t 

(:"-II 

;J '. 
[ 

RCC Study Numb-:r B4Mb2 

PREFACE 

General Information 

Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Tedmical Coordinator: 

He<id of Lead QA: 

Schedule 

Experimental Starting Date: 

Experimental Completion Date 

Delivery of Animals: 

A eel irnat izat ion: 

Treatment: 

Observation: 

[ 

[ 

[ 

Report 

RCC Ltd 

Wolferstrasse 4 

4414 Flillinsdorf I Switzerland 

Dr. C. Simon 

G. Arcelin 

F. Frickert I M. Bernstein 

I. Wuthrich 

04~SEP-2007 

27-SEP-2007 

04-SEP-2007 (females. 2000 mg/kg) 

06-SEP-2007 (females. 2000 mg/kg) 

Page6 

J 

J 

04-SEP-2007 to I 0-SEP-2007 (females, 2000 mg/kg) 

06-SEP-2007 to 12-SEP-2007 (females, 2000 mg/kg) 

ll-SEP-2007 (females, 2000 mglkg) 

13-SEP-2007 (females, 2000 mg/kg) 

04-SEP-2007 to 25-SEP-2007 (females, 2000 mg/kg) 

06-SEP-2007 to 27-SEP-2007 (females, 2000 mg/kg) 
l 

RCC S1udy Numh.:r 046462 Report (>age 7 

(;uidelines 

The study procedures described in this report meet or exceed the requirements of the following 

guidelint:"s: 

• Directive 2004/73/EC, B. I tris "Acute Oral Toxicity-Acute Toxic Class Method", April 

29, 2004. 

• OFCD Guidelines for the Testing of Chemicals, Number 423 "Acute Oral ToxicityA~:utc Toxic Class Method", adopted 17th December 200 I. 

Animal Welfare 

This study was perHmned in an AAALAC-approved laboratory in accordance with the Swiss 

Animal Protection Law under license no. 34. 

Archiving 

RCC Ltd (CII-4452 ltingcn I Switzerland) will retain the study plan, raw data, a sample of test 

item(s) and the linal repo11 of the present study for at least ten years. No data will be discarded 

without the Sponsor's written consent. 
Pag~ 8 

I SUMMARY 

Two groups. each of three female HanRcc:WlST (SPF) rats, were treated witl{ 

]by oral gavage administration at a dosage of 2000 mg/kg body weight. 

The tt'st item was diluted in vehicle (corn oil) at a concentration of0.2 g/mL and administered at 

a dosing volume of I 0 mL!kg. 

The animals were examined daily during the acclimatization period and mortality, viability and 

dinical ~igns were r~:cordcd. All animals were examined for clinical signs within the first 30 

mi11nles and approximately I, 2, 3 and S hours after treatment on day I and once daily during test 

days 2-1 ). tvlortality/viability was recorded at approximately 30 minutes, I. 2. 3 and 5 hours 

aftt·r administration on test day I (with the clinical signs) and twice daily during days 2-15. Body 

wei~.hts Wt~n: recorded on day l (prior to administration) and on days 8 and 15. All animals were 

necropsi~~d and examined macroscopically. 

A II animals survived until the end of the study period. 

No clinical signs were observed during the course of the study. 

The body weight of the animals was within the range commonly recorded tor this strain and age. 

No macroscopic findings were recorded at necropsy. 
Report Pag~: 9 

2 CONCLUSION 

Tile median lethal Jose o{ Jafter single oral administration 

to female rats, obscrwd over a period of 14 days is: 

LDSO (temale rat): greater than 2000 mg/kg body weight 

··\ -, 

.j'',v -~· ;;_: 
Page 10 

3 PlJRPOSE 

The purpose of this study was to assess the acute toxicity o~ ] 

when adm in istcrl~d hy a single oral gavage to rats. followed by an observation period of 14 days. 

This study provides inf(mnation for both hazard assessment and hazard classification purposes. 
[ R('C Sl.udv Number B.to462 Report Page 11 

4 MATERIALS AND METHODS 

4.1 Test System 

Animals: 

Rationale: 

Some~· 

Number of Animals per Group: 

Total number of Animals: 

Age when treated: 

I dent i fi~.:ation: 

Randomization: 

A eel i matiz.ation: 

4.2 Husbandry 

Room Number: 

Conditions: 

Accommodation: 

Rat, HanRcc: WIST(SPF) 

Recognized by international guidelines as a 

recommended test system. 

RCC Ltd 

Laboratory Animal Services 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

3 females 

6 females 

II weeks 

Unique cage number and corresponding color-coded 

spots on the tail. The animals were marked at 

acclimatization start. 

Selected by hand at time of delivery. 

No computer generated randomization program. 

Under laboratory conditions, after health 

examination. Only animals without any visible signs 

of illness were used for the study. 

0 I 05 I RCC Ltd, Fullinsdorf 

Standard Laboratory Conditions. Air-conditioned 

with 10-15 air changes per hour, and continuously 

monitored environment with ranges for room 

temperature 22 ± 3 oc and for relative humidity 

between 30-70 % (values above 70 % during 

cleaning process possible), automatically controlled 

light cycle of 12 hours light and 12 hours dark, 

music during the daytime light period. 

In groups of three in Makrolon type-4 cages with 

wire mesh tops and standard softwood bedding 

('Lignocel' Schill AG, CH-4132 

Muttcnz/Switzerland). 
[ 

RCC Study Number fHMfi2 

Diet: 

Report Page 12 

Pelleted standard Provimi Kliba 3433 rat/mouse 

maintenance diet, batch no. 23/07 (Provimi Kliba 

AG, CH-4303 Kaiseraugst/Switzerland) ad libitum. 

Results of analyses for contaminants are archived at 

RCC Ltd. 

Water: Community tap water from Fiillinsdorf ad libitum. 

4.3 Test Item 

4.3.1 Tt•st Item 

Results of bacteriological, chemical and contaminant 

analyses are archived at RCC Ltd. 

The following information was provided by the sponsor: 

Identification 

Synonym 

Description 

Batch number 

Purity I Formulation 

Stability of test item 

Expiry date 

Stability oftest item dilution 

Storage coml it ions 

Safetv precautions 

4.3.2 V chicle 

[ 

[ 

Solid; light brown 

] 

1 00% active polymer 

Stable under storage conditions. 

31-MAR-2008 

J 

Unknown in corn oil: is excluded fi·om the statement of 

compliance. 

At room temperature (range of20 :1 5 °C), light protected. 

Routine hygienic procedures were used to ensure the health 

and satcty ofthe personnel. 

Corn oil was found to be a suitable vehicle. The vehicle was chosen after a non-GLP solubility 

trial which was performed before the study initiation date. This formulation trial is excluded 

from the GLP statement of compliance. 
[ RCC Sludy Numher B4M62 Page 13 

The f(JIIowing information was provided by RCC Ltd: 

ldenlilica1iou 

Dcscripl ion 

Batch number 

Source 

Stability of vehicle 

Storage conditions 

Safety precautions 

4.4 Dose Formulation 

Corn oil 

Yellowish oily liquid 

27572782 

Carl Roth GmbH & Co. D-76185 Karlsruhe I Germany 

Stable under storage conditions; 

expiration date: February 2008 

At room temperature (range of20 ± 5 °C}, light protected. 

Routine hygienic procedures were used to ensure the health 

and safety of the personnel. 

l>osc levels are in terms of the test item as supplied by the sponsor. 

The dose formulations were made shortly before each dosing. 

First the vehicle was heated up to I 00-11 ooc and the determined amount of the test item was 

added while stirring on a magnetic stirrer. Additionally, an Ultra-Turrax (Janke & Kunkel, D7921 q Stauti.!n) was used to get a homogeneous suspension. Thereafter, the formulation cooled 

down 'vhik still stirring on the magnetic stirrer and was applied at a temperature of 20-30°C. 

The kmpcraturc was measured before each treatment and was found to be 26.4 and 25.8°C. 

respt:·.:tivcly. 

Homogcnt>ity of the test item in the vehicle was maintained during administration using a 

magnetic stirrer. 

4.5 Treatment 

The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg 

body weight alter being fasted t{)r approximately 17 to 18 hours (access to water was permitted). 

Food \vas provided again approximately 3 hours after dosing. 

The dosing volume was I 0 mL/kg body weight 

Rationale: Oral administration was considered to be an appropriate application method as it is a 

possible route of human exposure during manufacture, handling and use of the test item. 
[ RCC Sru.Jy ·I'Jwnher l:H6462 

4.6 Observations 

Mortality I Viability 

Body Weights 

Clinical Signs 

4. 7 Pathology 

4.7.1 Nc,:rotJsy 

Report Page 14 

Daily during the acclimatization period, during the tirst 30 

minutes and at approximately t, 2, 3 and 5 hours after 

administration on test day 1 (with the clinical signs) and 

twice daily during days 2-15. 

On test days I (prior to administration), 8 and 15. 

Daily during the acclimatization period, during the first 30 

minutes and at approximately 1, 2, 3 and 5 hours after 

administration on test day I. Once daily during days 2-15. 

All abnormalities were recorded. 

All animals were killed at the end of the observation period by carbon dioxide asphyxiation and 

discarded attcr macroscopic examinations were performed. No organs or tissues were retained. 

4.8 Statistical Analysis 

No statistical analysis was used. 

4.9 l>a1a Compilation 

Body \vcighls \vcre recorded on-line. 

Clinical sigus were recorded on data sheets. 

Mortality/viability were recorded on data sheets. 

Macroscopic findings were compiled into the RCC Tox Computer System during recording. 

The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data 

collcdion. storage and rctrievability. 
RCC Studv Numhcr B4M62 Report Page 15 

[ 

5 RESULTS 

5.1 Mortality 

No deaths occurred during the study. 

5.2 Clinical Signs 

No clinical signs were observed during the course of the study. 

5.3 Body Weights 

The body weight of the animals was within the range commonly recorded tor this strain and age. 

5.4 Macroscopic Findings 

No macroscopic findings were recorded at necropsy. 

5.5 Median Lethal Dose 

The median lethal dose o{ 

to female rats, observed over a period of 14 days is: 

}fter single oral administration 

LDSO (1emale rat): greater than 2000 mg/kg body weight 
L 

RCC Study Numhcr B4M62 

6 INDIVIDUAL FINDINGS 

Table 1 Mortality I Clinical Signs 

r)~~~~---~1\ 

mg/kg 111 

N ~---------t 

. -------- f!l- Test days 

al Sex Si. I 2 3 4 5 6 7 8 9 10 

fL 0.5* I* 2* 3* 5* 

2000 F No clinical signs -.} ~ ~ ~ " -.} ~ ~ ~ " " ~ ~ " -· ·f--··- c---------

2 F No clinical signs " " " " " " " " " " " " " " F No clinical signs " " " " " " " " " " " " -.} " -----F No clinical signs " " " " " " " " " ~ " ~ " " c-------· F No clinical signs ,j " -~ ,j " " " " " ~ " ~ ~ " ·-·--

F No clinical signs ~ " " -.} " ~ " " " ~ " " I -.} " 

* Examinations wen: pcrtimned within the tirst 30 minutes and L 2. 3 and 5 hours alter tr..:atment. 

No climcal signs were evident in any animal during the acclimatization period. 

Table 2 Body Weights 

IDos;·--~ mal 

~~g_ __ ft<_l._ 

~---

·-

-·-

--·~~·~ 

-2-lli)o·---·],i --

s 

6 ---

·-·---

--

-· 

Sex Dav 1 

( tr~atment) --

F 203.1 -

F 197.0 -·---- F 187.0 -· 

F 180.2 

F 176.9 ~--

F 180.0 

Body weights are presented in grams. 

Day 8 Day 15 

215.1 224.7 

212.4 229.5 

201.6 212.6 

195.1 205.6 

192.9 196.5 

198.7 207.2 

Page 16 

11 12 13 14 15 

~ ~ -.} ~ " " -.} " -.} " " " " " " " " " " " " " " ~ 

,j ~ ~ " " ,j 
[ 

RCC Stud~ Number 1346..162 Report Page 17 

Table 3 Macroscopic Findings ~·---

Dose Animal Sex Mode of Findings 

~~g!~__g ____ f.-----··-

No. f.---- ~ath 

2000 I F s No macroscopic findings f-· 

2 F s No macroscopic findings 

.\ r s No macroscopic findings -·-·-· -·-·--·- ··~ ·- -----·-· -- 2000 .~ F s No macroscopic findings ---·------ --·--

::; F s No macroscopic findings 

l__ ______ ---'~~--~~~~-= (. s No macroscopic lindings 

S: scheduled necropsy 
[ 

RCC Study Numbt:r B4M62 Report 

7 GLP-CERTIFICATE 

l"he Sw1ss GLP Monitoring Authorities 

sw~ss Agency for the 

Environment, Forests 

and Uodteapa 

;' . :=::~;!~cy for 

SWISsmedlC TnorapoutcProduots 

Statement of GLP Compliance 

ll1s hereby confirmed that 

during the period of 

the following Facilities of 

April 22. 25 - 29. 2005 

May 09- 13, 2005 

RCC ltd 

4452 ltingen 

Switzerland 

we1e mspected by the Federal Office of Public Health. the Swiss Agency for Therapeutic 

Products and the Sw1ss Agency for the Environment. Forests and Landscape with respect to 

the compliance with the Swiss legtslation on Good Laboratory Practice. 

Facilities 

-Test Facility: Toxicology 

-Test Facility: Environmental Chemistry 

& Pharmanalytics 

·Archive Facilities 

Areas of expertise • 

TOX, ACC, OTH (Safety 

Pharmacology, Alternative Test 

Systems) 

ACC, ECT, ENF, EMN, PCT, 

RES, OTH (Animal Metabolism) 

The tnspections were performed in agreement with the OECD Guidelines for National GLP 

Inspections and Audits. It was tound that the aforementioned test facilities were operating in 

compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5) at 

the hme they were inspected. 

Bern. November 2005 

Federal Office of Public Health 

The Director 

Prof. Th. Zeltner 

·fOX=- Ton:olog:y, ACC"" APatyhcal and CluW·.at Chem1tUY: ECT = Env1ronmental tmuclty on aquatic and terrcstnal 

orgamsrn~: Et-.F" Behavtour m wattt(, toil .and 111. BioaccumvlatJcm: EMN = Studlfll on .affect& on mesoc.osms and nah.;ral 

eeosy!>tt!'tn$. PCl.:::. Physlcat.chemtcal testtng :RES" Residue stuclies; OTH; Other, to be spec;1fft!d 

Page 18 
ATTACHMENT HEADER SHEET 

Attachment Number 014 

Attachment Name 

Health Effects Data on the New Chemical Substance - Skin Irritation Test 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP1 01105088166808 
TS-CMTP01 

Attachment #14 

Health Effects Data on the New Chemical Substance 

Skin Irritation Test 
REPORT 

J 

Primary Skin Irritation Study in Rabbits 

( 4-Hour Semi-Occlusive Application) 

Study Director: 

Test Facility: 

Sponsor: [ 

Dr. C. Simon 

Harlan Laboratories Ltd. 

Wolferstrasse 4 

4414 Fiillinsdorf I Switzerland 

Study Identification: Harlan Laboratories Study Cl5654 

Version: Final 

Study Completion Date: 06-Mar-2009 

Page 1 of32 

harlanTM .... 

J 
Hadan Laboratories Study C25654 

r J Report 

/ --------·- ----~ 

CONTENTS 

C()NTENTS .. ....... ...... ................................... ... ... ........................................................ .......... 2 

SICiNATURES ...................................................................................................................... 4 

STATEMENT OF COMPLIANCE .............. ........ .... ............ ...... ... ....... ..... .. .. ... ... ...... .... .. .. ... 5 

QUALITY ASSURANCE GLP STATEMENT ................................................................... 6 

PREFACE ............................................................................................................................. 7 

General Infonnation ... . . . .... . .. ... .. ... . . ... ... .. . .. ..... ....... .... .. . .... .. .. ... . . . . .... . . ..... ... ... . . .. ... .. . .... ... . 7 

Responsibilities ......... ........ ...... ...... ......... ..................... ............. ............................ .......... 7 

Schedule ......................................................................................................................... 8 

Data Requirements I Test Guidelines ............................................................................. 8 

Animal Welfare .............................................................................................................. 8 

Archtving . ...... ..... ......... ............................. .................... ....................................... ........... 8 

Classification Guidelines ................................................................................................ 9 

l SUMMARY ..................................................................................................................... 10 

2 PlJRPOSE ......................................... .................................................................... ........... 11 

3 MATERIALS AND METHODS .......... ..... ....... ........... ...... ..... . ........ ... .. ......... ... .... ... .. ...... 12 

3.1 Test System ............................................................................................................ 12 

3.2 Allocation ............................................................................................................... 12 

3.3 liusbandry .. ............................. ....... ............. ..................... ...................................... 13 

3.4 Test Item ................................................................................................................. 13 

3.5 Preparation of Dose Formulations .......................................................................... 14 

3.6 Test Iten1 Administration ....................................................................................... 14 

3.7 Rationale ................................................................................................................. 14 

3. 8 Observations . ..... ... ... . .. .... .. . ...... ... . . .. ... .. .. ... . ....... ......... .......... .... . ...... .. ..... . . . ... . .. .... . . .. 15 

3.9 Pathology ................................................................................................................ 15 

3.9. 1 Necropsy ...................................................................................................... 15 

3.10 Statistical Analysis ............................................................................................... 15 

3.11 Data Cotuplilation ................................................................................................ 15 

3.12 Determination of Skin Irritating Potential ............................................................ 16 

3 .12.1 Observation and Scoring ... .... ............................. ...................... ............. ..... 16 

4 RESULTS AND DISCUSSION ...................................................................................... 17 

4.1 (Jbservations ........................................................................................................... 17 

4.1.1 Viability I Mortality ..................................................................................... 17 

4.1.2 Clinical Signs ............................................................................................... 17 

4.1.3 Body Weights ............................................................................................... 17 

4.2 Pathology ................................................................................................................ 17 

4.2.1 Macroscopic Findings .................................................................................. 17 

4.3 Skin Reactions ......................................................................................................... 17 

4.3.1 Irritation ........................................................................................................ 17 

4.3.2 Coloration ..................................................................................................... 18 

4.3.3 Test Item Remnants ...................................................................................... 18 

09009e53805ec755 2 
Harlan Laboratories Study C25654 Report 

[ J 

4.3.4 Corrosion ...................................................................................................... 18 

5 CONCLUSION ................................................................................................................ 19 

6 SUMMARY TABLES ..................................................................................................... 20 

Skin hTitation Scores .................................................................................................... 21 

Skin Irritation scores- Mean Values After 24,48 and 72 Hours ................................ 21 

Skin Irritation Scores Assessment according to EC Guidelines .. . . .. ... .. .... .. .... . . .. ...... 21 

7 lNDIVII>tJAL TABLES .................................................................................................. 22 

Skin R.eactions .. . . . . .. . .. . ....... ..... .... .. .. ...... ... .... . .. .. .. . .. .... . ....... .. .... . .. . ... . . ... . . .. .... . . . .. . .. . ...... . . 23 

Body Weights . ... . .. .. . . . ... . ... . ......... ... . ..... . .. . ... .. .. ... .. . ........ .... . .. .... . . ..... . .. . . ... .......... .. . . . .... . . . 26 

APPENDIX I- COMMISSION REGULATION ............................................................... 28 

Commission Regulation (EC) No 440/2008, 30 MAY 2008 ................................. ..... 29 

APPENDIX II - COMMISSION DIRECTIVE ......... ......... ........ ...... ........ ........... .... .... .. ... . .. 30 

Commission Directive 2001159/EC, August 2001 ....................................................... 31 

09009e53805~c755 3 
Harlan Laboratories Study C25654 [ J Report 

SIGNATURES 

Study Director: Dr. C. Simon 

Ma11agement: ( f'ru) Dr. H. f'ankhauser 

·?J~~l.vt. 

Date: 0 {) .-~ ~za?j 

09009e5:18063517(i 4 
Harlan Laboratories Study C25654 Report 

r J 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: 

Test Item: [ 

Study Director: 

Study Title: 

C25654 

J 

Dr. C. Simon 

Primary Skin Irritation Study in Rabbits 

(4-Hour Semi-Occlusive Application) 

The pH measurement of the test item was performed before the study initiation date. This 

procedure is, therefore, excluded from this statement. 

This study has been performed in compliance with the Swiss Ordinance relating to Good 

Laboratory Practice, adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

1997 by decision ofthe OECD Council [C(97)186/Final]. 

Study Director: 

09009e538063.St76 

Dr. C. Simon 

CJ(;---___ 

o~~~;c;;_;:·;:;;;:-:::(!(:0 !J 

5 
Harlan Laboralmics Study C25654 

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Report 

··---·---·--·-·--·-----

QUAl,ITY ASSURANCE GLP STATEMENT 

Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen I Switzerland 

Harlan Laboratories Study: 

Test Item: [ 

Study Director: 

Studv Title: 

C25654 

J 

Dr. C. Simon 

Primary Skin Irritation Study in Rabbits 

(4-Hour Semi-Occlusive Application) 

The general facilities and activities are inspected at least once a year and the results are reported 

to the responsible person and the management. 

Study procedures, with the exception of the pH measurement of the test item, were periodically 

inspected. The study plan and this report were audited by the Quality Assurance. The dates are 

given below. 

Dates of Reports to 

Dates and Types of QA Inspections the Study Director 

and Test Facility 

Management 

25-Nov-2008 Study Plan 25-Nov-2008 

17·-Nov-2008 Process Based 17-Nov-2008 

(test system, test item, raw data, dose 

preparation, treatment) 

17-Fcb-2009 Report 17-Feb-2009 

This statement also confirms that this final report reflects the raw data. 

Quality Assurance: N. Engelke 

2:..~~11; 

U9ll09c5380635t76 6 
Harlan Laboratories Study C25654 

[ l 

PREli'ACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Laboratory I Technical Coordinator: 

Quality Assurance: 

HcadofQA: 

09009e531!0635t76 

Report 

[ J 

Primary Skin Irritation Study in Rabbits ( 4-Hour 

Semi-Occlusive Application) 

Harlan Laboratories Ltd. 

WOlferstrasse 4 

4414 Filllinsdorf I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr. C. Simon 

G. Arcelin 

C. Weng 

T. Fink 

7 
Harlan Laboratories Study C25654 Report 

[ J ·-------------------------------------

Schedule 

Experimental Starting Date: 

Tetmination: 

Delivery of Animals: 

Acclimatization: 

Administration I Treatment: 

Experimental Completion Date: 

26-Nov-2008 

05-Dec-2008 

26-Nov-2008 

26-Nov-2008 to 30-Nov-2008 (one female) 

26-Nov-2008 to 01-Dec-2008 (one male and one 

female) 

01-Dec-2008 (one female) 

02-Dec-2008 (one male and one female) 

05-Dec-2008 

Data Requirements I Test Guidelines 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guidelines for Testing of Chemicals, Section 4, number 404 "Acute Dermal 

Irritation I Corrosion", adopted April24, 2002. 

• Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods 

pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the 

Cmmcil on the Registration, Evaluation, Authorisation and Restriction of Chemicals 

(REACH), B.4 ACUTE TOXICITY: DERMAL IRRITATION/CORROSION (Official 

Journal No L 142,31/05/2008 p. 0182-0190. 

Animal Welfare 

This study was performed in an AAALAC-accredited laboratory in accordance with the Swiss 

Animal Protection Law under license no. 55. 

Archiving 

Harlan Laboratories Ltd. ( 4452 Itingen/Switzerland), will retain the study plan, raw data, a 

sample of the test item and the final report of the present study for at least ten years. No data will 

be discarded without the Sponsor's written consent. 

09009eS380635f76 8 
Harlan Laboratories Study C25654 Report 

[ J --·----·-···-~·--·---- --------------------------

Classitlcation Guidelines 

Commission Directive 2001/59/EC adapting to technical progress for the 28th time Council 

Directive 67/548/EEC on the approximation of the laws, regulations and administrative 

provisions relating to the classification, packaging and labelling of dangerous substances, August 

06, 20()1 (Official Journal of the European Communities Nr. L 225/1, August 21, 2001). 

09009e.S:l806 3 ~fl6 9 
Harlan Laboratories Study C25654 Report 

[ J 

1 SUMMARY 

The primary skin irritation potential o{ lwas investigated according to OECD test 

guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 mL to 

the intact lett flank of each of three young adult New Zealand White rabbits. The duration of 

treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours 

after removal of the dressing. 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) 

for each animal for erythema/eschar grades and for oedema grades, separately. The mean 

erythema/eschar score of the three animals was 0.00, 0.33 and 0.00, respectively and the mean 

oedema score was 0.00 for each of the three animals. 

The application of[ ...,to the skin resulted in mild erythema in one animal. This 

effect was reversible and was no longer evident 48 hours after treatment. The test item caused no 

staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at 

any of the measuring intervals and no clinical signs were observed. 

Thus, the test item did not induce significant or irreversible damage to the skin. 

Based upon the referred classification criteria (Commission Directive 2001159/EC of August 

200 l )l Jis considered to be "not irritating" to rabbit skin. 

09009e5380635n6 10 
[ 

Harlan Laboratories Study C25654 Report 

--' 

-----------------------· --------------------------

2 PURPOSE 

The purpose of this pr_imary skin irritation study was to assess the possible irritation potential 

when a single dose of[ · · ]was placed on the skin of rabbits for approximately four 

hours. 

This study should provide a rational basis for risk assessment in man as skin contact is one of the 

possible routes of human exposure. 

09009e538063Sf76 11 
Harlan Laboratories Study C25654 

[ I J Report 

3 MATERIALS AND METHODS 

3.1 Test System 

Animals: 

Rationale: 

Breeder: 

Number of Animals per Test: 

Age (when treated): 

Body Weight Range (when treated): 

Identification: 

Acclimatization: 

Randomizatimt: 

3.2 Allocation 

The animals were distributed as follows: 

Sex: 

Male: 

Females: 

09009cS380635f76 

Young Adult New Zealand White Rabbit, SPF 

Recognized by international guidelines as a 

recommended test system. 

Harlan Laboratories B.V. 

Kreuzelweg 53 

NL-5961 NM Horst I The Netherlands 

Postbus 6174 

5960 AD Horst I The Netherlands 

3 (Animals of both sexes were used) 

17 weeks (male) 

16 weeks (females) 

2364 g (male) 

2120- 2369 g (females) 

By unique cage number and corresponding ear 

number. 

Under laboratory conditions after health 

examination. Only animals without any visual signs 

of illness were used for the study. 

Selected by hand at time of delivery. 

No computer generated randomization program. 

Animal Nos.: 

54 

55- 56 

:' ~ ! • (-; • .-:::) 

,- ;, ~., .. ;_ -.,./ ' 

12 
Harlan Laboratone~ Study C25654 

[ J 

3.3 Husbandry 

Room Number: 

Conditions: 

Accommodation: 

Diet: 

Water: 

3.4 Test Item 

Report 

0501 I Harlan Laboratories Ltd., Filllinsdorf 

Standard Laboratory Conditions. Air-conditioned 

with 10-15 air changes per hour, and continuously 

monitored environment with ranges for room 

temperature 17-23 oc and for relative humidity 

between 30-70 %, automatically controlled light 

cycle of 12 hours light and 12 hours dark, music 

during the daytime light period. 

Individually in stainless steel cages equipped with 

feed hoppers and drinking water bowls. Wood 

blocks (Harlan Laboratories Ltd, Fullinsdort) and 

haysticks 4642 (batch no. 69/08, Provimi Kliba AG) 

were provided for gnawing. 

Pelleted standard Provimi Kliba 3418 rabbit 

maintenance diet ad libitwn (batch no. 55/08) 

provided by Provimi K.liba AG, 4303 Kaiseraugst I 

Switzerland. Results of analysis for contaminants are 

archived at Harlan Laboratories Ltd. 

Community tap water from Filllinsdorf ad libitum. 

Results of bacteriological, chemical and contaminant 

analyses are archived at Harlan Laboratories Ltd. 

The following infonnation was provided by the sponsor: 

Identification: 

Description: 

Batch Number: 

Purity: 

Stability of Test Item: 

Expiry Date: 

Storage Conditions: 

Safety Precautions: 

09009e538063flb4 

[ J 

100% 

Stable under storage conditions. 

28-0ct-2009 

At room temperature (range of20 ± 5 °C}, light 

protected. 

Additionally to routine hygienic procedures, a 

helmet mask was used to ensure the health and 

safety of the personnel. 

13 
Harlan Laboratodc~ Study C25654 Report 

[ J 

r 

L 

- ----~- ·-----------------------------

3.5 Preparation of Dose Formulations 

jwas used as delivered by the Sponsor. 

The pH of the test item was measured before the study initiation date and was found to be 4. 

According to Commission Regulation (EC) No 440/2008, 8.4. and OECD Guidelines 404, a test 

item is not required to be tested if the pH-value is less than 2 or greater than ll.S, owing to its 

predictable corrosive properties. 

3.6 Test Item Administration 

The test item was administered at 0.5 mL/animal, the dose specified in the test guidelines for a 

liquid test item. 

Four days before treatment, the left flank was clipped with an electric clipper, exposing an area 

of approximately 100 cm

2 (10 em x 10 em). The skin of the animals was examined one day 

before treatment, and regrown fur of all animals was clipped again. 

Animals with overt signs of skin injury or marked irritation which may have interfered with the 

interpretation ofthe results were not used in the test. 

On the day of treatment, 0.5 mL ot{_ }as placed on a surgical gauze patch (ca. 2.5 

em x 2.5 em) held in contact with the skin by means of an adhesive hypoallergenic aerated semiocclusive dressing and a restrainer bandage wrapped around the abdomen. 

The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed 

with lukewarm tap water to clean the application site so that any reactions (erythema) were 

clearly visible at that time. 

As it was suspected that the test item might produce irritancy, a single animal (one female) was 

treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 4-

hour exposure, the test was completed using the two remaining animals for an exposure period of 

four hours. 

3.7 Rationale 

The application form and dose were used to detect a possible irritating potential of the test item 

applied. 

09009c53SO!'d5t76 14 
Harlan Laboratories Study C25654 

l J 

3.8 Observations 

Viability I Mortality: 

Clinical Signs (systemic): 

Body Weights: 

Skin Reactions: 

3.9 Pntbology 

3.9.1 Necropsy 

Report 

Daily from acclimatization of the animals to the 

termination of test. 

Daily from acclimatization of the animals to the 

tennination of test. 

At start of acclimatization, on the day of application 

and at tennination of observation. 

See Section 3.12 

No necropsy was perfonned on the animals sacrificed at termination of observation. 

All rabbtts were sacrificed by an intravenous injection of pentobarbitone into the ear vein at a 

dose of at least 1 mL!kg body weight and discarded. 

3.10 Statistical Analysis 

No statistical analysis was perfonned. 

3.11 Uata Complilation 

Viability/mortality, clinical signs and skin reactions were recorded on data sheets and transcribed 

for compilation and analysis. 

Body weights were recorded on-line. 

The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data 

collection, storage and retrievability. 

09009e5~81l635!76 15 
[ 

Harlan Laboratories Study C25654 Report 

J 

3.12 Determination of Skin Irritating Potential 

3.12.1 Observation and Scoring 

The skin reaction was assessed according to the numerical scoring system listed in the 

Commission Regulation (EC) No 440/2008, B.4, at approximately 1, 24, 48 and 72 hours after 

exposure (removal of the uressing, gauze patch and test item). 

(See Appendix 1 on p. 28 ) 

Data were summarized in tabular form, showing for each individual animal the irritation scores 

tor erythema and oedema at all measurement intervals. Any lesions were described, including the 

degree and nature of irritation, corrosion or any other toxic effects observed, and their 

reversibility. 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) 

for each animal for erythema/eschar grades and for oedema grades, separately. An animal is 

positive when the mean score is 2 or greater. The test is positive for irritation when at least 2 

animals are positive for the same endpoint (erythema/eschar or oedema). 

(See Appendix IJ on p. 30 ) 

090fl9e~3806J5fl6 16 
Harlan Laboratories Study C25654 

[ J Report 

·------~----------------------------

4 RESULTS AND DISCUSSION 

4.1 Observations 

4.1.1 Viability I Mortality 

No mortality occurred. 

4.1.2 Clinical Signs 

No clinical signs were observed during the course of the study. 

4.1.3 Body Weights 

(See Individual Tables on p. 26 ) 

One female lost slight body weight (1.3%) during the acclimatization period but recovered until 

the end of the study. Otherwise, the body weights of all rabbits were considered to be within the 

nonnal range of variability. 

4.2 Pathology 

4.2.1 Macroscopic Findings 

No necropsy was performed at the end of the study. 

4.3 Skin Reactions 

4.3.1 Irritation 

(See Summary Tables on p. 21 and Individual Tables on p 23 ) 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) 

for each animal for erythema/eschar grades and for oedema grades, separately. The mean 

ctythemaleschar score of the three animals was 0.00, 0.33 and 0.00, respectively and the mean 

oedema score was 0.00 for each of the three animals. 

A very slight erythema was observed in the first treated female 1 hour after test item exposure 

and persisted up to the 24-hour reading. Otherwise, no local signs were observed in any animal 

at any observation. 

No abnom1al findings were observed on the treated skin of one animal48 hours after treatment. 

09009e5380635f76 17 
Harla•1 Laboratories Study C25654 

[ J Report 

4.3.2 Coloration 

No staining produced by the test item of the treated skin was observed. 

4.3.3 Test Item Remnants 

Test item rcnmants were evident on the application site of one female l hour after test item 

exposure. 

4.3.4 Corrosion 

Neither alterations of the treated skin were observed nor were corrosive effects evident on the 

skin. 

09009e5JROf..l5f76 18 
Ha1lan Lab01atoric~ Study C25654 

[ J Report 

5 CONCLUSION 

Based upon the referred classification criteria (Conunission Directive 2001/59/EC of August 

200 l )~ 1is considered to be "not irritating" to rabbit skin. 

09009e51806J~f76 19 
Harlan Laboratories Study C25654 Report 

[ J 

6 SUMMARY TABLES 

{)900~Jd3&(!(.35f76 20 

.n. j . ·~ \..,. .,.'}' .... ~ 
Hatlan Laboratc•ries Study C25654 Report [_ J 

Skin Irritation Scores 

Animal Evaluation 

Number Su Interval* Erythema Oedema -------" "" 

54 M 0 0 

55 F I 

I l hour 1 0 I 

56 --~~t-- 0 0 ·- .. 

54 ,MI 0 0 I . 

55 i F I 24 hours l 0 I 

56 i F 0 0 I ------·~~- " r l'vr ·---- . """"' t-· 

54 0 0 

55 1 F 41i hours 0 0 S6 i F i 0 0 

- s-4 ···1M1 -- --- c···"· 

0 0 

ss I f ! 72 hours 0 0 

56 F l 0 0 

* Exammatwm; were performed at the specified times after removal of the dressing. 

Skin Irritation scores- Mean Values After 24, 48 and 72 Hours 

Animal! ! 

~-tl_l!l_l_J_~lJl;Se._~LE~)'!!t~l~~- _Q~_em __ a_-'-N-1 

54 M i 0.00 3 0.00 3 I 

55 I f: II 0.33 3 0.00 3 

56 [< 0.00 3 0.00 3 

N =number of available data points. 

Skin Irritation Scores Assessment according to EC Guidelines 

Evaluated I , ___ it!t<~rvals !--·- Et")t~~~~~_a __ -+-_O_e __ d_e_m_a_-1 

24 hon.-s 1 

48 hours I Not Irritating Not Irritating 

_]!.hou~l__--------~--------~ 

'·:. ~--, 

<;,..,! 'v -'-. -· 

21 
Harlan Laboratories Study C25654 Report 

[ J --- -~·-----

7 INDIVIDUAL TABLES 

09009c53~06J5f76 22 
Harlan Labor atone~ Study C25654 Report 

[ J ~--"'-~---"-------~--

Skin Reactions 

Animal No. 54, Male 

After I hour: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

Aftc:r 24 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 48 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 72 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

09009e538063~f76 23 
Harlan Laboratories Study C25654 Report 

r "1 L . .J 

.. --.---~-- -·- ----·----~--~-""~-----

Animal No. 55, Female 

After 1 hour: Erythema: Very slight erythema 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

Remnants: Test item remnants evident on the application site 

After 24 hours: Erythema: Very slight erythema 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 48 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 72 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

09!10'1eS3806~5fl6 24 

,, ?~ ,, \ ; . f".j, 

· t -;.j · ......... t, r-~~ 
Harlan Labor atones Study C25654 Report 

[ J ~---- ---~-~~- --------"----·· 

Animal No. 56, Female 

Atkr l hour: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 24 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 48 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

After 72 hours: Erythema: NO ABNORMAL FINDINGS NOTED 

Oedema: NO ABNORMAL FINDINGS NOTED 

Scaling: NO ABNORMAL FINDINGS NOTED 

Staining: NO STAINING PRESENT 

09009eS3~06J5f76 25 
I I allan Laboralorieh Study C256S4 Report 

[ ] --··------·---------------------

BODY WEIGHTS {GRAM) 

MALES 

GROUP 1 (GROUPl) 

DAY(:~ 

WFB!<S 

ANlMI\.L 

l 

1 

2 

1 

5 

1 

------·-·· -------·····-·---------------···---------------------

2:1:15 2364 2458 

09009e'iJS05c9ef.9 26 
HarLm I. aboratorics Study C25654 Report 

[ 1 ···-··---·--------------------------------------

BODY WEIGHTS (GRAM) 

FEMAI,ES 

GROUP l (GROUPl) 

-----------------------------------------

LJAYS 

WEEKS 

ANIMAl, 

IVTL 11>11\T.l ?.ATln-' TREATMENT 

1 

1 

2 

1 

s 

1 

------·-----·------------------------ ---------------------- 214? 

2170 

O'l009e5330:\e9e69 

2120 

2369 

2242 

2427 

27 
Harlan Laboratories Study C25654 

[ l Report 

APPENDIX l- COMMISSION REGULATION 

090H9c53N06J.Sf!8 28 
Harlan Laborat\,ries Study C25654 

[ J Report 

Commission Regulation (EC) No 440/ 2008, 30 MAY 2008 

Grading of Skin Reactions 

ERYTliEMA AND ESCHAR FORMATION 

No erythema 

Very slight erythema (barely perceptible) 

Well-def:ined erythema 

Modcrak to severe erythema 

Severe erythema (beef redness) or eschar 

formation (i1~jurics in depth preventing erythema) reading 

OEDEMA FORMATION 

No oedema 

Very slight oedema (barely perceptible) 

Slight oedema (edges of area well-defined by definite raising) 

Moderate oedema (edges raised approximately 1 mm) 

Sl:lvere oedema 

(raised more than l rnm and extending beyond the area of exposure) 

09009e5J80635fl8 

0 

I 

2 

3 

4 

0 

1 

2 

3 

4 

29 
Harlan Laboraturies Study C25654 

[ J Report 

APPENDIX II- COMMISSION DIRECTIVE 

30 
[ 

Harlan Labmatoncs Study C25654 J . Report 

Commission Oirective 2001159/EC, August 2001 

Inflammation of the skin 

The following risk phrase shall be assigned in accordance with the criteria given: 

"R38 Irritating to skin" 

Substances and preparations which cause significant inflammation of the skin which persists 

for at least 24 hours after an exposure period of up to four hours determined on the rabbit 

according to the cutaneous irritation test method cited in Annex V. 

lnflanmmtion of the skin is significant if: 

(a) the mean value of the scores for either erythema and eschar formation or oedema 

formation, calculated over all the animals tested, is 2 or more, or 

(b) in the case where the Annex V test has been completed using three animals, either 

erythema and eschar formation or oedema formation equivalent to a mean value of 2 or more 

calculated for each animal separately has been observed in two or more animals. 

Jn both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should 

be used in calculating the respective mean values. 

Inflammation of the skin is also significant if it persists in at least two animals at the end of 

the observation time. Particular effects e.g. hyperplasia, scaling, discoloration, fissures, scabs 

and alopecia should be taken into account. 

Substances and preparations which cause significant inflammation of the skin, based on 

practical observation in humans on immediate, prolonged or repeated contact. 

Organic peroxides, except where evidence to the contrary is available. 

Cm·rosivc 

The substances or preparation shall be classified as corrosive and assigned the symbol 'C' and 

lhe indication of danger 'corrosive' in accordance with the following criteria: 

•a substance or a preparation is considered to be corrosive if, when it is applied to healthy 

intact animal skin, it produces full thickness destruction of skin tissue on at least one animal 

during the test for skin irritation cited in Annex X or during an equivalent method, 

•classification can be based on the results of a validated in vitro test, such as that cited in 

AJmex V (B.40. Skin corrosion: rat skin transcutaneous declrical resistance assay and human 

skin model assay), 

•a substance or a preparation should also be considered corrosive if the result can be predicted, 

for example fwm strongly acid or alkaline reactions indicated by a pH of 2 or less or 11,5 or 

greater. However, where extreme pH is the basis for classification, acid/alkali reserve may 

also be taken into consideration. If consideration of alkali/acid reserve suggests the substance 

or preparation may not be corrosive then further testing should be carried out to confirm this, 

preferably by usc of an appropriate validated in vitro test. Consideration of acid/alkali reserve 

should not be used alone to exonerate substances or preparations from classification as 

corrosive. 

09009e53805r•;71a 31 
Harlan Laboratories Study C25654 

[ J Report 

Risk phrases shall be assigned in accordance with the following criteria: 

R35 Causes severe burns 

•if, when applied to healthy intact animal skin, full thickness destruction of skin tissue occurs 

as a result of up to three minutes exposure, or if this result can be predicted. 

R34 Causes burns 

•if. when appii;:d to healthy intact animal skin, full thickness destruction of skin tissue occurs 

as a result of up to four hours exposure, or if this result can be predicted. 

•Organic hydroperoxides, except where evidence to the contrary is available. 

Notes: 

Where. classificat.ion is based on results of a validated in vitro test R35 or R34 should be 

applied according to the capacity of the test method to discriminate between these. 

Where classification is based upon consideration of extreme pH alone, R35 should be applied. 

0900?r5:1805ec7! a 32 
ATTACHMENT HEADER SHEET 

Attachment Number 015 

Attachment Name 

Health Effects Data on the New Chemical Substance- Eye Irritation Test 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP 1 011 05088166808 
TS-CMTPOl 

Attachment #15 

Health Effects Data on the New Chemical Substance 

Eye Irritation Test 
L 

REPORT 

J 

Primary Eye Irritation Study in Rabbits 

Study Director: 

Test Facility: 

Sponsor: [ 

Dr. C. Simon 

Harlan .Laboratories Ltd. 

Wolferstrasse 4 

4414 Fullinsdorf I Switzerland 

Study ldentificatlon: Harlan Laboratories Study C2566S 

Version: Final 

Study Completion Date: 11-Mar-2009 

Page 1 of31 

J 
[ 

Harlan Laboratories Study C25665 Report 

..J 

CONTENTS 

CONTENTS .......................................................................................................................... 2 

SIGNATURES ...................................................................................................................... 4 

STATEMENT OF COMPLIANCE ...................................................................................... 5 

QUALITY ASSURANCE GLP STATEMENT ................................................................... 6 

PREFACE ............................................................................................................................. 7 

General Information ................................................. ...... ................................................ 7 

Responsibilities ..... .. . ........ ......... ... . . .. . .... . ........ ................ ..... .... . ....... ... ... . ... . .. .. . ....... ... ..... 7 

Schedule ......................................................................................................................... 7 

Data Requirements I Test Guidelines ............................................................................. 8 

Animal Welfare .............................................................................................................. 8 

Archiving . . .. . . . . .. .. . . . . . . . ... .. . .. . . .. . . . . . . . . .. .. . .. ... . .. . ... . . . . . . . . . . . . . . . . . ... . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 8 

Classification Guidelines ............. ............ ... .... ............ .. . . .. ... . ...... .......... ... . . . . . .. . ... ... ......... 8 

1 SUMMARY ....................................................................................................................... 9 

2 PURPOSE .. .. ... .... ...... ..... .. .. . . .... .. ................. ..... . .. ..... .... .............. .... . . .. .. .. .. ...... ........ ..... . .... 10 

3 MATERIALS AND METHODS ..................................................................................... 11 

3.1 Test System ............................................................................................................ 11 

3.2 Allocation .... . . .. .. .. .. ... ................ ... ....... .... ...... ...... ......... ......... .......... .......... .... ..... ..... 11 

3.3 liusbandry ............................................................................................... ............... 12 

3.4 Test Item ................................................................................................................. 12 

3.4.1 Test Item ....................................................................................................... 12 

3.5 Preparation of Dose Formulations .......................................................................... 13 

3.6 Test Item Administration ....................................................................................... 13 

3.7 Rationale ................................................................................................................. 13 

3.8 Observations ........................................................................................................... 14 

3.9 Pathology ................................................................................................................ 14 

3.9.1 Necropsy ...................................................................................................... 14 

3 .1 0 Statistical Analysis . .. .... ............... .... ... ............... ... .. . . . . . . . .. .. . ..... .. ... .. .... . .. .. ... .. .. .. .. .. 14 

3.11 Data Compilation ................................................................................................. 14 

3.12 Determination of Eye Irritating Potential . ......... ........... .... .. ... .... ... . .... ........ ....... .... 15 

3.12.1 Observation and Scoring ............................................................................ 15 

4 RESULTS AND DISCUSSION ...................................................................................... 16 

4.1 Observations ........................................................................................................... 16 

4.1.1 Viability I Mortality ..................................................................................... 16 

4.1.2 Clinical Signs ............................................................................................... 16 

4.1.3 Body Weights ............................................................................................... 16 

4.2 Pathology ................................................................................................................ 16 

4.2.1 Macroscopic Findings .................................................................................. 16 

4.3 Eye Reactions ......................................................................................................... 16 

4.3.1 Irritation ........................................................................................................ 16 

4.3.2 Coloration ..................................................................................................... 17 

09009e538060dc33 2 
Harlan Laboratories Study C25665 Report 

[ l 

4.3.3 Corrosion ...................................................................................................... 17 

5 CONCLUSION . ........................................ ........................................................ ............... 18 

6 SUMMARY TABLES . ... . ....... ............................ ... . .. .......... ............ ............... ... ............ ... 19 

Eye Irritation Scores ..................................................................................................... 20 

Eye Irritation Scores- Mean Values after 24, 48 and 72 Hours .................................. 20 

Eye Irritation Scores - Assessment According to EC Guidelines .... .. .......... ... ............ 20 

7 INDIVIDUAL TABLES .................................................................................................. 21 

Eye Reactions ............................................................................................................... 21 

Body Weight ................................................................................................................ 25 

APPENDIX I- COMMISSION REGULATION ............................................................... 27 

Commission Regulation (EC) No 440/ 2008, 30 May 2008 ........................................ 28 

APPENDIX II - COMMISSION DIRECTIVE ................................................................... 30 

Commission Directive 200l/59/EC, August 2001 ....................................................... 31 

09009eS38060dc33 3 
Harlan Laboratories Study C25665 

[ J 

SIGNATURES 

Study Director: 

Management: 

09009e$38060dd4b 

Report 

Dr. C. Simon 

c_·J·,er----__ 

n~i~;d(·:.:··Jrw·~·:zao 5 

(ff11) Dr. H. Fankhauser 

/>.~·tvL .......... ~ .... ~···················· 

Date: //~- h4'1(,... ~00!1 

4 
Harlan Laboratories Study C25665 

[ l Report 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

Harlan Laboratories Study: C25665 

Test Item: J 

Study Director: Dr. C. Simon 

Study Title: Primary Eye Irritation Study in Rabbits 

This study has been performed in compliance with the Swiss Ordinance relating to Good 

Laboratory Practice, adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the 

OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 

1997 by decision of the OECD Council [C(97)186/Final]. 

Study Director: 

09009e53806450f~; 

Dr. C. Simon 

C.}t\.t--

D~t~;;;jt::···'ffc:r··:::·zws 

5 
Harlan Laboratories Study C25665 Report 

[ J 

QUALITY ASSURANCE GLP STATEMENT 

Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen I Switzerland 

Harlan Laboratories Study: C25665 

Test Item: J 

Study Director: Dr. C. Simon 

Study Title: Primary Eye Irritation Study in Rabbits 

The general facilities and activities are inspected at least once a year and the results are reported 

to the responsible person and the management. 

Study procedures were periodically inspected. The study plan and this report were audited by the 

Quality Assurance. The dates are given below. 

Dates of Reports to 

Dates and Types of QA Inspections the Study Director 

and Test Facility 

Management 

04-Dec-2008 Study Plan 04-Dec-2008 

26-Nov-2008 Process based 26-Nov-2008 

(test system, test item, raw data, dose 

preparation, treatment) 

05-Mar-2009 Report 05-Mar-2009 

This statement also confirms that this final report reflects the raw data. 

Quality Assurance: 

09009e538064SOfc 6 
Harlan Laboratories Study C25665 

[ l 

PREFACE 

General Information 

Test Item: 

Study Title: 

Sponsor: 

Study Monitor: 

Test Facility: 

QA: 

Responsibilities 

Study Director: 

Deputy Study Director: 

Laboratory I Technical Coordinator: 

Quality Assurance: 

HeadofQA: 

Schedule 

Experimental Starting Date: 

Termination: 

Delivery of Animals: 

Acclimatization: 

Administration I Treatment: 

Experimental Completion Date: 

09009eS38060dd4b 

[ 

l 

Report 

Primary Eye Irritation Study in Rabbits 

Harlan Laboratories Ltd. J 

Wolferstrasse 4 

4414 FUllinsdorf I Switzerland 

Harlan Laboratories Ltd. 

Quality Assurance GLP 

Zelgliweg 1 

4452 Itingen I Switzerland 

Dr. C. Simon 

G. Arcelin 

C. Weng 

T. Fink 

10-Dec-2008 

22-Dec-2008 

03-Dec-2008 

10-Dec-2008 to 14-Dec-2008(one female) 

10-Dec-2008 to 15-Dec-2008 (one male and one 

female) 

15-Dec-2008 (one female) 

16-Dec-2008 (one male and one female) 

22-Dec-2008 

7 
[ 

Harlan Laboratories Study C2S665 Report 

J 

Data Requirements I Test Guidelines 

This study followed the procedures indicated by the following internationally accepted 

guidelines and recommendations: 

• OECD Guidelines for Testing of Chemicals, Section 4, number 405 "Acute Eye 

Irritation I Corrosion", adopted April 24, 2002. 

• COMMISSION REGULATION (EC) No 440/2008 of 30 May 2008, B.S. "Acute 

Toxicity Eye Initation!Corrosion". 

Animal Welfare 

TI1is study was perfonned in an AAALAC-accredited laboratory in accordance with the Swiss 

Animal Protection Law under license no. 54. 

Archiving 

Harlan Laboratories Ltd. (4452 Itingen!Switzerland), will retain the study plan, raw data, a 

sample of the test item and the final report of the present study for at least ten years. No data will 

be discarded without the Sponsor's written consent. 

Classification Guidelines 

Commission Directive 200 1159/EC adapting to technical progress for the 28th time Council 

Directive 67/548/EEC on the approximation of the laws, regulations and administrative 

provisions relating to the classification, packaging and labelling of dangerous substances, August 

06,2001 (Official Journal of the European Communities Nr. L 225/1, August 21, 2001). 

09009e5380624ce4 8 
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Harlan Laboratories Study C2566S Report 

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1 SUMMARY 

The primary eye irritation potential o1 !]was investigated according to OECD test 

guideline no. 405. The test item was applied by instillation ofO.l mL into the left eye of each of 

three young adult New Zealand White rabbits. Scoring of irritation effects was performed 

approximately 1, 24, 48 and 72 hours after test item instillation. 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for 

ea<.~h animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The 

individual mean scores for corneal opacity and iris were 0.00 for all three animals. The 

individual mean scores for the conjunctivae were 0.00 for reddening and for chemosis for each 

of the three animals. 

The instillation o{ =into the eye resulted in mild, early-onset and transient ocular 

changes, such as reddening of the conjunctivae and sclerae. These effects were reversible and 

were no longer evident 24 hours after treatment. No abnormal findings were observed in the 

cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the 

measuring intervals. No staining of the treated eyes by the test item was noted and no clinical 

signs were observed. 

Thus, the test item did not induce significant or irreversible damage to the rabbit eye. 

Based upon the referred classification criteria (Commission Directive 2001159/EC of August 06, 

2001 >[ ~s considered to be "not irritating" to the rabbit eye. 

0900!le5380624ce4 9 

J \ ' .~ . ;• ~. ~~ J ... .Y "-' -· 
[ 

Harlan Laboratories Study C25665 Report 

2 PURPOSE 

The purpose of this primary eye irritation study was to assess the possible irritation potential 

when a single dose ot · · Jvas placed in the conjunctival sac ofrabbit eyes. 

This study should provide a rational basis for risk assessment in man as ocular contact is one of 

the possible routes of human exposure. 

09009eS:J8000dd4b 10 
Harlan Laboratories Study C25665 Report 

[ I J 

3 MATERIALSANDMETHODS 

3.1 Test System 

Animals: 

Rationale: 

Breeder: 

Number of Animals per Test: 

Age (when treated): 

Body Weight Range (when treated): 

Identification: 

Acclimatization: 

Randomization: 

3.2 Allocation 

Young Adult New Zealand White Rabbit, SPF 

Recognized by international guidelines as a 

recommended test system. 

Harlan Laboratories B.V. 

Kreuzelweg 53 

5961 NM Horst I The Netherlands 

Postbus 6174 

5960 AD Horst I The Netherlands 

3 (Animals of both sexes were used) 

18 weeks (male) 

12 weeks (females) 

2700 g (male) 

2570-2797 g (females) 

By unique cage number and corresponding ear 

number. 

Under laboratory conditions after health 

examination. Only animals without any visual signs 

of illness were used for the study. 

Selected by hand at time of delivery. 

No computer generated randomization program. 

The animals were distributed as follows: 

Sex: Animal Nos.: 

Male: 57 

Females: 58-59 

09009e53R060dd4b 11 
Harlan Laboratories Study C25665 

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3.3 Husbandry 

Room Number: 

Conditions: 

Accommodation: 

Diet: 

Water: 

3.4 Test Item 

3.4.1 Test Item 

Report 

0501 I Harlan Laboratories Ltd., Ftillinsdorf 

Standard Laboratory Conditions. Air-conditioned 

with 10-15 air changes per hour, and continuously 

monitored environment with ranges for room 

temperature 17-23 °C and for relative humidity 

between 30-70 % (values above 70 % during 

cleaning process possible), automatically controlled 

light cycle of 12 hours light and 12 hours dark, 

music during the daytime light period. 

Individually in stainless steel cages equipped with 

feed hoppers and drinking water bowls. Wood 

blocks (Harlan Laboratories Ltd, Ftillinsdort) and 

haysticks 4642 (batch no. 69/08, Provimi Kliba AG) 

were provided for gnawing. 

Pelleted standard Provimi Kliba 3418 rabbit 

maintenance diet ad libitum (batch no. 55/08) 

provided by Provimi Kliba AG, 4303 Kaiseraugst I 

Switzerland. Results of analysis for contaminants are 

archived at Harlan Laboratories Ltd. 

Community tap water from Ftillinsdorf ad libitum. 

Results ofbacteriological, chemical and contaminant 

analyses are archived at Harlan Laboratories Ltd. 

The following information was provided by the sponsor: 

Identification: 

Description: 

Batch Number: 

Purity: 

Stability of Test Item: 

Expiry Date: 

Storage Conditions: 

Safety Precautions: 

09009c53806450fe 

[ J 

100% 

Stable under storage conditions. 

28-0ct-2009 

At room temperature (range of20 ± 5 °C), light 

protected. 

Additionally to routine hygienic procedures, a 

helmet mask was used to ensure the health and 

safety of the personnel. 

12 
Harlan Laboratories Study C2S665 Report 

C I 

[ 

3.5 Preparation of Dose Formulations 

]was measured with a syringe and applied undiluted as it was delivered by the 

Sponsor. 

The pH of the test item was measured for Harlan Laboratories Study No. C25654 and was found 

to be 4. 

According to Commision Regulation (EC) No 440/2008, B.5. and OECD Guidelines 405, a test 

item needs not to be tested if the pH-value is less than 2 or greater than 11.5, owing to its 

predictable corrosive properties. 

3.6 Test Item Administration 

The test item was applied at 0.1 mL/animal, the dose specified in the test guidelines for a liquid 

test item. 

The eyes of the animals were examined one day prior to test item administration. 

Animals with overt signs of ocular injury or irritation which may have interfered with the 

interpretation of the: results were not used in the test. 

On the day of treatment, 0.1 mL o{ was placed in the cof\iunctival sac of the left 

eye of each animal after gently pulling the lower ltd away from the eyeball. The lids were then 

gently held together for about one second to prevent loss of test item. The right eye remained 

untreated and served as the reference control. The treated eyes were not rinsed after instillation. 

As it was suspected that the test item might produce irritancy, a single animal (one female) was 

treated first. As neither a corrosive effect nor a severe irritant effect was observed after the land 24-hour examinations, the test was completed using the two remaining animals. 

3. 7 Rationale 

The application form and dose were used to detect a possible irritating potential of the test item 

applied. 

13 
Harlan Laboratories Study C25665 

l J 

3.8 Observations 

Viability I Mortality: 

Clinical Signs (systemic): 

Body Weights: 

Eye Reactions: 

3.9 Pathology 

3.9.1 Necropsy 

Report 

Daily from acclimatization of the animals to the 

termination of test. 

Daily from acclimatization of the animals to the 

termination of test. 

At start of acclimatization, on the day of application 

and at termination of observation. 

See Section 3.12 

No necropsy was performed on the animals sacrificed at termination of observation. 

The animals were killed by intravenous injection of pentobarbitone into the ear vein at a dose of 

at least l mL/kg body weight (162 mg sodium pentobarbitone/kg body weight) and discarded. 

3.10 Statistical Analysis 

No statistical analysis was performed. 

3.11 Data Compilation 

Viability/mortality and local I clinical signs were recorded on data sheets and transcribed for 

compilation and analysis. 

Body weights were recorded on-line. 

The RCC Tox Computer System (RCC-Tox-Lims) had been validated with respect to data 

collection, storage and retrievability. 

09009e538060dd4b 14 
Harlan Laboratories Study C25665 

[ ] 

Report 

3.12 Determination of Eye Irritating Potential 

3.12.1 Observation and Scoring 

(See Appendix I on p. 27 ) 

The eye reactions were assessed according to the numerical scoring system listed in the 

Commision Regulation (EC) No 440/2008, B.5, at approximately l, 24, 48, 72 hours after 

administration. Scleral reddening and ocular discharge were also assessed. Eye examinations 

were made with a Varta Cliptrix diagnostic-lamp (Roth AG, CH-4153 Reinach/Switzerland). 

Data were summarized in tabular form, showing for each individual animal the irritation scores 

for the designated observation time, a description of the degree and nature of irritation, the 

presence of serious lesions and non-ocular effects. The scores of each animal at the following 

reading times (24, 48, 72 hours) were used in calculating the respective mean values (with the 

exception of the sclerae) for each type oflesion. 

(See Appendix II on p. 30 ) 

The classification system was based upon the referred classification criteria Commission 

Directive 2001/59/EC of August 06, 2001. 

09009e5380624ce4 15 
Harlan Laboratories Study C25665 

[ l Report 

4 RESULTS AND DISCUSSION 

4.1 Observations 

4.1.1 Viability I Mortality 

No mortality occurred. 

4.1.2 Clinical Signs 

No clinical signs were observed during the course of the study. 

4.1.3 Body Weights 

{See Individual Tables on p. 25 ) 

The body weights of all rabbits were considered to be within the normal range of variability. 

4.2 Pathology 

4.2.1 Macroscopic Findings 

No necropsy was performed at the end of the study. 

4.3 Eye Reactions 

4.3.1 Irritation 

(See Summary Tables on p. 19 and Individual Talbes on p. 21 ) 

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for 

each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The 

individual mean scores for corneal opacity and iris were 0.00 for all three animals. The 

individual mean scores for the conjunctivae were 0.00 for reddening and for chemosis for each 

of the three animals. 

No abnormal· findings were observed in the cornea or iris of any animal at any of the 

measurement intervals. 

A slight reddening of the conjunctivae was noted in the three animals 1 hour after test item 

instillation. Additionally, one female was observed with a slight reddening of the sclerae at the 

same observation time point. 

09009eS38060dd4b 16 
Harlan Laboratories Study C25665 Report 

L l 

No abnom1al findings were observed in the treated eye of any animal 24 hours after treatment. 

4.3.2 Coloration 

No staining produced by the test item of the treated eye was observed. 

4.3.3 Corrosion 

No corrosion of the cornea was observed at any of the reading times. 

09009eS38060dd1b 17 

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Harlan Laboratories Study C25665 Report 

[ J 

5 CONCLUSION 

Based upon the referred classification (Commission Directive 2001/59/EC of August 06, 2001), 

[ }is considered to be "not irritating" to the rabbit eye. 

09Q09e538060dd4b 18 
Harlan Laboratories Study C25665 Report 

[ J _______ .. ____ . __________________________ _ 

6 SUMMARY TABLES 

09009e53ROfiOdd4b 19 
Harlan Laboratories Study C25665 

[ J Report 

Eye Irritation Scores 

Animal Evaluation Corneal Area or Conjunctivae -~ 

t-~U_!II_!!~t:_ Set Interval* Opacity Corneal Opacity Iris Redness Chemosis Sclera ~M- ··-----·~~---·- -·- 57 0 0 0 I 0 0 

5!1 F 1 hour 0 0 0 I 0 0 

f--~_5~- F 0 0 0 I 0 1 ··-·-~ -~--- ·- --~~.-- --- 57 M 0 0 0 0 0 0 

58 F 24 hours 0 0 0 0 0 0 

59 ! __ . 0 0 0 0 0 0 - 57 M 0 0 0 0 0 0 

58 

-fJ-':''""~ 0 0 0 0 0 0 

59 0 0 0 0 0 0 

-· 57 0 0 0 0 0 0 

58 F \ 72 hours 0 0 0 0 0 0 

59 l F 0 0 0 0 l 0 I 0 

• Exammat1ons were performed at the specified times after mstlllatJon ofthe test 1tem. 

Eye Irritation Scores- Mean Values after 24,48 and 72 Hours 

Animal ! Corneal Conjunctivae 

Number Sex Opacity N Iris N Redness N Chemosis N ---~------- -·-~ !--·------·· 

57 M 0.00 3 0.00 3 0.00 3 0.00 3 

58 F 0.00 3 0.00 3 0.00 3 0.00 3 

59 F 0.00 3 0.00 3 0.00 3 0.00 3 

N ""' number of available data points. 

Eye Irritation Scores - Assessment According to EC Guidelines 

Evaluated Corneal Conjunctivae 

intervals Opacity Iris Redness Chemosis 

24 hours 

48 hours Not Irritating Not Irritating Not Irritating Not Irritating 

72 hours 

09009e53S060dd4b 20 
Harlan Laboratories Study C25665 Report 

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7 INDIVIDUAL TABLES 

09009e538060dd4b 21 
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Harlan Laboratories Study C25665 Report 

l 

..J 

Eye Reactions 

Animal No. 57, Male 

After l hour Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: Slight reddening 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 72 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

Observations included: cornea, conjunctivae (including nictitating membrane), sclera and iris. The presence (or 

absence, as appropriate} of opacity, vascularization, reddening, oedema, discharge, staining and test item remnants 

were assessed. 

09009e538060d,l4b 22 
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Harlan Laboratories Study C25665 Report 

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Animal No. 58, l<'emale 

After 1 hour: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: Slight reddening 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 72 hours: Co mea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

Observations included: cornea, conjunctivae (including nictitating membrane), sclera and iris. The presence (or 

absence, as appropriate) of opacity, vascularization, reddening, oedema, discharge, staining and test item remnants 

were assessed. 

09009e.538ll60dd4b 23 
[ 

Harlan Laboratories Study C25665 Report 

l --.... ~------·-·--

Animal No. 59, Female 

After t hour: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: Slight reddening 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: Slight reddening 

Test item: NO REMNANTS EVIDENT 

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

After 72 hours: Cornea: NO ABNORMAL FINDINGS NOTED 

Iris: NO ABNORMAL FINDINGS NOTED 

Conjunctivae: NO ABNORMAL FINDINGS NOTED 

Discharge: NO ABNORMAL FINDINGS NOTED 

Sclerae: NO ABNORMAL FINDINGS NOTED 

Test item: NO REMNANTS EVIDENT 

Observations included: cornea, conjunctivae (including nictitating membrane), sclera and iris. The presence (or 

absence, as appropriate) of opacity, vascularization, reddening, oedema. discharge, staining and test item remnants 

were ussessed. 

09009e538060dd4b 24 
Hatlan Lahorntories Study C25665 

l J 

BODY WEIGHTS (GRAM) 

MALES 

GROUP 1 (GROUPl) 

IICCl·lMA'I'IZ11.T!ON 

DAYS 1 

WEI':KS l 

ANIMA I. 

---~--·-" 

57 2620 

(19009e5 3 8060d9cc 

TREATMENT 

1 

l 

Report 

2 8 

1 2 

2700 2716 

25 
[ 

Harlan Laboratories Study C25665 

J 

BODY WEIGHTS (GRAM) 

FEMALES 

GROUP 1 (GROUPl) 

1\CCT,UIAT!ZATION TREATMENT 

DAYS 1 l 

WEEKS 1 l 

ANIMAL 

·---~·· ---~·-~-·---

SB 2745 2797 

59 249l 

0900'Jc 538060d9C<: 

Report 

2 a l 2 

3041 

2570 2655 

26 
Harlan Laboratories Study C2566S 

[ J Report 

APPENDIX I- COMMISSION REGULATION 

27 

09009eS380624cfll 
Harlan Laboratories Study C25665 

[ l Report 

---------------------------------

Commission Regulation (EC) No 440/2008, 30 May 2008 

Grading of Ocular Lesions 

CORNEA 

Opacity: degree of density (area most dense taken tor reading) 

No ulceration or opacity 0 

Scattered or diffuse areas of opacity (other than slight dulling of normal luster), 

details of iris clearly visible 1 

Easily discernible translucent area, details of iris slightly obscured 2 

Nacreous area, no details of iris visible, size of pupil barely discernible 3 

Opaque cornea, iris uot discernible through the opacity 4 

Area of cornea involved 

Zero 0 

One quarter (or less) but not zero 1 

Greater than one quarter, but less than half 2 

Greater than half, but less than three quarters 3 

Greater than three quarters, up to whole area 4 

IRIS 

Normal 

Markedly deepened mgae, congestion, swelling, moderate circumcorneal hyperemia, 

or injection, any of these or combination of any thereof, iris still reacting to light 

(sluggish reaction is positive) 

No reaction to light, hemorrhage, gross destruction (any or all of these) 

CONJUNCTIVAE 

Redness (refers 1o most severe reading of palpebral and bulbar conjunctivae 

when compared with control eye) 

Blood vessels normal 

Some blood vessels definitely hyperemic (injected) 

Diffuse, crimson color, individual vessels not easily discernible 

Diffuse beefy red 

Chemosis: lids and/or nictitating membranes 

No swelling 

Any swelling above normal (including nictitating membranes) 

Obvious swelling with partial eversion of lids 

Swelling with lids about half-closed 

Swelling with lids more than half-closed 

09009e5380624~ffi 

0 

1 

2 

0 

1 

2 

3 

0 

1 

2 

3 

4 

28 
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Harlan Laboratories Study C25665 

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.J 

Report 

ADDITIONALLY, OCULAR DISCHARGE, REDDENING OF THE SCLERAE AND 

STAINING 01<, CONJUNCTIVAE, SCLERAE AND CORNEA BY THE TEST ITEM 

WILL BE ASSESSED ACCORDING TO THE FOLLOWING SCHEME: 

OCULAR DISCHARGE 

No discharge 0 

Slight: Any amount different to nonnal (does not include small amount observed 

in inner canthus of normal animal) 1 

Moderate: Discharge with moistening of the lids and hair just adjacent to the lids 2 

Marked: Discharge with moistening of the lids and hairs, and a considerable area 

around the eye (rUJming) 3 

SCLERAE 

Redness 

Nonnal: Blood vessels nonnal 0 

Slight reddening: Some blood vessels definitely hyperemic (injected) l 

Moderate reddening: Diffuse, crimson color, individual vessels not easily discernible 2 

Marked reddening: Diffuse beefy red 3 

ST AfNJNG OF CONJUNCTIVAE, SCLERAE AND CORNEA BY THE TEST ITEM 

Not observed 0 

Slight staining 1 

Marked staining 2 

09009e538060c:ut4 29 
Harlan Laboratories Study C25665 

[ J 

Report 

APPENDIX II- COMMISSION DIRECTIVE 

09009e538060dc2e 30 
Harlan Laboratories Study C25665 

[ J Report 

Commission Directive 2001/59/EC, August 2001 

The following risk phrases shall also be assigned in accordance with the criteria given: 

"R36 ~ Irritating to eyes" 

Substances and preparations which, when applied to the eye of the animal, cause significant 

ocular lesions which occurred within 72 hours after exposure and which persist for at least 24 

hours. 

Ocular lesions are significant if the mean scores of the eye irritation test cited in Annex V have 

any of the following values: 

• •comea opacity equal to or greater than 2 but less than 3; 

• •iris lesion equal to or greater than 1 but not greater than 1,5; 

• •redness of the conjunctivae equal to or greater than 2,5; 

• •oedema of the conjunctivae (chemosis) equal to or greater than 2; 

or, in the case where the Annex V test has been completed using three animals if the lesions, on 

two or more animals, are equivalent to any of the above values except that for iris lesion the 

value should be equal to or greater than l but less than 2 and for redness of the conjunctivae the 

value should be equal to or greater than 2,5. 

In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be 

used in calculating the respective mean values. 

Substances or preparations which cause significant ocular lesions, based on practical experience 

in humans. 

Organic peroxides except where evidence to the contrary is available. 

"R41 ~Risk of serious damage to eyes" 

Substances and preparations which, when applied to the eye of the animal, cause severe ocular 

lesions which occur within 72 hours after exposure and which persist for at least 24 hours. 

Ocular lesions are severe if the mean scores of the eye irritation test in Annex V have any of the 

following values: 

• •comea opacity equal to or greater than 3; 

• •iris lesion greater than 1,5. 

The same shall be the case where the test has been completed using three animals if the lesion, 

on two or more animals, have any of the values: 

• •cornea opacity equal to or greater than 3; 

• •iris lesion equal to 2. 

In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be 

used in calculating the respective mean values. 

Ocular lesions are also severe when they are still present at the end of the observation time. 

Ocular lesions are also severe if the substance or preparation causes irreversible colouration of 

the eyes. 

Substances and preparations which cause severe ocular lesions, based on practical experience in 

humans. 

09009e538061.ldc2e 31 
ATTACHMENT HEADER SHEET 

Attachment Number 016 

Attachment Name 

Health Effects Data on the New Chemical Substance- Ames (Bacterial Reverse Mutation) Test 

Associated PMN Section Number 

N/A 

Does not contain CBI 

Report Number 

CMTP 1011 05088166808 
TS-CMTP01 

Attachment #16 

Health Effects Data on the New Chemical Substance 

Ames (Bacterial Reverse Mutation) Test 
r 

RCC - CCR STUDY NUMBER 1111700 

SALMONELLA TYPHIMURIUM 

AND 

ESCHERICHIA COLI 

REVERSE MUTATION ASSAY 

WITH 

FINAL REPORT 

STUDY COMPLETION DATE: 

September 20, 2007 

] 
[ 

RCC-CCR Study Number 1111700 

J 

Final Report Page 2 of 33 

1 COPY OF GLP CERTIFICATE 

• HESSEN 

• 

st4ecdr.doc 

Gute Laborpra:ds/Good Laboratory Praetiee 

GLP-BeseheiDigaag/Statemeat of GLP Compliance 

(gemllllliCcording to § 19b Abs. 1 Chtmlk.alierogesctz) 

Einc GLP-IMpektion rur Oberwac:hung der Einhaltung 

dcr (lLP-Grundsltze gemllll Chetnikaliengesetz bzw. 

Rlchtlinic ~8/320/EG wurde durchgefllhrt in 

1:1!:1 Prllfeinrichtung/Test facility 

Assessment of confonnity with GLP accordillg to 

Chemikalifngesetz and Directive 881320/EEC at: 

0 Prllf•tandottrresl site 

RCC-C)totestCel RoMa.U G•ltH 

RCC- Cytolcst Cell Resean:b GmbH 

In den Lcppsleinswiesen 19 

64380 Rossdorf 

(Unverwecbselbm Beuichrumg und Adrcstc!Untquivocal name and ~~dress) 

Prt ..... aado Kaleprieii/ANU of Espenlle 

(gemlll/according chemVwV-GLP Nr. 5.3/0I!CO guidance) 

:a Prf1funatn t.lll llt'.aU.nununc d~r tox.ikolopac.:hen 

K-igf!n!\{, htdteo 

3 ,..,.nruogt-n wr Bll"'ahmmunc drr erbl'l'tverin~ 

df!!rndrn Eigf'nschaften (in vitro und in vivo) 

6 PrClfung~n aur BeMimmuna von Rllc:kaU\nden 

• Ann.lytt!lcht: PrUfuncen an biolostar.hc:n Materialien 

t VttUI'J!Iichc-rhdtopnlfungen 

;;a Toxi<.tty atudit-a 

3 Mutagenicity studies 

6 Relliduea 

a Analytical studJ~s on biologital materials 

9 Virua validation atudies 

02.09.%GI6 Datum der lnspcktion/Datc of lnsptttlon 

(Tag Mona! Jahrlday month year) 

Die gc....,nlc l'rUfeinri,btung bellndel si<:h im nationalen GLP-Obc:rwachungsverfabnm und wird regelmllllig auf Einhalt1111g der GLP-Grundsll7.e l!berwacbt. 

Auf .Jer Gmrnllage de• lnspektioosbericllte& wird hiermit 

ooMJ!tigt, da-. in dieser Prtlfeinrichturog die oben genannlen Prtlfungen unter F.inhaltung der GLP- Grund-

~611.< durchgcfUhrt werden lconnen, 

The above mentioned lett facility is included 

in the national GLP Compliance Programme and is 

inspected on a regular basis. 

Based on lhe inspection report it can be confllTiled, 

that this test facility is able to cond~~<:t the 

aforementioned studies in compliance with the 

Principles of GLP. 

lm Aul\rag 

Th. z· c:rmann, Referent, Wiesbaden, den 19. Jallur %0117 

(Name und Funlttioll der Ye1'11ltwortlicbcn Person/ 

Name and function of responsible pmon) 

Hen. Mlollter1a• mr U•wclt, lllldkllea Raam alld Verbraudoenchlz, 

Mai~~ZB Stnle 841 065119 Wlcsbadea 

(Name und Adresse der OLP-ObetwaebungsbehOrde/Name and address ofthe GLP Monitoring Authority 
[ 

RCC-CCR Study Number 1111700 

2 CONTENTS 

1 COPY OF GLP CERTIFICATE 

2 CONTENTS 

3 PREFACE 

3.1 General 

3.2 Responsibilities 

3.3 Schedule 

34 Project Staff Signatures 

3.5 Good Laboratory Practice 

3 6 Guidelines 

3. 7 Archiving 

3.8 Deviations to Study Plan 

4 STATEMENT OF COMPLIANCE 

Final Report 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

6 SUMMARY OF RESULTS 

6.1 Conclusion 

7 OBJECTIVE 

7. 1 A1ms of the Study 

7.2 Reasons for the Study 

8 MATERIALS AND METHODS 

8.1 Controls 

8.2 Test System 

8.3 Mammalian Microsomal Fraction S9 Mix 

84 Pre-Experiment for Toxicity 

8.5 Dose Selection 

8.6 Experimental Performance 

8 7 Data Recording 

8.8 Acceptability of the Assay 

8. 9 Evaluation of Results 

8.10 Biometry 

9 DISCUSSION OF RESULTS 

10 REFERENCES 

11 DISTRIBUTION OF THE REPORT 

12 SUMMARY OF RESULTS 

12.1 Summary of Results Pre-Experiment and Experiment I 

12.2 Summary of Results Pre-Experiment and Experiment I a 

12.3 Summary of Results Experiment II 

·13 HISTORICAL CONTROL DATA 

14 ANNEX 1: TABLES OF RESULTS (9 PAGES) 

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Page 3 of 33 

2 

3 

4 

4 

4 

4 

5 

5 

5 

6 

6 

7 

8 

9 

9 

10 

10 

10 

11 

12 

13 

14 

15 

16 

16 

16 

17 

17 

17 

18 

19 

19 

20 

20 

21 

22 

23 

24 
[ RCC-CCR Study Number 1111700 

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Final Report Page 4 of 33 

3 PREFACE 

3.1 General 

Title Salmonella typhimurium and Escherichia coli 

Reverse Mutation Assay with 

[ J 

Sponsor 

Study Monitor 

Test Facs!ity· 

Contracting Institute: 

Reference Number: 

3.2 Responsibilities 

Study Dtrector. 

Deputy Study Director: 

Management 

Head of Quality Assurance Unit: 

3.3 Schedule 

Date of the Study Plan 

Experimental Starting Date: 

Experimental Completion Date: 

Date of Draft Report: 

Date of Final Report: 

s4c2drdoc 

[ ] 

] 

RCC 

Cytotest Cell Research GmbH (RCC-CCR) 

In den Leppsteinswiesen 19 

D-64380 Rossdorf 

R C C Ltd 

CH-4452 ltingen 

846473 

Dipl. Bioi. Andrea Sokolowski 

Dr. Hans-Eric Wollny 

Dr. Wolfgang VOikner 

Frauke Hermann 

July 

July 

August 

September 

September 

02,2007 

10.2007 

07,2007 

05,2007 

20,2007 
[ r~CC·CCH Study Niimber 1111700 Final Repo1i Page !:i of 33 

3.4 Project Staff Signatures 

Stttdy Director Dip!. Bioi. Andrea Sokolowski 

Date: September 20, 2007 

Manaqement Dr. Wolfgang Volkner 

Date: September 20. 2007 

3.5 Good Laboratory Practice 

The study was performed in compliance with: 

"Chenukaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" 

(Annex 1) dated July 25, 1994 ("BGBI. I 1994", pp. 1703), last revision dated ,June 27, 

2002 

"OCCD Principles of Good Laboratory Practice", as revised in 1997 f C(97) 186/Final] 

3.6 Guidelines 

Tlli~, ~,tudy followed the procedures indicated by the following internationally accepted guidelines and recommendations: 

"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 47·1: 

"Bncteri::ll Heverse Mutation Test", adopted July 21, 1997 

"Commission Directive 2000/32/EC, L 1362000, Annex 4D", dated May 19, 2000 

Japanese GuidelinP.s: 

"Kanpoan No. 281 -- Environment Protection Agency" 

"Eisei No. 127 --Ministry of Health & Welfare" 

"Ht;lisei mJ/10/31 Kikyoku No.2-- Ministry of International Trade & Industry" 

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RCC-CCR Study Number 1111700 

; J Final Report Page 6 of 33 

3. 7 Archiving 

RCC Cytotest Cell Research GmbH will archive the following data for 15 years: 

Raw data, study plan, final report, and a sample of the test item. 

No data will be discarded without the sponsor's consent. 

3.8 Deviations to Study Plan 

There were no deviations to the study plan. 

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J 

Final Report Page 7 of 33 

4 STATEMENT OF COMPLIANCE 

Study Nurnher: 1111700 

Test !tern [ J 

Sllrdy Di1 ector: Dip!. Bioi. Andrea Sokolowski 

Title: Salmonella typhimurium and Escherichia coli 

[ Reverse Mutation Assay with J 

This study petiormed in the test facility of RCC Cytotest Cell Research GmbH was 

conducted in cornphance with Good Laboratory Practice Regulations: 

"Chemikalien9esetz" (Chemicals Act) of the Federal Republic of Gem1any. "Anhang 1" 

(Annex l) dated July 25, 1994 ("BGBL I 1994", pp. 1703), last revrsion dated June 27, 

2002. 

"OECD Princ:ipk;s of Good Laboratory Practice'', as revised in 1997 [C(97)186/Finall 

There were no circumstances that may have affected the quality or integrity of the study. 

f.>t11dy Direc;tor RCC-CCR 

Dip!. Bioi. Andrea Sokolowski 

.• ~:~-! ....... ~···············~·········· ·•·•••••·····•• 

Date: September 20, 2007 
l RCC-CCI~ Study Number 

L 

111 1'/00 

5 J Final Report Page 8 of 33 

'' -----------~---------------

5 STATEMENT OF QUALITY ASSURANCE UNIT 

Gtudy Numlwt: 

SttHiV Du edor 

Tttle 

1111700 

[ J 

Dipl. BioL Andrea Sokolowski 

Salmonella typhimurium and Escherichia coli 

Reverse Mutation Assay with [ J 

The g(mer;:~l facilities and activities of RCC Cytotest Cell Research GmbH are inspected 

penodically and the results are reported to the responsible person and the management 

Study procedures were inspected periodically. The study plan and this report were audited 

by !IH~ Cluality Assurance Unit. The dates are given below. 

r-· 

PhaS£':S attd DRtes of OAU Inspections/ Audits Dates of Reports to the Study 

Director and to Management ----- ·~---·-- ---·-·-·------··---

Study Pl<ln (Drt.~ft): June 29,2007 June 29, 2007 1~-- ------~-~-

Study Plan: ,July 03,2007 .. ···-· ·----- - ------ ··---- Process ln~.;pr:ctinrr July 19, 2007 July 19, 2007 

August 01, 2007 August 01, 2007 --- '"'' '. ·-·-·-" . - -·---·-·~ . 

Draft Repo1i September 13,2007 September 13. 2007 

T hts ::;t;1ten 1ent is to contirm that the present final repoti reflects the raw data. 

Head of Quality Assurance Unit Frauke Hermann 

l '·''·- ". _;; .. , . . . . . . . . . . ' . ' ' '~ .... ' ..... ' .. ' . ' .... ' .... 

Date: September 20. 2007 
[ 

RCC-CCR Study Number 1111700 

>] 

Final Report Page 9 of33 

6 SUMMARY OF RESULTS 

This study was performed to investigate the potential o( 

]to induce gene mutations in the plate incorporation test (experiment I) and 

the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, 

TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. 

The assay was performed in two independent experiments both with and without liver 

microsomal activation. The plate incorporation test with strain TA 1537 with metabolic 

activation was repeated, since questionable results were obtained (reported as experiment 

Ia). In the pre-incubation test data were not evaluate for strain TA 1537, due to 

contamination of the strain. This part was repeated and is reported as part of experiment II. 

Each concentration, including the controls, was tested in triplicate. The test item was 

tested at the following concentrations: 

Pre-Experiment/Experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 IJg/plate 

Experiment II: 33; 100; 333; 1000; 2500; and 5000 IJg/plate 

The plates incubated with the test item showed normal background growth up to 

5000 IJg/plate with and without metabolic activation in both independent experiments. 

No toxic effects, evident as a reduction in the number of revertants (below the indication 

factor of 0.5), occurred in all experiments with and without metabolic activation. 

No substantial increase in revert?Dt colony numbers of any of the five tester strains was 

observed following treatment wit'L_ ]at any dose level, 

neither in the presence nor absence of metabolic activation (S9 mix). 

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 

6.1 Conclusion 

In conclusion, it can be stated that during the described mutagenicity test and under the 

experimental conditions reported, the test item did not induce gene mutations by base pair 

changes or frameshifts in the genome of the strains used. 

Therefore.[ ;Js considered to be non-mutagenic in this 

Salmonella typhimurium and Escherichia coli reverse mutation assay. 

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Final Report Page 10 of 33 

7 OBJECTIVE 

7.1 Aims of the Study 

The experiments were performed to assess the potential of the test item to induce gene 

mutations by means of two independent Salmonella typhimurium and Escherichia coli 

reverse mutation assays. Experiment I was performed as a plate incorporation assay. 

Since a negative result was obtained in this experiment, experiment II was performed as a 

pre-incubation assay. 

7.2 Reasons for the Study 

The most widely used assays for detecting gene mutations are those using bacteria (3). 

They are relatively simple and rapid to perform, and give reliable data on the ability of an 

agent to interact with DNA and produce mutations. 

Reverse mutation assays determine the frequency with which an agent reverses or 

suppresses the effect of the forward mutation. The genetic target presented to an agent is 

therefore small, specific and selective. Several bacterial strains, or a single strain with 

multiple markers are necessary to overcome the effects of mutagen specificity. The 

reversion of bacteria from growth-dependence on a particular amino acid to growth in the 

absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely 

used marker. 

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his· ~ his• and trp- ~ trp+ reversions, respectively. The S. typhimurium 

and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, 

TA 100. and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations. 

According to the direct plate incorporation or the pre-incubation method the bacteria are 

exposed to the test item with and without metabolic activation and plated on selective 

medium. After a suitable period of incubation, revertant colonies are counted. 

To establish a dose response effect at least 6 dose levels with adequately spaced intervals 

were tested. The maximum dose level was 5000 j.Jg/plate. 

To validate the test, reference mutagens were tested in parallel to the test item. 

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RCC-CCR Study Number 1111700 ;J Final Report Page 11 of 33 

8 MATERIALS AND METHODS 

Internal RCC-CCR Test Item Number: S 768911 

The test item and the information concerning the test item were provided by the 

sponsor. 

Identity: 

Batch No.: 

Aggregate state at 

room temperature: 

Colour: 

Purity: 

Stability in solvent: 

Storage: 

Expiration Date: 

[ 

[ J 

Solid 

Light brown 

100 % active polymer 

Not indicated by the sponsor 

At room temperature, light protected 

March 31 , 2008 

J 

On the day of the experiment, the test iten( Jwas 

suspended in THF (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was cnosen 

because of its solubility properties and its relative non-toxicity to the bacteria (5}. 

Precipitation of the test item occurred in the test tubes (all experiments) and in the overlay 

agar on the incubated agar plates in experiment II from 2500 J.Jg/plate up to 5000 J,Jg/plate 

with and without metabolic activation. The undissolved particles had no influence on the 

data recording. 

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RCC-CCR Study Number 11117()0 Final Report Page 12 of 33 ;J 

8.1 Controls 

8.1.1 Negative Controls 

Concurrent untreated and solvent controls were performed. 

8.1.2 Positive Control Substances 

Without metabolic activation 

Strains: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentration: 

Strains 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentration: 

Strain: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentration: 

With metabolic activation 

Strains: 

Name: 

Supplier: 

Catalogue No.: 

Purity: 

Dissolved in: 

Concentration: 

TA 1535, TA 100 

sodium azide, NaN3 

SERVA, D-69042 Heidelberg 

30175 

at least 99% 

water deionised 

1 0 IJQ/plate 

TA 1537, TA 98 

4-nitro-o-phenylene-diamine, 4-NOPD 

SIGMA, D-82041 Deisenhofen 

N 9504 

> 99.9% 

DMSO (MERCK, D-64293 Darmstadt; purity> 99 %) 

10 IJQ/plate in T A 98, 50 IJQ/plate in TA 1537 

WP2 uvrA 

methyl methane sulfonate, MMS 

Merck-Schuchardt. D-85662 Hohenbrunn 

820775 

> 99.0% 

water deionised 

3 IJL/plate 

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA 

2-aminoanthracene, 2-AA 

SIGMA, D-82041 Deisenhofen 

A 1381 

97.5% 

DMSO (MERCK, D-64293 Darmstadt; purity> 99 %) 

2.51Jg/plate (TA 1535, TA 1537, TA 98, TA 100), 

10 tJg/plate (WP2 uvrA) 

The stability of the positive control substances in solution is unknown but a mutagenic 

response in the expected range will be sufficient evidence of biological stability. 

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RCC-CCR Study Number 1111700 

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Final Report Page 13 of 33 

8.2 Test System 

8.2.1 Characterisation of the Salmonella typhimurium Strains and E. coli 

Strain 

The histidine dependent strains are derived from S. typhimurium strain L T2 through a 

mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation 

they possess a faulty lipopolysaccharide envelope which enables substances to penetrate 

the cell wall more easily. A further mutation causes a reduction in the activity of an excision 

repair system. The latter alteration includes mutational processes in the nitrate reductase 

and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the 

strains TA 98 and T A 100 the R-factor plasmid pKM 101 carries the ampicillin resistance 

marker (6). 

Strain WP2 (4) and its derivatives all carry the same defect in one of the genes for 

tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise 

either by a base change at the site of the original alteration or by a base change elsewhere 

in the chromosome so that the original defect is suppressed. This second possibility can 

occur in several different ways so that the system seems capable of detecting all types of 

mutagen which substitute one base for another. Additionally, the uvrA derivative is 

deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain 

may be more readily mutated by agents. 

When summarised the mutations of the T A strains and the E. coli strain, used in this study 

can be described as follows: 

.-----·--· 

--·---- Salmonella typhimurium 

Strains Genotype Type of mutations indicated 

TA 1537 his C 3076; rta·; uvrs·: frame shift mutations -- ITA98 his D 3052; rta·; uvrB-;R-factor " " 

TA 1535 his G 46; rta·; uvrs·: base~air substitutions 

TA 100 his G 46; rta·; uvrB-;R-factor " II 

Escherichia coli ~--·-~·-·-·~-~--~~·~-

WP2 uvrA trp·; uvrA·: base-pair substitutions and others 

Regular checking of the properties of the strains regarding the membrane permeability and 

ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest 

Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames (6). In this way it 

was ensured that the experimental conditions set down by Ames were fulfilled. 

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from 

Trinova Biochem GmbH (35394 GieBen, Germany). 

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RCC-CCR Study Number 1111700 

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Final Report Page 14 of33 

8.2.2 Storage 

The strain cultures were stored as stock cultures in ampoules with nutrient broth 

+ 5% DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen. 

8.2.3 Precultures 

From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml 

Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 !JL ampicillin 

(25 ~Jg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per 

litre: 

8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 

5 g NaCI (MERCK, D-64293 Darmstadt) 

The bacterial cultures were incubated in a shaking water bath for 4 hours at 3r C. 

8.2.4 Selective Agar 

The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt 

8.2.5 Overlay Agar 

The overlay agar contains per litre: 

for Salmonella strains: 

6.0 g MERCK Agar Agar• 

6.0 g NaCI* 

10.5 mg l.-HistidinexHCixH20* 

12.2 mg Biotin* 

* (MERCK, D-64293 Darmstadt) 

for Escherichia coli: 

6.0 g MERCK Agar Agar* 

6.0 g NaCI* 

2.5 mg Tryptophan* 

Sterilisations were performed at 121° C in an autoclave. 

8.3 Mammalian Microsomal Fraction 59 Mix 

The bacteria used in this assay do not possess the enzyme systems which, in mammals, 

are known to convert promutagens into active DNA damaging metabolites. In order to 

overcome this major drawback an exogenous metabolic system is added in form of 

mammalian microsome enzyme activation mixture. 

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8.3.1 S9 (Preparation by R C C - C C R) 

Phenobarbitai/~-Naphthoflavone induced rat liver 59 is used as the metabolic activation 

system. The 59 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight 

approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; 

D-22335 Hamburg) and B-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three 

consecutive days. The livers are prepared 24 hours after the last treatment. The 59 

fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) 

followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in 

ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one 

week. Each batch of 59 mix is routinely tested with 2-aminoanthracene as welt as 

benzo(a)pyrene. 

The protein concentration in the 59 preparation was 27.6 mg/ml (lot no. R 210607) in all 

experiments. 

8.3.2 S9 Mix 

Before the experiment an appropriate quantity of 59 supernatant was thawed and mixed 

with 59 co-factor solution. The amount of 59 supernatant was 10% v/v in the 59 mix. 

Cofactors are added to the 59 mix to reach the following concentrations in the 59 mix: 

8 mM MgCI2 

33 mM KCI 

5 mM Glucose-6-phosphate 

5 mM NADP 

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. 

During the experiment the 59 mix was stored in an ice bath. The 59 mix preparation was 

performed according to Ames et al.(1 ). 

8.4 Pre-Experiment for Toxicity 

To evaluate the toxicity of the test item a pre-experiment was performed with strains 

TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA Eight concentrations were tested for 

toxicity and mutation induction with three plates each. The experimental conditions in this 

pre-experiment were the same as described below for the experiment I (plate incorporation 

test). 

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a 

clearing of the bacterial background lawn. 

The pre-experiment is reported as experiment I, if the following criteria are met: 

Evaluable plates (>0 colonies) at five concentrations or more in all strains used. 

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Final Report Page 16 of 33 

8.5 Dose Selection 

In the pre-experiment the concentration range of the test item was 3- 5000 1-1gfplate. The 

pre-experiment is reported as experiment I. Since no toxic effects were observed 

5000 j.lgfplate were chosen as maximal concentration. 

Tile concentration range included two logarithmic decades. The following concentrations 

were tested in experiment II: 

33; 100; 333; 1000; 2500; and 5000 J,Jg/plate 

8.6 Experimental Performance 

For each strain and dose level including the controls, three plates were used. 

The following materials were mixed in a test tube and poured onto the selective agar 

plates: 

251JL Test solution at each dose level, solvent control 

100 IJL Reference mutagen solution (positive control}, 

500 !JL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test 

without metabolic activation), 

100 IJL Bacteria suspension (cf. test system, pre-culture of the strains), 

2000 IJL Overlay agar 

In the pre-incubation assay 25 IJL test solution, solvent control, 100 J.il reference mutagen 

solution (positive control), 500 JJL S9 mix I S9 mix substitution buffer and 100 IJL bacterial 

suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After preincubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured 

on selective agar plates. 

After solidification the plates were incubated upside down for at least 48 hours at 3r C in 

the dark (2). 

8. 7 Data Recording 

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk 

CB 7BN, UK) with the software program Ames Study Manager. The counter was 

connected to an IBM AT compatible PC with printer to print out the individual values and 

the means from the plates for each concentration together with standard deviations and 

enhancement factors as compared to the spontaneous reversion rates (see tables of 

results) Due to precipitation of the test item and air bubbles some plates were counted 

manually. 

~h.h, _;_ ', 

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Final Report Page 17 of33 

8.8 Acceptability of the Assay 

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered 

acceptable if it meets the following criteria: 

regular background growth in the negative and solvent control 

the spontaneous reversion rates in the negative and solvent control are in the range of 

our historical data 

the positive control substances should produce a significant increase in mutant colony 

frequencies 

8.9 Evaluation of Results 

A test item is considered as a mutagen if a biologically relevant increase in the number of 

revertants exceeding the threshold of twice {strains TA 98, TA 100, and WP2 uvrA) or 

thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control 

is observed (3}. 

A dose dependent increase is considered biologically relevant if the threshold is exceeded 

at more than one concentration (2). 

An increase exceeding the threshold at only one concentration is judged as biologically 

relevant if reproduced in an independent second experiment. 

A dose dependent increase in the number of revertant colonies below the threshold is 

regarded as an indication of a mutagenic potential if reproduced in an independent second 

experiment. However, whenever the colony counts remain within the historical range of 

negative and solvent controls such an increase is not considered biologically relevant. 

8.1 0 Biometry 

According to the OECD guideline 471, a statistical analysis of the data is not mandatory. 

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RCC-CCR Study Number 1111700 

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Final Report Page 18 of33 

9 DISCUSSION OF RESULTS 

The test item[ )was assessed for its potential to induce 

gene mutations in the plate incorporation test \experiment I) and the pre-incubation test 

(experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and 

TA 100, and the Escherichia coli strain WP2 uvrA 

The assay was performed in two independent experiments both with and without liver 

microsomal activation. The plate incorporation test with strain T A 1537 with metabolic 

activation was repeated, since questionable results were obtained (reported as experiment 

Ia). In the pre-incubation test data were not evaluate for strain TA 1537, due to 

contamination of the strain. This part was repeated and is reported as part of experiment II. 

Each concentration, including the controls, was tested in triplicate. The test item was 

tested at the following concentrations: 

Pre-Experiment/Experiment I and Ia; 3; 10; 33; 100; 333; 1000; 2500; and 5000 !Jglplate 

Experiment II: 33; 100; 333; 1000; 2500; and 5000 J.Jg/plate 

The plates incubated with the test item showed normal background growth up to 

5000 J.Jg/plate with and without metabolic activation in both independent experiments. 

No toxic effects, evident as a reduction in the number of revertants (below the indication 

factor of 0.5), occurred in all experiments with and without metabolic activation. 

No substantial increase in revertant colony numbers of any of the five tester strains was 

observed following treatment with[ ]at any dose level, 

neither in the presence nor absence of metabolic activation (S9 mix). In Experiment I with 

metabolic activation strain TA 1537 showed a minor increase. The number of colonies 

exceeded the threshold of thrice the number of the corresponding solvent control at 

concentration 1000 IJQ/plate. To clarify the results a confirmatory experiment was 

performed under identical conditions. In the confirmatory experiment no increase was 

observed. Therefore this minor increase is judged ad biologically irrelevant. 

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies. 

In conclusion, it can be stated that during the described mutagenicity test and under the 

experimental conditions reported, the test item did not induce gene mutations by base pair 

changes or frameshifts in the genome of the strains used. 

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Final Report Page 19 of33 

10 REFERENCES 

1. Ames, B.N, J. McCann, and E. Yamasaki (1977) 

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian 

microsome mutagenicity test 

In: B J Kilbey et al. (Eds.)"Handbook of Mutagenicity Test Procedures" Elsevier, 

Amsterdam, 1-17 

2. de Serres F.J. and M.D. Shelby (1979) 

Recommendations on data production and analysis using the Salmonella/microsome 

mutagenicity assay 

Mutation Res. 64, 159-165 

3 Hollstein,M., J. McCann, FA Angelosanto and W.W. Nichols (1979) 

Short-term tests for carcinogens and mutagens 

Mutation Res. 65, 133-226 

4. Green, M.H.L and WJ. Muriel (1976) 

Mutagen Testing Using TRP+ Reversion in Escherichia Coli 

Mutation. Res. 38, 3- 32 

5. Maron D.M., J. Katzenellenbogen and B.N. Ames, (1981) 

Compatibility of organic solvents with the Salmonella/Microsome Test 

Mutation Res. 88, 343-350 

6. Maron D.M., Ames, B.N. (1983} 

Revised methods for the Salmonella mutagenicity test 

Mutation Res. 113, 173-215 

11 DISTRIBUTION OF THE REPORT 

Sponsor 

Study Director 

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3 x (2x copy, 1x electronic copy (pdf)) 

1 x (original) 
[ RCC~CCR Studv Number 1111700 Final Report Page 20 of 33 

J 

12 SUMMARY OF RESULTS 

12.1 Summary of Results Pre-Experiment and Experiment I 

Study Name 1111700 Study Code: RCC-CCR 1111700 

Experiment: 1111700 W Plate Date Plated: 10/07/2007 

Assay Conditions Date Counted: 1310712007 

Metabolic Test Dose Level Revertant Colony Counts (Mean ±SD) 

ACIIVUII.Q!! Group {b!glplate} 

TA 1535 TA 1537 TA98 TA 100 WP2 uvrA 

Without THF 20±4 12 ± 5 26 ±6 162 ± 16 49 ± 4 

Activation Untreated 19 ± 3 10 ±6 34±17 153 ± 10 45 ± 8 

[ J 3119 16 ± 1 10 ± 4 34±7 143 ± 10 40±6 

10 1J9 17 ± 7 8±3 28 ± 12 171 ± 9 47 ± 11 

33119 17 ± 9 11 ± 2 30 ± 13 153 ± 7 56 ±4 

100 119 22 ±8 12 ± 1 33±8 155 ± 9 57 ±7 

333 1J9 17 ± 3 10 ± 2 28 ± 2 151 ± 10 46 ±4 

1000 119 20±2 14 ± 2 29 ± 2 159 ± 30 52 ±8 

2500119 20 ±3 10 ±4 36 ±3 160 ± 10 51± 21 

5000119 15 ± 2 9±3 36±4 145 ± 19 43 ± 2 

NaN3 10119 2019±55 2218 ± 80 

4--NOPD 10119 464 ± 17 

4-NOPD 50119 108 ± 16 

MMS 3.0 11L 1197 ± 72 

With THF 20 ± 3 11 ±4 41 :I: 10 188 :I: 12 76 ± 15 

Activation Untreated 22 ±3 16 :t 3 28 ±3 170 :t 15 59±3 1 .. 

J 3 1J9 18 ± 7 14 ± 1 36 ± 7 164 ± 13 62 ±4 i 

r 10 119 17:1:5 10:1:2 40±9 152 ± 25 65 ± 11 L 33119 19 ±4 15 ± 3 47:1:2 163 ± 10 67 ± 7 

100 119 17 ± 5 14 ± 3 40 ± 5 174 ± 11 67 ±6 

333119 20±4 16 ±2 43 ±7 146 ± 10 61 ± 8 

1000 ll9 26 ± 10 37 ± 6 46:1:10 171 :1:5 77 ± 6 

2500119 28 :t 8 20±7 51± 9 175±15 69 ± 10 

5000 1J9 23±2 23 ± 1 43 ± 2 174 ± 13 62 ± 12 

2-AA 2.5 1J9 289±8 252 :t 29 1467 ± 2128 ± 

138 196 

2-AA 10.0 119 296 ± 18 

~~~Posit~lv~e~C~o~nt~rocl~s~---------------------------------------------------

NaN3 

2-AA 

4-NOPD 

MMS 

sodium azide 

2-aminoanthracene 

4-nitro-o-phenylene-diamine 

methyl methane sulfonate 

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Final Report Page 21 of 33 

12.2 Summary of Results Pre-Experiment and Experiment I a 

Study Name: '1111700 

Experiment. 1111700 Wa Plate 

Assay Conditions: 

Metabolic Test 

Activation Qroup 

With THF 

Actl'"l'on [ Uot.eatod 

2-AA 

Dose Level 

lug/plate) 

J 31Jg 

10 1-19 

331Jg 

100 IJ9 

3331JQ 

1000 1-19 

2500 IJ9 

50001-19 

2.51JQ 

Study Code: RCC-CCR 1111700 

Date Plated: 17/07/2007 

Date Counted: 20!07/2007 

Revertant Colony Counts (Mean ±SO) 

TA 1537 

15±4 

16 ± 4 

19 ±4 

14 ±6 

15 ±2 

18 ± 1 

16 ± 6 

16 ± 3 

16 ± 1 p 

18 ± 2p 

363 ± 15 

~X~~ositiveCon~t~ro~ls~--------------------~K~ey~ro~P~Ia~t~e~P~os~~~·x~C~o~d~e~s ______________ __ 

4-NOPD 4-nitro-o-phenylene-diamine p Precipitate 

2-AA 2-aminoanthracene 

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RCC-CCR Study Number 1111700 Final Report Page 22 of 33 [ 5 J 

12.3 Summary of Results Experiment II 

Study Name: 1111700 Study Code: RCC-CCR 1111700 

Expenment 1111700 HV2 Pre Date Plated 26/07/2007 I 02/0812007* 

Assay Condttions: Date Counted: 01108/2007 f 0710812007* 

Metabolic Test Dose level Revertant Colony Counts (Mean ±SD) 

8.c;tiy9tion QlQY.Q (yg/(21ate} 

TA 1535 TA 1537 TA98 TA 100 WP2 uvrA 

Without THF 15 ± 5 9±1 39 ±4 140 ± 18 35±6 

Activation Untreated 13 ±4 10 ±3 41 ± 7 132 ± 15 58± 13 

[ l 331J9 12 ± 3 11 ± 3 27:1:3 135 ± 9 39 ± 5 

100 IJ9 14 ±2 8±4 37 ± 8 125 ± 10 45 ± 13 

333 IJ9 15 ±4 9±3 29±6 130 ± 13 41 ± 9 

1000 IJ9 16 ±4 13 ± 2 34 ±4 129 ±0 44 ±4 

2500 IJ9 13 ± 3p 9 ± 3p 28 ±4 p 79 ± 9P 42 ± 3p 

5000 IJ9 14 ± 9P 10 ± 2p 32 ± sP 106±2p 41 ± aP 

NaN3 10 IJ9 1919 ± 67 1845 ± 48 

4-NOPD 10 IJ9 741 ±67 

4-NOPD 50 IJ9 145 ± 16 

MMS 3.0 !JL 344 ± 13 

With THF 18 ::1: 1 18 ±4 47 ±8 142 ± 8 62 ± 10 

Activation Untreated 20±7 12 ±2 49±8 159 ± 11 62±9 

[ J 

331Jg 19:1:4 18 ±4 44:1:10 150 ± 20 63 ± 12 

100 IJ9 18 ±2 15:1: 1 42:1:11 136 ± 16 59:1:3 

333j.lg 20 ± 1 16 ±2 42 ±6 145:1: 13 55:1:1 

1000 IJ9 16:1:3 15±2UM 34:1:3 143 ± 4 61 ± 3 

2500 IJ9 18:1: 3p 12 ± 4 PM 45 ± 5p 117±14p 53:1:5 PM 

M 

5000 IJ9 18 ± 2 p 9 ± 1 PM 41 ±2p 109±14p 54 :1:6PM M 

2-AA 2.5 IJ9 233:1:3 159:1:8 1277:1:85 1939 ± 

144 

2-AA 10.0 IJ9 415 ± 22 

~eyjo_F:()~'!}~~gon~!<!l? Ke:f to Plate Postfix Codes 

NaN3 sodium azide p Precipitate 

2-AA 2-aminoanthracene M Manual count 

4-NOPO 4-nttro-o-phenylene-diamine u Air bubbles 

MMS methyl methane sulfonate 

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[ RCC-CCR Study Number 1111700 J Final Report Page 23 of33 

13 HISTORICAL CONTROL DATA 

These data represent the laboratory's historical control data from May 2005 until June 

2006 representing approx. 200 experiments (WP2 uvrA the historical data are based on 

approx. 100 experiments). 

Strain without S9 mix with S9 mix 

Mean so Min Max Mean so Min 

Solvent control 20.8 4.7 9 35 24.7 5.9 7 

TA 1535 Negative control 20.4 4.4 11 31 24.2 5.5 10 

Max 

43 

38 

Positive control 1422.0 464.7 781 4900 332.0 95.3 107 695 

Solvent control 11.2 3.7 5 28 16.2 5.0 6 36 

TA1537 Negative control 11.6 4.0 4 28 17.1 5.4 7 34 

Positive control 99.8 32.5 53 425 276.8 132.6 59 746 

Solvent control 28.1 6.1 15 49 37.9 7.4 20 57 

TA98 Negative control 30.2 6.6 16 60 39.0 7.5 18 64 

Positive control 439.0 155.2 176 1818 1839.4 898.6 407 4891 

Solvent control 130.7 20.8 87 197 147.0 25.5 84 255 

TA 100 Negative control 138.2 21.6 86 216 150 1 24.2 96 214 

Positive control 2083.1 281.3 616 2872 2372.9 958.4 417 5230 

Solvent control 55.8 7.2 31 74 63.9 9.1 34 84 

WP2uvrA Negative control 55.9 8.6 36 76 65.6 10.4 33 91 

Positive control 991 0 522.9 249 1810 3194 84.8 211 930 

Mean = mean value of revertants/plate 

SD = standard deviation Min = minimal value/Max = maximal value 

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RCC-CCR Study Number 1111700 [ J Final Report Page 24 of 33 

14 ANNEX 1: TABLES OF RESULTS (9 PAGES) 

Pre-Experiment and Experiment 1: 1111700 W Plate Incorporation (4 pages) 

Pre-Experiment and Experiment I a: 1111700 W Plate Incorporation ( 1 page) 

Experiment II 1111700 HV2 Pre-Incubation (4 pages) 

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Final Report Page 25 of 33 

----------------------------------------------

Study Name 1111700 Study Code RCC-CCR 1111700 

Expemnent 1111 700 W Plate Date Plated: 10/07/2007 

Assay Condit•ons. Date Counted: 13107/2007 

Without metabolic activation 

-----'sti ain _______ COmpound Dose level- Mean Standard Individual revertant 

per plate revertants Deviation counts 

TA 1535 [ 3 ~g 16.3 0.6 0.8 16, 17, 16 

10 IJ9 17.0 7.2 0.9 19,9, 23 

331J9 16.7 9.1 0.8 27, 13, 10 

100j.Jg 22.0 8.2 1 1 13, 29, 24 

3331J9 17.0 35 0.9 19, 19, 13 

1000 IJQ 20.3 1.5 1.0 19,22.20 

2500 IJ9 19.7 2.5 1.0 20, 17, 22 

5000 tJQ 14.7 2.3 07 16, 12. 16 

THF 19.7 4.0 19,16,24 

Untreated Control 18.7 2.5 16 

---·~~··- -·----···-------

TA 1537 [ l 3JJg 10.0 40 0.8 6, 14. 10 

101.19 8.0 3.5 0.7 6, 12,6 

33!-19 113 2.1 0.9 12, 13. 9 

100 1-19 12.3 0.6 1.0 12, 13. 12 

3331J9 10.3 1.5 09 10, 9, 12 

10001-19 13.7 1.5 1.1 14, 15, 12 

25001-19 10.0 3.6 08 7, 14, 9 

50001-19 9.0 2.6 0.8 12, 7, 8 

THF 12.0 50 12, 7. 17 

Untreated Control 9.7 5.8 13, 3, 13 ---~------·---

------ TA 98 

J 

31J9 34.3 6.7 13 31,30,42 

10 1-19 28.3 119 1.1 23,42.20 

331Jg 29 7 12.9 1.1 44,26,19 

100119 33.3 8.1 1.3 24,38,38 

3331JQ 28.0 1.7 1.1 26,29,29 

1000 IJQ 28 7 2.1 1.1 28,31,27 

2500 IJQ 36.3 32 14 34, 35,40 

5000 IJ9 363 4.0 1.4 41, 34, 34 

THF 26.0 6.2 21,33,24 

Untreated Control 34.0 16.7 19, 52, 31 

----------

TA 100 ] 3 f-19 143.0 101 0.9 152, 145, 132 

10 IJ9 171.0 9.2 1.1 161, 173, 179 

331JQ 153.3 7.4 0.9 156, 159, 145 

1001-JQ 154.7 9 1 1.0 165, 151, 148 

3331J9 151.3 10.1 09 150, 142, 162 

1000 IJQ 158.7 297 1.0 126, 184, 166 

2500 IJQ 159.7 10.1 1.0 148, 166, 165 

5000 fJQ 144.7 186 0.9 125, 162, 147 

THF 162.0 157 155, 180, 151 

Untreated Control 153.0 9.5 154. 143, 162 --- ·----~~-~~-

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Page 26 of 33 

--·-·---~--------·------------------------------

Study Name: 1111 700 

Experiment 1111700 W Plate 

Assay Conditions 

----strai;,-----·- Compound 

WP2uvrA 

THF 

Untreated Control 

TA 1535 NaN3 

TA 1537 4-NOPD 

TA 98 4-NOPD 

TA 100 NaNJ 

WP2 uvrA MMS 

] 

Dose level 

per plate 

31.19 

101.19 

331JQ 

1001J9 

3331.19 

10001.19 

25001.19 

5000 IJ9 

10 IJ9 

501JQ 

10 IJ9 

101-19 

3.01JL 

4-NOPD 4-nitro-o-phenylene-diamine 

MMS methyl methane sulfonate 

s4c2dr.doc 

Study Code RCC-CCR 1111700 

Date Plated 10/0712007 

Date Counted 1310712007 

Without metabolic activation 

Mean Standard Ratio Individual revertant 

revertants Deviation treated I colony counts 

per plate solvent 

40.0 5.6 0.8 41, 34,45 

46 7 11.0 1.0 34,52,54 

55.7 4.2 1.1 51, 59, 57 

57.3 6.5 1.2 64, 51,57 

45.7 3.8 0.9 44,50,43 

51 7 8.4 1.1 42, 57, 56 

51.0 207 10 74,34,45 

42.7 1.5 0.9 44,43,41 

48.7 42 50,52,44 

450 8.2 38, 43,54 

2019.0 54.6 102.7 1988,2082,1987 

108.0 15.9 9.0 120,114,90 

464.3 16 7 179 474,445,474 

2217.7 79.9 13.7 2183, 2309, 2161 

1197.3 71.8 24.6 1224, 1116, 1252 
[ 

RCC-CCR Study Number 1111700 

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Final Report Page 27 of 33 

Study Name 1111 700 Study Code: RCC-CCR 1111700 

Experiment 1111700 W Plate Date Plated 1 0107/2007 

Assay Conditions Date Counted. 13/07/2007 

With metabolic activation 

--Strain --··~--COmpound Dose level Mean Standard Ratio Individual revertant 

per plate revertants Deviation treated I colony counts 

TA 1535 31J9 18.0 7.2 0.9 16,12,26 

10119 17.0 4.6 0.9 16,22,13 

331Jg 18.7 4.0 0.9 21, 21, 14 

100 ll9 17.3 5.1 0.9 16,23,13 

333119 19 7 4.0 1.0 22,15,22 

1000119 260 9.5 1.3 20,37,21 

2500119 28.0 8.2 1.4 35, 30, 19 

5000119 22.7 1.5 1.1 23,21,24 

THF 20.0 3.0 17,20,23 

Untreated Control 22.0 2.6 19,24, 23 

--~-----·-·~----

TA 1537 [ 3119 14 0 10 1.3 14, 13, 15 

10119 10.0 1.7 0.9 9,9, 12 

331J9 15.3 2.9 1.4 17, 17, 12 

100119 14.0 3.5 13 10, 16, 16 

3331Jg 15.7 1.5 1.4 17, 16, 14 

1000 119 37.3 59 3.4 44,33,35 

2500119 20.3 65 18 27, 14, 20 

5000 IJ9 22.7 1.2 2.1 22,22,24 

THF 110 3.6 8, 10, 15 

Untreated Control 16.0 3.0 19. 16 

TAM [ 3119 35.7 7.1 0.9 28,42,37 

10 IJ9 39.7 8.5 1.0 31.48,40 

331Jg 47.0 1.7 1.2 48,48,45 

1001Jg 39.7 4.5 1.0 40,35,44 

3331J9 42.7 6.5 1.0 43,36,49 

1000 1!9 46.3 9.7 1.1 44,38, 57 

2500 ll9 510 8.9 1.3 58, 41, 54 

5000 IJ9 43.3 2.1 1.1 44,45,41 

THF 40.7 10.0 51,31,40 

Untreated Control 28.0 3.5 30, 30 

TA 100 

1 

3119 163.7 13.3 0.9 157, 155, 179 

10 IJ9 152.3 24.7 08 125, 173, 159 

33119 163.0 10.4 09 168, 170. 151 

100119 173.7 11.4 0.9 177, 161' 183 

333119 146.3 10.0 0.8 156, 136, 147 

1000 IJ9 171.0 4.6 0.9 172, 166, 175 

2500 l.l9 174.7 153 0.9 169. 163, 192 

5000 IJ9 1737 13.1 09 159, 184, 178 

THF 188.0 12.2 182,180,202 

Untreated 170.0 15.0 170 

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RCC-CCR Study Number 

[ 

1111700 

_] 

Final Report Page 28 of 33 

---------------------------------------------------

Study Name_ 1111700 

Experiment 1 1 11700 W Plate 

Assay Condttions_ 

Study Code: RCC-CCR 1111700 

Date Plated: 10/07/2007 

Date Counted: 13/07/2007 

With metabolic activation 

- ---COmpound ----Dose level Mean 

per plate revertants 

---------

WP2•"A [ l 3119 62_0 

10119 65.0 

33119 66 7 

100119 67.3 

3331J9 61.0 

1000119 77.3 

2500 IJ9 69.0 

5000 I.JQ 61.7 

THF 75.7 

Untreated Control 59.0 -------··~·--.-

TA 1535 2-AA 2.51.JQ 2887 

TA 1537 2-AA 2.51.JQ 252.0 

TA98 2·AA 2.5119 1467.0 

TA 100 2-AA 2.51J9 2127.7 

WP2 uvrA 2-AA 10.0 IJQ 295.7 

Key to Posillve Controls 

2-M 2-aminoanthracene 

s4c2dr.doc 

Standard 

Deviation 

3_6 

10.5 

67 

5.5 

7.9 

6.1 

9.5 

12.3 

14.8 

3.5 

8.1 

28.6 

137.8 

1963 

18.1 

....... ,. 

Ratio Individual revertant 

treated I colony counts 

o_a 65,58,63 

0.9 55,64, 76 

09 65,74,61 

09 61, 70, 71 

08 55,58, 70 

1.0 84, 76, 72 

0.9 70,59, 78 

08 72,48,65 

92,63, 72 

63, 57, 57 

14.4 285,298,283 

22.9 249,282,225 

36.1 1562, 1530, 1309 

11.3 1925,2317.2141 

3.9 279. 293, 315 
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Final Report Page 29 of 33 

-·----····----~~----------------------------------------

Study Name 1111700 

Experiment: 1111700 Wa Plate 

Assay Condi!lons 

TA 1537 

THF 

Untreated Control 

TA 1537 2·AA 

Key to Positive Controls 

2-AA 2-aminoanlhracene 

s4c2drdoc 

J 31JQ 

10 ~IQ 

331Jg 

1001-19 

3331-Jg 

1000 1-19 

25001-19 

5000 IJ9 

2.51)9 

With metabolic activation 

19.3 

14.3 

14.7 

17.7 

163 

16.0 

16.0 

17.7 

15.3 

15.7 

363.3 

Standard 

Deviation 

3.5 

5.7 

2.3 

12 

6.0 

2.6 

1.0 

2.1 

4.2 

4.0 

15.1 

1.3 

09 

1.0 

1.2 

1.1 

1.0 

1.0 

1.2 

23.7 

Study Code RCC-CCR 1111700 

Date Plated: 26/07 1200·1 I 

Date Counted: 01/08/2007 

Individual revertant 

counts 

19, 16, 23 

8, 16, 19 

16. 16. 12 

19, 17, 17 

10, 17, 22 

19, 14. 15 

15P,16P.17P 

16P, 20 P, 17 P 

14,12,20 

12,20 

374,346,370 

Key to Plate Postfix Codes 

p Preapitate 
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RCC·CCR Study Number 1111700 

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Final Report Page 30 of 33 

Study Name 1111 700 Study Code RCC-CCR 1111700 

Expenment 1111 700 HV2 Pre Date Plated 26/07/2007 I 0210612007* 

Assay Comlltions Date Counted: 01/08/2007 I 0710812007* 

Without metabolic activation 

----· ------- ~-~-. 

Stra•n coifiiXlillid Dose level Standard Ratio Individual revertant 

Deviation treated I counts 

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

TA 1535 f J 331.19 12.0 2.6 08 14, 9. 13 

L 1001-19 14.0 1.7 0.9 13, 13. 16 

333119 14.7 4.0 1 0 17, 17, 10 

1000 119 16.3 4.0 1.1 20, 12, 17 

2500119 13.3 2.9 0.9 10P,15P,15P 

5000119 140 8.5 0.9 23 P, 6 P. 13 P 

THF 15.0 4.6 19, 10. 16 

Untreated Control 13.3 3.5 17 

-------- TA1537l 

1 

331J9 11.3 3.1 1.2 14, 12, 8 

100 IJQ 8.3 40 0.9 6,6. 13 

3331.19 9.3 2.5 1.0 12, 9, 7 

10001-19 12.7 2.3 1.4 14, 14. 10 

2500119 8.7 31 0.9 8 P, 12 P, 6 P 

5000 IJ9 10.3 1.5 1.1 12 P, 10 P, 9 P 

THF 9.3 12 10, 8, 10 

Untreated Control 103 25 10,13,8 ---------·---~ 

·--------.. ~-·· ~--~- ----~----

TA 98 ] 33 IJQ 270 3.5 0.7 23.29,29 

100 1.19 37.3 8.3 1.0 44,40, 28 

3331)g 28.7 5.9 0.7 31,22,33 

10001.19 33.7 4.2 0.9 37.35,29 

2500 IJQ 27.7 3.5 0.7 31 P. 28 P, 24 P 

5000119 32.0 5.2 08 29P, 29 P, 38 P 

THF 387 3.8 37,43, 36 

Untreated Control 40.7 7.0 48,40,34 __ , _______ ·-----~-----~---------~·-· 

TAiOor- ---- 331J9 134.7 8.7 1.0 142, 137, 125 

100 IJ9 1250 96 0.9 129, 132, 114 

3331.19 130.3 12.7 0.9 128, 144, 119 

10001.19 129.0 00 0.9 129, 129. 129 

25001.19 78 7 9.1 0.6 87 P, 80 P, 69 P 

5000 IJQ 105.7 2.1 0.8 105 P, 108 P, 104 P 

THF 139.7 17.9 149,119,151 

Untreated Control 131.7 15.3 140, 141' 114 

Key to Plate Postfix Codes 

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[ RCC-CCR Study Number 

Study Name 1111700 

Expenment. 1111700 HV2 Pre 

Assay Cond•tions 

1111700 

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Final Report 

Without metabolic activation 

Page 31 of 33 

Study Code RCC-CCR 1111700 

Date Plated 2610712007 I 02108/2007* 

Date Counted 01/08/2007 I 07/08/2007• 

---Sirain-- --------Compound -·- Dose level Mean Standard Ratio Individual revertant 

per plate revertants Deviation treated I colony counts 

WP2 uvrA 33f.l9 38.7 5.1 1 1 33,40,4~ 

100 f.l9 45.3 12.5 1.3 54, 51, 31 

3331Jg 41.0 8.7 1.2 51,36,36 

1000 ll9 43.7 38 1.2 42,41,48 

2500 il9 42.0 2.6 1.2 45 P, 40 P, 41 P 

50001-19 410 7.5 1.2 34 P. 40 P, 49 P 

THF 35.0 6.2 37,28,40 

Untreated Control 58.0 13.0 50, 73, 51 -----·__.~.------

TA 1535 NaN3 101-19 1918.7 67.2 127.9 1996, 1886, 1874 

TA 1537* 4-NOPD 501Jg 145.3 16.2 15 6 137, 164, 135 

TA96 4-NOPD 10 ll9 740.7 673 19.2 709,695,818 

TA 100 NaN3 101Jg 1844.7 47.6 13.2 1802. 1836, 1896 

WP2 uvrA MMS 301JL 344.0 13.5 9.8 355,329,348 

Key to Positrve Co•1lrols Key to Plate Postfix Codes 

--------~··----·-

NaN3 sodrum azide p Prectprtate 

4-NOPD 4 -n1tro-o-phenylene-diam1ne 

MMS methyl methane sulfonate 

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RCC-CCR Study Number 1111700 

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Final Report Page 32 of 33 

Study Name 1111700 Study Code: RCC-CCR 1111700 

Experiment 1111700 HV2 Pre Date Plated 26/07/2007 I 0210812007* 

Assay Conditions Date Counted 01/08/2007 I 0710812007* 

With metabolic activation 

------strair\~--- compound Dose level Mean Standard Ratio Individual revertant 

per plate revertants Deviation treated I colony counts 

TA 1535 33119 19.0 4.4 1.1 21,22,14 

100119 18.3 2.1 1.0 19,16,20 

333 pg 19.7 06 1.1 19,20, 20 

1000119 16.3 3.1 0.9 19. 17, 13 

2500119 17.7 29 1.0 21 P, 16 P, 16 P 

5000 IJ9 17 7 2.1 1.0 17P,16P.20P 

THF 17.7 1.2 17, 19, 17 

Untreated Control 203 7.4 23 

TA 1537* 33119 18.0 4.4 1.0 23, 15. 16 

100119 15.3 1.2 0.8 16, 14, 16 

333119 16.0 1.7 0.9 17, 14, 17 

10001-19 15.3 1.5 0.8 15, 17 U M. 14 

2500119 12.3 3.5 0.7 16 PM, 12 PM, 9 PM 

5000119 9.3 0.6 0.5 1 0 P M, 9 P M. 9 P M 

THF 18.3 3.8 21, 20, 14 

Untreated Control 12.0 2.0 12, 10. 14 ------

TA98 331Jg 43.7 9.8 0.9 38,38, 55 

100 IJ9 42.0 11 4 0.9 29,47, 50 

3331Jg 42.3 5.7 09 36,44, 47 

1000 IJ9 34.0 2.6 0.7 31, 35, 36 

2500 IJ9 44.7 4.5 1.0 49 P, 45 P, 40 P 

5000119 41.3 2.3 09 44 P, 40 P, 40 P 

THF 46.7 8.1 42,42, 56 

Untreated Control 48.7 7.6 40,54 

TA 100 [ 331-19 150.3 19.7 1.1 158, 128. 165 

1001-19 136.3 16.3 1.0 122, 133, 154 

333 IJ9 144.7 12.5 1.0 132, 157. 145 

10001-19 1433 35 1.0 143, 147. 140 

2500 IJ9 117.0 14.4 0.8 121 PM, 101 PM, 129 PM 

5000 f-19 109.0 14.2 0.8 125 PM, 98 P M, 104 PM 

THF 142.0 7.5 141, 135, 150 

Untreated Control 159.3 10.5 170 

Key to Plate Postfix Codes 

M Manual count 

u Air bubbles 

s4c2dr.doc 
[ RCC-CCR Study Number 

Study Name 1111700 

Expenment 1111700 HV2 Pre 

Assay Cond1\lons· 

THF 

J 

Untreated Control 

TA 1535 2-AA 

TA 1537* 2-AA 

TA98 2-AA 

TA 100 2-AA 

WP2 uvrA 2·AA 

Key to Positive Controls 

2-AA 2-aminoanthracene 

s4c2dr.doc 

1111700 

J 

Final Report 

With metabolic activation 

Page 33 of33 

Study Code: RCC-CCR 1111700 

Date Plated 26/07/2007 I 02/08/2007* 

Date Counted: 01/08/2007 I 0710812007* 

Dose level Mean Standard Ratio Individual revertant 

per plate revertants Deviation treated I colony counts 

331J9 63.3 11.5 1.0 70, 70, 50 

100 IJ9 59.3 3.2 10 63,57,58 

3331Jg 55.0 1.0 0.9 55,54,56 

1000 IJ9 61.0 3.5 1.0 65,59, 59 

2500 IJ9 53.0 5.3 0.9 49 PM, 51 PM, 59 PM 

5000 IJ9 53.7 5.5 0.9 54 PM, 48 PM, 59 PM 

61.7 9.5 62, 71, 52 

61.7 9.3 54 

2.51J9 2327 2.5 13.2 235, 230, 233 

251)9 159.0 8.2 8.7 152, 157, 168 

2.51Jg 1277.3 85.3 27.4 1291, 1355, 1186 

2.51Jg 19393 144.0 13.7 2055, 1778, 1985 

10.0pg 415.3 22 1 6.7 418,392,436 

Key to Plate Postfix Codes 

p Precipitate 

M Manual count