U.S. ENVIRONMENTAL PROTECTION AGENCY

• You must provide all information requested in this form to the extent that it is !mown to or reasonably ascertainable by you. Make reasonable estimates ifyou do not have actual data. Before you complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from the Toxic Substances Control Act (TSCA) Information Service by calling 202-554-1404, or faxing 202-554-5603). Ifa user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the IS-user fee identification number you have generated. Remember, your user fee ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM-226), P.O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee.

Part I- GENERAL INFORMATION You must provide the currently correct Chemical Abstracts (CA) Name of the new chemical substance, even if you claim the identity as confidential. You may authorize another person to submit chemical identity information for you, but your submission will not be complete and the review will not begin until EPA receives this information. A letter in support of your submission should reference your TS user fee identification number. You must submit an original and two copies of this notice including all test data. Ifyou claimed any information as**confidentia~** a single sanitized copy must also be submitted.

Part II- HUMAN EXPOSURE AND ENVTIWNMENTAL RELEASE
Ifthere are several mannfacture, processing, or use operations to be described in Part n, sections A and B ofthis notice, reproduce the sections as needed.

Part ill-LIST OF ATTACHMENTS Attach additional sheets if there is not enough space to answer a question fully. Label each continuation sheet with the corresponding section heading. In Part 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 described pollution prevention and recycling information you may have regarding the new substance. So-called "binding" boxes are included throughout this form for you to indicate your willingness to be bound to certain statements you make in this section, such as use, production volume, .Protective equipment

... This option is intended to reduce delays that routinely accompany the development of consent orders or Significant New Use Rules. Except in the case of exemption applications (such as TMEA, LVE, LOREX) where certain information provided in such notification is binding on the submitter when the Agency approves the exemption application, checking a binding box in this notice does not by itselfprohibit the submitter from later deviating from the information (except chemical identity) reported in the form.

CONFIDENTIALITY CLAIMS You may**cl~im** 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. Ifyou 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.

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

EPA FORM 7710-25 (Rev. 5-95) 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 !mown to or reasonably ascertainable by you, ifthese data are related to the health and environmental effects on the manufacture, processing, distribution in commerce, use, or disposal 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 ifthey do not appear in the open literature. You should clearly identify whether test data is on the substance or on an analog. Also, the chemical composition of the tested material should be characterized. Following are examples oftest data and other data. Data should be submitted according to the requirements of §720.50 of the Premanufacture Notification Rule (40 CFR Part 720).

Test Data (Check Below any included in this notice) Environmental fate data D Yes Health effects data 181 Yes Environmental effects data D Yes PhysicaVChemical Properties • 181 Yes Other data 

### I"-,.:J81 Yes**(;;,~.:J.** Risk assessl!i'fpts -;;::: Structure/ati,t!wty relailbnships ·:;o**-c~~·-~~~** Test data not-in the**pos~~ssi6n** or control oftbl subtiiiUer:• ·'

• A physical and chemical properties worksheet is located on the laswage of this

### f;r~.**~-·** r ··- TYPE OF NOTICE (Check Only One)
PMN (Premanufacture Notice)**-~"'"** (
C":J N

### ~ 

# D D D INTERMEDIATE PMN (submitted in sequence with final product PMN)
SNUN (Significant New Use Notice)
TMEA (Test Marketing Exemption Application) 

# D L VE (Low Volume Exemption) @ 40 CFR 723 .50(c)(!) 

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

# D L VE Modification
IS THIS A CONSOLIDATED PMN?
# of chemicals 

# D D LOREX Modification
Yes
(Prenotice Communication # required, enter # on page 3)

Replaces previous editions of EPA Form 7710-25.\ -pu\>lic reporting burden for this collection of information is estimated to average 110 hours per response, including time for reviewing irttructions, searching existing data sources, gathering. and maintaining the data**nee~ed,** and**~omple~ing** and**.revi~wing ~he** **collecti~n** of information. Send comments regarding the burden estimate or any other aspect of this collectiOn of mformatwn, mcludmg suggestwns for reducing this burden, to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W., Washington, D.C. 20460; and to the Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION

I certify that to the best of my knowledge and belief:
1. The company named in Part I, section A, subsection la of this notice form intends to manufacture or import for a commercial purpose, other than in small quantities solely for research and development, the substance identified in Part I, Section B.
2. All information provided in this notice is complete and truthful as of the date of submission.
3. I am submitting with this notice all test data in my possession or control and a description of all other data known to or reasonably ascertainable by me as required by §720.50 of the Premanufacture Notification Rule.

Additional Certification Statements:
If you are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following user fee certification statement that applies: 

### 1:8] The Company named in Part I, Section A has remitted the fee of$2500 specified in 40 CFR 700.45(b), or 

# D The Company named in Part I, Section A has remitted the fee of $1000 for an Intermediate PMN (defined @ 40 CFR 700.43) in accordance with 40 CFR 700.45(b), or 

# D The Company named in Part I Section A is a small business concern under 40 CFR 700.43 and has remitted a fee of$100 in accordance with 40 CFR 700.45(b).
If you are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(c)(l) or a Low release and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check the following certification statements: 

# D The manufacturer submitting this notice intends to manufacture or import the new chemical substance for commercial purposes, other than in small quantities solely for research and development, under the terms of 40 CFR 723.50. 

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

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

# D Ifthis application is for an LYE in accordance with 40 CFR 723.50(c)(l), 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 misinterpretation is subject to criminal penalty pursuant to 18 USC 1001. Signature and title of Authorized Official (Original Signature Required) Date

Signature of agent

- (if applicable) Date

EPA FORM 7710-25 (Rev. 5-95) Page 2 Confidentialla. Person Submitting Notice (in U.S.)

b. Agent (if applicable)

Joint Submitter (if applicable) I Company
Mailing address

State, ZIP Code

### N~~;of auth;rized

City, State, ZIP Code. Telephone

Contact (in U.S.) 

# _______________,_·...;;·_·--.

. ,•..

3. Company

If

you have (PC) concerning this and EPA assigned a PC Number to the notice, enter the number.

for the chemical substance covered by this notice, enter the notice assigned by EPA.

EPA FORM 7710-25 (Rev. 5-95) Page3 

### .,, ...

Mark (X) ifnone

Mark (X) ifnone

Mark (X) ifnone**·---------------·-------------~--------·--·--·--·-----·---·---··-**1. 

# D Manufacture 2. 

## lZI Import 

# D 6. Type ofNotice

- Mark (X) Only Only 3. Both 

# D Binding Option 

# D Binding Option Mark (X) Mark (X)

EPA FORM 7710-25 (Rev. 5-95) Page41.

b. name correct Name TSCA Inventory

c. For Class I substances a CA Index Name must be provided. For Class 2 substances either a CA Index Name orCA Preferred Name must be provided, which ever is appropriate based on CA 9CI nomenclature rules and conventions). 

## Pyridine, 4-decylPlease 

# D which method you used to develop or obtain the Method I (CAS Inventory Expert Service- a copy of the Identification report obtained from the CAS Inventory Expert Services must be submitted as an attachment to ·this information reported in this 

# IZ! Method 2 (Other Source)

and CAS Registry for the substance)

C15H25N 1815-99-2

e. For a class I provide a complete and correct structure diagram. For a class 2 substance- (1) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature of the reaction or process. (3) Indicate the range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure diagram. as comolete as can be known. if one can be reasonably ascertained.**:~··** . . . 

# N



# - Me

EPA FORM 7710-25 (Rev. 5-95) Page 5a. Indicate the number-average weight ofthe lowest molecular weight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement or the basis for your estimates: GPC 

# D Other 

# 0: (Specify) i) lowest number average molecular weight: ii) maximum weight% below 500 molecular weight: iii) maximum weight % below 1000 molecular weight:

You must separate claims monomer or "Confidential" box next to any item you claim as confidential Confidential

(X) the (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. Mark (X) this column if entry in column (1) is confidential. Indicate the typical weight percent of each monomer or other reactant in the polymer. (2)

- (3)

- (4)

- Mark (X) the identity column ifyou want a monomer or other reactant used at two weight percent or less to be listed as part ofthe polymer description on the TSCA Chemical Substance Inventory. Mark (X).this column ifentries in columns (3) and (4) are confidential. (5)

- (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

d. The commercial purposes. this column

(CAS Inventory Expert Service- a copy ofthe identification report obtained from CAS Inventory Expert Service must be submitted as as attachment % % % % %
% %
reported in this notice (check one). Method 2 (other source)

e. Provi de a correct or partial chemical structure diagram, as complete as can be known, if one can be reasonably ascertained.

this box if you attach a continutation sheet. EPAFORM7710-25 (Rev. 5-95) Page6 % % % % % % %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." Estimate the maximum % of each If there are unidentified estimate their total %.



### Unknown impurities

subsection 1 or 2.


# D Mark (X) this box ifyou attach a continuation sheet 5. Trade trade names for the new 

### Symmarine 

# D Mark (X) this box ifyou attach a continuation sheet. you must a generic name for your substance that reveals the specific chemical identity ofthe 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.

use, or disposal ofthe new chemical substance.

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

%

%

%

%

%Part 

### I

-- GENERAL INFORMATION

-- Continued Section C --PRODUCTION, IMPORT, AND USE INFORMATION: Mark (X) the "Confidential" box next to any item you claim as confidential. 1. Production volume- Estimate the maximum production volume during the first 12 months ofproduction. 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-montb. production (kg/yr) 

### Maximum 12-month production (kg/yr) Confi- Binding (100% new chemical substance basis) (100% new chemical substance basis) dential Option Mark(x)



# - -- X
2. Use Information- You must make separate confidentiality claims for the description ofthe category of use, the percent of production volume devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim 

### as confidential. a. (1)

-- Pescribe each intended category ofuse ofthe new chemical substance by function and application

.. (2)

-- Mark (X) this column ifentry 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 ofthe 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 ifentry 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 ifentry(ies) in column (8) is (are) confidential business information (CBI). Category ofuse (1) CBI Binding Produc- CBI %in CBI % of substance expected per use CBI Option tion% Form- (8) (by function and application i.e. a dispersive dye Mark(x) ulation Site- Con-* Indus- Com- Binding for finishing polyester fibers) (2) (3) (4) (5) (6) (7) limited surner trial mercia! Option (9) 

### r-:---



### - .L. 

# X 100% 100% 

### X 

### X X X

% %
% %

% %
% %
% %
y.,_ % * Ifyou have identified a "consumer" use, please provide on a continuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumer products.

In addition include estimates ofthe concentration ofthe new chemical substance as expected in consumer products and describe the chemical reactions by which this substance loses its identity in the consumer product. 0Mark (X) this box ifyou attach a continuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2a as confidential, enter a generic description ofthat category. Read the use Instructions Manual for examples of generic use descriptions. description 

### Addictive for consumer use products; Dispersive use



# D Mark (X) this box ifyou attach a continuation sheet. 3. Hazard Information

- Include in the notice a copy ofreasonable facsimile of any hazard warning statement, label, material safety data sheet, or other Binding information which will be provided to any person who is reasonably likely to be exposed to this substance regarding protective equipment or practices Option for the safe handing, transport, use, or disposal ofthe new substance. List in part ill hazard information you include. Mark(x) 

# lXI Mark (X) this box ifyou attach hazard information.

EPA FORM 7710-25 (Rev. 5-95) PageSComplete section A for each type control. Importers do not have to cornp],ete there are further industrial 1.

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

additional sites on a continuation sheet, and if any of the sites have significantly different production rates or operations, include all the information requested in this section for those sites as attachments.

1. Batch

2. Continuous 

### 50 Maximum kg/batch (100% new chemical substance) 8

d. Process description Mark (X) to indicate your willingness to have your process description binding. Batches/year 

### 20 Batches/year you

Confidential



# X

(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 ofthe new chemical substance.

EPA FORM 7710-25 (Rev. 5-95) Page9A. Pure· 

### or diluted PMN substance

___ _.,.



### .,. Mix 

### Tank __ _.,._..., wash<D

Other (fragrance) ingredients 

# I Containers 

# J

Processing of PMN substance will involve only mixing it with other (fragrance} ·ingredients and then drumming 

### it. ·---·-· .... 

# _________ ,....

!Pure or diluted P:MN substance, either in pure form or diluted with other (fragrance) materials is blended with other (fragrance) !materials in a mixing vessel (maximum SOkg/batch). The blended (fragrance) product is then collected into 5 gallon pails to 55 gallon 

### drums.



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

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

### Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE-- Continued Section B --INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section B for typical processing or use operations involving the new chemical substance at sites you do not control. Importers do not have to complete this section for operations outside the U.S.; however, you must report any processing or use activities after import. See the Instructions Manual. Complete a separate section Bfor each type of processing, or use operation involving the new chemical substance. Ifthe same operation is performed at more than one site describe the typical operation common to these sites. Identify additional sites on a continuation sheet. 1. Operation Description

-- To claim information in this section as confidential, circle or bracket the specific information that you claim as confidential. (1) ··Diagram the major unit operation steps and chemical conversions, including interim storage and transport containers (specify- e.g. 5 gallon pails, 55 gallon drums, rail cars, tank trucks, etc). On the diagram, identify by letter and briefly describe each worker activity. (2)- Provide the identity, the approximate weight (by kg/day or kg/batch, on an 100% new chemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily or per batch). (3) --Identify by number the points of release, including small or intermittent releases, to the environment ofthe new chemical substance. (4) Please enter the# of sites (remember to identify the locations of these sites on a continuation sheet):

A Pure or diluted PMN substance O!her (fragrance) ingredients 

### Mix Tan:k 

# I



# ... J Containers ] 

### 2 #of sites

The PMN substance (pure) or as a component of a blended mixture (present between 0.01% and 0. 75%) will be further diluted into a finished product (at a concentration between 0.001% and 0.375%). There will be essentially no environmental release during this process. Environmental release will occur during final use of consumer products.

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

# D Mark (X) this box ifyou attach a continuation sheet. 2. Worker Exposure/Environmental Release (1)- From the diagram above, provide the letter for each worker activity. Complete 2-8 for each worker activity described. (2) --Estimate the number of workers exposed for all sites combined. (4) --Estimate the typical duration of exposure per worker in (a) hours per day and (b) days per year. (6) --Describe physical form of exposure and% new chemical substance (if in mixture), and any protective equipment and engineering controls, if any, used to protect workers. (7) --Estimate the percent of the new substance as formulated when packaged or used as a final product. (9) --From the process diagram above, enter the number of each release point. Complete 9-13 for each release point identified. (10) --Estimate the amount ofthe new substance released (a) directly to the environment or (b) into control technology to the environment (in kg/day or kg/batch). (12) --Describe media ofrelease i.e. stack air, fugitive air (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, or other (specify) and control technology, if any, that will be used to limit the release ofthe new substance to the environment. (14) --Identify byproducts which may result from the operation.

(3), (S), (8), (11), (13) and (IS)-- Mark (X) this column if any ofthe proceeding entries are confidential business information (CBI). Letter #of CBI Duration CBI Protective Equip. I %in CBI Release Amount of

of Workers of Engineering Controls/ Form- Number New Act- Exposed Exposure Physical Form and% ulation Substance ivity new substance Released CBI

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

A 

### 1

(14)

-- Byproducts: 

### 8.00 20.00 Rubber gloves and safety glasses



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

EPA FORM 7710-2S (Rev. S-9S) 

### 0.01%- 15.00%

Page 13 

### 1 0.0002 kg/day Media of Release & Control Technology
(12)

unknown CBI

(13)

(IS)

## OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle or bracket the specific information that you claim as confidential. 

### In this section you may provide information not reported elsewhere in this form regarding your efforts to reduce or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal of the PMN substance. Please include new information pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due to the new chemical substance. Source reduction includes the reduction in the amount or toxicity of chemical wastes by technological modification, process and procedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated or released as air emissions or water discharges, or land disposal. Descriptions of pollution prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance with existing regulatory requirements and can be either quantitative or qualitative. The EPA is interested in the information to assess overall net reductions 

### in toxicity or environmental releases and exposures, not the shifting of risks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). 

### In addition, information on the relative cost or performance characteristics of the PMN substance to potential alternatives may be provided. 

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



# D Mark (X) this box ifyou attach a continuation sheet.
EPA FORM 7710-25 (Rev. 5-95) Page 14

## Part Ill
-- LIST OF ATTACHMENTS
Attach continuation sheets for sections of the form and test data and other data (including physical/chemical properties and structure/activity information), and optional information after this page. Clearly identify the attachment and the section of the form to which it relates, if appropriate. Number consecutively the pages of the attachments. In the column below, enter the inclusive page numbers of each attachment Mark (X) the "Confidential" box next to any attachment name you claim as confidential. Read the Instructions Manual for guidance on how to claim any information in an attachment as confidential. You must include with the sanitized copy ofthe notice form a sanitized version of any attachment in which you claim information as confidential. Attachment name Attachment Confipage number(s) dential' CAS & Name of material 

### 18 MSDS 

### 19-23 Gas Chromatography 

### 24-26 FTIR Spectrum 

### 27 Mass Spectrometer 

### 28-29 NMR 

### 30-31 UV Spectrum 

### 32-33 fA-cute toxicity test in Rats 

### 34-53

Acute dermal irritation in rabbits 

### 54-69 Local Lymph Node assay in Mouse 

### 70-90 Reverse Mutation Assay-Ames Test 

### 91-112



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

EPA FORM 7710-25 (Rev. 5-95) Page 15PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical and chemical properties data, please complete the following worksheet for data you provide and include it in the notice. Identify the property measured, the page ofthe notice on which the property appears, the value of the property, the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. Properties that are measured for mixtures or formulations should be so noted (% PMN substance in_). You are not required to submit this worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential information is properly protected. You should submit this worksheet as a supplement to your submission of test data. This worksheet is not a substitute for submission oftest data. · Property Mark Page Value !Measured o Conti- (X) if number Estimate dential (a) provided (b) (c) Mark(X) (MorE) (d) X 19 M Physical state of neat substance**(~)** X (1) (g) X Vapor pressure @Temperature 50 

# oc <lkpa Torr E Density/relative density g/cm3 E Solubility @ Temperature 

# oc

Solvent giL X 21 Immiscible E Solubility in water @ Temperature 25 

# oc giL Melting temperature 

# oc E Boiling 

### I sublimation temperature@ torr pressure 

# oc E Spectra
Dissociation constant
Particle size distribution
Octanol/ water partition coefficient Henry's Law constant
Volitalization from water
Volitalization from soil pH@ concentration X 21 123C M Flammability Explodability Adsorption 

### I coefficient
Other

- Specify

EPA FORM 7710-25 (Rev. 5-95) Page 16

### Premanufacture
. .Notice Electronic Con'tinuation Sheet Continued From: Part II section A 1 a, pg 9 Continuation Title: Section A-Industrial Sites Controlled 

### by Submitter 2 Insert narrative continuation here ConfiSecond site at which PMN substance will be processed q.enti!U 

# D

-

i

# I

- I: Operations at this facility will be the same as given on page 9 and 

### 12-



# -

..

. '
Insert any picture here (PDF)**Confi~** dential ' 

# D·

..
' 

### ..

'•

'

.. 

# D**Pag~17** 

### n.'Jun 15 10 08:35p f-'· r

# I~ IIIII

# ~lflllllllllllllllllllllllll!lllll~lllll SANITIZED SUBMISSION PMN2010P1 A-1



## . Continuation Sheet
ID P10ASB2(6)C R1 Safety glasses/Respirat Protectiori wtth a NIOSH certified mask with a minimum APF rating of10 I pH Monitoring/PoWder dlssolved in water

EPA FORM 7710-25 (Rev. 6-09) Replaces previous editions of EPA Fom1 7710.25 Page 19CAS-Nr. zu Symmarine

L3 ANSWER 1 OF 1 REGISTRY COPYRIGHT 2009 ACS on STN RN 1815-99-2 REGISTRY ED Entered STN: 16 Nov 1984 CN Pyridine, 4-decyl- (CA INDEX NAME) OTHER NAMES: CN 4-Decylpyridine MF C15 H25 N CI COM LC STN Files: BEILSTEIN*, CA, CAPLUS, CASREACT, CHEMCATS, CSCHEM, USPATFULL, USPATOLD (*File contains numerically searchable property data) 

# CN)

x2)9-Me

**PROPERTY DATA AVAILABLE IN THE 'PROP' FORMAT** 8 REFERENCES IN FILE CA (1907 TO DATE) 8 REFERENCES IN FILE CAPLUS (1907 TO DATE)

### Material Safety Data Sheet

Version 1 Revision Date 12 02/2009 Print Date 12/04/2009

1. PRODUCT AND COMPANY IDENTIFICATION
Product name Pyridin, 4-DecylProduct code 902601 Company

For information call 800-422-1559 or evenings, call: 973-256-2930 Emergency Phone Number Chemtrec 1-800-424-9300 or 1-703-527-3887 The emergency contact should only be used in the event of chemical emergencies involving a spill, leak, fire exposure or accident involving chemicals.
2. HAZARDS IDENTIFICATION
The following evalutation is in compliance with 29 CFR 1910.1200. Emergency Overview This product is Water solubility Odour Flash point Flammability (DOT173) Flammability (OSHA) OSHA PHYSICAL HAZARD (OTHER): Acute Health Hazard Eyes Skin Inhalation LD50 Oral LD50 Dermal LC50 Inhalation

Chronic Health Hazard: liquid

- colourless immiscible characteristic

> 212 °F 

### I > 100 °C

none None. None.

Mild Eye Irritant Moderate skin irritant Mild Inhalation Irritant Toxic Not Toxic Not Toxic

This material has not been found to be carcinogenic per NTP. This material has not been found to be carcinogenic per !ARC. This material has not been found to be carcinogenic per OSHA.

### Material Safety Data Sheet

Version 1 Revision Date 12/02/2009 Print Date 12/04/2009

3. COMPOSITION/INFORMATION ON INGREDIENTS
CAS-No. Product Type

4. FIRST AID MEASURES Raw material

Emergency and First Aid Procedures: Eye contact

Skin contact

Inhalation
Ingestion

5. FIRE-FIGHTING MEASURES
Flash point HMIS Rating

NFPA Classification Flammability (OSHA) Suitable extinguishing media

Unsuitable media Special protective equipment and precautions for firefighters Specific hazards arising from the chemical Irrigate thoroughly with plenty of water until discomfort is relieved. Consult a physician for proper medical care. Take off all contaminated clothing immediately. Wash off immediately with soap and plenty of water. If symptoms persist, call a physician. If signs of dizziness and/or nausea occur, then remove the person immediately to fresh outside air. If accidentally swallowed obtain immediate medical attention.

> 212 °F 

### I > 100 °C Health: 2 Reactivity: 0 Flammability: 1

Class IIIB None. Dry chemical Carbon dioxide (C02) Water may be ineffective. 0 = Insignificant 1 =Slight 2 =Moderate 3 =Serious 4 =Severe

Follow NFPA Chapter 30 Fire Prevention Methods.

None.

6. ACCIDENTAL RELEASE MEASURES
This product is liquid

- colourless

### Material Safety Data Sheet

· .. ;:·
....**~.:·:~'E.-~::. ·.:.:/.::;~·::,:/.{.** **_:_:~;~~:** :: Version 1
Water solubility Odour Methods for cleaning up
Disposal Considerations



# 1: HANDLING AND STORAGE Revision Date 12/02/2009 Print Date 12/04/2009
immiscible characteristic Cover with an inert absorbent material and remove to a disposal container. Mop up and then wash with soapy water. Dispose of in accordance with local regulations. Observe local, state and federal regulations. Incineration is the most recommended disposal method.

Use only in well-ventilated areas. Handle in accordance with good industrial hygiene and safety practice. For industrial use only. Keep containers tightly closed in a dry, cool and well-ventilated place. Keep away from heat and flame. Flammability (OSHA) None. OSHA PHYSICAL HAZARD None. (OTHER):

8. EXPOSURE CONTROLS/PERSONAL PROTECTION
Exposure Guidelines
Although no TLV/TWA is established for this product by Federal standards, we recommend a lowest achievable TLV by means of sufficient ventilation. Respiratory protection Respirator with filter for organic vapour Ventilation protection Ensure adequate ventilation. Eye protection Chemical resistant goggles must be worn. Hand protection Wear suitable gloves.

9. PHYSICAL AND CHEMICAL PROPERTIES
Form liquid Color colourless Odour characteristic Flash point > 212 °F 

### I > 100 °C Water solubility immiscible

10. STABILITY AND REACTIVITY

### Material Safety Data Sheet

..

# ~**-~.**

Version 1 Revision Date 12/02/2009 Print Date 12/04/2009 Chemical Stability Possibility of Hazardous reactions Conditions to avoid Materials to avoid Hazardous decomposition products Stable Will Not Occur None. None. None.

11. TOXICOLOGICAL INFORMATION
Acute oral toxicity

Acute dermal toxicity Acute inhalation toxicity

12.ECOLOGICALINFORMATION LDSO rat Dose: > 300- < 2,000 mg/kg Method: Fixed Dose Method Please refer to section 2 for toxicological information. Please refer to section 2 for toxicological information.

There is currently no ecological information available at this time.

13. DISPOSAL CONSIDERATIONS Dispose of in accordance with local regulations. Observe, local, state and federal regulations. Incineration is the most recommended disposal method.

14. TRANSPORT INFORMATION Not classified as dangerous in the meaning of transport regulations.
15. REGULATORY INFORMATION
Toxic Substances Control Act {TSCA) Inventory Status This product is not acceptable under TSCA rules

SARA Title III Section 313 This product contains no chemicals subject to the reporting requirements of section 313 of SARA.

US. EPA CERCLA Hazardous Substances (40 CFR 302) ·In accordance with SARA CERCLA 302.4 Regulations, if a release occurs in a quantity equal to or exceeding the reportable quantity, notification must be immediately made to the National Response Center at 800-424-8802. A release is considered any spilling, leaking, pumping, pouring, emitting, emptying, discharging, escaping, dumping or disposing into the environment.

### Material Safety Data Sheet ''··. ·::: 

### .... \

,_. '. '.·, :/ : 

## ". \**~**

Version 1 Revision Date 12/02/2009 Print Date 12/04/2009

Contains no CERCLA reportable ingredients.

California Prop. 65

This product does not contain any chemicals known to State of California to cause cancer, birth, or any other reproductive defects.
16. OTHER INFORMATION

Further information These safety guidelines are based on information obtained from reliable sources. Prudent use and handling as part of good manufacturing practices is advised. Employees must be trained to handle material safely and wear recommended P.P.E. This new MSDS was created to conform with OSHA hazard communication standard 29CFR 1910.1200. Please discard previous form. This MSDS was prepared by the Regulatory Affairs Department.Timebase:FGCTB06 Sequence:FGC831_2008-1 0-20

PI 24832 Oe 082 337 FrS-8 WD
Operator: Via/Number: Control Program: Quantif. Method: UJWEV 68 FGC831 FGC831_Proben Path: Injection Volume: Sample Type: Run Time (min): 

### ·- ·'.1 '1/C,·..: ,.,. ,· RD\CHROM\FGCTB06\FGC831 1,0 unknown 30,00 21.10.2008 11 :33 AM

# ~~.~;:

..**~**

### ..

Quantif. Method: 21.10.200810:54 Column Info: 20m DS 0.181Jm df:0.18mm /60-9-240• KAS Nr185 Channel: Channei_A (Channei_A =DB-WAX Channei_B = DB-1)

150 FGC831 2008-10-20 PI 24832 Oe 082 337 Fr6-8 WD pP

100 

### ,_

50

,_

-20,0

No.

2 3 4 5 6 7 8 9 10 11 Total: I 5,0

Ret. Time Ret.lndex

min 1,195 1025,75 10,297 1932,42 10,414 1943,01 11,668 2061,39 11,842 2078,47 12,925 2186,21 13,327 2227,39 14,593 2361,28 15,333 2442,55 15,772 2491,86 17,233 2663,53

PI 24832 Oe 082 337 Fr6-8 WD GC_Area%_QM/Integration I 10,0

Area Type pA*min

0,003 BMB 0,012 BMB 0,002 BMB 0,016 BMB 0,012 BMB 0,008 BMB 6,770 BMB 0,010 BMB 0,003 BMB 0,004 BMB 0,002 BMB 6,843 I 15,0

Rei.Area % 0,047 n.a. 0,171 n.a. 0,031 n.a. 0,236 n.a. 0,179 n.a. 0,120 n.a. 98,934 n.a. 0,144 n.a. 0,048 n.a. 0,066 n.a. 0,025 n.a. 100,000

Page 1/1 I 20,0

Peak Name I 25,0 30,0

Chromeleon (c) Dlonex 1996-2001 Version 6.70 SP3 Build 1884Timebase:FGCTB06 Sequence:FGC831_2008-1 0-20

PI 24832 Oe 082 337 Fr6-8 WD
Operator: Vial Number: Control Program: UIWEV 68 FGC831 Path: Injection Volume: Sample Type: RD\CHROM\FGCTB06\FGCo31 1,0 unknown Quantif. Method: FGC831_Proben Run Time (min): 30,00 21.10.2008 11:34AM

Quantif. Method: 21.10.200810:54 Column Info: 20m DS 0.181Jm df:0.18mm /60-9-240• KAS Nr185 Channel: Channei_B (Channei_A =DB-WAX Channei_B = DB-1)

150 FGC831 2008-10-20 Pl24832 Oe 082 337 Fr6-8 WD p,l!

100

1-

50



## ,_

-2 0,0

No.

2 3 4 5 6 7 8 Total: I 5,0

Ret.Time Ret. Index

min 7,981 1372,07 11,605 1655,89 12,202 1706,49 12,437 1726,63 12,816 1759,57 14,653 1927,24 16,010 2059,56 16,228 2081,72

PI 24832 Oe 082 337 Fr6-8 WD GC_Area%_QM/Integration I 10,0

Area Type pA*min

0,007 BMB 0,012 BMB 0,021 BMB 0,020 BMB 8,190 BMB 0,002 BMB 0,006 BMB 0,005 BMB 8,264 I 15,0

Rei.Area % O,Q79 n.a. 0,143 n.a. 0,257 n.a. 0,248 n.a. 99,107 n.a. 0,026 n.a. 0,077 n.a. 0,063 n.a. 100,000

Page 1/1 I I 20,0 25,0

Peak Name ..min 30,0

Chromeleon (c) Dlonex**1996~2001** Version 5.70 SP3 Build 1884ChromatographicConditionsGCHP6890(Agilent)HP6890(Agilent)ColumnTypeDB-1DB-WaxPhaseDimethylpolysiloxanePolyethylenglycoleCarrierGasHydrogenHydrogenFlow1.3ml(17min)-0.5mUmin2-3.0mUmin1.3ml(17min)-0.5mUmin2-3.0mUminDetectorTypeFIDFIDDetectorTemperature275°C275°CInjectionVolume1J..LL(Split1:70;1:300inMTBE*)1J..LL(Split1:70;1:300inMTBE*)ColumnLength20m20mColumnDiameter(i.D.)0.18mm0.18mmFilm0.18J..Lm0.18J..LmInjector·CISCISInjectorTemperature80°C-12°C/s-250oc80°C-12°C/s-250ocOvenTemperatureandHeating60°C-9°C/min-220oc60°C-9°C/min-220ocRate*MTBE=Methyl-t-butylether1.31.21.10.90.80.70.60.50.40.1v(aliph.CH2,CH3)I380036003400320030002800260024002200200018001600140012001000800Instrument:BioRadFTS575CResolution: 1cm-1Technique:FTIR-NaCIFilmPl24832Charge:OE082337_Fr.6-8_WD100%2040*601069380100120162176140160180IN190204200Pl24832Charge:OE082337_Fr.6-8_WDmlz=176mlz=134mlz=190JW•219220mlz=106240mlz=162260280300320EquipmentandConditionsThermoFinniganSSQ7000/6890GasChromatographGasChromatograph:GCColumn:CarrierGas:Flow:TemperatureProgram:Injector:InjectorProgram:Autosampler:MassSpectrometer:ScanRange:DataSystem:Agilent6890·60mZB-1,id0.25mm,film0.25helium2.0mllmin80°C(start)-4°C/min.(rate)-280°CGerstelCIS3aooc(start)-12°C/sec.(rate)-280°CAgilent7683SeriesinjectorThermoFinniganSSQ7000m/z25-450ICIS2Software(;))oe-xp2ProtonS1111PU:.PEe.l\'TdateOct212008dfxq100.695colventCOC13dnCllfile/links/mercur-dpn:·Uy/daily/350636t_P'I.._2t832_h.£idelmACQVISX'l':IONc:lrrcftsfxq400.4ZOdmf18500tnKltemp25.0at6.000POOC£SS1NGnp?&8'14wt.filefbbstpvrpwd.ltof6406.1pl:OC!3600fn•GOwe:.:r:6.0wexp0...,.401.3wntntl.28ct128aloctnqainnotusedFLAGSily!o•dpyDISPLAY1.P-100.1vpU99.990we2<5.....15.85is&54.79•fl798.G<fpthins2.000cdcphftnotused5a.V<!;•av•vftPl24832Charge:OE082337_Fr.6-8_WDIN0III,-..i iIIIIIII.-Ii109L-..,----12.0087L,--J2.036543L,---J2.122[}(l14.862.1813.12ppmNMR-DataSubstance:PI24832,Charge:OE082337_Fr.6-8_WDbcadaShiftinppma8.47b7.10c2.59d1.62e1.40-1.20f0.88SpectrometerFrequencyRangeScansAcquisitionTimeSolventReferencefeMultiplicityFunctionalitymultipletaromaticprotonsmultipletaromaticprotonstripletaliphaticprotonsquintetaliphaticprotonsmultipletaliphaticchainprotons'-------triplet400MHz-NMR-Spectrometer400.420MHz6406.1Hz1286.000secDeuterochloroform(CDCh)Tetramethylsilane(TMS)methylgroupNumberofProtons2222143INPl24832Charge:OE082337_Fr.6-8_WDalwaysinspirlngmora_UV-Tesflabor1::1ICl--I-I-1·.-----1-IiII!_jI.f--!----0,7JIIIIIIIIII1IIIIiIiiIIII!I0,5jIII\IlA0,4IIJi0,31IIIIIII1r-------·-:----[1ii·I''EtII_' 'I•f--Ii---t-II210,0250300350400450500nm550--081046ASP-03.11.2008-IO,OOmginEthanolOe082337Fr.6-8WDMA.XIMA/MNMALISTEDaten-File:081046A11forrration:10,00rrgi1BhanolOe082337Fr.6-8WJSchweltNert0,0010Abszissen-BnheR:nmQ-dinaten-Bnhett:Art.Abszisse()dilateTyp1261,920,0677Peak2255,830,0902Peak600650700750800,0MethodfordeterminationofUV-absorptionSubstanceOe082337Fr.6-8WDMeasuringrange210-800nmConcentration10.00mg1100mlSolventEthanolPathlength1mmExtinctionIAbsorptionmaximumE1%11cm68IAmax.262nmE1%11cm90IAmax.256nmEffectsbyadditionofacidorbasePAGE 1 OF 20 PAGES



### STUDY SPONSOR:

2082..0230.doc!JS 

### P124832:
ACUTE ORAL TOXICITY IN THE RAT ·FIXED DOSE METHOD
PROJECT NUMBER: 2082/0230 

# harlanTM 6,.



### AUTHOR: 

### J Bradshaw



### TEST FACILITY: 

### Harlan Laboratories Ltd Shardlow 

### Business Park Shard low Derbyshire 

### DE72 2GD 

## UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER: 2082/0230 PAGE2



### QUALITY ASSURANCE REPO.RT 

### This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA 

### Standard Procedure.
This report. has been audited by the Quality Assurance Unit, and is considered to be an accurate account of the data generated

## ~nd.ofthe procedures followed.

In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule of this study were as follows:
12 November 2008 13, 28 January 2009 06 January 2009 06 January 2009 06·January 2009 15 January 2009 

### § 18 February 2009 

### § Date of QA 

### Signature Standard Test Method Compliance Audit Test Material Preparr;:ttion Animal Preparation Dosing**Asses~ment** 

### of**Respon~e** 

### Necropsy Draft Report'Audit Final Report Audit §. Evaluation specific to this study

# ~·")/\-\.~~.

...**~**

# ................................................................. . . 17 MAR 2009 DATE

................................................ . For the Quality Assurance Unit*

*Authorised QA Signatures: Deputy Head of Department JM Crowther MIScT MRQA Senior Audit Staff: G Wren ONC MRQAPROJECT NUMBER: 2082/0230 PAGE3



### GLP COMPLIANCE STATEMENT


### The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 512004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/1 0/EC.

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



### J Bradshaw SSe (Hons) Study Director DATE:

.........

# (P.J-1AR..?.~~~ 

# ................... .

This report may be presented In final form as a digital (pdf} document. Such documents are prepared by scanning the paper original,

and are considered of equivalent Integrity and authenticity to versions produced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, Is the definitive document.PROJECT NUMBER: 2082/0230 PAGE4



### CONTENTS QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 6 2. TEST MATERIAL AND EXPERIMENTAL PREPARATION 6 

### 2.1 Description,. Identification and Storage Conditions 6 

### 2.2 Experimental Preparation 6 3. METHODS 7 3.1 Animals and Animal Husbandry 7 3.2 Procedure 8 3.3 Evaluation of Data 9 4. ARCHIVES 9 5. RESULTS 10 5.1 Dose Level

- 2000 mg/kg 1 0 5.2 Dose Level

- 300 mg/kg 1 0 6. CONCLUSION 11 Table 1 Individual Clinical Observations and Mortality Data- 2000 mg/kg 12 Table 2 Individual Bodyweights and Bodyweight Changes

- 2000 mg/kg 13 Table 3 Necropsy Findings- 2000 mg/kg 14 Table 4 Individual Clinical Observations and Mortality Data- 300 mg/kg 15 Table 5 lnd.ividual Bodyweights and Bodyweight Changes

- 300 mg/kg 16 Table 6 Individual Necropsy Findings- 300 mg/kg 17 Appendix 1 Flow**C~art** 

### for the Sighting Test 18 Appendix 2 Flow Chart for the Main Test · · 19 Appendix 3 Statement of**GL~** 

### Compliance in Accordance with Directive 2004/9/EC 20

•'l.PROJECT NUMBER: 2082/0230



### P124832: ACUTE ORAL TOXICITY IN THE RAT

- FIXED DOSE METHOD
SUMMARY PAGE5



### Introduction. The study was performed to assess the acute oral toxicity of the test material in the Wistar strain rat. The method was designed to meet the requirements of the following:

• OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity- Fixed Dose Method" (2001)

• Method 

### 81 bis Acute Toxicity (Oral) of Commission Directive 2004fi3/EC


### Method. Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test material, as a solution in arachis oil BP, at a dose level of 300 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. 

### Mortality. The animal treated at a dose level of 2000 mg/kg was killed in extremis seven days after dosing. There were no deaths noted at a dose level of 300 mg/kg.


### Clinical Observations. Signs of systemic toxicity noted in the animal treated at a dose level of 2000 mg/kg were hunched posture, pilo-erection, decreased respiratory rate, lethargy, hypothermia and dehydration. There were no signs of systemic toxicity noted in animals treated at a dose level of 300 mg/kg. 

### Bodyweight. Surviving animals showed expected gains in bodyweight.



### · Necropsy. Abnormalities noted at necropsy of the animal treated at a dose level of 2000 mg/kg were patchy pallor of the liver and haemorrhage of the gastric mucosa. No abnormalities were noted at necropsy of animals treated at a dose level of 300 mg/kg.


### Conclusion. The acute oral median lethal dose (LD5o) of the test material in the female Wistar strain rat was estimated to be in the range of 300

- 2000 mg/kg bodyweight (Globally Harmonised Classification System -Category 4).PROJECT NUMBER: 2082/0230 PAGES



### Pl24832: ACUTE ORAL TOXiCITY IN THE RAT



### . . .



### - FIXED DOSE METHOD
1. INTRODUCTION
The study was performed to assess the acute oral toxicity of the test material in the Wistar strain rat. The method was designed to meet the requirements of the following:

• OECD Guidelines for Te.sting of Chemicals No 420 "Acute Oral Toxicity**~** 

### Fixed Dose



# .. . . 

### Method" (2001)

• Method B1 

### bis Acute Toxicity (Oral) of Commission Directive 2004/73/EC
The test system was chosen because the rat has been shown to be a suitable model for this type of study and is recommended in the test method. The re!?ults of the study are believed to be of value in predicting the likely toxicity of the test material to man.
The study was performed between 07 January 2'0Q9 and 04 February 2009.


### 2. TEST MATERIAL AND EXPERIMENTAL PREPARATION
2.1 Description, Identification and Storage Conditions Sponsor's identification Pl24832 Description

•
. • l • • 

### Batch number Date received Storage conditions straw coloured liquid 

# OE 082

## 33i.i=r.6~8 WD 17 November 2008 room temperature in the dark
The**int~grity** 

### of S!Jpplied data relating to th.e**id~ntity,** 

### purity and stability of the test ' ' I' ' • ''

... l:. ' 

### material is the responsil)ility of t,he**~ponsor.** 

### 2.2 Experimental Preparation
For the purpose 

# of the 2000 mgikg

## ·d~se level the test material was used as supplied. The specific gravity was determined and used to calculate the appropriate dose volume for the required dose level. For the purpose of the 300 mg/kg dose level the test material



## . ) 

# (PROJECT NUMBER: 2082/0230 PAGE7



### was freshly prepared, as required, as a solution in arachis oil BP. Arachis oil BP was used because the test material did not dissolve/suspend in distilled water.
Determination by analysis of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.


### 3. METHODS 3.1 Animals and Animal Husbandry
Female Wistar (HsdRccHan®TM:WIST®TM) strain rats were supplied by Harlan Laboratories UK Limited, Bicester, Oxen, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The bodyweight variation did not exceed 

## ± 20% of the initial/mean bodyweight of any previously dosed animal(s).
The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxen, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.PROJECT NUMBER: 2082/0230 PAGES


### 3.2 Procedure


### In the absence of data regarding the toxicity of the test material, 300 mg/kg was chosen as the starting dose.

A single animal was treated as follows:
Dose Level Concentration Dose Volume Number of Rats

(mg/kg) (mg/ml) (ml/kg) Female

300 30 

### 10 1 

### In the absence of toxicity at a dose level of 300 mglkg, an additional animal was treated as follows:
Dose Level Specific Gravity Dose Volume Number of Rats

(mg/kg) (ml/kg) Female

2000 0.886 2.26 1 

### Due to mortality and signs of systemic toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:
Dose Level Concentration Dose Volume Number of Rats

(mg/kg) (mg/ml) (ml/kg) Female



### 300 

### 30 10 4


### A total of five animals were therefore treated at a dose level of 300 mg/kg in the study.

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.

Clinical observations were made 

### %, 1, 2, and 4 hours after dosing and then daily for up to fourteen days. Morbidity and mortality checks were made twice daily.

Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 14 or at death.PROJECT NUMBER: 2082/0230 PAGE9


### At the end of the ob$ervation period the surviving animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.


### 3.3 Evaluation of Data
Data evaluations inciuded the relationship, if any, between the animals' exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects. If possible the signs of evident toxicity were also identified. Evident toxicity is defined as the toxic effects which are of a severity such that administration at the next highest level could result in mortality. Using the mortality data obtained, an estimate of the acute oral median lethal dose (LDso) of the test material was made.


### 4. ARCHIVES
Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which instructions will be sought as to further retention or disposal.PROJECT NUMBER: 2082/0230 PAGE10



### 5. RESULTS
5.1 

### Dose Level**~** 

### 2000 mg/kg
Individual clinical observations and mortality data are given in Table 1.

5.1.1 Mortality
The animal was killed 

## in extremis seven days after dosing.
5.1.2 Clinical Observations Signs of. systemic toxicity noted were hunched posture, pilo-erection, decreased respiratory rate, lethargy, hypothermia and dehydration.
5.1.3 

### Bodyweight
The bodyweight on Day 0 and at death are given in Table 2.


### 5.1.4 Necropsy Necropsy findings are given in Table 3.
Abnormalities noted were patchy pallor of the liver and haemorrhage of the gastric mucosa.
5.2 Dose 

### Level**~** 

### 300 mg/kg
Based on the results at a dose level of 2000 mg/kg, a dose level of 300 mg/kg bodyweight was investigated.
Individual clinical observations and mortality data are given in Table 4.

5.2.1 Mortality
There were no deaths.PROJECT NUMBER: 2082/0230 PAGE 11


### 5.2.2 Clinical Observations
No signs of systemic toxicity were noted during the observation period.


### 5.2.3 Bodyweight
Individual bodyweights and bodyweight changes are given in Table 5.

All animals showed expected gains in bodyweight over the observation period.


### 5.2.4 Necropsy
Individual necropsy findings are given in Table 6.

No abnormalities were noted at necropsy.


### 6. CONCLUSION
The acute oral median lethal dose (LD 50) 

### of the test material in the female Wistar strain rat was estimated to be in the range of 300

- 2000 mg/kg bodyweight (Globally Harmonised Classification System- Category 4).Pl24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODTable1IndividualClinicalObservationsandMortalityData-2000mg/kgEffectsNotedAfterDosingEffectsNotedDuringPeriodAfterDosingDoseAnimal(Hours)LevelNumbermglkgandSex%120002-000Female0=NosignsofsystemictoxicityH=HunchedpostureP=Pilo-erectionRd=DecreasedrespiratoryrateL=LethargyHo=HypothermiaDH=DehydrationX*=Animalkilledinextremis2040(Days)123456780HPRdHPRdHPHPHPHPLRdHoDhX*PROJECTNUMBER:2082/0230'91011121314PAGE12Pl24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODTable2IndividualBodyweightsandBodyweightChangesN2000mg/kgDoseLevel.AnimalNumberBodyweight(g)atDayBodyweightGain(g)Bodyweight(g)DuringWeekmg/kgandSex0714atDeath1220002-0Female193--181-------·-····------------=AnimaldeadPROJECTNUMBER:2082f0230PAGE13r.NPI24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODTable3NecropsyFindings-2000mg/kgDoseLevelAnimalNumberTimeofDeathMacroscopicObservationsmg/kgandSexLiver:patchypallor20002-0FemaleKilledDay7Gastricmucosa;haemorrhagic-------------PROJECTNUMBER:2082/0230PAGE14@PI24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODTable4IndividualClinicalObservationsandMortalityData-300mg/kgDoseAnimalEffectsNotedAfterDosingEffectsNotedDuringPeriodAfterDosingILevelNumber(Hours)(Days}mg/kgandSex%12 41 2345678910111213141-0000000000000000000Female3-0'Female0000000000000000003003-1000000000000000000Female3-2000000000000000000Female3-3000000000000000000Female0=NosignsofsystemictoxicityPROJECTNUMBER:2082/0230PAGE15(iPI24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODTable5BodyweightsandBodyweightChanges-300mg/kgDoseBodyweight(g)atDayLevelAnimalNumberandSexmg/kg07141-0Female2172302403-0Female2002082113003-1Female2042142213-2Female1811932053-3Female186200207----------------------PROJECTNUMBER:208210230BodyweightGain(g)DuringWeek121310331071212147--------PAGE16Q_PI24832:ACUTEORALTOXICITYINTHERAT-F!XEDDOSEMETHODTable6IndividualNecropsyFindings-300mg/kgDoseLevelAnimalNumberTimeofDeathMacroscopicObservationsmg/kgandSex1-0FemaleKilledDay14Noabnormalitiesdetected3-0FemaleKilledDay14Noabnormalitiesdetected3003-1FemaleKilledDay14Noabnormalitiesdetected3-2FemaleKilledDay14Noabnormalitiesdetected3-3FemaleKilledDay14Noabnormalitiesdetected----PROJECTNUMBER:208210230PAGE17(®Pl24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODAppendix1FlowChartfortheSightingTestSTART,...Death*foroutcomeEvidenttoxicityproceduretoconfirmtheGHSclassificationNodeathsorevidenttoxicityPROJECTNUMBER:2082/0230PAGE18Appendix2PI24832:ACUTEORALTOXICITYINTHERAT-FIXEDDOSEMETHODFlowChartfortheMainTest1withevidenttoxicityandlor::;;1deathNodeathsorevidenttoxicity,:1START.....GroupsizeThe5animalsineachmainstudygroupwillincludeanyanimaltestedatthatdoselevelinthesiahtinastud*AnimalwelfareoverrideIfthisdoselevelcauseddeathinthesightingstud,thennofurtheranimalswillbetested.Godirectlytooutcome..·,••PROJECTNUMBER:2082/0230PAGE19®PROJECT NUMBER: 2082/0230 PAGE20


### Appendix3 Statement of GLP Compliance in Accordance with Directive 

### 2004/9/EC


# f 

# oH) Department 

# '7.:: ofHealth

THE DEPA.l{TMENT OF HEALTH OF 

### THE GOVERNMENT OF 

### THE UNITED KINGDOM GOOD LABORATORY PRA<;:TICE

STATEMENT OF COMPLIANCE iN ACCORDANCE WITH DmECTIVE 200419/EC

TEST FACILI'IY

Harlan Laboratj)ries Ltd. SliardJow Business P.ark London Road 'Sh.ardlow Derby DE272GD

.D!-TE OF INSPEcrraN 19th August 2008 TEST TYPE
Annlyticiti/Ciinical Chemistry Environmental Fate Environmental Toxicology**M:ut~t;eniclty** Phys/Chem Toxicology

A general in$pection for compliance with the Principles of Oood Laboratory Practice was canied**ou~** at the above test facility as pan ·ofthe UK**GL~** Compliance**Progra~e.** At the time of**inspecti~J?.** no deviations were found of sufficient magnitude to affect the validity of**non~clinical** st)Jdies perfonned at these facilitles.

Dr. Andrew J. Gray Head, UK GLP Monitoring AuthorityPAGE 1 OF 16 PAGES



### STUDY SPONSOR:

,..,., ·.

. •, .. •,, ......**~·**.....

2082-0231.doc!JS 

### P124832:

ACUTE DERMAL IRRITATION IN THE RABBIT
PROJECT NUMBER: 2082/0231 

# harlan
...


# ....

AUTHOR: J 

### Bradshaw

TEST FACILITY: 

### Harlan Laboratories Ltd Shardlow Business Park Shard low Derbyshire DE722GD 

### UK Telephone: +44 (0) 1332 792896 

### Facsimile: +44 (0) 1332 799018PROJECT NUMBER: 208210231 PAGE2



### QUALITY ASSURANCE REPORT
This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure.

This report has been audited by ttJe Qual_ity Assurance Unit, and is considered to be an accurate account of the data generated and 

### of the procedures followed.

In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule of this study were as follows: 14 Noveml;>er 2008 13 January 2009 13 January 2009 13 January 2009 13 January 2009 

### § 25 February 2009 

### § Date of QA 

### Signature Standard**Te~t** 

### Method Compliance Audit Test Material Preparation Animal Preparation Dosing·· · ' Assessment of Response ·Draft

### ~eport Audit Final Report Audit 

### § Evaluation specific to this study


# G 02 MAR 2009



# ...........................**:.l..D..s::~** 

# ...................... ,............. DATE:

......... :
..... :
.............................. .. For the Quality Assurance Unit*

*Authorised QA Signatures: •· • Deputy Hea.d of Department: JM Crowther MIScT MRQA Senior Audit Staff: G Wren ONC MRoAPROJECT NUMBER: 2082/0231 PAGE3



### GLP COMPLIANCE STATEMENT


### The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (Sl 1999/3106 as amended by 812004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.

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



### J Bradshaw BSc (Hons) Study Director DATE:

....

## tZ..f~~~.JQ~~ 

# ........................ .

This report may be presented in final form as a digital (pdf) document. Such documents are prepared by scanning the paper original,

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, In all cases the hand-signed paper original, held in secure archives, 

### rs the definitive document.PROJECT NUMBER: 2082/0231



### QUALITY ASSURANCE REPORT GLP COMPLIANCE STATEMENT CONTENTS SUMMARY 1. INTRODUCTION 

### 2. TEST MATERIAL CONTENTS

2.1 Description, Identification and Storage Conditions



## 3.

4. 

## 5. 2.2 Preparation of Test Material 2.3 Measurement of pH 

### METHODS 3.1 Animals and Animal Husbandry 3.2 Procedure 3.3 Interpretation of Results 

### ARCHIVES RESULTS 5.1 Skin Reactions 5.2 Bodyweight 

### 6. CONCLUSION Table 1 Table 2 Table 3 Individual Skin Reactions Following**3~Minute** 

### and 1-Hour Exposures Individual Skin Reactions Following 4-Hour Exposure Individual Bodyweights and Bodyweight Change PAGE4



### 2 

### 3 

### 4 

### 5 6 

### 6 

### 6 6 

### 7 

### 7 7 8 9 

### 10 11 11 12 

### 12 13 14 15 Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 16PROJECT NUMBER: 208210231 PAGE5



### Pl24832: ACUTE DERMAL IRRITATION IN THE RABBIT
SUMMARY
Introduction. The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

• OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal ) Irritation/Corrosion" (adopted 24 April 2002)

• Method 84 Acute Toxicity (Skin Irritation) of Commission Directive 2004n3/EC


### Results. 3-minute and 1-hour semi;.occluded applications of the test material to the intact skin of one rabbit produced no corrosive effects.
A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced well-defined erythema and slight oedema. Other skin reactions noted were light brown discolouration of the epidermis, loss of skin flexibility and elasticity and crust formation, which prevented accurate evaluation of erythema and oedema. Slight desquamation was noted at one treated skin site and two treated skin sites appeared normal at the 14-day observation. No corrosive effects were noted.


### Conclusion. The test material produced a primary irritation index of 3.7 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme.PROJECT.NUMSER: 2082/0231 PAGES



### Pl24832:. ACUTE DERMAL IRRITATION IN THE RABBIT

1. INTRODUCTION The study was performed to assess the irritancy potential of the test material following single, 3-minute, 1 and 4-hour, semi-occluded applications to the intact rabbit skin. The method was designed to meet the requirements of the following:

• OECD

.Guidel_ines for the Testing ·of Chemicals No. 404 "Acute Dermal lrritation/Corrosion 1' 

### (adopted 24 Apri12002)

• Method 84 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC
The albino rabbit has been shown to be a suitable model for this type of study and is recommended in the test method. The results of the study are believed to be of value in predicting the likely skin irritancy potential of the test material to man.
The study was performed between 17 December 2008 and 20 January 2009.


### 2. TEST MATERIAL 

### 2.1 Description, ldenti'ijcation and Stqrage. Conditions Sponsor's identification PI 24832 Description straw coloured liquid Batch number OE 082 337 Fr. 6-8 WD Purity 98.8%. CAS Number 1815-99-2 Date received Storage conditions 17 November'2008 room temperature in the dark The ·integrity of. supplied data· relat)ng

..to 

# the**id~ntity,** p·urity and stability of the test material is the responsibility of the Sponsor.

. 

### 2.2 Preparation of Test**Ma~rial** 

### For the purpose of the study the test material was' used as supplied.PROJECT NUMBER: 2082/0231 PAGE7


### The absorption of the test material was not det.ermined.


### 2.3 Measurement of pH
The pH of the test material was determined _prior to cor11mencement of the study and found to be as follows:
Preparation pH Measurement immediately after 10 minutes after 20 minutes Undiluted as Supplied approximately 8.5 not applicable not applicable 90% v/v aqueous preparat!on

· of the test material**appro~imately** 6.6 approximately 6.5 approximately 6.6


### 3. METHODS 

### 3.1 Animals and Animal Husbandry
Three New Zealand White rabbits were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK. At the start of the study the animals were in the weight range of 2.0 to 3.5 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to ma.ins drinking water and food (2030 Teklad Global Rabbit diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxen, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study._
The temperature and relative humidity were set to.achieve limits of 17 to 23°C and 30 to 70% respectively. Any**occa~ional** 

### deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was cbntrolled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to**c~ntain** 

### any contaminant of a level that might have affected the purpose or integrity of the study.

-----··-------------·----···---··--··-·--···-·--·PROJECT NUMBER: 2082/0231 PAGES


### 3.2 Procedure
On the day before the test each rabbit was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
One rabbit was initially treated. Three suitable sites were selected on the back of the rabbit At each test site a quantity of 0.5 ml 

### of the test material was introduced under a 2.5 em x 2.5 em cotton gauze patch and placed in position on the shorn skin. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
One patch was removed at each of three time points: 3 minutes, 1 hour and 4 hours after application. Any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water.
After consideration of the skin reactions pr<?duced in the first animal, two additional animals were treated with 0.5 ml of test material. One patch was applied to the back of each rabbit, and was allowed to remain in contact with the skin for a period of four hours. Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the following scale:
EVALUATION OF SKIN REACTIONS 

### Erythema and Eschar Formation No erythema

..................................................................................................... Very slight erythema (barely perceptible)

....................................................... .. Well-defined erythema

..................................................................................... Moderate to severe erythema

........................................................................ .. Severe erythema (beef redness) to eschar formation preventing grading of erythema

.......................................................................................................... Value 

### 0 

### 1 2 3


## 4PROJECT NUMBER: 208210231



### Oedema Formation No oedema

....................................................................................................... Very slight oedema (barely perceptible)

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

...................... .. Moderate oedema (raised approximately 1 millimetre)

................................... . Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure)

..................................................................................................... Any other skin reactions, 

### if present, were also recorded. PAGES



### Value 0 

### 1 2 3
4

Additional obseJVations were made on Days 7 and 14 to assess the reversibility of skin reactions.

Individual bodyweights were recorded ori Day 

### 0 (the day of dosing) and at the end of the observation period.


### 3.3 Interpretation of Results
Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test material. The test material was classified according to the following scheme devised by Draize 

### J H (1959) "Dermal Toxicity" In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States, Austin, Texas, p.46-59: 

### Primary Irritation Index 0
> Oto2


### > 

### 2 to 

### 5

> 5to8 

### Classification of Irritancy Non-irritant Mild irritant Moderate irritant Severe irritant I



### If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the testPROJECT NUMBER: 208210231 PAGE10


### material is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.


### 4. ARCHIVES
Unless instructed otherwise by the Sponsor,· all original data and the final report will be retained in the Harlan Laboratories Ltd, Shard low, UK archives for five years, after which instructions will be sought as to further retention or disposal.PROJECT NUMBER: 2082/0231 PAGE 11



### 5. RESULTS
5.1 Skin Reactions
5.1.1 3-Minute Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 1.
No evidence of skin irritation was noted during the study.


### 5.1.2 1-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 1. Very slight erythema was noted at the treated skin site at the 24-hour observation with well-defined erythema and very slight oedema noted at the 48 and 72-hour observations. Light brown discolouration of the epidermis and loss of skin elasticity were also noted at the treated skin site at the 72-hour observation. Crust formation, which prevented accurate evaluation of erythema and oedema, was noted at the treated skin site at the 7-day observation. The treated skin site appeared normal at the 14-day observation.


### 5.1.3 4-Hour Exposure Period
The individual scores for erythema/eschar and oedema are given in Table 2.
Well-defined erythema and very slight oedema were noted at one treated skin site and very slight erythema was noted at one other treated skin site one hour after patch removal. Well-defined erythema and very slight to slight oedema were noted at all treated skin sites at the 24, 48 and 72-hour observations. Light brown discolouration of the epidermis and loss of skin flexibility and/or elasticity were noted at two treated skin sites at the 48-hour observation and at all treated skin sites at the 72-hour observation. Crust formation, which prevented accurate evaluation of erythema and oedema, was noted at 

### all treated skin sites at the 7-day observation. Slight desquamation was noted at one treated skin site and two treated skin sites appeared normal at the 14-day observation.PROJECT NUMBER: 208210231 PAGE12 

### 5.2 Bodyweight
Individual bodyweights and bodyweight changes are given in Table 3.

All animals showed expected gain in bodyweight during the study.


### 6. CONCLUSION
The test material produced a primary irritation index of 3. 7 and was classified as, a MODERATE IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.PROJECT NUMBER: 2082/0231 PAGE13
PI 24832 : ACUTE DERMAL IRRITATION IN THE RABBIT


### Table 1 Individual Skin Reactions Following 3-Minute and 1-Hour Exposures
Individual Scores

- Rabbit Number and Sex Skin Reaction Observation Time 68067 Male 3-Minute Exposure 1 Hour 24 Hours Erythema/Eschar 48 Hours Formation 72 Hours ?Days 14 Days 1 Hour 24 Hours Oedema Formation .48Hours 72 Hours

?Days 14 Days

Br 

### = light brown discolouration of the epidermis Le 

# = Loss of skin elasticity Cf 

### = Crust formation 0 0 

### 0 0 0 0 0 0 0 0 0 0

?e 

### = Adverse reaction prevented accurate evaluation of erythema ?od 

### = Adverse reaction prevented accurate evaluation of oedema 1-Hour Exposure 0 

### 1 2 2BrLe ?eCf 0 0 0 1 1 ?od 0

---------------·--------

.. ------------·-------------------··-----··-·-·-------···-PROJECT NUMBER: 2082/0231
PI 24832 : ACUTE DERMAL IRRITATION IN THE RABBIT


### Table 2 Individual Skin Reactions Following 4-Hour Exposure

Individual Scores- Rabbit Number and Sex Skin Reaction Observation Time 68067 Male 68102 Male
1 Hour 0 

### 1 24 Hours 2 2 Erythema/Eschar 48 Hours 2 2BrLe Formation 72 Hours 2BrLe 2BrLeLf 7 Days ?Cf ?Cf 14 Days 

### OD 0 1 Hour 0 0 24 Hours 1 1 Oedema 48 Hours 2 2 Formation 72 Hours 2 2 7 Days ?od ?od 14 Days 0 0 Sum of 24 and 72-hour Readings {S) 22 Primary Irritation Index (S/6) 22/6= 3.7 Classification MODERATE IRRITANT

( ) =Total values not used for calculation of primary irritation index Br 

### = light brown discolouration of the epidermis Le_ 

### = Loss of skin elasticity Lf 

### = Loss of skin flexibility Cf 

### = Crust formation D 

### = Slight desquamation ?e = Adverse reaction prevented accurate evaluation of erythema ?od 

### = Adverse reaction prevented accurate evaluation of oedema 68103 Maie
2 

### 2 2BrLe 2BrLeLf ?Cf 

### 0 1 

### 2 2 2 ?od 0 PAGE 14

Total

( 3)

6 (6)

6 ( ()..12)

( 0) ( 1 )

4 { 6)

6 ( 0-12}

( 0)PROJECT NUMBeR: 2082/0231 PAGE15 Pl24832 :ACUTE DERMAL IRRITATION IN THE RABBIT


### Table 3 Individual Bodyweights and Bodyweight Change

Rabbit Number Individual Bodyweight (kg) Bodyweight Change (kg) 

### and Sex DayO Day 14
68067 Male 2.76 2.99 0.23

68102 Male 2.51 2.62 0.11

68103 Male 2.31 2.60 0.29**--~--------------------------~---- ----~-**  - -**~----** ---- - ---- - - - -- ·-· ·- ·---· ·-···--···--PROJECT NUMBER: 2082/0231 PAGE16


### Appendix 1 Statement of GLP Compliance in Accordance with Directive 2004/9/EC


# f

# nli) Department

### ~ ofHealth



### THE DEP:MITMENT OF HEALTH OF THE GOVERNMENT OF 

## THE UNITED KINGDOM GOOD LABORATORY 

### PRACTICE

STATEMENT OFCOMPLIANCE 

### iN ACCORDANCE WITH DJRECTIVE 2004/9/EC

TESr FACIUTY
Harlan Laboratj)ries Ltd. Sllardlow Business Park London Road 'SIJardlow Derby DE272GD

D,:\TE OFINSPECl'IQN 19th August 2008 TESTTYPE
Ailalyucid/CJinical Cbemistry Environmental Fate Environmental Toxicology Mn

...geriicity Phys/Chem Toxicology

A general.**in~pection** for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part ofthe UK**GL~** Compliance**Progra~e.** Ji.t the time**of.inspcctip~** no deviations were found of sufficient magnitude to affect**~e** validlty of non-clinical stj.ldies performed at these facilities.

Dr. Andrew J, Gray Head, UK GLP MonitoriugAuthorityPAGE 1 OF 21 PAGES



### Pl24832: 

# harlanTM



# ....

LOCAL LYMPH NODE ASSAY IN THE MOUSE

STUDY SPONSOR:

.. . .'- • ·.' t

2082-0232.doc!JS .· ....**~** 

# .. · 

### PROJECT NUMBER: 2082/0232

AUTHOR: 

### J Bradshaw

TEST FACILITY: 

### Harlan Laboratories Ltd Shardlow Business Park Shard low Derbyshire DE72 2GD 

## UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER: 208210232 PAGE2



### QUALITY ASSURANCE REPORT


### This study type is classed as short-term. The standard test method for this study type ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure.

This report has been audited by the Quality Assurance Unit, and is considered to be an accurate account of the data generated and of the procedures followed.

In each case, the outcome of QA evaluation is reported. to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule of this study were as follows: 

### 12 November 

### 2008 16 February 

### 2009 23 February 

### 2009 19 February 

### 2009 16 February 

### 2009 19 February 

### 2009 13 March 

### 2009 Standard Test Method Compliance Audit



### § § §' 

### Date of QA 

### Signature .. 

### Test Material Preparation Test System Preparation Animal Preparation Dosing Assessment of Response Draft Report Audit Final**R~port** 

### Audit Evaluation specific**~o** 

### this study


## 2 7 MAR 2009 DATE:

............................................... . For the Quality Assurance Unit*

*Authorised QA Signatures: Deputy Head of Department: JM Crowther MIScT MRQA Senior Audit Staff: G Wren ONC MRQAPROJECT NUMBER: 2082/0232 PAGE3



### GLP COMPLIANCE STATEMENT


### The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by Sl 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/1 0/EC.

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

.



### J Bradshaw BSc (Hons) Study Director ............................ DATE:

.........

### 2.5..MAR~.tQa9

## .................. ..

This report may be presented In final form as a digHal (pdf) document. Such documents are prepared by scanning the paper original,

and are considered of equivalent integrity and authenticity to versions proCiluced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, is the definitive document.PROJECT NUMBER: 2082/0232



### QUALITY ASSURANCE REPORT GLP COMPLIANCE STATEMENT CONTENTS SUMMARY 1. INTRODUCTION 2. TEST MATERIAL CONTENTS

2.1 Description, Identification and Storage Conditions 2.2 Preparation of Test Material 3. METHODS 3.1 Animals and Animal Husbandry 3.2 Procedure 3.3 Interpretation of Results 4. ARCHIVES 5. RESULTS 5.1 Preliminary Screening Test 5.2 Main Test 6. CALCULATION OF EC 3 

### VALUE 7. CONCLUSION Table 1 Clinical Observations, Bodyweight and Mortality Data

- PAGE4



### 2 

### 3. 

### 4 5 

### 6 6 6 7 

### 7 7 8 

### 10 11 

### 12 12 12 

## 13 13 Preliminary Screening Test 14 Table2 Table 3 Table4 Appendix 1 Appendix2 Appendix 3 Appendix4 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index 15. Individual Clinical Observations and Mortality Data 16 Individual Bodyweights and Bodyweight Changes 17 Current Positive Control Study for the Local Lymph Node Assay 18 Summary of Positive Control Data for the Local Lymph Node Assay 19 Vehicle Determination Record 20 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 21PROJECT NUMBER: 2082/0232 PAGE5


### Pl24832: LOCAL LYMPH NODE ASSAY IN THE MOUSE
SUMMARY


### Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBNCa strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay'' (adopted 24 April 2002)

• Method 842 Skin Sensitisation (Local Lymph ·Node Assay) of Commission Directive 2004/73/EC


### Methods. Following a preliminary screening test in which clinical signs of toxicity were noted at a concentrations above 25% v/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 JJI (25 lJI per ear) of the test material as a solution in acetone/olive oil 4:1 at concentrations 

### of 25%, 10% or 5% v/v. A further group of four animals was treated with acetone/olive oil4:1 alone.


### Results. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: Concentration {% v/v) in Stimulation Index Result

acetone/olive oil4:1 

## .. ' 

### 5 2.14 Negative 

### 10 3.91 Positive 25 

### 6.65 Positive


### The concentration of test material expected to cause a 3 fold increase in 3 

### HTdR incorporation (EC3 value) was calculated to be 7.4% v/v in acetone/olive oil4:1.


### Conclusion. The test material was considered to be a sensitiser under the conditions of the test.PROJECT NUMBER: 2082/0232 PAGE6



### PI 24832: LOCAL LYMPH NODE ASSAY IN THE MOUSE

1. INTRODUCTION
A study was performed to assess the skin sensitisation potential of the test material in the CBNCa strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay 11 

### (adopted 24 April 2002)

• Method 842 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC
The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.
The strain of mouse used in these laboratories has been shown· to produce satisfactory responses using known sensitisers and non-sensitisers during the in-house validation. The results of routine positive control studies are shown in Appendix 1 and Appendix 2. The results of the study are believed to be of value in predicting the sensitisation potential of the test material to man.
The study was performed between 21 January 2009 and 17 February 2009.


### 2. TEST MATERIAL
2.1 Description, Identification and Storage Conditions Sponsor's identification Description Batch number Purity CAS Number Date received Storage conditi-ons P124832 straw coloured liquid OE 082 337 Fr. 6-8 WD 98.8% 1815-99-2 17 November 2008 room temperature in the darkPROJECT NUMBER: 208210232 PAGE7


### The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.



### 2.2 Preparation of Test Material
For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section. The vehicle determination record is included as Appendix 3.
Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.



### 3. METHODS
3.1 Animals and Animal Husbandry
Female CBNCa (CBNCaOiaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, 

### UK On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to ·mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxen, UK) was allowed throughout the study.

The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlledPROJECT NUMBER: 2082/0232 PAGES 

### by a time switch to give twelve hours continuous light 

### (06.00 to 

### 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.


### 3.2 Procedure
Animals in which any adverse effects were noted that were considered to approach the moderate severity limit set forth in the UK Home Office Project Licence, were humanely killed.


### 3.2.1 Preliminary Screening Test Using available information regarding the irritancy potential of the test material, a preliminary screening test was performed using two mice. The mice were treated by daily application of 25 (JI of the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6.


### 3.2.2 Main Test


### 3.2.2.1 Test Material Administration Groups of four mice were treated with the test material at concentrations of 25%,, 10% or 5% v/v in.acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 J.ll of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.PROJECT NUMBER: 2082/0232 PAGE9


### A further group of four mice received the vehicle alone in the same manner.
3.2.2.2 3 

### H-Methyl 

### Thymidine Administration
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 !JI of phosphate buffered saline (PBS) containing 3 

### H-methyl thymidine eHTdR: 80 !JCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 JJCi to each mouse.
3.2.2.3 

### Observations


### Clinical Observations: All animals were observed twice daily on Days 1 , 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of 

### ill health during the test were recorded. 

### Bodyweights: The bodyweight of each mouse :was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
3.2.2.4 

### Terminal Procedures


### Termination: Five hours following the administration of 3 

### HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes. Preparation of Single 

### Cell Suspension: 

# A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).PROJECT NUMBER: 2082/0232 PAGE10



### Determination of 3 

### HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3 

### HTdR incorporation was measured by**~-scintillation** 

### counting. The "Poly QTMn 

### vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence,

. which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

3.3 Interpretation of Results
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3 

### HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser 

### if at least one concentration of the test material results in a threefold or greater increase in 3 

### HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3 

### HTdR incorporation will be classified as a "non-sensitiser".
At the request of the study sponsor the EC 3 

### value was calculated. The 

### EC3 value is the concentration of test material expected to cause a 3 fold increase in 3 

### HTdR incorporation. The equation used for the calculation of 

### EC3 is:


### EC3 

# = c 

### + [(3-d)/(b-d) x (a-c)]

a 

# = lowest concentration giving stimulation index >3 b 

# = actual stimulation index caused by a c 

# = highest concentration failing to produce a stimulation index of 3 

### d 

# = actual stimulation index caused by cPROJECT NUMBER: 2082/0232 PAGE 11


### 4. ARCHIVES
Unless instructed othetwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which instructions 

### will be sought as to further retention or disposal.PROJECT NUMBER: 2082/0232 PAGE 12



### 5. RESULTS 5.1 Preliminary Screening Test Clinical observations, bodyweight and mortality data are given in Table 1.
The animal treated with the test material at a concentration of 50% v/v in acetone/olive oil 4:1 was humanely killed, on Day 6, due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence. Signs of systemic toxicity noted in this animal were hunched posture, lethargy, ptosis and splayed gait. Redness to the base of the ears and the neck of the animal were also noted.
No signs of systemic toxiqity were noted in the animal treated with the test material at a concentration of 25% vlv in acetone/olive oil 4:1

.



## .. Based on this information the dose levels selected for the main test were 25%, 10% and 5% v/v in acetone/olive oil 4: 1.


### 5.2 Main Test 5.2.1 Estimation of the Proliferative Response of Lymph Node Cells
The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2.
A stimulation index of**~reater** 

### than 3 was recorded for the test material at concentrations of25% or 10% v/v in acetone/olive oi14:1.
A stimulation index of less than 3 was recorded for the test material at a concentration of 5% v/v in acetone/olive oil 4:1.


### 5.2.2 Clinical Observations and Mortality Data
Individual clinical observations and mortality data for test and control animals are given in Table 3.
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.PROJECT NUMBER: 2082/0232 PAGE13


### 5.2.3 Bodyweight
Individual bodyweights and bodyweight changes for test and control animals are given in Table4. Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.


### 6. CALCULATION OF 

### ECa VALUE
EC3 

# = c 

### + [(3-d)/(b-d) X 

### (a-c)]



### a= 10 

### b 

### = 3.91 c=5 

### d 

### = 2.14
ECs 

# =5 

### + [(3-2.14)/(3.91-2.14) x (10-5}] 

# = 7.4
The concentration of test material expected to cause a 3 fold increase in 3 

### HTdR incorporation (EC 3 

### value) was calculated to be 7.4% v/v in acetone/olive oil4:1.


### 7. CONCLUSION The test material was considered to be a sensitiser under the conditions of the test.

a 

### = lowest concentration giving stimulation index >3 b 

### = actual stimulation index caused by a c 

# = highest concentration failing to produce a stimulation index of 3 d 

### = actual stimulation index caused by cPROJECT NUMBER: 2082/0232 PAGE14 Pl24832: LOCAL LYMPH NODE ASSAY IN THE MOUSE


### Table 1 Clinical Observations, Bodyweight and Mortality Data

- Preliminary Screening Test

Concentration Bodyweight Day

(% v/v) in Animal (g) acetone/olive Number 1 2 3 oil4:1 Day Day 

### Pre- Post Pre- Post Pre- Post 

### 4 1 

### 6 Dose Dose Dose Dose Dose Dose
( 

### 50 5-1 17 14 0 0* O* 

### 0* 0* 0* 0*

25 S-2 17 17 

### 0 0 0 0 0 0 0

0 

### = No signs of systemic toxicity H 

# = Hunched posture L= Lethargy Pt= Ptosis Ws = Splayed gait * 

### = Redness to the base of the ears and the neck 5



### 0*

0

X 

# = Animal humanely killed due to the occurrence of clinical signs of toxicity that approached the moderate severity limit set forth in the UK Home Office Project Licence 6
HLPt Ws*

X
0PROJECT NUMBER: 2082/0232 PAGE15
PI 24832 : LOCAL LYMPH NODE ASSAY IN THE MOUSE


### Table 2 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index Concentration

(% v/v) in dpm dpm/Node a stimulation Index b Result acetone/olive oil 4:1 Vehicle 6081.88 760.24 na na

5 13014.61 1626.83 2.14 Negative

10 23796.63 2974.58 3.91 Positive

25 40450.85 5056.36 6.65 Positive

dpm 

### = Disintegrations per minute a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes) b 

### = Stimulation Index of 3.0 or greater indicates a positive result na 

# = Not applicablePROJECT NUMBER: 2082/0232 PAGE16
PI 24832 : LOCAL LYMPH NODE ASSAY IN THE MOUSE


### Table3 Individual Clinical Observations and Mortality Data

Concentration Day 1 Day2 Day3

(% v/v) in Animal Day Day Day acetone/olive oil Number Pre- Post Pre- Post Pre- Post 4 5 

### 6 4:1 Dose Dose Dose Dose Dose Dose 1-1 0 

### 0 0 0 

### 0 0 0 0 0
1-2 0 0 0 0 0 0 0 0 0 Vehicle 1-3 0 0 0 0 0 0 0 0 0


### 1-4 0 0 0 0 0 0 0 0 0
2-1 0 0 0 0 0 0 0 0 0
2-2 0 0 0 0 0 0 0 0 0 5 2-3 0 0 0 0 0 0 0 0 0
2-4 0 0 0 0 0 0 0 0 0
3-1 0 0 0 0 0 0 0 0 0
3-2 0 0 0 0 0 0 0 0 0 10 3-3 0 0 0 0 

### 0 0 0 0 0
3-4 0 0 0 0 0 

### 0 0 0 0
4-1 0 0 0 0 0 0 0 0 0

4-2 0 0 0 0 0 0 0 0 0 25 4-3 

### 0 0 0 

### 0 0 0 0 

### 0 0

4-4 0 0 0 0 0 0 0 0 0

0 

# = No signs of systemic toxicityPROJECT NUMBER: 208210232 PAGE17 Pl24832 : LOCAL LYMPH NODE ASSAY IN THE MOUSE


### Table4 Individual Bodyweights and Bodyweight Changes Concentration Bodyweight (g)

(% v/v) in Animal Number Bodyweight acetone/olive oil Day1 Day6 Change (g) 4:1 1-1 19 

### 19 

### 0

1-2 16 

### 17 1 Vehicle 1-3 18 19 1
1--4 

### 18 19 

### 1

2-1 18 19 1

2-2 17 18 1 5 2-3 15 16 1
2--4 16 18 2
3-1 18 20 2
3-2 17 17 0 10 3-3 16 17 1
3-4 15 16 1
4-1 19 19 0
4-2 19 

### 19 0 25 4-3 

### 17 17 0

4--4 20 

### 20 0PROJECT NUMBER: 2082/0232 PAGE18
PI 24832 : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Appendix 1 Current Positive Control Study for the Local Lymph Node Assay


### Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The method was designed to meet the requirements of the following:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted ·24 April2002)

• Method 842 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Test Material: Project number: Study dates: a.-Hexylcinnamaldehyde, Tech, 85% 0039/1057 17 October 2008 to 23 October 2008


### Methods. One group of five animals was treated with 50 JJI (25 JJI per ear) of a.-Hexylcinnamaldehyde, Tech, 85% as a solution in .acetone/olive oil4:1 at a concentration of 15% v/v. A further group offive animals was treated with acetone/olive oil4:1 alone.


## Res.u/ts. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: Concentration % vlv in Stimulation Index Result

acetone/olive oil4:1 

### 15 6.55 Positive 

### Conclusion. a.-Hexylcinnamaldehyde, Tech, 85% was cons!idered tope a sens!tiser under the conditions of the test.PI24832:LOCALLYMPHNODEASSAYINTHEMOUSEAppendix2Summaryof.PositiveControlDatafortheLocalLymphNodeAssay•ProjectStartDateFinishDateTestMaterialNumber0039/101706/03/0812/03/08a.-Hexylcinnamaldehyde0039/101807/03/0813/03/08a.-Hexylcinnamaldehyde0039/102828/03/0803/04/08Phenylacetaldehyde(90%)0039/102910/04/0816/04/082,4-Dinitrobenzenesulfonicacid,sodiumsalt0039/103211/04/0817/04/08a.-Hexylcinnamaldehyde0039/103723/05/0829/05/08a.-Hexylcinnamaldehyde0039/103805/06/0811/06/08a.-Hexylcinnamaldehyde0039/105717/10/0823/10/08a.-Hexylcinnamaldehyde0039/105829/10/0804/11/08a.-Hexylcinnamaldehyde0039/105929/10/0804/11/08a.-Hexylcinnamaldehyde0039/106005/11/0817/12/08a.-Hexylcinnamaldehyde0039/106105/11/0811/11/08a.-Hexylcinnamaldehyde0039/106205/11/0811/11/08a.-Hexylcinnamaldehyde-------------•=a=b=StandardTestMethod599('Individual'nodes)RatiooftesttocontrollymphocyteproliferationStimulationindexgreaterthan3.0indicatesapositive_r_esultConcentration5%,10%,25%v/v5%,10%,25%v/v1%,2.5%,5%v/v1%,5%,10%w/w15%vlv5%,10%,25%v/v5%,10%,25%v/v15%v/v15%v/v15%v/v15%v/v15%v/v15%v/vPROJECTNUMBER:2082/0232Vehicle·acetoneethanoVdistilledwater7:3propyleneglycol1%pluronicl92indistilledwateracetone/oliveoil4:1liquidparaffinBPpolyethyleneglycol300acetone/oliveoil4:1dimethylformamidebutanonedimethylsulphoxideacetoneethanol/distilledwater7:3StimulationlndexaClassificationbI5.52,5.05,12.89PositiveII4.70,10.57,17.76PositiveI2.23,4.25,23.51PositiveI0.96,4.29,13.15PositiveI.10.91PositiveI1.73,2.32,4.58Positive2.07,4.22,10.92Positive6.55Positive4.55Positive4.05Positive5.73Positive4.21Positive9.49PositivePAGE19PROJECT NUMBER: 2082/0232 PAGE20 Pl24832: LOCAL LYMPH NODE ASSAY IN THE MOUSE 

### Appendix3 Vehicle Determination Record
Vehicle Concentration Method of Description of Suitability* Preparation Formulation acetone/olive oil 50% (4:1} 0.5 ml test material Vortex mixer solution suitable for dosing + 0. 5 ml vehicle

* 

# = Suitable for dosing if formulation is a solution or fine homogenous suspension which can be administered via a micropipette

### Appendix4 PROJECT NUMBER: 208210232



### Statement of GLP Compliance in Accordance with Directive 2004/9/EC 

# toH) Department 

## \:..: ofHealth

THE DEPAaTMENI.' OF 

## :aEAL'i"B OF 

### 'tHE GOWRN'MENT OFTBE UNITED KINGDOM GOOD LABORATOR:YPRA.CTlCE!

STATEMENT OF COMPLIANCE IN .i\CCORDANCB WITifDIREcTrVE 2004/9iEC

Harlan· Laboratories Ltd. Sllardlow Business Park l.«<ml!ln Road. Shardlow Detby D.E72 2GD.**Q~TE** QF INSPECTION 19th.August 200S .An!\'ly"ticai!Ciinical**Clt~mistcy** Environmenta!Fate · Environmental Toxicology Mutagenicity Phys/Chem T<lxieology

A.gener.al**~pection for·c;omplilll}~e**  wi_fr! th,e Pcincjp!es ofOood Laboratory**?ra~tice \'\!~** **carr,i~** out -at the ab,ove test facility aspnrt ofth-e UK GLP Gompliaru:e Pr!)"gramme. At**~e** time of"inspecti.on no·**4~viations** ·were foond of .S\Iffi!!iem ma!P.Jitade 

## to affect 

### tbe. valiP:ity of "!)on-cliniCal studies peri'Onned .at these facilitie.'h

Dr. Andrew· J. C.ray Head, UK GLP'Monltoring:Authocity PAGE21PAGE 1 OF 22 PAGES



### Pl24832:
REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPH/MURIUM

STUDY SPONSOR:**···,~r<r-** · .. .

### ·A·: t: 

### .·:···r'

2.082.-02.33.doc!MSOffice 

### PROJECT NUMBER: 2082/0233

AUTHOR: PWThompson



### TEST FACILITY:· Harlan Laboratories Ltd Shardlow Business Park Shard low Derbyshire DE72 2GD UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER: 2082/0233 PAGE2



### QUALITY ASSURANCE REPORT


### This study type is classed as short-term. The standard test method for this study type

. ("General Study Plan" in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure.

This report has been audited by the Quality Assurance Unit, and is considered to be an accurate account of the data generated and of the procedures followed.

In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule of this study were as follows: 

### 20 November 

### 2008 14, 19 January 

### 2009 19 January 

### 2009 Standard Test Method Compliance Audit Test Material Preparation


### 14 January 

### 2Q09 26 January 

### 2009 § 20 February 

### 2009 § Date of 

### QA Signature Test System Preparation Exposure Assessment -of Response Draft Report Audit Final Report Audit 

### § Evaluation specific to this study

\

# .~·.b

# ...**~~**

# ..................... . DATE: For the Quality Assurance Unit*

*Authorised QA Signatures: Deputy Head of Department JM Crowther MIScT MRQA Senior Audit Staff: G Wren ONC MRQA 

# 1 8 MAR 2009PROJECT NUMBER: 2082/0233 PAGE3



### GLP COMPLIANCE STATEMENT


### The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by Sl 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98}17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.

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

PWThompson HNC Study Director DATE:
.....

### 1.7...N.~R::2~0.9

# ........................ .

This report may be presented in final form as a digital (pdf) document Such documents are prepared by scanning the paper original,

and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, is the definitrve document.PROJECT NUMBER: 2082/0233



### QUALITY ASSURANCE REPORT GLP COMPLIANCE STATEMENT CONTENTS SUMMARY 

### 1. INTRODUCTION 

### 2. TEST MATERIAL 

### 3. METHODS 

### ·3.1 Tester Strains CONTENTS



### 3.2 Preparation of Test and Control Materials



### 4. 5.



### 6. 

## 7. 3.3 Microsomal Enzyme Fraction 3.4 

### S9-Mix and Agar 

### 3.5 Test Procedure 

### 3.6 Acceptance Criteria 

### 3. 7 Evaluation Criteria ARCHIVES RESULTS 

### 5.1 Preliminary Toxicity Test 

### 5.2 Mutation Test CONCLUSION. REFERENCES PAGE4



### 2 3 

### 4 5 

### 7 8 

### 8 8 9 10 10 10 12 12 

### 13 14 14 14 

### 15 15 Table 1 Table2 Table 3 Table 4 Table 5 Appendix 1 Appendix 2 Spontaneous Mutation Rates (Concurrent Negative Controls) 16 Test Results: Experiment 1- Without Metabolic Activation 17 Test Results: Experiment 1- With Metabolic Activation 18 Test Results: Experiment 2 -Without Metabolic Activation 19 Test Results: Experiment 2 -With Metabolic Activation 20 History Profile of Vehicle and Positive Control Value 21 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 22PROJECT NUMBER: 2082/0233



### Pl24832: REVERSE MUTATION ASSAY "AMES TEST" USING 

### SALMONELLA TYPHIMURIUM

SUMMARY PAGES



### Introduction. The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method 813/14 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. 

### Methods. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (1 0% liver 89 in standard co-factors). The dose range for Experiment 1 was determined in a preliminary toxicity assay and was 0.5_ to 150 and 1.5 to 500 J,Jg/plate for plates dosed. in the absence and presence of 89, respectively. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose level and an expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material. 

### Results. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive· control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the 89-mix were validated.
The test material caused a visible reduction in the growth of the bacterial background lawns of all of the 

### Salmonella strains, initially from 50 and 150 j.Jg/plate in the absence and presence of 89, respectively. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without 89-mix and experiment number. The test material was, therefore, tested up to the toxic limit. A globular precipitate was observed in the preliminary toxicity assay at 5000 j.Jg/plate. There was no precipitate noted in Experiments 1 and 2 due to the toxicity of the test material preventing testing up to 5000 j.Jg/plate.PROJECT NUMBER: 2082/0233 PAGE6


### No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Conclusion. 

### The test material was considered to be non-mutagenic under the conditions of this test.PROJECT NUMBER: 2082/0233



### ?124832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM



### 1. INTRODUCTION PAGE7



### This study was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers {1, 2, 3) and Garner et 

### a/ {4) in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella 

### typhimurium to various concentrations of the test material. The method used conforms with the OECD Guidelines for the Testing of Chemicals, No. 471, Method 813/14 in Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines. A copy of the Certificate of Compliance with GLP, issued by the UK Department of Health, is included.
The mutant strains of Salmonella 

### are incapable of synthesising histidine and are, therefore, dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation to histidine independent forms which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene 

### (uvrB: except TA 1 02) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal preparation (89) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.
The experimental phase of this study was performed between 23 December 2008 and 02 February 2009.

### 2. TEST MATERIAL Sponsor's identification Description Batch number Date received Storage conditions PROJECT NUMBER: 208210233



### P124832 Straw coloured liquid OE 082 337 Fr.6-8 WD 17 November 2008 Room temperature in the dark PAGES



### The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.


### 3. METHODS
3.1 Tester Strains
The strains used in this assay were all mutants derived from Salmonella typhimurium 

### L

T2 and were those recommended for general screening.
TA100 TA1535 sensitive to agents inducing base-pair substitution TA102

TA 1537 sensitive to agents inducing frame-shift mutations TA98
The strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, 

### R factors, uvrB 

### mutation and the spontaneous reversion rate (5}.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient proth (Oxoid Limited; lot number 629250 02/13) and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable· count analysis on nutrient agar plates.PROJECT NUMBER.: 2082/0233 PAGE9


### 3.2 Preparation of Test and Control Materials
The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.2%) of the test material. Analysis for concentration, homogeneity and stability of the test material formulations is n'ot a requirement of the test guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 1 0 4 

### microns.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials in the absence of 89 were as follows: N-ethyi-N'-nitro-N-nitrosoguanidine (ENNG):

. 3 jJg/plate for TA 100 and 5 JJQ/plate for TA1535 9-Aminoacridine (9AA): Mitomycin C (MMC): 4-Nitroquinoline-1-oxide (4NQO): 

### 80 JJQ/plate for T A 1537 0.5 JJQ/plate for TA102 0.2 jJg/plate for TA98
In addition, 2-Ar:ninoanthracene {2M), Benzo(a)pyrene (BP) and 1,8-Dihydroxyanthraquinone (DAN), which are non-mutagenic in the absence of metabolising enzymes, were used in the S9 series of plates at the following concentrations:

2AA: 1 J.JQ/plate forTA100 2M: 2 J.JQ/plate for TA 1535 and TA 1537 BP: 5 J,Jg/plate for TA98 DAN: 10 J.JQ/plate for TA102PROJECT NUMBER: 208210233 PAGE10


### 3.3 Microsomal Enzyme Fraction
S9 was prepared in-house on 23 November 2008 from the livers of male rats weighing

- 200g. These had each orally received three consecutive daily doses of**phenobarbitone/~-naphthoflavone** 

### (80/1 00 mg per kg per day) prior to S9 preparation. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at -196°C.


### 3.4 89-Mix and Agar
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9 1.65 M KCI/0.4 M MgCI2 

### 0.1 M Glucose-6-phosphate 0.1 

### M NADP 5.0ml 1.0ml 2.5ml 2.0ml 0.2 M Sodium phosphate buffer (pH 7.4)25.0 ml Sterile distilled water 14.5 ml
A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the 89-mix. This procedure was repeated, in triplicate, on the day of each experiment. Top agar was prepared using 0.6% Difco Bacto agar (lot number 7284259 08/12) and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin solution added to

. each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS

Limited {lot number 1051721-02 07/13).


### 3.5 Test Procedure
3.5.1 Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 JJQ/plate. The test was performedPROJECT NUMBER: 2082/0233 PAGE 11 

### by mixing 0.1 ml of bacterial culture (T

### A 1 00), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation, 0.5 ml of 89-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control {dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 

### 48 hours incubation at 3r'C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.


### 3.5.2 Mutation Test- Experiment 1
Six concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. Dose ranges were allocated as follows:
Salmonella 

### strains {without 89): 0.5, 1.5, 5, 15, 50, 150 !Jg/plate. Salmonella 

### strains (with 89): 1.5, 5, 15, 50, 150, 500 !Jg/plate. An additional dose level and an expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar; 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of 89-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without 89-mix.

All of the plates were incubated at 37°C for approximately 

### 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.


### 3.5.3 Mutation Test- Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test materialPROJECT NUMBER: 208210233 PAGE12


### dose range was the same as Experiment 1 (0.5 to 150 and 1.5 to 500 !Jglplate for the Salmonella 

### strains dosed in the absence and presence of 89, respectively).

An additional dose level and an expanded dose range were again selected in order to achieve both four non-toxic doses and the toxic limit of the test material.


### 3.6 Acceptance Criteria
The reverse mutation assay may be considered valid 

### if the following criteria are met:

All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2006 and 2007 presented in Appendix 1.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 

### bacteria per mi.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the 89-mix. The historical control ranges for 2006 and 2007 are presented in Appendix 1.
There should be a minimum of four non-toxic test material dose levels.

There should be no evidence of excessive contamination.


### 3.7 Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biologicai relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation,PROJECT NUMBER: 2082/0233 PAGE 13 

### however, statistical significance will 

### not be the only

.. determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.


### 4. ARCHIVES
Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories**~td,** 

### Shardlow, UK archives for five years, after which instructions will be sought as to further retention or disposal.PROJeCT NUMBER: 2082/0233 PAGE14



### 5. RESULTS
5.1 Preliminary Toxicity Test


### The test material was initially toxic to the strain of Salmonella 

### used (TA100) at 50 and 150 jJg/plate in the absence and presence of S9, respectively. The test material formulation and the 89-mix used in this experiment were both shown to be sterile.

The number of revertant colonies for the toxicity assay were:

With(+) or Dose ()Jg/plate) without(-) Strain Metabolic 

### 0

. 0.15 

### ·o.s 1.5 5 15 50 150 500 1500 5000 Activation



# - TA100 

### 91 89 97 

### 86 

### 92 97 92S DV 

## ov ov OVP
+ TA100 

### 75 84 90 87 96 86 73 788 

## ov ov OVP



### . S; Sparse bactenal background lawn V: Very weak bacterial background lawn P: Precipitate 

### 5.2 Mutation Test


### Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the 89-mix used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material; positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 to Table 5.

A history profile of vehicle and positive control values is presented in Appendix 1.

The test material caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella 

### strains, initially from 50 and 150 J-Ig/plate in the absence and presence of 89, respectively. The sensitivity of the tester strains to the toxicity of the

## ··~PROJECT NUMBER: 2082/0233 PAGE15



### test material varied slightly between strain type, exposures with or without 89-mix and experiment number. The test material was, therefore, tested up to the toxic limit. A globular precipitate was observed in the preliminary toxicity assay at 5000 !Jg/plate. There was no. precipitate noted in Experiments 1 and 2 due to the toxicity of the test material preventing testing up to 5000 IJg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, 

### at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the 89-mix and the sensitivity of the bacterial strains.

6. CONCLUSION


### The test material was considered to be non-mutagenic under the conditions of this test.


### 7. REFERENCES
1. Ames B N, Durston WE, Yamasaki E and Lee F D (1973.b) Proc. Nat/. Acad. Sci. (USA), 

### 70,2281-2285.

2. Ames B N, McCann 

### J and Yamasaki E (1975b) Mutation Research, 

### 31, 347-364.
3. Maron DM and Ames BN (1983) Mutation Research, 

### 113, 173-215.

4. Garner R C, Miller E C, and Miller 

### J A (1972) Cancer Res., 

### 32, 2058-2066.

5. Mortelmans K, Zeigler E (2000) Mutation Research, 

### 455,**29~60.** 

### 6. Kirkland D 

## J (Ed) (1989} Statistical Evaluation of Mutagenicity Test Data. UKEMS 

### Sub-committee on Guidelines for Mutagenicity TesUng, 

### Report

- Part Ill, Cambridge University Press.

### Table 1

EXPERIMENT 1

TA100 85 108 {96)

95

EXPERIMENT 2

TA100 79 100 {99) 118 PROJECT NUMBER: 2082/02.33 P124832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHJMURJUM 

### Spontaneous Mutation Rates (Concurrent Negative Controls)

Number 

### of revertants (mean number 

### of colonies per plate) Base-pair substitution type Frameshift type TA1535 TA102 TA98 TA1537 21 210 23 14 PAGE16

23 (20)

. 200 (207) 21 (21) 11 (13) 16 210 20 

### 13

Number of revertants ·(mean numberofcolon ies per plate) Base-pair substitution type Frameshift type TA1535 TA102 TA98 TA1537 20 247 22 22 

### 25 (22) 235 (248) 20 (21) 10 (16) 20 263 21 16PROJECT NUMBER: 2082/0233 PI 24832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM


### Table 2 Test Results: Experiment 1 -Without Metabolic Activation
Test Period From: 24 January 2009 

### I To: 27 Januarv 2009 With or Test Number of revertants (mean number of colonies per plate) without substance Base-pair substitution type S9-Mix concentration TA10D TA1535 fl.!g/plate) 113 (108) 30 (31)



# - 0 111 30 101 6.4# 34 2.3 99 (108) 27 (25)



# - 0.5 102 23 123 13.1 24 2.1 114 (107) 23 (26)



# - 1.5 104 24 104 5.8 31 4.4 122 (119)

. 29 (32}



# - 5 124 36 111 7.0 31 3.6 104 (103) 20 {23)



# - 15 104 18 101 1.7 30 6.4 99 (107) 15 (23)



# - 50 112 23 110 7.0 32 8.5 878 (78) OV (0)



### - 150 82S 

## ov 65S 11.5 

## ov 0.0 Positive Name ENNG ENNG controls Concentration 3 5 S9-Mix (!Jg/plate) 486 (510} 726 No. colonies 522 1087



# -

ENNG 

### 4NQO 9AA MMC 

# s T 

# v # per plate 523 21.1 1118

N-ethyi-N'-nitro-N-nitrosoguanidine 4-Nitroquinoline-1-oxide 9-Aminoacridine Mitomycin C Sparse· bacterial background lawn Toxic, no bacterial background lawn Very weak bacterial background lawn Standard deviation (977) 217.9 TA102 230 (266) 291 278 32.1 247 (274} 280 294 24.1 255 (261) 252 277 13.7 242 (270) 285 283 24.3 257 (247) 243 240 9.1 273 (258) 241 261 16.2 97 

### s (92) 96 

## s 848 7.2 MMC 0.5 771 (891) 1041 862 137.4 Frameshift type TA98 TA1537 20 (21) 11 (10) 20 9 24 2.3 10 1.0 22 (20} 14 (11) 21 10 16 3.2 9 2.6 24 (20) 7 (8) 21 9 16 4.0 9 1.2 24 (20) 10 (10) 16 11 20 4.0 8 1.5 25 (23) 

### a (10) 19 14 26 3.8 8 3.5 19 (20) 8 (7) 23 5 18 2.6 9 2.1 10 

### s (10) OT (0) 

### as OT 13 

### s 2.5 OT 0.0

4NQO 9AA

0.2 80 166 (218) 1150 (1825) 245 1715 ·242 44.8 2610 736.2 PAGE 17PROJECT NUMBER: 2082/0233 Pl24832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM 

### Table 3 Test Results: Experiment 1 -With Metabolic Activation
Test Period From: 24 Janu'!tY 2009 

### I To: 27 January 2009 With or Test Number of revertants {mean number of colonies**~r** plate} without substance Base-pair substitution type Frameshift type S9-Mix concentration TA100 TA1535 TA102 TA9B. TA1537 (f.lg/plate) 90 (100) 10 (12) 370 (337) 23 (24) 8 (B) + 0 BB 13 339 24 9 121 18.5# 12 1.5 302 34.0 24 0.6 B 0.6 B4 (89) 11 (12) 331 (352) 24 (24) 12 (11) + 1.5 101 13 365 22 B 81 10.8 12 1.0 361 18.6 27 2.5 12 2.3 B4 (89) 15 (14) 333 (351) 23 (23) 9 (11) + 5 92 12 355 25 9 92 4.6 16 2.1 365 16.4 20 2.5 15 3.5 100 (87) 9 (9) 306 (323) 20 (23) 7 (10) + 15 89 9 333 25 9 73 13.6 10 0.6 330 14.8 24 2.6 13 3.1 70 (68) 11 (11) 318 (327) 21 (24) 7 (7) + 50 71 7 343 27 7 64 3.8 16 4.5 319 14.2 23 3.1 8 0.6 565 (62) 13S (10) 321 (257) 15 {15) 10 (7) + 150 68S BS 247 18 7 62S 6.0 10S 2.5 203 59.6 12 3.0 5 2.5 OV (0) 

## ov (0) 898 (94) 18 

### s (15) OT (0) + 500 

## ov OV 1045 158 OT 

## ov 0.0 

## ov 0.0 898 6,7 128 3.0 OT 0.0 Positive Name 2AA 2AA DAN BP 2AA controls Concentration 1 2 10 5 2 S9-Mix (!Jg/plate} 1529 (1949) 132 (257) 1286 (1379) 241 (238) 367 (375) No. colonies 1528 356 1446 252 288

per plate 2789 727.8 283 114.2 1405 83.1 221 15.7 471 91.8 +

2AA 2-Aminoanthracene BP Benzo(a)pyrene DAN 1,8-Dihydroxyanthraquinone S Sparse bacterial background lawn T Toxic, no bacterial background lawn V Very weak bacterial background lawn # Standard deviation PAGE 18PROJECT NUMBER: 2082{0233 Pl24832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURJUM 

### Table 4 Test Results: Experiment 2- Without Metabolic Activation
Test Period From: 30 January 2009 

### I To: 02 February 2009 With or Test Number of revertants (mean number of colonies per plate)_ without substance Base-pair substitution type S9-Mix concentration TA100 TA1535 _((Jg/plate) 101 (98) 22 (20)



# - 0 106 18 88 9.3# 19 2.1 93 (109) 15 (23)

. 0.5 104 25 129 18.4 29 7.2 99 (100) 20 (20)



## . 1.5 101 22 

## c 1.4 17 2.5 95 (97) 16 {14)



# - 5 107 13 90 8.7 14 1.5 96 {94) 12 (14)



# - 15 80 18 106 13.1 11 3.8 

## ov (0) 

## ov (0)



# - . 50 

## ov ov ov 0.0 

## ov 0.0 

## ov (0) 

## ov (0)



# - 150 

## ov ov ov 0.0 

## ov 0.0 Positive Name ENNG ENNG controls Concentration 3 5 S9-Mix (IJg/plate) 1142 (1132) 658 No. colonies 1135 583



# -

ENNG 4NQO 9AA MMC 

## c 

# s T 

# v # per plate 1118 12.3 505

N-ethyi-N'-nitro-N-nitrosoguanidlne 4-Nitroquinoline-1-oxide 9-Aminoacridine Mitomycin C Contaminated Sparse bacterial background lawn Toxic, no bacterial background lawn Very weak bacterial background lawn Standard deviation (582) 76.5 TA102 216 (211) 208 208 4.6 183 (227) 263 236 40.7 199 {196) 177 212 17.7 202 (204) 221 190 15.6 206 (200) 188 206 10.4 126 (120) 116 119 5.1 558 (65) 75S 648 10.0 MMC 0.5 860 (811) 761 813 49.5 Frameshlft type TASB TA1537 13 (20) 9 {10) 26 10 20 6.5 11 1.0 25 (32) 11 (10) 28 8 42 9.1 10 1.5 17 (17) 10 (7) 16 6 18 1.0 6 2.3 24 (21) 8 {7) 13 7 25 6.7 6 1.0 13 (13) 5 (6) 8 7 18 5.0 7 1.2 25S (19) 

## ov (0) 17 

## s 

## ov 158 5.3 

## ov 0.0 158 (14) OT (0) 21 

## s OT 7S 7.0 OT 0.0

4NQO 9AA

0.2 80 148 (150) 1660 (1243) 125 994 178 26.6 1074 363.6 PAGE19PROJECT NUMBER: 2082/0233 ?124832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM 

### Table 5 Test Results: Experiment 2- With Metabolic Activation
Test Period From: 30 January 2009 

### I To: 02 February 2009 With or Test Number of revertants (mean number of colonies per plate) without substance Base-pair substitution type Frameshift type 89-Mlx concentration TA100 TA1535 TA102 TA98 TA1537 (lJg/Q1ate) 107 (102) 14 (15) 263 (235) 22 (24) 10 (9) + 0 103 11 186 20 14 97 5.0# 21 5.1 255 42.3 31 5.9 4 5.0 89 (99) 7 (10) 220 (239) 31 (34) 13 (11) + 1.5 96 9 248 46 13 111 11.2 15 4.2 250 16.8 26 10.4 8 2.9 91 (89) 19 (18) 256 (243) 25 (24) 10 (10) + 5 91 12 223 26 10 85 3.5 22 5.1 249 17.4 22 2.1 9 0.6 84 (78) 8 (10) 221 (226) 21 (21) 4 (10) + 15 89 9 240 25 15 60 15.5 12 2.1 218 11.9 17 4.0 11 5.6 121 (101) 6 (6) 214 (218) 16 (19) 9 (9) + 50 88 4 226 20 8 93 17.8 7 1.5 213 7.2 22 3.1 11 1.5 OV (0) 

## ov (0) 94 (127) 108 (14) OV (0) + 150 OV OV 141 138 

## ov OV 0.0 

### OV 0.0 145 28.4 188 4.0 

## ov 0.0 OT (0) OT (D) 41 

### s (43) 

## ov (D) OT (0) + 500 OT OT 488 

## ov OT OT 0.0 OT 0.0 398 4.7 

## ov 0.0 OT 0.0 Positive Name 2AA 2AA DAN BP 2AA controls Concentration 1 2 10 5 2 89-Mix (!Jglplate) 3243 (2356) 233 (216) 950 (805) 247 (245) 515 (410) No. colonies 224D 216 950 247 318

per plate 1584 835.5 200 16.5 516 250.6 242 2.9 397 99.1 +

2AA 2-Aminoanthracene BP Benzo(a)pyrene DAN 1,8-Dihydroxyanthraquinone S Sparse bacterial background lawn T Toxic, no bacterial background lawn V Very weak bacterial background lawn 

### # Standard deviation PAGE20PROJECT NUMBER: 2082/0233 Pl24832: REVERSE MUTATION ASSAY "AMES TEST" USING SALMONELLA TYPHIMURIUM 

### Appendix 1 History Profile of Vehicle and Positive Control Value COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2006 Strain TA100 TA1535 · WP2wrA' TA102 TA98 TA1537 $9-Mlx ·89 +$9 -$9 +59 -89 +$9 -59 +S9 -$9 +59 -59 +$9 Mean 99 90 20 13 25 30 286 337 22 26 12 14 5D 15.6 13.7 5.6 3.2 4.5 5.4 34.7 31.2 5.7 5.7 3.2 4.6 Min 66 62 8 6 12 14 197 245 11 13 4 3 Max 160 147 39 31 48 50 381 393 77 48 28 27 Values 897 733 880 716 787 632 151 88 906 732 868 703 POSITIVE CONTROL VALUES 2006 Strain TA100 TA1535 WP2wrA· TA102 TA98 TA1537 59-Mix ·59 +59 ·59 +59 -59 +59 -$9 +59 -89 +59 -S9 +S9 Mean 578 1585 352 245 855 541 1458 917 218 222 1023 498 

### so 218.2 566.9 229.5 82.2 325.1 222.1 267.7 166.1 64.3 67.0 502.1 138.8 Min 228 363 111 108 276 199 919 621 101 102 175 186 Max 1955 2969 1448 621 1942 1991 2074 1430 480 417 2718 917 Values 162 162 164 164 159 158 64 64 164 164 164 164 COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2007 TA100 TA1535 WP2uvrA· TA102 TA98 TA1537 WP2uvrA· Strain pKM101 89-Mix -59 +59 -59 +59 ·S9 +59 -$9 +59 ·S9 +$9 -89 +59 -$9 +59 Mean 94 87 21 13 26 28 273 309 21 26 11 13 132 162 80 16.0 14.8 5.2 3.6 5.8 6.4 40.4 43.2 5.9 6.0 3.8 4.1 48.5 47.8 Min 63 50 9 6 12 11 194 208 7 11 2 4 65 76 Max 156 152 38 28 46 49 390 395 99 57 28 27 278 263 Values 807 629 767 601 541 384 159 107 1282 609 1245 569 197 128 POSITIVE CONTROL VALUES 2007 Strain TA100 TA1535 WP2uvrA" TA102 TA98 TA1537 WP2uvrA· pKM101 59-Mix -89 +59 -89 +S9 -59 +59 -S9 -t59 -89 +S9 -89 +59 -59 +S9 Mean 558 1231 343 231 652 505 1281 960 256 308 1697 339 960 1103 PAGE21

WP2uvrA· pKM101 -59 +59 133 157 31.3 28.4 77 121 234 216 81 22

WP2uvrA· pKM101 -59 +$9 1962 1850 839.1 889.8 553 110 3438 3053 19 20

WP2pKM101 -S9 +$9 100 128 21.8 21.4 67 96 136 165 27 15

WP2pKM101 -S9 +59 2134 997 50 313.5 651.1 864.2 476.5 707.9 535.2 458.9 303.6 289.7 280.5 1193.6 254.4 1679.61010.6 1047.0 586.4 Min 258 320 79 Max 1634 3777 2067 Values 196 197 197

SD 

## = Standard deviation Min= Minimum value ·Max= Maximum value 73 138 139 567 661 2023 1185 2422 203 159 159 60 401 78 106 117 107 332 355 1091 285 1441 1483 2055 5618 677 2522 3023 3934 2024 61 419 202 414 198 64 64 9 11PROJECT NUMBER: 208210233


### Appendix 2 Statement of GLP Compliance in Accordance with Directive 

### 2004/9/EC



# I 

# nli) Department**~** ofHeafth

THE DEPARTlVIEN'l' OF HEALTH OF 

### THE GOVERNMENT 

### OF TBE UNITED KINGDOM. GOOD I.ABORATORY PRACTICE**~""l.'j.\."l"EJ'vffiNT** OF COMPLIANCE IN ACCORDANCE \"¥TI':l'I DIRECTIVE 2004t91EC

Rul'lnn**Lnbo.-..ttoric~:~** Ltd. Shardlow Business Pm:·I-. London Road Shat·dlow Derby 'l>"E72 2Gl>

19th August· 2008 Annlytienl/Ciinical Chcmist1-y E1wh·onn1CnUll Fat'e Environmclttnl Toxicology Mutagenicity Phys/Chcm Toxico!Qgy

A general inspection .for compliance with ·the**Prlncit~Les** of Ch>Od .Laborato1·y Practice ww; carr.ied out at the above test facility as part of the UK GLP COJ:npiiance Prog1·amme. At the time of inspection no deviations were found of suffi.cien.r magnitude to affect the validity of non-clinical studies performed a't these fadlitic!',

'P1·. Andrew J. Gray Hea.a, UK GLP 1\.icnil.toring Authority PAGE22