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 if you do 

not have actual data. 

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

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

If a user fee has been remitted for this notice (40 CFR 700.45), indicate in the boxes above the 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. 

If you claimed any information as confidentia~ a single sanitized copy must also be 

submitted. 

Part II-HUMAN EXPOSURE AND ENVTIWNMENTAL RELEASE 

If there are several mannfacture, processing, or use operations to be described in Part n, 

sections A and B of this 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, L VE, 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 itself prohibit 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. If you claim information in the notices as confidential you must 

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

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

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, if these 

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

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

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

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

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

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

composition of the tested material should be characterized. Following are 

examples 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 

Confidential 
la. 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) 

if none 

Mark (X) 

if none 

Mark (X) 

if none 

·---------------·-------------~--------·--·--·--·-----·---·---··-
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) Page4 
1. 

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 5 
a. Indicate the number-average weight of the lowest molecular weight composition of the 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 of that composition. 

Describe the methods of measurement 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 if you want a monomer or other reactant used at two weight percent or less to be listed as part of the polymer 

description on the TSCA Chemical Substance Inventory. 

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

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

d. The 

commercial purposes. 

this column 

(CAS Inventory Expert Service- a copy of the 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. Pro vi 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 if you attach a continuation sheet 

5. Trade trade names for the new 

Symmarine 

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

you must a generic name for your substance that reveals 

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

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

use, or disposal of the 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 of production. Also estimate the maximum 

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

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

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

Maximum first 12-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 of the category of use, the percent of production volume 

devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item 

you claim as confidential. 

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

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

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

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

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

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

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

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

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

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

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

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

Option tion% Form- (8) 

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

for finishing polyester fibers) (2) (3) (4) (5) (6) (7) limited surner trial mercia! Option (9) r-:--- - .L. X 100% 100% X X X X 

% % 

% % 

% % 

% % 

% % 

y.,_ % 

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

In addition include estimates of the concentration of the new chemical substance as expected in consumer products and describe the chemical reactions by which this 

substance loses its identity in the consumer product. 

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

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

use Instructions Manual for examples of generic use descriptions. 

description Addictive for consumer use products; Dispersive use 

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

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

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

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

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

EPA FORM 7710-25 (Rev. 5-95) PageS 
Complete 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 of the new chemical substance. 

EPA FORM 7710-25 (Rev. 5-95) Page9 
A. 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 if you 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. If the same operation is performed at more than one site describe the 

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

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

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

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

(by kg/day or kg/batch, on an 100% new chemical substance basis), and entry point of all feedstocks (including reactants, solvents and catalysts, etc) and all 

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

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

these sites on a continuation sheet): 

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 if you attach a continuation sheet. 

2. Worker Exposure/Environmental Release 

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

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

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

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

protect workers. 

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

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

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

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

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

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

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

Letter #of CBI Duration CBI Protective Equip. I %in CBI 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 if you 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 of useful chemical components from wastes that would 

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

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

with existing regulatory requirements and can be either quantitative or qualitative. The EPA is interested in the 

information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting 

of risks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In 

addition, information on the relative cost or performance characteristics of the PMN substance to potential 

alternatives may be provided. 

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

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

examples. 

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

volume manufactured; (3) a reduction in the generation of waste materials through recycling, source reduction or other means; (4) a 

reduction in potential toxicity or human exposure and/or environmental release; (5) an increase in product performance, a decrease in 

the cost of production and/or improved operation efficiency of the new chemical substance in comparison to existing chemical 

substances used in similar application; or (6) the extent to which the new chemical substance may be a substitute for an existing 

substance that poses a greater overall risk to human health or the environment. 

D Mark (X) this box if you 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 of the 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 15 
PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET 

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

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

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

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

Properties that are measured for mixtures or formulations should be so noted (% PMN substance in_). You are not required to 

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

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

is not a substitute for submission of test data. · 

Property Mark Page Value !Measured o 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 19 
CAS-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 ,_ 

-2 

0,0 

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 1884 
Timebase: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 1884 
Chromatographic Conditions 

GC HP 6890 (Agilent) HP 6890 (Agilent) Column Type DB-1 DB-Wax 

Phase Dimethylpolysiloxane Polyethylenglycole 

Carrier Gas Hydrogen Hydrogen 

Flow 1.3 ml (17 min) - 0.5 mUmin2 - 3.0 mUmin 1.3 ml (17 min) - 0.5 mUmin

mUmin 

Detector Type FID FID 

Detector Temperature 275°C 275°C 

Injection Volume 1J..LL (Split1 :70; 1:300 in MTBE*) 1J..LL (Split 1 :70; 1:300 in MTB

Column Length 20m 20m 

Column Diameter (i. D.) 0.18 mm 0.18 mm 

Film 0.18J..Lm 0.18J..Lm 

Injector · CIS CIS Injector Temperature 80°C - 12°C/s - 250 oc 80°C - 12°C/s - 250 oc 

Oven Temperature and Heating 60°C- 9°C/min - 220 oc 60°C - 9°C/min - 220 oc 

Rate 

*MTBE = Methyl-t-butylether 
1.3 

1.2 

1.1 

0.9 

0.8 0.7 

0.6 0.5 

0.4 

0.1 

v (ali ph. CH2, CH3) 

I 

3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 

Instrument : BioRad FTS575C 

Resolution : 1 cm-1 

Technique: FTIR- NaCI Film 

Pl24832 

Charge: OE 082 
100% 

20 40 

* 

60 

106 

93 

80 100 120 

162 17 6 

140 160 180 

I 

N 

190 204 

200 

Pl24832 

Charge: OE 082 337 _Fr.6-8

mlz

mlz = 134 

JW• 

219 

220 

mlz= 106 

240 

mlz= 16

260 2
Equipment and Conditions 

Thermo Finnigan SSQ 7000/ 6890 Gas Chromatograph 

Gas Chromatograph: GC Column: Carrier Gas: 

Flow: 

Temperature Program: 

Injector: Injector Program: 

Autosampler: 

Mass Spectrometer: 

Scan Range: 

Data System: 

Agilent 6890 · 

60 m ZB-1, id 0.25 mm, film 0.25 

helium 

2.0 mllmin 

80°C (start)- 4°C/min. (rate)- 280°C 

Gerstel CIS3 

aooc (start)- 12°C/sec. (rate)- 280°C 

Agilent 7683 Series injector 

Thermo Finnigan SSQ 7000 

m/z25 -450 

ICIS 2 Software 
oe-xp2 Proton 

S1111PU:. PEe. l \'T 

date Oct 21 2008 dfxq 100. 695 

colvent COC13 dn Cll 

file /links/mercur- dpn: ·U 

y/daily/350636t_P'I .. 

_2t832_h.£id elm 

ACQVISX'l':ION c:lrrcft 

sfxq 400.4ZO dmf 18500 

tn Kl temp 25.0 

at 6.000 POOC£SS1NG 

np ?&8'14 wt.file 

fb 

bs 

tpvr 

pw 

d.l 

tof 

6406.1 pl:OC! 

3600 fn 

• 

GO we:.:r: 

6.0 wexp 

0 ...,. 

401.3 wnt 

nt l.28 

ct 128 

aloct n 

qain not used 

FLAGS 

il y 

!o • 

dp y 

DISPLAY 

1.P -100.1 

vp U99. 9 

90 

we 2<5 

..... 15.85 

is &54.79 

•fl 798.G 

<fp 

th 

ins 2.000 

cdc ph 

ft 

not used 

5a.V<!; 

•av• vft 

Pl24832 

Charge: OE 082 337 _Fr.6-8_WD 

I 

N 0 

I I I ,-.. i i I I I I I I I . -I i 

10 9 

L-..,----1 

2.00 

8 7 

L,--J 

2.03 

6 5 4 3 

L,---J 

2.12 

2 

[} 

2.18 
NMR-Data 

Substance: PI 24832, Charge: OE 082 337 _Fr.6-8_WD 

b c 

a 

d 

a 

Shift in ppm 

a 8.47 

b 7.10 

c 2.59 

d 1.62 

e 1.40-1.20 

f 0.88 

Spectrometer 

Frequency Range 

Scans 

Acquisition Time 

Solvent 

Reference 

f 

e 

Multiplicity Functionality 

multiplet aromatic protons 

multiplet aromatic protons 

triplet aliphatic protons 

quintet aliphatic protons 

multiplet aliphatic chain protons 

'-------triplet 

400 MHz-NMR-Spectrometer 

400.420 MHz 

6406.1 Hz 

128 

6.000 sec 

Deuterochloroform (CDCh) 

Tetramethylsilane (TMS) 

methylgroup 

Number of Protons 

2 2 

2 2 

14 

3 
I 

N 

Pl24832 

Charge: OE 082 337 _Fr.6-8_WD 

U

1

::

1 

I Cl-- I - 

· .-----1- I i 

I I ! 

I .f- -! 

0,7 J I I I I I I 

I I I 

I I I I i I i 

I I I I ! 

0,5j I I I \ I 

A 0,4 I I J i 

0,31 I I I I I 

1 r------ -·-: 

i i 

''Et I I_ ' ' 

• f -- I i---t- 

210,0 250 300 350 400 450 500 

nm 

550 

--081046ASP- 03.11.2008- IO,OOmg in Ethanol Oe 082 337 Fr.6-8 WD 

MA.XIMA/MNMA LISTE 

Daten-File: 081046A 11forrration: 10,00rrg i1 Bhanol Oe 082 337 Fr.6-8 WJ 

SchweltNert 0,0010 

Abszissen-BnheR: nm 

Q-dinaten-Bnhett: A 

rt. Abszisse ()dilate Typ 

1 261,92 0,0677 Peak 

2 255,83 0,0902 Peak 

600 
Method for determination of UV-absorption 

Substance Oe 082 337 Fr.6-8 WD 

Measuring range 210- 800nm 

Concentration 10.00mg 1100ml 

Solvent Ethanol 

Path length 1 mm 

Extinction I Absorption maximum E1%11cm 68 I Amax. 262nm E1%11cm 90 I Amax. 256nm 

Effects by addition of acid or base 
PAGE 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 799018 
PROJECT 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 

~·")/\-\.~~. ... ~ ................................................................. . DATE . 1 7 MAR 2009 ................................................ . 

For the Quality Assurance Unit* 

*Authorised QA Signatures: 

Deputy Head of Department JM Crowther MIScT MRQA 

Senior Audit Staff: G Wren ONC MRQA 
PROJECT 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 (LD50) 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: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 1 Individual Clinical Observations and Mortality Data- 2000 mg/kg 

Effects Noted After Dosing Effects Noted During Period After Dosing 

Dose Animal (Hours) Level Number 

mglkg and Sex % 1 

2000 2-0 0 0 Female 

0 = No signs of systemic toxicity 

H = Hunched posture 

P = Pilo-erection 

Rd = Decreased respiratory rate L =Lethargy 

Ho = Hypothermia 

DH = Dehydration 

X* = Animal killed in extremis 

2 

0 

4 

0 

(Days) 

1 2 3 4 5 6 7 8 

0 HPRd HPRd HP HP HP HPLRd HoDhX* 

PROJECT NUMBER: 2082/0230 

9 
Pl24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 2 Individual Bodyweights and Bodyweight Changes N 2000 mg/kg 

Dose Level. Animal Number Bodyweight (g) at Day 

Bodyweight (g) 

mg/kg and Sex 0 7 14 at Death 

2000 2-0 Female 193 - - 181 

-- ---·-···· ------ --- --

-= Animal dead 

PROJECT NUMBER: 2082f0230 
PI 24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 3 Necropsy Findings - 2000 mg/kg 

Dose Level Animal Number Time of Death Macroscopic Observatio

mg/kg and Sex 

Liver: patchy pallor 2000 2-0 Female Killed Day7 Gastric mucosa; haem

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

PROJECT NUMBER: 2082/0230 
PI 24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 4 Individual Clinical Observations and Mortality Data - 300 mg/kg 

Dose Animal Effects Noted After Dosing Effects Noted During Period After Dosing 

Level Number (Hours) (Days} 

mg/kg and Sex % 1 2 4 1 2 3 4 5 6 7 8 9 10 

1-0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Female 

3-0 

Female 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

300 3-1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Female 

3-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Female 

3-3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Female 

0 = No signs of systemic toxicity 

PROJECT NUMBER: 2082/0230 
PI 24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Table 5 Bodyweights and Bodyweight Changes - 300 mg/kg 

Dose Bodyweight (g) at Day 

Level Animal Number and Sex 

mg/kg 0 7 14 

1-0 Female 217 230 240 

3-0 Female 200 208 211 

300 3-1 Female 204 214 221 

3-2 Female 181 193 205 3-3 Female 186 200 207 

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

PROJECT NUMBER: 208210230 

Bodyweigh

1 

13 

3 

10 

12 

14 
PI 24832: ACUTE ORAL TOXICITY IN THE RAT- F!XED DOSE METHOD 

Table 6 Individual Necropsy Findings - 300 mg/kg 

Dose Level Animal Number Time of Death Macroscopic Observatio

mg/kg and Sex 

1-0 Female Killed Day 14 No abnormalities detecte

3-0 Female Killed Day 14 No abnormalities detecte

300 3-1 Female Killed Day 14 No abnormalities detecte

3-2 Female Killed Day 14 No abnormalities detecte

3-3 Female Killed Day 14 No abnormalities detecte

----

PROJECT NUMBER: 208210230 
Pl24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Appendix 1 Flow Chart for the Sighting Test 

START 

,... 

Death * for outcome 

Evident toxicity procedure to confirm the GHS classification 

No deaths or evident toxicity 

PROJECT NUMBER: 2082/0230 
Appendix2 

PI 24832: ACUTE ORAL TOXICITY IN THE RAT- FIXED DOSE METHOD 

Flow Chart for the Main Test 

1 with evident toxicity andlor ::;; 1 death 

No deaths or evident toxicity 

,:1 

START 

..... 

Group size 

The 5 animals in each main study group will include any animal tested 

that dose level in the siahtina stud 

* Animal welfare override 

If this dose level caused death in the sighting stud , then no further 

animals will be tested. Go directly to outcome .. · 

, •• 

PROJECT NUMBER: 2082/0230 
PROJECT NUMBER: 2082/0230 PAGE20 

Appendix3 Statement of GLP Compliance in Accordance with Directive 

2004/9/EC 

f oH) Department 

'7.:: of Health 

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 ·of the 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 Authority 
PAGE 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 799018 
PROJECT 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 MRoA 
PROJECT 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 16 
PROJECT 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/Corrosion1

' (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 

4 
PROJECT 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 test 
PROJECT 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 OF COMPLIANCE 

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 

TEST TYPE 

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 of the 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 MonitoriugAuthority 
PAGE 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 799018 
PROJECT 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 MRQA 
PROJECT 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 EC3 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 21 
PROJECT 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 3HTdR 

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 Assay11 (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 dark 
PROJECT 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 controlled 
PROJECT 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 3HTdR 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 3HTdR 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'). 3HTdR 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 3HTdR 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 EC3 value was calculated. The EC3 value is the 

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

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 c 
PROJECT 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 (EC3 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 c 
PROJECT 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 

0 
PROJECT 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 applicable 
PROJECT 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 toxicity 
PROJECT 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 0 
PROJECT 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. 
PI 24832 : LOCAL LYMPH NODE ASSAY IN THE MOUSE 

Appendix2 Summary of.Positive Control Data for the Local Lymph Node Assay• 

Project Start Date Finish Date Test Material Number 

0039/1017 06/03/08 12/03/08 a.-Hexylcinnamaldehyde 0039/1018 07/03/08 13/03/08 a.-Hexylcinnamaldehyde 0039/1028 28/03/08 03/04/08 Phenylacetaldehyde (90%) 

0039/1029 10/04/08 16/04/08 2,4-Dinitrobenzenesulfonic acid, sodium salt 

0039/1032 11/04/08 17/04/08 a.-Hexylcinnamaldehyde 

0039/1037 23/05/08 29/05/08 a.-Hexylcinnamaldehyde 

0039/1038 05/06/08 11/06/08 a.-Hexylcinnamaldehyde 0039/1057 17/10/08 23/10/08 a.-Hexylcinnamaldehyde 0039/1058 29/10/08 04/11/08 a.-Hexylcinnamaldehyde 0039/1059 29/10/08 04/11/08 a.-Hexylcinnamaldehyde 0039/1060 05/11/08 17/12/08 a.-Hexylcinnamaldehyde 

0039/1061 05/11/08 11/11/08 a.-Hexylcinnamaldehyde 

0039/1062 05/11/08 11/11/08 a.-Hexylcinnamaldehyde 

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

•= 

a= 

b= 

Standard Test Method 599 ('Individual' nodes) 

Ratio of test to control lymphocyte proliferation 

Stimulation index greater than 3.0 indicates a positive_r_esult 

Concentration 

5%, 10%, 25% v/v 5%, 10%, 25% v/v 

1%, 2.5%, 5% v/v 

1%,5%,10% w/w 

15%vlv 

5%, 10%, 25% v/v 

5%, 10%,25% v/v 

15% v/v 

15% v/v 

15% v/v 

15%v/v 

15%v/v 

15% v/v 

PROJECT NUMBER: 2082/0232 

Vehicle· 

acetone 

ethanoVdistilled water 

7:3 

propylene glycol 

1% pluronic l92 

in distilled water 

acetone/olive oil 4:1 

liquid paraffin BP 

polyethylene glycol 300 

acetone/olive oil4: 1 

dimethyl formamide 

butanone 

dimethyl sulphoxide 

acetone ethanol/distilled water 

7:3 

Stimu

5.52

4.70, 

2.23

0.96

1. 73

2.07
PROJECT 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 \:..: of Health 

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. 

Shard low 

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 of th-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 

PAGE21 
PAGE 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 799018 
PROJECT 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 2009 
PROJECT 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 22 
PROJECT 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 

TA1537 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 1 0 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 04 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 TA102 
PROJECT 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 1 00 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 performed 
PROJECT 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 material 
PROJECT 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 16 
PROJECT 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 17 
PROJECT 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 18 
PROJECT 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 

PAGE19 
PROJECT 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 

PAGE20 
PROJECT 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 11 
PROJECT 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