TSCA NON-CONFID)ENTIAL BUSINESS INFORMATION

DOCUMENT DESCRIPTION DOCUMENT CONTROL NUMBER DATE RECEIVED

p- 17-- 3 151D20 0060 / , 62

COMMENTS: DEC LASS

DOUS NOT CONTAIN CBI
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460

OFFICE OF

&F( CHEMICAL SAFETY AND

POLLUTION PREVENTION

Notice of Change in CBI Chemical Identity Status

Pre-Manufacture Notice (PMN) P-02-0833

Notice of commencement (NOC) received: 9/16/2003

Please be advised that the Confidential Business Information (CBI) claim for the

above referenced substance was dropped by the information submitter in the

NOC.

The identified non-confidential chemical name for this substance as provided in

the NOC is:

I ,5-DIOXA-9-AZASPIRO[5.5]UNDECANE, 3,3,8,8, 10,1 0-H EXAM ETHYL-9-[1 -14-

(OXIRANYLMETHOXY)PHENYL]ETHOXY]-

The current TSCA Inventory name is

1, 5-Dioxa-9-azaspiro[5. 5]undecane, 3,3,8,8,10,1 0-hexamethyl-9-[l1-14-(2-

oxiranylmethoxy)phenyl]ethoxy]-

CASRN: 434898-80-3

This notice as submitted to the NCIC files on 9/1/2011

Name of staffer: Jeffrey Santacroce

Position: Asst. NOC Coordinator

Branch: RDMB/IMD/OPPT/OCSPP

CONTAINS NO CB1
_____________________________________________________ Form Approved. O.M.B. No. 2070-0012. Approval Expires 10-31-96.

U.S. ENVIRONMENTAL PROTECTION AGENCY AGENCY USE ONLY

Date of receipt

PREMANUFACTURE ~f- K

L NOTICE 2

FORUNW CHEMICL SUBSTANCE Company Sanitized

OCUNEN CONTROL OFFICERCE

OFFICE OF POLLUTION PREVENTION

U.S. E.P.A. 401 M STREET, SW

WASHINGTON, D.C. 20460

nte. etoanubropa s Document control number EP ase number, in the Premanufacture Notice 150 j 1X A)f?32

GENERAL INSTRUCTIONS T

* You must provide all information requested in this form to the extent that it is known to or reasonably ascertainable by you. Make reasonable estimates if you do not have actual

data.

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

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

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

ID number must also appear on your corresponding fee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM-226), P.O. 360399M, Pittsburgh, PA 15251-

6399, Attn. TSCA User fee. SANITIZED

Pait I - GENERAL INFORMATION ^ OPy TEST DATA AND OTHER DATA

You must provide the currently correct Chemical Abstracts (CA) Name of the new chemical You are required to submit all test data in your posseassion or control and to provide a substance, even if you claim the identity as confidential. You may authorize another person to description of all other data known to or reasonably ascertainable by you, if these data are submit chemical identity information for you, but yosr submission will not be complete and the related to the health and envirounmenta effects on the manufacture, processing, distribution in

review will not begin until EPA receives this information. A letter in support of your submiassion commerce, use, or disposal of the new chemical substance. Standard literature citations may be

should reference your TS user fee identification number. You must submit an original and two submitted for data in the open scientific literature. Complete teat data (written in English). not

copies of this notice including all test data. If you claimed any information as confidential, a sunmmaries of data. must be submitted if they do not apnar in the olen liteatr. You should

single saniticed copy must also be submitted, clearly identify whether test data is on the substance or on an analog. Also, the chemical

composition of the tested masterial should be characterized. Following are examples of teat data

and other data. Data should be submitted according to the requirements of §720.50 of the

Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Premanufacmure Notification Rule (40 CFR Part 720).

If there are several manufacture, processing, ur use operations to be described is Part It, sections

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

Part Ill- LIST OF ATTACHMENTS Test Data (Check Below any included in this notice)

Attach additional sheets if there is not enough space to answersa question fully. Label each * Environmental fate data 0 Yes *Other data 0 Yes

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. * Health effects data 0 Yes Risk assessments

Environmental effects data 0 Yes Structure/activity relationships

OPTIONAL INFORMATION Physical/Chemical Properties* Yes Test data not in the possession or

control of the submitter

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

substance. On page I1I of this form, space has been provided for you to described pollution *A physical and chemical properties worksheet is located on the last page of this form.

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

TYPE OF NOTICE (Check Only One)

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,PN(reaucteNoi)

protective equipment ... This option is intended to reduce delays that rutinely accompany the M (PeauctrNoi)

development of consent orders or Significant New Use Rules. Except in the case of exemption

applications (such as TMEA, LVE, LOREX) where certain information provided in such UINTERMEDIATE PMN (submitted in sequence with final product PMN)

notification is binding on the submitter when the Agency approves the exemption applicstion,

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.U SNUN (Significant New Use Notice)

CONFIDENTIALITY CLAIMS ED TMEA (Test Marketing Exemption Application) C=

You may claim any information in this notice as confidential. To assert aclas J LVE (Low Volume Exemption) @ 40 CFR 723.50(c)(1) C,. mark (X) the confidential box next to the infurmation that you clam 1 IIA Ed 0 

claim in an attachment, circle or bracket the information you claim a~brfifilniayt y Ifyoi~s~n ~ ,

information in the notices as confidential, von must also provide a sanitized1 sE'P y LOREX (Low Release/Low Exposure Exemption) (40.C 723 cg

notice. (including attachments). For additional instructions on claiming sntormuf Was 4 9

confidential, read the Instructions Manual. D V oiiainLRXMfcto~ i

tLf\i Mark (x) if any information in this notice is claimed as confidential. Fl

IS THIS A CONSOLIDATED PMN? YesYes

# of chemicals __________

(Prenotice Conmmunication # required, enter # on page 3) 0%

EPA FORM 77 10-25 (Rev. 5-95) Replaces previous editions of EPA Form 7710-25.
Public reporting burden for this collection of information is estimated to average 110 hours per response, including time for reviewing

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

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

I. The company named in Part 1, section A, subsection I a 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. 1 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:

ZThe Company named in Part 1, Section A has remitted the fee of $2500 specified in 40 CFR 700.45(b), or

Di 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

LIThe 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 (LYE) application in accordance with 40 CFR 723.50(c)(1) or a Low release and low

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

LIThe manufacturer submidtting 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.

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

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

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

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

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

described herein. Any knowing and willful misinterpretation Is subject to criminal penalty pursuant to 18 Usc 1001. Confidential

Signature and titleiof Authorized Offic(or I Signs Requird) /Date

-> ~7 1 - , Naeem H. Mady, Head, Regulatory Affairs ____________ ______

Signature of agent - (if applicable) Date

EPA FORM 77 10-25 (Rev. 5-95) Page 2
Part I -- GENERAL INFORMATION

Section A - SUBMITTER IDENTIFICATION ConfiMark 0) the "Confidential" box next to any subsection you claim as confidential dential

I a. Person Name of authorized official Position

Submitting

Notice (in U.S.) Naeemn H. Madv Head, Regulatory Affairs

Company

b.Agent (if aeo authoized officia l Positionio

applicable)(umeran sret

c. ~ ~ ~ 4 Wfyuaehumtinihsnte sPaofaint subiso, Box mar thi0bo.5

b.ointmt (if Name of authorized official Position

applicable)

ContactCinmU.S.

3. Ifou hae bad a ng ipre commnictio (PC)be cocnn thisnoicean

EAasgeaPCimrto Ste nIotie enteron thea number.be

4. If you revil submitt ed ahsnot exempto ajicts fork bisin thechemical.

EPA.t If youpevioul sumte aPMNzd fritissanc enteriheiPM

~~~~Malnadrs(number byEAsietwtdawrrinop assine et) **ifn e

5. f ouhae ub itted a te, f B P os de intenton toe manfacur oruimpor

f Thech eical susace covee byuthois otficel enertesotctnmbrMoknX

6.hoa Typee ofacer Noticet - Mrk(nMnuacura2Imor

Ce Elb Bindlt ing COp~tion l BnigOto

ansret Markin (ddes Markbe 

EPAytwn 779-90 FOR 10-2 5954Pae1 785. 
Part I -- GENERAL INFORMATION -- Continued

Section B -- CHEMICAL IDENTITY INFORMATION: You must provide a currently correct Chemical Abstracts (CA) name of the substance based on

Ithe ninth Collective Index (9C1) of CA nomrenclature rules and conventions.

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

Complete either item 1 (Class 1 or 2 substances) or 2 (Polymers) as appropriate. Complete all other items.

If another person will submit chemiical identity information for you (for either Item I or 2), mark (X) the box at the right. ConfiIdentify the name, company, and address of that person in a continuation sheet. C dential

I.Class I or 2 chemical substances (for definitions of class 1 and class 2 substances, see the Instructions Manual)

a. Class of substance - Mark (X) I [D Class 1 or 2 []Class 2

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

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

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

C. Please identify which method you used to develop or obtaln the specified chemical identity information reported in this notice: (check

one).

Method I (CAS Inventory Expert Service - a copy of the Identification LI Method 2 (Other Source)

report obtained from the CAS Inventory Expert Services must be

submitted as an attachment to this notice)

e. For a class I substance, provide a complete and correct chemical 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 X

range of composition and the typical composition (where appropriate). (4) Provide a correct representative or partial chemical structure

diagram, as complete as can be known, if one can be reasonably ascertained.

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

EPA FORM 7710-25 (Rev. 5-95) Page 4
Part I -- GENERAL INFORMATION -- Continued

Section B -- CHEMICAL IDENTITY INFORMATION -- Continued

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

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

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

1,000 absolute molecular weight of that composition.

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

i) lowest number average molecular weight:

ii) maximum weight % below 500 molecular weight: N/A

iii) maximum weight % below 1000 molecular weight:

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

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

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

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

polymer.

(2) - Mark (X) this column if entry in column (1) is confidential.

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

(4) - 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 commercial

purposes.

(7) - Mark_(X)_this column if entry in column_(6)_is confidential. ______

Monomer or other reactant and CAS Registry Number Confi- Typical Identity Confi- Maximum Confidential composition Mark (X) densial residual dential

(1) ()(3) (4 (5) (6) (7)~

Not Applicable%%

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

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

LIMethod 1 (CAS Inventory Expert Service - a copy of the identification report []Method 2 (other source)

obtained from CAS Inventory Expert Service must be submitted as

as attachment to this notice)

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

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

Not Applicable

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

EPA FORM 77 10-25 (Rev. 5-95) Page 5
Part I -- GENERAL INFORMATION -- Continued

Section B -- CHEMICAL IDENTITY INFORMATION -- Continued

3. Impurities

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

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

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

Impurity and CAS Registry Number Maximum Confipercent dential

(a) (b) _____

x

x

x

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

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

x

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

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

CGX PR 774

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

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

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

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

Aromatic ether derivative

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

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

Number if available._____

Byproduct CAS Registry Number Confi-

(1) (2) dential

None expected

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

EPA FORM 77 10-25 (Rev. 5-95) Page 6
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-month production (kg/yr) Maximum 12-month production (kglyr) Confi- Binding

(100% new chemical substance basis) (100% new chemical substance basis) dential Option

__________________________________________ ____________________________________ Markar((x

2. Use Informnation -- 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) -- Describe each intended category of use of the new chemical substance by function and application..

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

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

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

(5)-- Mark (X) this column if entry in column (4) is confidential business information (CB1).

(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 infonmation (CBI).

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

have the use type provided in (8) binding.

(9) -- Mark (X) this column if entry(ies) in column (8) is (are) confidential business information (CR1).

Category of use (1) CR1 Binding Produc- CRI % 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- Corn- Binding

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

Polymerization initiator and regulator 100% 100% 100%

for thermoplastics and elastomers ___ ______

*If you have identified a "consumner" 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. EMark (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

LIMark (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

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

for the safe handing, transport, use, or disposal of the new substance. List in part Ill hazard information you include.

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

EPA FORM 77 10-25 (Rev. 5-95) Page 7
Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

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

Iclaim as confidential

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

do not have to complete this section for operations outside the U.S.; however, you may still have reporting requirements if there are further industrial processing

or use operations after import. You must describe these operations. See instructions manual

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

Name

Site address (number and street)

No US manufacturing, processing, or use operation planned at this time

City, County, State, ZIP code

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

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

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

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

b. Type --

Mark (X) [D Manufacturing Processing Use

C. Amount and Duration -- Complete 1 or 2 as appropriate ________________________________

Maximum kg/batch (100% new chemical Hours/batch Batches/year

substance)

1. Batch

Maximum kg/batch (I100% new chemicai Hours/batch Batches/year

substance)

12. Continuous

d. Process description 1 Mark (X) to indicate your willingness to have your process description binding.

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

Not applicable

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

EPA FORM 77 10-25 (Rev. 5-95) Page 8
Part H-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued

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

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

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

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

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

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

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

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

time of exposure.

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

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

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

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

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

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

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

Mark (x) substance Mark (x) Exposed

(1) (2) (3) (4) (5) (6) (M) (8) (9) (tO) (11) (12)

Not applicable

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

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

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

(1) -- Enter the number of each release point identified in the process description, part 11, section A, subsection ld(3).

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

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

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

the new substance will be released from that release point.

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

on land, characterize the disposal method and state whether it is approved for disposal of RCRA hazardous waste. On a continuation sheet, for each site describe any

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

environment after control technology (in kg/day).

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

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

numbers of the POTW (Publicly OwniedTretatment Works). Mark (X) if the POTW name or NPDES # is confidential business information (CRI).

Release Amount of new substance CBI Media of Control technology and efficiency (you may wish to optionally attach efficiency data)CB

Number released release

(1) (2a) (2b) ,-k ani Riding

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

Not applicable

(7) Mark (X) the LIPOTW provide name(s) below: CBI l Navigable Other - Specify provide NPDES # CBI

destination(s) of waterway

releases to water.

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

EPA FORM 77 10-25 (Rev. 5-95) Page 9
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

processi .ng, 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. Identif additional sites on a continuation sheet.

1. Operation Description -- To clalm 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 contalners (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 intermtittent releases,

to the environment of the new chemical substance. (4) Please enter the # of sites (remember to identify the locations of these sites on a continuation sheet):

# of sites

Please see attached continuation sheet

ZMark (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) directiy 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 alr (optional-see Instructions Manual), surface water, on-site or off-site land or incineration, POTW, or other (specify)

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

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

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

Letter # of CR1 Duration CB1 Protective Equip. / % in CBI Release Amount of CBI Media of Release CBI

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

Act- Exposed Exposure Physical Form and % ulation Substance Technology

ivity new substance Released

(1) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (10a) (l0b) (11) (12) (13)

Eye protection, gloves, Waste to offA 1 2 10.25 12 respirators x 1 X Isite incinerator

(14) -- Byproducts: (15)______

None expected

DMark (X) this box if you attach a continuation sheet,

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

No information available

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

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

Material Safety Data Sheet (MSDS) 14 -20

Chemical Abstracts Inventory ExDert Services Reiort 21 X

Part 11 Section 1) Continuation Sheet 22 X

Analytical Certificate Rerort for CGX PR 774 23 -53

Salmonella TvDhimurium Reverse Mutation Assay Screenina Version with TKA 40231 54 -73 ____

Acute Toxicity of TKA 40231 to Daphnia Maana in a 48-Hour Screeninci Test 74 -80 ____

TKA 40273 (CGX PR 774) Acute Oral Toxicity Study in Rats 81 - 99 ____

TKA 40273 (CGX PR 774) Acute Eve Irritation Study in Rabbits 100 -125

TKA 40273 (CGX PR 774) Primary Skin Irritation Study in Rabbits (4-Hour Semi-Occlusive

Apolication) 126 -150

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

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

(X if number Estimiate dentmi

(a) provided (b)(c ()

Physical state of neat substance X 14 X (s) (1) *..(g) M

Vapor pressure

- @ Temperature 0CTorr

Density/relative density g/cm3___

Solubility 9/L

@ Temperature 0

Solvent

Solubility in water @ Temperature ___ g/L

Melting temperature oC

Boiling/I sublimation ternperature@ torr pressure ___ C

38,

Spectra (NMR, MS, IR) X 49-531 See attached spectra M x

Dissociation constant

Particle size distribution

Octanol / water partition coefficient

Henry's Law constant

Volitalization from water

Volitalization from soil

pH @ concentration

Flammability Flash Point

Explodability

Adsorption / coefficient

Other - Specify (HPLC) X 41 See attached chromatograms M X

lemental Analysis X 32 __ __________ M X

Page 13
Ciba Specialty Chemicals Corporation

USA

540 White Plains Road..::*

P.O. Box 2005

Tarrytown, NY 10591-9005 C ib a

8am to 5pmn Phone: (800) 431-2360

24-Hour Health/Environmental Emergency Phone: (800) 873-1138

Value beyond chemistry

***DRAFT * **Material Safety Data Sheet MSDS No: 7340

1. PRODUCT IDENTIFICATION Health 2

Trade Name: CGX PR 774 2 1 lmablt 1

Intended Use: R & D Product Reactivity 0

> Protective Equipment

NFPA RATING HMIS RATING

Important Use Information:

This material is not intended for use in products for which prolonged contact with mucous membranes or abraded skin, body fluids,

or implantation within the human body, is specifically intended, unless the finished product has been tested in accordance with

national and international applicable safety regulations. Because of the wide range of such potential uses, Ciba Specialty Chemicals

Corporation is not able to recommend this material as safe and effective for such uses and assumes no liability for such uses.

2. COMPOSITION / INFORMATION ON INGREDIENTS

0 CAS No. CHEMICAL IDENTITY EXPOSURE LIMITS CARCINOGEN

S ______ _____ __STATUS___

H ACG1H OSHA MFR. IARC NTP OSHA

A TWA ISTEL PEL STEL __ __

Confidential Aromatic ether derivative Not Not Not Not Not Not Not Not

Estab- Estab- Estab- Estab- Estab- Rev- Rev- Revlished lished lished lished lished iewed iewed iewed

Concentration 1 - 100 % by wt_____

3. HAZARDS ]IDENTIFICATION

Emeruency Overview

Appearance:

White solid

Flammability:

Printed: 07/09/2002 Page 1 of 7

14
CGX PR 774

***DRAFT***

Use proper grounding techniques when emptying this product from containers weighing more than 1 pound. A build-up of

hazardous electrostatic charges could cause a flash fire or explosion when contents are emptied into a flammable atmosphere. See

Section 7.

Environmental:

The aquatic toxicity of this product has not been fully assessed. Avoid spillage or leakage to a body of water.

Health:

Limited testing information is available for this product, however, this product is expected to present little or no immediate hazard

to people if spilled or released.

Disposal:

Sweep or shovel spilled material and place into a sealable container. Dispose in accordance with local, state and federal

regulations. This product is not a hazardous waste under RCRA.

Primary Route of Entry:

Dermal, inhalation, ingestion

Eye:

This product is not expected to cause eye irritation.

Skin:

This product is not expected to cause skin irritation. Its ability to cause allergic skin reactions is unknown as no test data exist.

Swallowing:

Small amounts, if swallowed, are not expected to cause injury; avoid swallowing.

Inhalation:

This product has not been tested for inhalation toxicity; avoid inhalation.

Note: Refer to Section 11, Toxicological Information for details

4. FIRST AID MEASURES

First Aid for Swallowing:

If swallowed, give at least 3-4 glasses of water, but DO NOT induce vomiting. Do not give anything by mouth to an unconscious or

convulsing person.

First Aid for Skin:

Following skin contact, wipe away excess material with a dry towel. Then wash affected areas with plenty of water and soap, if

available, for several minutes. Get medical attention if irritation occurs.

First Aid for Inhalation:

If inhaled, remove from area to fresh air. Get medical attention if respiratory irritation develops, or if breathing becomes difficult.

First Aid for Eye:

Following eye contact, flush eyes with plenty of water for at least 15 minutes. Get medical attention if irritation occurs.

Note to Physician:

Specific identity of this product or component(s) of this product is withheld as a trade secret. A more complete description will be

provided to a doctor or nurse in the event of a medical emergency. Aggravated Medical Conditions: None known.

Printed: 07/09/2002 Page 2 of 7

15
CGX PR 774

***DRAFT***

5. FIRE FIGHTING MEASURES

Extinguishing Media:

Carbon dioxide, dry chemical, foam, water mist.

Unusual Hazards:

Decomposition and combustion products may be toxic.

Fire Fighting Instructions:

Use self-contained breathing apparatus.

6. ACCIDENTAL RELEASE MEASURES

Spill or Release Procedures:

Sweep or vacuum and place into sealable container for disposal. Wear protective equipment as specified below.

7. HANDLING AND STORAGE

Handling:

Health and environmental effect test data are limited or do not yet exist for this chemical. As with all industrial chemicals of

unknown toxicity, use prudent practices when handling. Avoid eye, skin and clothing contact. Do not inhale. Do not taste or

swallow. Use only with adequate ventilation.

Storage:

Keep container tightly closed when not in use and during transport.

Explosion Hazards:

* For All Packages:

DANGER! EXPLOSION RISK

- Risk of explosion if an air-dust mixture forms

- Avoid creating dusty conditions

- Empty only into grounded containers

- If container is larger than 550 US gallons (2m3) in volume, or when flammable solvents are present, the container must be

inerted or the system otherwise designed to prevent or contain an explosion - seek expert advise.

* In addition, for products packaged in fused-lined (coated) fiber drums, fiber drums with conductive liners, steel drums,

steel pails, and Ciba supplied bulk bags, the following instructions also apply:

- Always ground this package before emptying

* For products which have external protective packaging, discharge product only from the primary product packa ging, not

from the external container or its liner.

The user is responsible for designing a system that safely handles solid additives and to ensure proper training of

employees in the system's use.

Printed: 07/09/2002 Page 3 of 7

16
CGX PR 774 ***DRAFT***

8. EXPOSURE CONTROLS / PERSONAL PROTECTION

Engineering Controls:

Work in well-ventilated areas. Do not breathe dust.

General Protection:

Wear coveralls.

Eye/Face Protection:

In cases where there is likelihood of eye contact, wear chemical goggles.

Skin Protection:

Wear impervious gloves as a standard handling procedure.

Respiratory Protection:

Wear dust mask or respirator in conditions where dust arises from use.

9. PHYSICAL AND CHEMICAL PROPERTIES

Appearance:

White solid

10. STABILITY AND REACTIVITY

Chemical Stability:

Stable.

Incompatibility with other Materials:

Strong oxidizing agents, strong acids, strong bases

Hazardous Decomposition Products:

Thermal decomposition and burning may produce carbon monoxide, carbon dioxide, nitrogen oxides and other toxic organic

compounds.

Hazardous Polymerization:

Will not occur.

Conditions to Avoid:

None known.

11. TOXICOLOGICAL INFORMATION

Acute Oral Toxicity:

Confidential Aromatic ether derivative

(Rat) LD5O: 2,000 mg/Kg

Acute Dermal Toxicity:

Not known

Acute Inhalation Toxicity:

Not known

Printed: 07/09/2002 Page 4 of 7

V7
CGX PR 774 ***DRAFT **

Skin Irritation:

Confidential Aromatic ether derivative

(Rabbit) Not an irritant

Eye Irritation:

Confidential Aromatic ether derivative

(Rabbit) Not an irritant

Sensitization:

Not known

Mutagenicity:

Confidential Aromatic ether derivative

Ames test: Not mutagenic

12. ECOLOGICAL INFORMATION

Acute Toxicity to Invertebrates:

Confidential Aromatic ether derivative

(Daphnia magna) Screen: > 100 mgfL (WAF)

13. DISPOSAL CONSIDERATIONS

Disposal Considerations:

Dispose in accordance with federal, state and local regulations.

14. TRANSPORT INFORMATION

This product is not regulated by any means of transport.

15. REGULATORY INFORMATION

US Federal Regulations:

Clean Air Act -Hazardous Air Pollutants (HAP):

This product contains no hazardous air pollutants (HAP), as defined by the U.S. Clean Air Act.

Clean Air Act - Ozone Depleting Substances (ODS):

This product neither contains, nor was manufactured with, a Class I or Class II ozone depleting substance (ODS). We have relied

on our suppliers labeling their products. None have done so.

Clean Water Act - Priority Pollutants (PP):

This product contains no chemicals listed under the U.S. Clean Water Act Priority Pollutant List.

Occupational Safety and Health Act (OSHA):

This product is considered to be a hazardous chemical under the OSHA Hazard Communication Standard (29 CFR 1910.1200). Its

hazards are: Not known.

Resource Conservation and Recovery Act (RCRA):

Printed: 07/09/2002 Page 5 of 7

18
CGX PR 774

***DRAFT***

This product is not considered to be a hazardous waste under RCRA (40 CFR 261).

SARA Title III: Section 302 - Extremely Hazardous Substances (EHS):

This product contains no chemicals regulated under Section 302 as extremely hazardous substances.

SARA Title M: Section 304 - CERCLA:

This product contains no chemicals regulated under Section 304 as extremely hazardous chemicals for emergency release

notification ("CERCLA" List).

SARA Title III: Section 311/312 - Hazard Communcation Standard (HCS):

This product is regulated under Section 311-312 (40 CFR 370).

SARA Title Ill: Section 313 Toxic Chemical List (TCL):

This product does not contain a toxic chemical for routine annual Toxic Chemical Release Reporting' under Sec. 313 (40 CFR

372).

TSCA Section 5(e) - Consent Order / SNUR:

This product is not subject to a Section 5(e) Consent Order or Significant New Use Rule (SNUR).

TSCA Section 8(b) - Inventory Status:

This product contains a chemical(s) not included on the TSCA inventory and is transmitted to you solely for R&D purposes and

may not be used, processed or distributed for commercial purposes.

TSCA Section 12(b) - Export Notification:

This product does not contain any chemicals subject to Section 12(b) export notification.

Additional Federal Information:

This product is not subject to a Section 5(f0/6(a) rule.

International Remulations:

Canadian Inventory Status:

Not all components listed on the Domestic Substance List (DSL)

European Inventory Status (ElNECS):

This product contains one or more chemicals currently not on the European Inventory of Existing Commercial Chemical

Substances (ELNECS) or Pre-market notified.

Chemical Name: Aromatic ether derivative

CASRN: Confidential

Percent in Composition: 1 - 100 % by wt

State Re2ulations:

California Proposition 65:

This product does not contain any chemicals currently on the California list of Known Carcinogens and Reproductive Toxins.

Massachusetts Right-to-Know:

This product does not contain any chemicals which are subject to Massachusets Right-to-Know disclosure requirement.

New Jersey Right-to-Know:

The following is required composition information:

Printed: 07/09/2002 Page 6 of 7

19
CGX PR 774

***DRAFT***

Chemical Name: Aromatic ether derivative

NJ Registry #: 29943300001-6542

Pennsylvania Right-to-Know:

The following is required composition information:

Chemical Name: Aromatic ether derivative

CASRN: Confidential

Comment: Not on Pennsylvania Hazardous Substance List

16. OTHER INFORMATION

MSDS No: 7340

Disclaimer:

The information and recommendations contained herein are based upon data believed to be correct. However, no guarantee or

warranty of any kind expressed or implied is made with respect to the information contained herein.

Information Contact:

For technical information contact your technical sales representative. For additional health / safety Iregulatory information,

contact the Product Safety Customer Coordinator at (914) 785-4288.

Printed: 07/09/2002 Page 7 of 7

20
crsInvyentory Expert Service

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

Fax: 614-447-3747

E-mail: answers@cas.org

Internet: http://www.cas.org/Support/client~html

INVENTORY EXPERT SERVICE REPORT

IES-ORDER NUMER: 68035

REGISTRY NUMBER:3

CA INDEX NAME

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

flIf thiu box is checked, CAS has made correction(s) marked in red to

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

submitting it to the EPA.

21

2540 Olentangy River Road P.O. Box 3343 Columbus, Ohio 432 10-0334
-C

CD

rI)

22
~ ~' Ciba Specialty Chemiicals Inc., Analytics Process Development Schweizerhalle

Ciba

Study Report

Study Title: Development of an Analytical Certificate, inc. IR-, NMVR- and

MS- spectra

Test Item: CGX PR 774

Study Number 14022921

Test Facility: Ciba Specialty Chemicals Inc.,

Analytics Process Development Schweizerhalle

CE 6.22, CH-4133 PRATTELN 1

Test Facility Dr. J. M. Higel

Management: Analytics Process Development Schweizerhalle

CE 6.22, CH-4133 PRATTELN 1

Study Director: Dr. P. Fux

Analytics Process Development Schweizerhalle

CE 6.22, CH-4133 PRATTELN 1

Signature:

Completion Date: ... z

External Test Ciba Specialty Chemicals Inc.

Facilties/Structure Elucidation

Principal CH-4002 BASEL

Investigators: Principal investigator: Dr. S. Rudolph-B36hner

Ciba Specialty Chemicals Inc.

Analytics R&D CE, Service Center Elemental Analysis

CH-4002 BASEL

Principal investigator: H. Waldner

Property of Ciba Specialty Chemicals Inc 1402292WA

23
_______p 2 of 12-

Study Number: 14022921 1

Table of Contents

Page

1 . Title Page 1

2. Table of Contents 2

3. Abstract 3

4. General Information 4

4.1 Sponsor 4

4.2. Test Faciiity, Study Director and Study Personnel 4

4.3. External Test Facilities and Principal Investigators 4

4.4. Test Period 4

4.5. Archive 4

4.6. Distribution 4

5. Materials and Methods 5

5.1. Identification of the Test Item 5

5.2. Test Methods 5

6. Results 8

6.1 Identity by NMR and MS 8

6.2 Identity by IR 10

6.3 Elemental composition (C, H, N, and 0) and traces of Cl, Cu 10

and K by elemental analysis.

6.4 Content of main component and by products by HPLC 11

6.5 Content of residual methanol by GC 11

6.6 Content of residual ethyl acetate, dimethylformamide and 11

toluene by GC

6.7 Content of epoxide by titration 11

7. Appendices 12

Appendix 1: Analytical Method 12 pages

Appendix 2: 1H-NMR and 13C-NMR Spectra 4 pages

Appendix 3: El and ESI-MS Spectra 2 pages

Appendix 4: IR Spectrum 1 page

1402292 lAZProperty of Ciba speciary Chemnicals Inc. 14022921AZ

24
Study Number: 14022921

3. Abstract

1402292 I AZProperty of Ciba Specialty Chemicals In.10221A

25
Study Number: 14022921[~jf l

4. General Information

4.1. Sponsor

Ciba Specialty Chemicals Inc.,

Product safety and regulatory affairs PA 3.4

CH-4002 BASEL

4.2. Test Facilities, Study Director and Study Personnel

Test Facility: Ciba Specialty Chemicals Inc.,

Analytics Process Development Schweizerhalle

CE 6.22, CH-4133 PRATTELN 1

Study Director: Dr. Pierre Fux, WS-2093.P.08

Studyv Personnel: 0. John, WS-2093.P.17

4.3. External Test Facilities and Principal Investigators

Test Facility: Ciba Specialty Chemicals Inc.,

Structure Elucidation

CH-4002 Basel

Principal Investigator:, Dr. S. Rudolph-B36hner, K-403. P.09

Test Facility: Ciba Specialty Chemicals Inc.,

Analytics R&D CE, Service Center Elemental Analysis,

CE709

CH-4002 Basel

Principal Investigator: H. Waldner, R-1 059.4.15

4.4. Test Period

Experimental Starting Date: 16.05.2002

Experimental Completion Date: 11.06.2002

4.5 Archive

The originals of the test order, study plan, raw data and the final report will be

archived in the test facility archive WS-2093.

4.6 Distribution

Copies of the final report were distributed after study completion to sponsor, principal

investigators (on request) and to the laboratory staff concerned.

14O22921AZPropetty Of Ciba Specialty Chemicals Inc 14022921AZ

26
Study Number: 14022921 'i

5. Materials and Methods

5.1. Identification of Test Item

L _ 5.2 Test Methods

The test item has been characterised according to the analytical method KBB-493/1.

This method covers the quantitative determination of components, by-products and

the IR spectrum (see appendix 4). The main methods are:

- Reversed phase liquid chromatographic determination of the content of main

component and by-products.

- Gas chromatographic quantitative determination of residual solvents

The determination of the identity was performed by 'H-NMR and '3C through

assignment of the signals in the proton spectrum, by El-S and ESI-MS, by IR and

by elemental composition.

1402292 IAZproperIWof Ciba Specialty Chemicals Inc. 14022927AZ

27
Study Number: 14022921

NMR:

The NMR spectra were acquired on an UNITY INOVA 500 NMR spectrometer (Varian

Inc., Palo Alto, USA) operating at 500MHz proton frequency. The NMR experiments

were performed at 2700 using a solution of the sample in CDC13. Chemical shift

values were referenced towards the solvent peak ODC13 (5 1H- = 7.24 ppm, 8 130C

77.0 ppm)

EW-S:

The direct inlet El-MS was acquired on a MAT 95 mass spectrometer (Finnigan MAT

Inc., Bremen, Germany) operating at an ion energy of 70 eVolt. Sample was

introduced via direct inlet probe.

ESI-MS:

The direct inlet ESI mass spectrum was acquired in the positive ionization mode on a

Time-of-Flight mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA).

Sample was introduced as a solution in Methanol / THE of approximately 0.01%. To

enhance adduct ion formation Potassiumformate was added.

IR: The determination of identity by IR-spectrum is part of KBB-493/1.

Elemental Composition:

Carbon, Hydrogen and Nitrogen:

Method Dumas combustion method

Principle A well-defined amount of the test sample is combusted at about

100000C under an atmosphere containing oxygen. The formed

combustion gases (002, H20 and NO) are then quantitatively

detected by infrared absorption and thermal conductivity (N2)

respectively.

Equipment Leco CHNS-932 or equivalent

Measurement The following operating parameters are valid for the determination of

C, H and N.

Operating parameters for Leco CHNS-932

Furnace Oxidation Reduction

Temperature [00] 1000 650

Sample preparation About 1 to 3 mg of the test sample is wrapped in a capsule

consisting of Ag and directly combusted.

Calibration The calibration is done using pure organic substances containing C,

H and N with known elementary composition: e.g. EDTA (Ethylenediaminetetraacetic acid; C 40.96 %, H 5.50 % and N 9.56 %).

1402292 1 AZProperty of Ciba Specialty Chemicals Inc. 1402292 1 AZ

28
Study Number: 14022921

Oxygen: Conversion / Cracking to CO.

Principle A well defined amount of the test sample is combusted at about

1 300 0C under an atmosphere containing nitrogen / hydrogen. The formed

combustion gas (CO) is then quantitatively detected by infrared absorption.

Equipment Leco RO-478 or equivalent

Sample About 1 to 3 mg of the test sample are wrapped in a capsule

Preparation consisting of Sn and directly combusted.

Calibration The calibration is done using pure organic substances containing 0

with known elementary composition: e.g. Sulfamethazine (11.50% 0).

Chlorine: Combustion followed by titration (Sch6niger method).

For the combustion according to Sch6niger (02-flask-method) a few milligram of the test

sample to be analysed are completely burned in an Erlenmeyer flask filled with oxygen. An

absorbent solution provides the absorption of the reaction gases. Afterwards, this solution is

being analysed by argentometric titration to determine the content of halogens in the test

sample.

Potassium: ICP-OES at 766.491 nm

Copper: ICP-OES at 327.395 nm

14022921lAZPropefly of Ciba Specialty Chemicals Inc 14022921AZ

29
Study Number: 14022921

6. Results

6.1 Idniyb NRadM

30
* Study Number: 14022921c'

1402292 IAZPropefly ol Coba Specialty Chemicals Inc ?022A

31
Study Number: 14022921

6.2 Identity by JR

The identity is confirmed by JR spectrum.

See appendix 4 for spectrum.

14022921lAZProperty of Ciba Specialty Chemicals Inc. 1402292 1AZ

32
Study Number: 14022921

6.4 Content of Main Component and BV Products by HPLC

33
Study Number: 14022921

7. Appendices

Appendix 1 Copy of the analytical method KBB-493/1

12 pages

Appendix 2 'H-NMR and 13C-NMR Spectra

4 pages

Appendix 3 El and ESl-MS Spectra

2 pages

Appendix 4 IR Spectrum

1 page

1402292 lAZProperty ot Ciba Specialty Chemicals Inc 1402221AZ

34
Analytical Method Ciba

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 1 of 12

Distributed by Analytics Process Development Schweizerhalle Issue: 0 1. MAY 2002

This docuiment is generated from an electronic file which is controlled in compliance Print 12.06.2002

with the ISO 9001 Qiialit standard, date:

Authors:1 0JonManacrement of test Dr. J.M. Higel

Dr. P. Fux, CE 6. facility: CE 6.22

Main component:

1. Identity by NMR and MS spectroscopy

2. Identity by IR-spectrum

3. Elemental composition (C, H, N, and 0)

and traces of Cl, Cu and K by elemental analysis.

4. Content of main component and by products by HPLC

5. Content of residual methanol by GC

6. Content of residual ethyl acetate, dimethylformamide and toluene by GC

7. Content of epoxide by titration

Proprietary ii-ormatiofl of Ciba specialty Chemicals Inc. Not to be disclosed Value beyond chemistry to third parties without previous consent of Ciba Specialty Chemicals Inc.

35
Analytical Method&

Ciba..

Revision: 00 CGX PR 774 KBB-493/1

Date: --- ________

initial: Page 2 of 12

Structures of starting materials

Propneiary iftrmiUilof or Ciba Specialty Chernicals Inc. Not to be disclosed Value beyond chemistry

to third panilc-' without previous consent of Ciba Specialty Chemnicals Inc.

36
Analytical Method

Ciba. . Revision: 00 CGX PR 774 KBB-493/1

Initial:Page 3 of 12

Structures of by-products

Propriearzy informfationl of Ciba Spe±cialty Chemicals Inc. Not to be disclosed Value beyond chemistry

to third partis without previous consenti of Ciba Specialty Chemicals Inc.

37
Aiiii1Ty-Tia1Method

___ ___ ___ ___ ___ C ba

Revision: 00 CGX PR 774 KBB-4931

Date: --

Initial: Page 4 of 12

1. Identity by NMR and MS spectroscopy

Recording and interpretation of 'H-NMR, '

3C-NMR and MS- spectra by Structure

Elucidation, CH 4002 Basle.

2. Identity by IR-Spectrumn

Check IR spectrum of the sample for qualitative congruence with the reference spectrum

below.

FT-IR-Spectrometer: Nicolet FT-1R 5 10

Range of spectrum: 4000 - 550 cm-'

Transmission: 0 -100 %

Resolution: 4 cm'1

Reference: air

Recording technique: ATR

No. of scans: 64

Waveriumbers (cm-i)

Proprietary information of Ciba Specialty Chemicals Inc. Not to be disclosed value beyond chemistry

to third panics without previous consent of Ciba Specialty Chemicals Inc

38
* Ciba Specialty Chemicals Inc.

Analytical Method

____ ____ ____ ___ ____ ___ Ciba

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 5 of 12

3. Elemental analysis

Determination of the C, H, N, Cl and 0-content according to the Dumas combustion

method and traces of K (ICP) and Cu (ICP) by Service Center Elemental Analysis

(SCEA), CH-4002 BASEL.

4. Content of main component and by products by HPLC

Principle of separation: liquid chromatography

Calculation : external standard method for Ethyiphenylacetate, CA 32-0099, CG 41-1212,

CG 41-0350, CG 41-1742 and main component.

The other by-products are given as total area percent.

Suggested chromatographic conditions

Column dimensions: 250 x 4.6 mm

Stationary phase: Nucleosil, C 18, 5p m

Mobile phase: A: Acetonitrile with 0. 1% Ammonia solution 25 %

B: 20 % methanol + 80 % water with

0. 1% Ammonia solution 25 %

Gradient: t [min] A [] B[%

0 65 35

5 65 35

25 99 1

40 99 1

40.1 65 35

1 50 1 65 35

Flow rate: 1.0 ml/min

Column temperature: 25 0

C

Injection volume: 5pl

Detector: UV- detector at 230 nm

Proprietary information of Ciba Specilty Chemicals Inc. Not to be disclosed Value beyond chemistry

to third panics without previous consent of Ciba Specialty Chemicals Inc.

39
Analytical Mto

Ciba ::

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 6 of 12

Test solution

Prepare a sample solution in acetonitrile with a final concentration of approx.

20 - 25mg/25m1.

Standard solution

Prepare a solution in acetonitrile with a final concentration of 0.5 - 1.5 mgI25ml of

4-Ethylphenyl acetate, CA 32-0099, CG 41-12 12, CG 41-0350, CG 41-1742 and 20.0 -

25 .0mg of CGX PR 774 (batch FF226/9 F1 purifi6).

Table of data

Typical chromatogramns: see on page 7

Propriary information of Ciba Specialty Chemicals Inc. Not to be disclosed Valuc beyond chemistry to third panics without previous consent of Ciba Specially Chemicals Inc.

40
Analytical Method 64

Ciba :?

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 7 of 12

Proprieary information of Ciba Spcialty Checalsi Inc. Not to be disclosed Value beyond chemistry

to third panics without previous consent of Ciba Specialty Chec.als Inc.

41
Ciba Specialty Chemicals Inc.

Analytical Method ___ ___ ___ ___ ___ C ba

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 8 of 12

5. Content of residual methanol by GC

Principle of separation: gas chromatography with flush technique and flame ionisation

detector.

Calculation : external standard method.

Suggested chromatographic conditions

Column: fused silica, length 10 m, i.d. 0.53 mm

Stationary phase: Silicone, DB-1, film thickness 5.0 gim or equivalent

Injection volume: I jil

Carrier gas: He, 2 mI/mmn

Temperatures: Injector: 0.5 min at 320*C

Oven: 2 min at 40'C,

with 10'C/min to 320'C

Detector: 340'C

Standard solution

Prepare a methanol solution in ethyl acetate with a final sample concentration of 0.2 - 0.5

mg/mi.

Test solution

Prepare a solution in ethyl acetate with a final sample concentration of 80.00 - 120.00

mg/ml.

Table of data____________

component retention time

Residual methanol 1.9___________

Ethyl acetate5.

Propneciary infortnation of Ciba Specialty Chemkials Inc Not to be disclosed Value beyond chemistry ito third panics without previous consent of Ciba Specialty Cheinicals Inc.

42
*Ciba Specialty Chemicals Inc. A ayia

AnaytialMethod Ciba

Revision: 00 CGX PR 774 K"B-493/1

Date: --

Initial: Page 9 of 12

6. Content of residual ethyl acetate, dimethylformamide and toluene by

GC

Principle of separation: gas chromatography with flush technique and flame ionisation

detector.

Calculation : external standard method.

Suggested chromatographic conditions

Column: fused silica, length 10 m, i.d. 0.53 mm

Stationary phase: Silicone, DB- 1, film thickness 5.0 g~m or equivalent

Injection volume: I A.1

Carrier gas: He, 2 ml/min

Temperatures: Injector: 0.5 min at 320'C

Oven: 2 min at 40'C,

with 10'C/min to 320'C

Detector: 340'C

Standard solution

Prepare an ethyl acetate + dimethylformamide + toluene solution in methanol with a final

concentration of 0.05 - 0.2 mg/mI each.

Test solution

Prepare a solution in methanol with a final sample concentration of 80.00 - 120.00

mg/mI.

Table of data __________

component retention time

min

Methanol 1.9

Residual ethyl acetate 5.1

Residual dimethylforrnamide 8.2

Residual toluene 8.5

Proprietary information of Ciba Specialty Chemicals Ic. Not to be disclosed Value beyond chemistry

to third panics without previous consent of Ciba Specialty Chemicals Inc.

43
Ciba Specialty Chemicals Inc.

Analytical Method Ciba

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 10 of 12

7. Content of epoxide by titration

7.1 Content of amine

Principle of determination: potentiometric titration of the amine functional group with

perchioric acid.

Procedure

Weigh 0.4500 - 0.6500cg of a sample into a titration vessel, dissolve with a mixture of 20

ml of glacial acetic acid and 20 ml of acetone and titrate potentiometrically as follows:

Instrument: 736 GP? Titrino, Metrohm or equivalent

Electrodes: Glass; Ag-AgCI, electrolyte: LiCI in glacial acetic acid, saturated

Titration solution: 0. 1 mol/I perchloric acid in glacial acetic acid

(exactly adjusted normal solution

Titration parameters: according to method '>DET U, Amine"

Calculation

The content of Amine in the sample is calculated as:

a-*0.1 - f

W

where A = content of amine in Eq/Kg

a = consumption of perchloric acid-solution I ml] for the sample after temperature

correction

f =factor of the perchloric acid solution*

W =sample weight [a]

Proprietry informiation of ciba Specialiy Chemicals Inc Not to be disclosed Value beyond chemistry

it) third pantics without previous consent of ciba Specialty Chemicals Inc.
'Ciba Specialty Chemicals Inc. A ayia

AnaytialMethod z

____ _ ____ __ ___ ____ ____ ___Ciba. .

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 11 of 12

7.2 Content of the sum epoxide + amine:

Principle of determination: potentiometric titration of the epoxide functional group with

perchioric acid after reaction with TAAB-reagent*.

Preparation of the TAAB-reagent:

Add 400 ml] of glacial acetic acid and 500 ml of acetone to 70g of

tetraethylammoniumbromide. The absence of consumption of perchloric acid for that

reag~ent must be verified regularly. If it is not the case, a fresh solution has to be prepared.

Procedure

Weigyh 0.2500 0 .3500a of a sample into a titration vessel, dissolve with 30 ml of

TAAB-reagent *and titrate potentiometrically as follows:

Instrument: 736 GP Titrino, Metrohm or equivalent

Electrodes: Glass; Agy-AgGI, electrolyte: LiCI in glacial acetic acid, saturated

Titration solution: 0. 1 mol/l perchioric acid in glacial acetic acid

(exactly adjusted normal solution

Titration parameters: according to method '5DET U, Epoxid"

Calculation

The sum of the content of Epoxide and Amine in the sample is calculated as:

a-0.1 *f

S -

w

where S = sum of epoxide and amine in Eq/Kg

a = consumption of perchloric acid-solution [ml] for the sample after temperature

correction

f = factor of the perchloric acid solution

W = sample weight [g]

Proprietary information ofCeba Specialty Chemicals Inc. Not to be disclosed Value beyond chemistry

to third parties without previous consent of ciba Specialty Chemicals Inc

45
Ciba Specialty Chemicals Inc. M to

Analytical Mto

Ciba

Revision: 00 CGX PR 774 KBB-493/1

Date: --

Initial: Page 12 of 12

7.3 Content of epoxide in the sample

The content of epoxide (Eq/Kg) in the sample is calculated as the difference of the two

titrations carried out according to pos. 7.2 and 7. 1.

Propritry information of Ciba Specialty Chemicals Inc Not to be disclosed Value beyond chemistry

to third panics without previous consent of Ciba Specialty Chemicals Inc.

46
Appendix 2

Study Number: 14022921

'H- NMR and 13 C-NMR spectrum

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-1 87

47
Appendix 2

Study Number: 14022921

Property of' Ciba Specialty Chemicals Inc.

48
Appendix_2 Page 3ofW-- 4----

Study Number: 14022921

1H-NMR spectrum (in CDCI3) expanded

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-1 87

e4

Property of Ciba Specialty Chemicals Inc.

49
Appendix 2-ag P f 4~

Study Number: 14022921

13C-NMR in CDC13: overview

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-187

Property of Ciba Specialty Chemnicals Inc.

50
Appendix 3 ~L I~

Study Number: 14022921

MS spectra

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-187

Chemical Structure Icomposition:

Property of Ciba Specialy Chovicals Inc.

51
Appendix 3 PStudy Number: 14022921

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-187

Property of Ciba Specialty Chemnicals Inc.

52
Study Number: 14022921

IR spectrum

Product Designation: TK A 40273

Product Name: CGX PR 774

Batch Number: Vs. 836-1 87

Chemical Structure Icomposition:

Property of Ciba Specialty Chemicals Inc.

53
'~ Li~ f~ / ~(J *7% S./

RCC-CCR PROJECT 692300

PROJECT ID OF THE CONTRACTING

INSTITUTE:

RCC PROJECT 803542

SALMONELLA TYPHIMURIUM

REVERSE MUTATION ASSAY

SCREENING VERSION

WITH

TKA 40231

REPORT

STUDY COMPLETION DATE:

March 09, 2001

54
Test Report RCC-CCR Project 692300

CONTENTS

PREFACE 3

General 3

Project Staff 3

Schedule 3

Project Staff Signatures 4

Guidelines 4

Archiving 4

Distribution of the Report 4

OBJECTIVE 5

Aims of the Study 5

Reasons for the Study 5

MATERIALS AND METHODS 6

Test Item 6

Controls 7

Test System 8

Mammalian Microsomal Fraction S9 Mix 9

Experimental Performance 11

Data Recording 11

Acceptability of the Assay 11

EVALUATION OF RESULTS 12

DISCUSSION OF RESULTS 13

CONCLUSION 13

REFERENCES 14

ANNEXE: TABLES OF RESULTS 15

Main-Experiment: Plate Incorporation Test 15

HISTORICAL CONTROL DATA 20

st4r -Page 2 of 20-

55
Test Report RCC-CCR Project 692300

PREFACE

General

Sponsor: Ciba Specialty Chemicals Inc.

Additives Division

P.O. Box

CH-4002 Basel, Switzerland

Study Monitor: Dr. H. J. Weideli

R- 1045. 1.32

Testing Facility: RC C

CYTOTEST CELL RESEARCH GMBH

In den Leppsteinswiesen 19

D-64380 Ro~dorf

RCC-CCR Project No.: 692300

Contracting Institute: RCC LTD.

REGISTRATION AND CONSULTING DIVISION

CH-4452 Itingen

RCC Project No.: 803542

Test Item: TKA 40231

RCC-CCR Test Item No.: 5236211

Title: Salmonella Typhimuriumn

Reverse Mutation Assay with TKA 40231

Project Staff

Study Director: Andrea Sokolowski

Management: Dr. Wolfgang Volkner

Schedule

Start of Experiments: March 01, 2001

End of Experiments: March 09, 2001

Date of Final Report: March 09, 2001

st4r -Page 3 of 20 -

56
Test Report RCC-CCR Project 692300

Project Staff Signatures

Study Director Andrea Sokolowski

Date: March 09, 2001

Management Dr. Wolfgang Volkner

Date: March 09, 2001

Guidelines

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:

"Ninth Addendum to OECD Guidelines for Testing of Chemicals", Section 4, No. 471:

"Bacterial Reverse Mutation Test", adopted July 21, 1997

EEC Directive 2000/32, L 1362000, Annexe V, B 13/14 dated May 19, 2000

Archiving

RCC Cytotest Cell Research GmbH, D-64380 Roldorf will archive the following data for

2 years:

Raw data, protocol and copy of report, and sample of the test item.

No raw data or material relating to the study will be discarded without the sponsor's prior

consent.

Distribution of the Report

Contracting Institute 2x (original, copy)

Study Director l x (copy)

st4r -Page 4 of 20 -

57
Test Report RCC-CCR Project 692300

OBJECTIVE

Aims of the Study

The experiment was performed to assess the potential of the test item to induce gene

mutations by means of Salmonella typhimurium reverse mutation assays. The experiment

was performed as a plate incorporation assay.

Reasons for the Study

The most widely used assays for detecting gene mutations are those using bacteria. They

are relatively simple and rapid to perform, and give reliable data on the ability of an agent

to interact with DNA and produce mutations.

Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is

therefore small, specific and selective. Several bacterial strains, or a single strain with

multiple markers are necessary to overcome the effects of mutagen specificity. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the

absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely

used marker.

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair

(TA 1535, TA 100, and TA 102) and frameshift (TA 1537, TA 98) mutations.

According to the direct plate incorporation method the bacteria are exposed to the test

item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.

To establish a dose response effect six dose levels with adequately spaced concentrations were tested. The maximum dose level was 5000 pg/plate.

To validate the test, reference mutagens are tested in parallel to the test item.

st4r -Page 5 of20 -

58
Test Report RCC-CCR Project 692300

MATERIALS AND METHODS

Test Item

The test item and the information concerning the test item were provided by the

sponsor.

Name: TKA 40231

Batch No.: 1/01

Aggregate State

at Room Temperature: solid

Colour: white

Stability in Solvent: not indicated by the sponsor

Storage: room temperature

Expiration Date: February 01, 2003

On the day of the experiment, the test item TKA 40231 was dissolved in acetone (purity >

99 %, MERCK, 0-64293 Darmstadt). The solvent was chosen because of its solubility

properties and its relative non-toxicity to the bacteria.

No precipitation of the test item occurred up to the highest investigated dose.

st4r -Page 6 of 20 -

59
Test Report RCC-CCR Project 692300

Controls

Negative Controls

Concurrent untreated and solvent controls were performed.

Positive Control Substances

Without metabolic activation

Strains: TA 1535, TA 100

Name: sodium azide, NaN 3

Supplier: SERVA, D-69042 Heidelberg

Catalogue No.: 30175

Purity: at least 99 %

Dissolved in: water deionised

Concentration: 10 pg/plate

Strains: TA 1537, TA 98

Name: 4-nitro-o-phenylene-dia mine, 4-NOPD

Supplier: SIGMA, D-82041 Deisenhofen

Catalogue No.: N 9504

Purity: > 99.9 %

Dissolved in: DMVSO (purity >99 %, MERCK, D-64293 Darmstadt)

Concentration: 50 pg/plate in TA 1537, 10 pg/plate in TA 98

Strain: TA 102

Name: methyl methane sulfonate, MMS

Supplier: MERCK-SCHUCHARDT, D-85662 Hohenbrunn

Catalogue No.: 820775

Purity: > 99.0 %

Dissolved in: water deionised

Concentration: 5.0 p/plate

With metabolic activation

Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102

Name: 2-aminoanthracene, 2-AA

Supplier: SIGMA, D-82041 Deisenhofen

Catalogue No.: A 1381

Purity: 97.5 %

Dissolved in: DMSO (purity >99 %, MERCK, 0-64293 Darmstadt)

Concentration: 2.5 pg/plate (10.0 pg/plate in TA 102)

The stability of the positive control substances in solution was unknown but a mutagenic

response in the expected range is sufficient evidence of biological stability.

st4r -Page 7 of 20-

60
Test Report RCC-CCR Project 692300

Test System

Characterisation of the Salmonella typhimurium Strains

The histidine dependent strains are derived from S. typhimuriumn strain LT2 through a

mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation

they possess a faulty lipopolysaccharide envelope which enables substances to penetrate

the cell wall more easily. A further mutation causes an inactivation of the excision repair

system. The latter alteration includes mutational processes in the nitrate reductase and

biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus'. In the

strains TA 98 and TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicillin

resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision

repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQI carrying the

hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene.

In summary, the mutations of the TA strains used in this study can be described as follows:

Salmonella typhimurium

TA 1537: his C 3076; rfa*; uvrB': frame shift mutations

TA 98: his D 3052; rfa-; uvrB';R-factor: frame shift mutations

TA 1535: his G 46; rfa-; uvrB-: base-pair substitutions

TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitutions

TA 102: his G 428; rfa'; uvrB+;R-factor: base-pair substitutions

Regular checking of the properties of the strains regarding the membrane permeability

and ampicillin resistance as well as spontaneous mutation rates is performed in RCCOCR according to Ames et al. (1). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

The bacterial strains TA 1535, TA 1537, TA 1 Ooand TA 102 were obtained from Ames

(University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained

from von E. Merck (D-64293 Darmstadt).

Storage

The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 %

DMVSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

st4r -Page 8 of20 -

61
Test Report RCC-CCR Project 692300

Precultures

From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into

250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 pl ampicillin

(25 pg/mI) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 pl tetracycline (2 .ig/ml) was added to strain TA 102. This nutrient medium contains per litre:

8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)

5 g NaCI (MERCK, D-64293 Darmstadt)

The bacterial culture was incubated in a shaking water bath for 4 hours at 370 C.

Selective Agar

The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar

The overlay agar contains per litre:

6.0 g MERCK Agar Agar*

6.0 g NaCI*

10.5mg L-HistidinexHClxH2O*

12.2 mg Biotin*

* (MERCK, D-64293 Darmstadt)

Sterilisations were performed at 1210 C in an autoclave.

Mammalian Microsomal Fraction S9 Mix

The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order

to overcome this major drawback an exogenous metabolic system is added in form of

mammalian microsome enzyme activation mixture.

S9 (Preparation by RCCCCR)

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male

rats, strain Wistar Hanlbm (RCC Ltd, Biotechnology and Animal Breeding Division, CH4414 F~llinsdorf; weight approx. 220 - 320 g) which received three applications of 80

mg/kg b.w. Phenobarbital i.p. dissolved in aqua deionised (Desitin; D-22335 Hamburg)

and j3-Naphthoflavone orally dissolved in corn oil (Aldrich, 0-89555 Steinheim). The livers

were prepared 24 hours after the last treatment.

st4r -Page 9 of 20 -

62
Test Report RCC-CCR Project 692300

After decapitation of the anaesthetised animals the livers of the animals were removed,

washed in 150 mM KOL and homogenised. The homogenate was diluted 1+3 in KCI and

centrifuged at 9,000 g for 25 minutes at 40 C. A stock of the supernatant containing the

microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -800 C. Small numbers

of the ampoules are kept at -20* C for several weeks before use. The protein content

was determined using an analysis kit of Bio-Rad Laboratories, D-80939 Munchen (BioRad protein assay, Catalogue No. 5000006).

The protein concentration in the S9 preparation was 27.4 mg/mI (lot no. 190101)

$9 Mix

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed

with S9 cofactor solution. The amount of S9 supernatant was 15% v/v. The composition

of the cofactor solution was chosen to yield the following concentrations in the S9 mix:

8 MM MgCI 2

33 mM KCI

5 mM Glucose-6-phosphate

5mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was

performed according to Ames et al. (2).

Dose Selection

The maximum concentration was 5000 pg/plate. The concentration range covered two

logarithmic decades. In this study eight adequately spaced concentrations were tested.

The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 pg/plate

st4r -Page 10 of 20-

63
Test Report RCC-CCR Project 692300

Experimental Performance

For each strain and dose level, including the controls three plates were used as a minimum.

The following materials were mixed in a test tube and poured onto the selective agar

plates:

100 pl Test solution at each dose level, solvent control, negative control or reference

mutagen solution (positive control),

500 pl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test

without metabolic activation),

100 pl Bacteria suspension (cf. test system, pre-culture of the strains),

2000pl Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 370 C in

the dark.

Data Recording

The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS J GmbH, D-61184 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each

concentration together with standard deviations and enhancement factors as compared to

the spontaneous reversion rates (see tables of results).

Acceptability of the Assay

The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets

the following criteria:

- regular background growth in the negative and solvent control

- the spontaneous reversion rates in the negative and solvent control are in the range of

our historical data - the positive control substances should produce a significant increase in mutant colony

frequencies

st4r -Page 11 of 20 -

64
Test Report RCC-CCR Project 692300

EVALUATION OF RESULTS

A test item is considered positive if a dose related increase in the number of revertants or

a biologically relevant increase for at least one test concentration is induced.

A test item producing neither no a dose related increase in the number of revertants nor a

biologically relevant positive response at any one of the test points is considered nonmutagenic in this system.

A biologically relevant response is described as follows:

A test item is considered mutagenic if the number of reversions is at least twice the

spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains

TA 1535 and TA 1537 (3, 4).

Also, a dose-dependent increase in the number of revertants is regarded as an indication

of possibly existing mutagenic potential of the test item regardless whether the highest

dose induced the above described enhancement factors or not.

st4r -Page 12 of20 -

65
Test Report RCCCCR Project 692300

DISCUSSION OF RESULTS

The test item TKA 40231 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535,

TA 1537, TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following

concentrations:

3; 10; 33; 100; 333; 1000; 2500 and 5000 pg/plate.

No toxic effects, evident as a reduction in the number of revertants, were observed in all

strains with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to

5000 pg/plate with and without S9 mix in all strains used.

No relevant substantial increase in revertant colony numbers of the five tester strains was

observed following treatment with TKA 40231 at any concentration level, neither in the

presence nor absence of metabolic activation (S9 mix). In strain TA 1535 a weak increase

of the revertant colony numbers was observed in the highest concentration with metabolic

activation. Since the threshold of three was not reached the test item is considered to benon-mutagenic in this assay.

Appropriate reference mutagens were used as positive controls. They showed a distinct

increase in induced revertant colonies.

CONCLUSION

In conclusion, it can be stated that during the described mutagenicity test and under the

experimental conditions reported, the test item did not induce gene mutations by base

pair changes and frameshifts in the genome of the strains used.

Therefore, TKA 40231 is considered to be non-mutagenic in this Salmonella typhimurium

reverse mutation assay.

st4r -Page 13 of 20 -

66
Test Report RCC-CCR Project 692300

REFERENCES

1 . Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973)

Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection

Proc. Nati. Acad. Sci. (USA) 70, 2281-2285

2. Ames, B.N., J. McCann, and E. Yamasaki (1977)

Methods for detecting carcinogens and mutagens with the salmonella/mammalian microsome mutagenicity test

In: B.J. Kilbey et al. (Eds.)

"Handbook of Mutagenicity Test Procedures"

Elsevier, Amsterdam, 1-17

3. Claxton, L.D., Allen, J., Auletta, A., Mortelmans, K., Nestmann, E. and Zeiger, E.

(1987)

Guide for the Salmonella typhimurium/mammalian microsome tests or bacterial mutagenicity

Mutation Res. 189, 83-9 1

4. Hollstein,M., J. McCann, F.A. Angelosanto and W.W. Nichols (1979)

Short-term tests for carcinogens and mutagens

Mutation Res. 65, 133-226

5. Grafe, A., I.E. Mattern and M. Green (1981)

A European collaborative Study of the Ames assay: ).Results and general interpretation Mutation Res. 85, 391-410

st4r -Page 14 of20 -

67
Test Report RCC-CCR Project 692300

ANNEXE: TABLES OF RESULTS

Main-Experiment: Plate Incorporation Test

Test item: TKA 40231

S9 mix from : Rat liver (Batch 190101)

Test strain: TA 1535

without S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 10 9 11 10 1.0

Solvent Control 9 8 11 9 1.5 1.0

Positive Control* 735 718 685 713 25.4 76.4

3 7 7 9 8 1.2 0.8

10 6 8 9 8 1.5 0.8

33 7 4 6 6 1.5 0.6

100 8 5 8 7 1.7 0.8

333 7 5 5 6 1.2 0.6

1000 6 12 7 8 3.2 0.9

2500 7 5 8 7 1.5 0.7

5000 12 15 13 13 1.5 1.4

with S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 9 11 9 10 1.2

Solvent Control 11 11 11 11 0.0 1.0

Positive Controlf 100 113 110 108 6.8 9.8

3 9 8 9 9 0.6 0.8

10 11 9 15 12 3.1 1.1

33 9 13 12 11 2.1 1.0

100 8 8 8 8 0.0 0.7

333 9 6 10 8 2.1 0.8

1000 20 15 8 14 6.0 1.3

2500 16 19 21 19 2.5 1.7

5000 24 26 20 23 3.1 2.1

*enhancement factor - revertants / concentr. test item

I revertants / solvent control

'Sodium azide 10.0 pg/plate

S2-aminoanthracene 2.5 pg/plate

st4r -Page 15 of 20 -

68
Test Report RCC-CCR Project 692300

Main-Experiment: Plate Incorporation Test

Test item: TKA 40231

S9 mix from: Rat liver (Batch 190101)

Test strain: TA 1537

without S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 11 15 6 11 4.5

Solvent Control 10 5 5 7 2.9 1.0

Positive Control* 47 63 47 52 9.2 7.8

3 8 5 9 7 2.1 1.1

10 7 4 6 6 1.5 0.9

33 9 10 15 11 3.2 1.7

100 11 12 7 10 2.6 1.5

333 6 19 9 11 6.8 1.7

1000 6 16 12 11 5.0 1.7

2500 11 14 11 12 1.7 1.8

5000 15 6 10 10 4.5 1.5

with S9 mix

Concentration Plate Revertants /plate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 9 6 6 7 1.7

Solvent Control 6 6 5 6 0.6 1.0

Positive Control" 120 146 133 133 13.0 23.5

3 4 6 5 5 1.0 0.9

10 8 8 6 7 1.2 1.3

33 8 8 4 7 2.3 1.2

100 3 4 5 4 1.0 0.7

333 5 7 8 7 1.5 1.2

1000 2 7 4 4 2.5 0.8

2500 4 7 6 6 1.5 1.0

5000 6 10 10 9 2.3 1.5

*enhancement fato revertants / concentr. test item

fa Ir revertants / solvent control

4 nitro-o-phenylene diamine 50.0 pg/plate

S2-aminoanthracene 2.5 pg/plate

st4r -Page 16 of 20 -

69
Test Report RCC-CCR Project 692300

Main-Experiment: Plate Incorporation Test

Test item: TKA 40231

S9 mix from: Rat liver (Batch 190101)

Test strain: TA 98

without S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 21 17 25 21 4.0

Solvent Control 40 21 20 27 11.3 1.0

Positive Control* 199 119 163 160 40.1 5.9

3 37 39 38 38 1.0 1.4

10 24 25 34 28 5.5 1.0

33 27 23 25 25 2.0 0.9

100 34 28 19 27 7.5 1.0

333 32 35 36 34 2.1 1.3

1000 24 33 30 29 4.6 1.1

2500 37 32 24 31 6.6 1.1

5000 31 35 29 32 3.1 1.2

with S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 48 51 48 49 1.7

Solvent Control 52 37 45 45 7.5 1.0

Positive Control" 795 805 837 812 21.9 18.2

3 39 31 35 35 4.0 0.8

10 44 39 38 40 3.2 0.9

33 44 35 41 40 4.6 0.9

100 47 34 44 42 6.8 0.9

333 38 35 35 36 1.7 0.8

1000 38 36 36 37 1.2 0.8

2500 36 29 168 78 78.3 1.7

5000 114 49 39 67 40.7 1.5j

*enhancement fato revertants / concentr. test item

fatr=~ revertants / solvent control

4 nitro-o-phenylene diamine 10.0pg/plate

S2-aminoanthracene 2.5 pg/plate

st4r -Page 17 of 20 -

70
Test Report RCC-CCR Project 692300

Main-Experiment: Plate Incorporation Test

Test item: TKA 40231

S9 mix from : Rat liver (Batch 190101)

Test strain: TA 100

without S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 151 135 147 144 8.3

Solvent Control 102 122 129 118 14.0 1.0

Positive Controls 718 736 777 744 30.2 6.3

3 118 93 124 112 16.4 0.9

10 154 125 115 131 20.3 1.1

33 108 99 101 103 4.7 0.9

100 99 93 94 95 3.2 0.8

333 99 109 86 98 11.5 0.8

1000 128 93 89 103 21.5 0.9

2500 125 91 88 101 20.6 0.9

15000 1 40 82 107 1 76 33.9 0.6

with S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 143 139 155 146 8.3

Solvent Control 132 171 144 149 20.0 1.0 IPositive Control" 1362 1594 1044 1333 276.1 8.9

3 110 127 40 92 46.1 0.6

10 il1 140 127 126 14.5 0.8

33 154 141 136 144 9.3 1.0

100 128 135 162 142 18.0 1.0

333 101 129 117 116 14.0 0.8

1000 91 110 114 105 12.3 0.7

2500 132 124 122 126 5.3 0.8

5000 122 136 105 121 15.5 0.8

*enhancement factor - Xrevertants / concenir. test item

2: revertants / solvent control

SSodium azide 10.0 pg/plate

U2-aminoanthracene 2.5 pg/plate

st4r -Page 18 of 20 -

71
Test Report RCC-CCR Project 692300

Main-Experiment: Plate Incorporation Test

Test item: TKA 40231

S9 mix from: Rat liver (Batch 190101)

Test strain: TA 102

without S9 mix

Concentration Plate Revertants Iplate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 257 279 258 265 12.4

Solvent Control 273 270 258 267 7.9 1.0

Positive Control* 1047 1611 1608 1422 324.8 5.3

3 266 291 292 283 14.7 1.1

10 299 261 271 277 19.7 1.0

33 198 195 210 201 7.9 0.8

100 279 289 273 280 8.1 1.0

333 280 209 286 258 42.8 1.0

1000 321 302 295 306 13.5 1.1

2500 303 303 298 301 2.9 1.1

5000 278 304 309 297 -16.6 1.1

with S9 mix

Concentration Plate Revertants /plate

pg/plate 1 2 3 mean s.d. factor*

Negative Control 342 381 342 355 22.5

Solvent Control 336 365 294 332 35.7 1.0

Positive Control" 702 850 905 819 105.0 2.5

3 348 392 334 358 30.3 1.1

10 139 144 137 140 3.6 0.4

33 144 134 103 127 21.4 0.4

100 125 159 159 148 19.6 0.4

333 114 139 107 120 16.8 0.4

1000 400 381 355 379 22.6 1.1

2500 335 364 330 343 18.4 1.0

5000 405 352 391 383 27.5 1.2

*enhancement factor = revertants / concentr. test item

Srevertants / solvent control

'Methyl methane sulfonate 5.0 p1/plate

S2-aminoanthracene 10.0 pg/plate

st4r -Page 19 of 20 -

72
Test Report RCC-CCR Project 692300

HISTORICAL CONTROL DATA

Negative Control

(without metabolic activation)

1535 1537 98 100 102

9-29 5-28 15-57 77 -189 121 -293

Solvent Control

(without metabolic activation)

1535 1537 98 100 102

9-25 4-28 14-58 77 -225I 121 -295

Positive Control

(without metabolic activation)

1535 1537 98 100 102

68-814~ 42-191I 86 -450J 460 -910 751 -1395

These values represent our historical control data since 1995.

st4r -Page 20 of 20 -

73
REPORT

Study Title:

ACUTE TOXICITY OF

TKA 40231

TO DAPHNIA MAGNA

IN A 48-HOUR SCREENING TEST

Study Director:

Dr. Birgit Seyfried

Study Completion Date:

March 19, 2001

Test Facility:

RCC Ltd

Environmental Chemistry &

Pharmanalytics Division

CH-4452 Itingen / Switzerland

Sponsor:

Ciba Specialty Chemicals Inc.

Additives Division

P.O. Box

CH-4002 Basel / Switzerland

RCC Study Number:

803621

Page 1 of 7

74
ROC Study Number 803621 REPORT Page 2

TKA 40231

SIGNATURES

Study Director: Dr. Birgit Seyfried

......... ......................

Date: 'V-./.~~~d

Management: Mr. Markus Arenz -

Dat:

75
RCC Study Number 803621 REPORT Page 3

TKA 40231

CONTENTS

SIGNATURES ................................................................................ 2

CONTENTS .................................................................................. 3

PREFACE .................................................................................... 4

1 MATERIALS AND METHODS......................................................... 5

1.1 EXPERIMENTAL CONDITIONS ................................................ 5

1.2 DOSAGE AND CONCENTRATIONS........................................... 6

2 RESULTS............................................................................... 7

Tabie 1: Influence of TKA 40231 on the mobility of Daphnia magna.................. 7

Table 2: Dissolved oxygen concentrations, pH values and temperature

in the test media at the start and end of the test ............................... 7

Table 3: Appearance of the test media during the test period ......................... 7

76
RCC Study Number 803621 REPORT Page 4

TKA 40231

PREFACE

GENERAL

Study Title: Acute toxicity of TKA 40231 to Daphnia magna in a

48-hour screening test

Sponsor: CIBA Specialty Chemicals Inc.

Additives Division,

P.O. Box

CH-4002 Basel / Switzerland

Monitoring Scientist: Dr. H. J. Weideli

Testing Facility: RCC Ltd

Environmental Chemistry & Pharmanalytics

DivisionZeigliweg 1

CH-4452 Itingen / Switzerland

RESPONSIBILITIES

Study Director: Dr. Birgit Seyfried

Technical Coordinator: Mrs. Elsbeth Ackermann

SCHEDULE

Experimental Starting Date: March 12, 2001

Experimental Completion Date: March 15, 2001

Study Completion Date: March 19, 2001

77
ROC Study Number 803621 REPORT Page 5

TKA 40231

1 MATERIALS AND METHODS

Test Item: TKA 40231 (CGX PR 774)

Batch No.: 783-068

Purity: >95%

Expiry Date: February 02, 2003

Storage conditions: Closed container, room temperature, in the dark

1.1 EXPERIMENTAL CONDITIONS

The acute toxicity of the test item TKA 40231 to Daphnia magna was determined in a 48-

hour static test based on the Commission Directive 92/69/EEC, C.2 (1992) and the OECD

Guideline for Testing of Chemicals, No. 202, Part 1, (1984).

In each treatment and the control, 20 daphnids were tested, divided into two groups of ten

animals, each group in 50 ml test medium in a 1 00-mi glass-beaker. At the start of the test

the used test animals were 6-24 hours old and were not first brood progeny.

Water temperature: 20-21 CC during the test period

Light conditions: The test was performed as far as possible in the dark to

avoid photolytic degradation of the test item

Test duration: 48 hours

Test water: Reconstituted water according to the test guidelines

78
RCC Study Number 803521 REPORT Page 6

TKA 40231

1.2 DOSAGE AND CONCENTRATIONS

Three WAFs (Water Accommodated Fractions) with test item loading rates of 1, 10 and

100o mg/I were prepared as follows:

Three individual mixtures were prepared by weighing adequate amounts of the test item by

means of an analytical balance:

- 3.05 mg suspended in 3000 mI test water - 10.0 mg suspended in 1000 ml test water

- 31.0 mg suspended in 300 ml test water

The test item was dispersed in the test media by ultrasonic treatment for 15 minutes. Then

the mixtures were stirred by a magnetic stirrer at room temperature in the dark over 96 hours

to dissolve respectively disperse a maximum concentration of the test item in the test media.

No auxiliary solvent or emulsifier was used.

After the stirring period just before the preparation of the test media, each mixture was

filtered through a membrane filter (pore size 0.45 pm). The three filtrates and a control (test

water without test item) were tested in parallel.

79
RCC Study Number 803621 REPORT Page 7

TKA 40231

2 RESULTS

Table 1: Influence of TKA 40231 on the mobility of Daphnia magna

Loading rate / No. of No. of % of immobilized

Treatment daphnids immobilized daphnids after

tested daphnids after

24h I48h 24 h 48 h

Control 20 0 0 0 0

1.0 mg/I filtered 20 0 0 0 0

10 mg/I filtered 20 0 0 0 0

100 mg/I filtered 20 0 0 0 0

Table 2: Dissolved oxygen concentrations, pH values and temperature in the test

media at the start and end of the test

Loading rate / Start (0 hours) End (48 hours)

Treatment

Oxygen pH Temperature Oxygen pH Temperature

_____________ (mg/I) ____ (OC) (mg/I) ____ (OC)

Control 8.3 17.8 1 21 8.2 7.8 20

1.0 mg/I filtered 8.1 7.8 21 8.1 7.8 20

10 mg/I filtered 8.1 7.9 21 8.1 7.8 20

100 mg/I filtered 8.2 7.9 21 8.1 7.8 20

Table 3: Appearance of the test media during the test period

Loading rate /Appearance of the test item

Treatment in the test media

1.0 mg/I filtered no remarkable observations, clear test solution

10 mg/I filtered no remarkable observations, clear test solution

100 mg/I filtered no remarkable observations, clear test solution

80
RCC Study Number 843786

TKA 40273 (CGX PR 774):

Acute Oral Toxicity Study in Rats

Report

Author: G. Arcelin

Sponsor: Ciba Specialty Chemicals Inc.

CH-4002 Basel / Switzerland

Page 1 of 19

81
RCC STUDY NUMBER 843786 Report Page 2

TKA 40273 (CGX PR 774)

Table of contents

1 PREFACE ....................................................................................... 4

1.1 GENERAL............................................................................... 4

1.2 RESPONSIBILITIES..................................................................... 4

1.3 SCHEDULE .............................................................................. 4

1.4 ARCHIVING.............................................................................. 5

1.5 SIGNATURE PAGE...................................................................... 6

1.6 QUALITY ASSURANCE UNIT........................................................... 7

1.7 STATEMENT OF COMPLIANCE ....................................................... 8

1.8 ACCREDITATION........................................................................ 9

1.9 TEST GUIDELINES...................................................................... 9

1.10 SUMMARY OF STUDY PLAN AMENDMENT ......................................... 9

2 SUMMARY ................................................................................... 10

3 CONCLUSION............................................................................... 10

4 PURPOSE...................................................................................... 11

5 MATERIALS AND METHODS................................................................ 11

5.1 TEST SYSTEM.......................................................................... 11

5.2 HUSBANDRY............................................................................ 11

5.3 TESTITEM ............................................................................. 12

5.4 VEHICLE............................................................................... 12

5.5 DOSE FORMULATION................................................................. 13

5.6 TREATMENT............................................................................ 13

5.7 OBSERVATIONS ....................................................................... 13

6 PATHOLOGY ................................................................................ 14

6.1 NECROPSY ............................................................................. 14

7 STATISTICAL ANALYSIS..................................................................... 14

8 DATA COMPILATION......................................................................... 14

9 RESULTS....................................................................................... 15

9.1 MORTALITY ............................................................................. 15

9.2 CLINICAL SIGNS ....................................................................... 15

9.3 BODY WEIGHTS........................................................................ 15

9.4 MACROSCOPIC FINDINGS ........................................................... 15

9.5 MEDIAN LETHAL DOSE ............................................................... 15

10 INDIVIDUAL FINDINGS....................................................................... 16

10.1 MORTALITY/ CLINICAL SIGNS....................................................... 16

10.2 BODY WEIGHTS........................................................................ 17

10.3 MACROSCOPIC FINDINGS ........................................................... 17

11 CERTIFICATIONS............................................................................. 18

82
RCC STUDY NUMBER 843786 Report Page 3

TKA 40273 (CGX PR 774)

Table of contents (cont'd)

11.1 ACCREDITATION! EUROPEAN STANDARD EN 45001.......................... 18

11.2 GLP -CERTIFICATION................................................................ 19

83
RCC STUDY NUMBER 843786 Report Page 4

TKA 40273 (CGX PR 774)

1 PREFACE

1.1 GENERAL

Title TKA 40273 (CGX PR 774):

Acute Oral Toxicity Study in Rats

Sponsor Ciba Specialty Chemicals Inc.

CH-4002 Basel / Switzerland

Study Monitor Mrs. Isabelle Frei

Test Facility RCC Ltd

Toxicology Division

W61ferstrasse 4

CH-4414 FRllinsdorf ISwitzerland

1.2 RESPONSIBILITIES

Study Director G. Arcelin

Deputy for Study Director M. Ot

Technical Coordinator N. Hostettler

Head of RCC Quality 1. W0thrich

Assurance

1.3 SCHEDULE

Experimental Starting Date 1 5-MAY-2002

Experimental Completion Date 07-JUN-2002

Delivery of Animals 1 5-MAY-2002 (females, 2000 mg/kg)

1 7-MAY-2002 (males, 2000 mg/kg)

Acclimatization 1 5-MAY-2002 to 21 -MAY-2002 (females, 2000 mg/kg)

1 7-MAY-2002 to 23-MAY-2002 (males, 2000 mg/kg)

Treatment (first dose) 22-MAY-2002 (females, 2000 mg/kg)

24-MAY-2002 (males, 2000 mg/kg)

Observation 15-MAY-2002 to 05-JUN-2002 (females, 2000 mg/kg)

17-MAY-2002 to 07-JUN-2002 (males, 2000 mg/kg)

Study Completion Date 04-JUL-2002

84
RCC STUDY NUMBER 843786 Report Page 5

TKA 40273 (CGX PR 774)

1.4 ARCHIVING

ROC Ltd (CH-4452 Itingen / Switzerland) will retain the study plan, raw data, amendment, a

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

will be discarded without the Sponsor's consent.

85
RCC STUDY NUMBER 843786 Report Page 6

TKA 40273 (CGX PR 774)

1.5 SIGNATURE PAGE

Study Director: G. Arce*

date

Management: S. Corney

dat

86
RCC STUDY NUMBER 843786 Report Page 7

TKA 40273 (CGX PR 774)

1.6 QUALITY ASSURANCE UNIT

RCC Ltd, Toxicology Division, CH-4452 Itingen ISwitzerland

STATEMENT

RCC STUDY NUMBER : 843786

TEST ITEM TKA 40273 (CGX PR 774)

STUDY DIRECTOR G. Arcelin

TITLE : TKA 40273 (CGX PR 774):

Acute Oral Toxicity Study in Rats

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

the responsible person and the management.

Study procedures, with the exception of the trial formulation, were periodically inspected. The

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

below.

Dates and Types of Dates of Reports to the Study Director

QAU Inspections and to Management

1 3-MAY-2002 Study Plan 1 3-MAY-2002

1 7-MAY-2002 Process Based 1 7-MAY-2002

18-JUN-2002 Report 18-JUN-2002

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

Quality Assurance: (b) S aqren

..............

date: 04LIU L-2co 2

87
RCC STUDY NUMBER 843786 Report Page 8

TKA 40273 (CGX PR 774)

GOOD LABORATORY PRACTICE

1.7 STATEMENT OF COMPLIANCE

RCC STUDY NUMBER : 843786

TEST ITEM . TKA 40273 (CGX PR 774)

STUDY DIRECTOR : G. Arcelin

TITLE . TKA 40273 (CGX PR 774):

Acute Oral Toxicity Study in Rats

The stability of the test item in the vehicle is unknown and is, therefore, excluded from this

statement. The trial formulation was performed before the study initiation date and is also

excluded from this statement.

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

Laboratory Practice, adopted February 2nd ,2000 [RS 813.01 6.5]. This Ordinance is based on

the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted th November 26 , 1997 by decision of the OECD Council [C(97)1 86/Final].

These procedures were consistent with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, the United States (EPA and

FDA), and Japan (MHLW, MAFF and METI).

Study Director: G. ArceJin-)

. . ... e. . ...

date: rU- z~

88
RCC STUDY NUMBER 843786 Report Page 9

TKA 40273 (CGX PR 774)

1.8 ACCREDITATION

The test facility ORCC Ltd, Toxicology Division' is accredited according to EN 45001 under

accreditation number STS 085 by the Swiss Accreditation Service.

1.9 TEST GUIDELINES

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

following guidelines:

OECD Guidelines for the Testing of Chemicals, Number 423 "Acute Oral Toxicity - Acute

Toxic Class Method", adopted 20 December 2001.

Directive 96/54/EEC, B.1 tris "Acute Oral Toxicity-Acute Toxic Class Method", September 30,

1996.

1.10 SUMMARY OF STUDY PLAN AMENDMENT

Study Plan Amendment No. 1:

Reference to the analytical certificate was added.

89
RCC STUDY NUMBER 843786 Report Page 10

TKA 40273 (CGX PR 774)

2 SUMMARY

Three male and three female HanBrl: WIST (SPF) rats were treated with TKA 40273 (CGX

PR 774) by oral gavage administration at a dosage of 2000 mg/kg body weight. The test

item was diluted in vehicle (PEG 300) at a concentration of 0.2 g/ml and administered at a

volume dosage of 10 mI/kg.

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

and clinical signs were recorded. All animals were examined for clinical signs at

approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days

2-15. Mortality/viability was recorded twice daily during test days 1-15. Body weights were

recorded on day 1 (prior to administration) and on days 8 and 15. All animals were

necropsied and examined macroscopically.

The following animals were treated and percentage of mortality was observed:

Males Females

2000 mg/kg 33 % 0%

One male animal was found dead on test day 3. All other animals survived until the end of

the study period.

Slightly ruffled fur and hunched posture was noted in one female animal and all male

animals treated at 2000 mg/kg from 1 to 5 hours after the test item administration.

The body weight of the animals was within the range commonly recorded for this strain and

age.

No macroscopic findings were recorded at necropsy.

3 CONCLUSION

The median lethal dose of TKA 40273 (CGX PR 774) after single oral administration to rats

of both sexes, observed over a period of 14 days is:

LD5 0 (RAT): GREATER THAN 2000 mg/kg BODY WEIGHT

90
ROC STUDY NUMBER 843786 Report Page 11

TKA 40273 (CGX PR 774)

4 PURPOSE

The purpose of this study was to assess the acute toxicity of TKA 40273 (CGX PR 774)

when administered by a single oral gavage to rats, followed by an observation period of

14 days.

This study provides information for both hazard assessment and hazard classification

purposes.

5 MATERIALS AND METHODS

5.1 TEST SYSTEM

Test system Rat, HanBrl: Wist (SPE)

Rationale Recognized by the international guidelines as a

recommended test system.

Source RCC Ltd, Biotechnology and Animal Breeding Division

CH-4414 F~jllinsdorf / Switzerland

Number of animals per group 3 males or 3 females

Total number of animals 3 males

3 females

Age when treated Males: 8 weeks

Females: 13 weeks

Identification Unique cage card and corresponding color-coded spots on

the tail.

Acclimatization Under laboratory conditions, after health examination. Only

animals without any visible signs of illness were used for the

study.

5.2 HUSBANDRY

Room no. 104 / RCC Ltd, Fujllinsdorf

Conditions Standard Laboratory Conditions. Air-conditioned with 10-15

air changes per hour, and continuously monitored environment with target ranges for temperature 22 ± 3 00 and for

relative humidity between 30-70 % (values above 70 %

during cleaning process possible), 12 hours fluorescent light/

12 hours dark, music during the light period.

91
RCC STUDY NUMBER 843786 Report Page 12

TKA 40273 (CGX PR 774)

Accommodation In groups of three per sex in Makrolon type-4 cages with

wire mesh tops and standard softwood bedding (tignocel'

Schill AG, CH-41 32 Muttenz/Switzerland).

Diet Pelleted standard Provimi Kliba 3433 rat/mouse

maintenance diet, batch no. 1/02 (Provimi Kliba AG, CH4303 Kaiseraugstl Switzerland) ad libitum. Results of

analyses for contaminants are archived at RCC Ltd, Itingen.

Water Community tap water from Fullinsdorf ad libitum. Results of

bacteriological, chemical and contaminant analyses are

archived at ROC Ltd, Itingen.

5.3 TEST ITEM

The following information was provided by the sponsor:

Identification TKA 40273 (CGX PR 774)

Product Name CGX PR 774

Description White powder

Batch number 836-1 87

Purity See Analytical Certificate No. 14022921, Ciba Specialty

Chemicals Inc.

Expiry date 30-APR-2004

Stability of test item dilution Unknown in PEG 300; is excluded from the statement of

compliance.

Storage conditions In the original container, at room temperature

(range of 20 ± 3 'C), away from direct sunlight.

Safety precautions Routine hygienic procedures were used to ensure the health

and safety of the personnel.

5.4 VEHICLE

The following information was provided by ROC Ltd:

Identification Polyethylene glycol 300 (PEG 300)

Description colorless viscous liquid

Lot number 433337/1 20602

Source FLUKA Chemie GmbH, CH-9471 Buchs

Stability of vehicle Stable under storage conditions;

expiration date: 28-FEB-2003

92
RCC STUDY NUMBER 843786 Report Page 13

TKA 40273 (CGX PR 774)

Storage conditions In the original container, at room temperature

(range of 20 ± 3 0C), away from direct sunlight.

Safety precautions Routine hygienic procedures were used to ensure the health

and safety of the personnel.

PEG 300 was found to be a suitable vehicle.

The vehicle was chosen after a non-GLP solubility trial which was performed before the

study initiation date. This trial formulation is excluded from the GLP statement of compliance.

5.5 DOSE FORMULATION

Dose levels are in terms of the test item as supplied.

The dose formulations were made shortly before each dosing occasion.

The test item was weighed into a tared glass beaker on a suitable precision balance and the

vehicle added (weight:volume). The formulations were prepared using a magnetic stirrer.

Homogeneity of the test item in the vehicle was maintained during administration using a

magnetic stirrer.

5.6 TREATMENT

The animals received a single dose of the test item by oral gavage administration at

2000 mg/kg body weight after being fasted for 17 to 18 hours (access to water was

permitted). Food was provided again 3 hours after dosing.

The application volume was 10 mI/kg body weight.

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

is a possible route of human exposure during manufacture, handling and use of the test

item.

5.7 OBSERVATIONS

Mortality / Viability Daily during acclimatization and twice daily during days 1 -15.

Body weights On test days 1 (prior to administration), 8 and 15.

Clinical signs Daily during acclimatization and at approximately 1, 2, 3 and

5 hours after administration on test day 1. Once daily during

days 2-15. All abnormalities were recorded.

93
ROC STUDY NUMBER 843786 Report Page 14

IKA 40273 (CGX PR 774)

6 PATHOLOGY

6.1 NECROPSY

Necropsy was performed in one male animal found dead on test day 3.

All surviving animals were killed at the end of the observation period by an intraperitoneal

injection of Vetanarcol at a dose of at least 2.0 mi/kg body weight (equivalent to at least

324 mg sodium pentobarbitone/kg body weight) and discarded after macroscopic examinations were performed. No organs or tissues were retained.

7 STATISTICAL ANALYSIS

No statistical analysis was used.

8 DATA COMPILATION

Body weights were recorded on-line.

Clinical signs were recorded on data sheets.

Mortality/viability were compiled into the ROC computer system during recording and/or

recorded on data sheets.

Macroscopic findings were compiled into the RCC computer system during recording.

94
RCC STUDY NUMBER 843786 Report Page 15

TKA 40273 (CGX PR 774)

9 RESULTS

9.1 MORTALITY

One male animal was found dead on test day 3. All other animals survived until the end of

the study period.

9.2 CLINICAL SIGNS

Slightly ruffled fur and hunched posture was noted in one female animal and all male

animals treated at 2000 mg/kg from 1 to 5 hours after the test item administration.

9.3 BODY WEIGHTS

The body weight of the animals was within the range commonly recorded for this strain and

age.

9.4 MACROSCOPIC FINDINGS

No macroscopic findings were recorded at necropsy.

9.5 MEDIAN LETHAL DOSE

The median lethal dose of TKA 40273 (CGX PR 774) after single oral administration to rats

of both sexes, observed over a period of 14 days is:

LD50 (RAT): GREATER THAN 2000 mg/kg BODY WEIGHT

95
ROC STUDY NUMBER 843786 Report Page 16

TKA 40273 (CGX PR 774)

10 INDIVIDUAL FINDINGS

10.1 MORTALITY/I CLINICAL SIGNS

______Test days

Dose An. Sex Signs 1 _;j2 13 4 5 6 17 8 9 10 11 12 13 14 15

mg/kg No. 1_____ 12* 13*-5

2000 1 F No clinical 4 4 4 4 4 4 4 4 4

signs

2 F No clinical 4 4 4 4 4 4 4 4 4

signs

3 F No clinical 4 4 4 4 4 4 4

signs

Ruffled fur 1 1 1 1

Hunched 4444

posture

2000 4 M No clinical 4 4 4 4 4 4 4

signs

Ruffled fur 1 1 1 1

Hunched 4444

posture

5 M No clinical 4 +

signs

Ruffled fur 1 1 1 1

Hunched 4444

posture

6 M No clinical 4 4 4 4 4 4 4

signs

Ruffled fur 1 1 1 1

Hunched 4444

posture I I I II I I

Key: 1 slight, found dead, 4noted

* Examination performed approximately 1, 2, 3 and 5 hours after treatment.

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

96
ROC STUDY NUMBER 843786 Report Page 17

TKA 40273 (CGX PR 774)

10.2 BODY WEIGHTS

Dose An. Sex Dayl1 Day 8 Dayl15

m 1kq N o. ___(treatment) _____

2000 1 F 194.6 207.0 214.7

2 F 174.1 184.1 195.7

3 F 173.9 188.1 203.2

2000 4 M 229.2 260.3 281.0

5 M 240.6----

6 M 239.4 279.9 315.2

Body weights are presented in grams

10.3 MACROSCOPIC FINDINGS

Dose An. Sex Mode Findings

mg/kg No. of ______death

2000 1 F S No macroscopic findings

2 F IS No macroscopic findings

3 F S No macroscopic findings

2000 4 M S No macroscopic findings

5 M D No macroscopic findings

6 M S No macroscopic findings

S: Scheduled necropsy, D found dead

97
RCC STUDY NUMBER 843786 Report Page 18

TKA 40273 (GGX PR 774)

11 CERTIFICATIONS

11.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

fh Eidgen6ssisches Amrt for Mesawesen S Schweizenische Akkreditierungssalle

Ih Office f~dhral de mizroiogie Service d'accriditation suisse

Ufficia federale di metrologia A~c Servizio d'accreditamento avizzero

Swiss Federal Office of Metrology S Swiss Accreditation Service

ACCREDITATION

Based on the Accreditation and Designation Ordinance

dated 17' June 1996

the Swiss Federal Office of Metrology grants the

RCC Ltd

Toxicology Division

Zelgliweg 1

4452 Itingen

accreditation as a

Testing Laboratory for toxicological investigation of

pharmaceuticals and medical devices, agrochemnicals, industrial

chemicals, food- and feed-additives

in accordance with SN EN 45001.

The accredited scope of testing is defined in the Official

Directory of the Accredited Testing Laboratories in Switzerland.

The relevant requirements of the ISO 9002 standard

are also covered by this accreditation.

Accreditation number: STS 085

Date of the accreditation: 16"e September 1994

Date of the last renewal of the accreditation: 20"' September 1999

The accreditation is valid until: 19"' September 2004

Wabern, 20"' September 1999 Swiss F dera Office of Metrology The Dicr The he o A

)Xl4.1 AS

Dr Wolfgang Schwitz Hanspeter lschi

SAS Is a sign" of ato mulfiaeal agivrenit ol to Europea co-operadm or cr edlall (EA) for calibralion. wsWQn and cerdlication ol pro*"t. personnel. quality anid awnvlrientl mianagernent sylim.

98
RCC STUDY NUMBER 843786 Report Page 19

TKA 40273 (CGX PR 774)

11.2 GLP - CERTIFICATION

The Swiss GLP Monitoring Authorities

SisFederal Swiss Agency for the Intircantonall Office

ofice o niomnFrssfrteCnrlo

Public health an Enciont FMet )frecntro o

Statement of GLP Compliance

It is hereby confirmed that

during the period of August 15 - 17, 2000

the following Test Facilities of RCC Ltd

4452 Itingen

Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for the

Environment, Forests and Landscape and the Intercantonal Office for the Control of

Medicines with respect to the compliance with the Swiss legislation on Good Laboratory

Practice.

Test Facilities areas of expertise*

- Toxicology Division TOX, ACC

- Environmental Chemistry and ACC, ECT, ENF, PCT,RES,

Pharmanalytics Division 0TH (Animal metabolism)

- Microbiological Diagnostics by 0TH (Microbiology)

Biotechnology & Animal Breeding Division

The inspection was performed in agreement with the OECD Guidelines for National GLP

inspections and Audits. It was found that the aforementioned test facilities were operating in

compliance with the Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.51 at

the time they were inspected.

Federal Office of Public Health

The Director

Bemn, November 2000 Prof. Th. Zeltner

*TOX - Toxicology . ACC -Analytical and Clinical Chemistry , ECT *Environmental toxicity on aquatic and terrestrial

organisms . ENF - Behaviour in water, soil and air. Bicaccumulation PCT a Physical-chemical tasting ;RES a Residue

studies 0 TH - Other, to be spectfied

99
RCC Study Number 843788

TKA 40273 (CGX PR 774):

Primary Eye Irritation Study in Rabbits

Report

Author: G. Arcelin

Sponsor: Ciba Specialty Chemicals Inc.

CH-4002 Basel / Switzerland

Page 1 of 26

100
ROC STUDY NUMBER 843788 Report Page 2

TKA 40273 (CGX PR 774)

Table of contents

1 PREFACE ....................................................................................... 4

1.1 GENERAL............................................................................... 4

1.2 RESPONSIBILITIES ..................................................................... 4

1.3 SCHEDULE .............................................................................. 4

1.4 ARCHIVING.............................................................................. 4

1.5 SIGNATURE PAGE ...................................................................... 5

1.6 QUALITY ASSURANCE UNIT........................................................... 6

1.7 STATEMENT OF COMPLIANCE ....................................................... 7

1.8 CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT ............... 8

1.9 ACCREDITATION ........................................................................ 9

1.10 TEST GUIDELINES ...................................................................... 9

1.11 ANIMAL WELFARE....................................................................... 9

1.12 SUMMARY OF STUDY PLAN AMENDMENT ......................................... 9

2 SUMMARY ................................................................................... 10

3 PURPOSE ...................................................................................... 11

4 MATERIALS AND METHODS ................................................................ 11

4.1 TEST SYSTEM.......................................................................... 11

4.2 HUSBANDRY............................................................................ 12

4.3 TESTITEM ............................................................................. 12

4.4 TEST ITEM PREPARATION ........................................................... 13

4.5 TREATMENT ............................................................................ 13

4.6 OBSERVATIONS........................................................................ 13

4.6.1 IRRITATION SCORES ....................................................... 13

5 PATHOLOGY................................................................................. 14

5.1 NECROPSY............................................................................. 14

6 TABULATION AND EVALUATION OF RESULTS .......................................... 14

7 DATA COMPILATION AND STATISTICAL ANALYSIS .................................... 14

8 RESULTS ....................................................................................... 15

8.1 VIABILITY/MORTALITY AND CLINICAL SIGNS ..................................... 15

8.2 IRRITATION............................................................................. 15

8.3 COLORATION........................................................................... 15

8.4 CORROSION ............................................................................ 15

8.5 BODY WEIGHTS........................................................................ 16

8.6 CONCLUSION........................................................................... 16

9 APPENDICES ................................................................................ 17

101
RCC STUDY NUMBER 843788 Report Page 3

TKA 40273 (CGX PR 774)

Table of contents (cont'd)

9.1 EYE IRRITATION SCORES............................................................ 17

9.2 INDIVIDUAL FINDINGS................................................................ 19

9.3 SUMMARY OF EVALUATION CRITERIA ............................................ 22

9.4 CERTIFICATION........................................................................ 24

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001 ............... 25

9.4.2 GLP - CERTIFICATION...................................................... 26

102
RCC STUDY NUMBER 843788 Report Page 4

TKA 40273 (CGX PR 774)

1 PREFACE

1.1 GENERAL

Title TKA 40273 (CGX PR 774):

Primary Eye Irritation Study in Rabbits

Sponsor Ciba Specialty Chemicals Inc.

CH-4002 Basel / Switzerland

Study Monitor Mrs. Isabelle Frei

Test Facility RCC Ltd

Toxicology Division

Wdlferstrasse 4

CH-441 4 F0llinsdorf ISwitzerland

1.2 RESPONSIBILITIES

Study Director G. Arcelin

Deputy for Study Director M. Ott

Technical Coordinator P. Reissbrodt

Head of RCC Quality 1. WOthrich

Assurance

1.3 SCHEDULE

Experimental Starting Date 23-MAY-2002

Experimental Completion Date 31 -MAY-2002

Delivery of the Animals 07-MAY-2002

Allocation 23-MAY-2002

(Animals were previously used for RCC Study 843787)

Acclimatization 23-MAY-2002 to 27-MAY-2002

Treatment 28-MAY-2002

Observation Throughout 72 hours after treatment.

Termination 31 -MAY-2002

Study Completion Date 03-JUL-2002

1.4 ARCHIVING

RCC Ltd (CH-4452 ltingenl/ Switzerland) will retain the study plan, raw data, amendment, a

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

will be discarded without the Sponsor's consent.

103
ROC STUDY NUMBER 843788 Report Page 5

TKA 40273 (CGX PR 774)

1.5 SIGNATURE PAGE

Study Director: G. Arceli

date: 6-JL ~z

Management: (//S. Cor y

date: 1-7

104
RCC STUDY NUMBER 843788 Report Page 6

IKA 40273 (CGX PR 774)

1.6 QUALITY ASSURANCE UNIT

RCC Ltd, Toxicology Division, CH-4452 Itingen ISwitzerland

STATEMENT

RCC STUDY NUMBER 843788

TEST ITEM . TKA 40273 (CGX PR 774)

STUDY DIRECTOR : G. Arcelin

TITLE . TKA 40273 (CGX PR 774):

Primary Eye Irritation Study in Rabbits

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

the responsible person and the management.

Study procedures were periodically inspected. The study plan and this report were audited

by the Quality Assurance Unit. The dates are given below.

Dates and Types of Dates of Reports to the Study Director

QAU Inspections and to Management

1 6-MAY-2002 Study Plan 1 6-MAY-2002

22-MAY-2002 Process Based 22-MAY-2002

1 9-JUN-2002 Report 1 9-JUN-2002

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

Quality Assurance:G.Ht

date: 03. j~UL-2eD62

105
ROC STUDY NUMBER 843788 Report Page 7

TKA 40273 (CGX PR 774)

GOOD LABORATORY PRACTICE

1.7 STATEMENT OF COMPLIANCE

ROC STUDY NUMBER: 843788

TEST ITEM : TKA 40273 (CGX PR 774)

STUDY DIRECTOR : G. Arcelin

TITLE : TKA 40273 (CGX PR 774):

Primary Eye Irritation Study in Rabbits

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

Laboratory Practice, adopted February 2"', 2000 [RS 813.01 6.5]. This Ordinance is based

on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted th November 26 , 1997 by decision of the OECD Council [0(97)186/Final].

These principles are consistent with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, the United States (EPA and

FDA), and Japan (MHLW, MAFF and METI).

Study Director: G. Arceifi

date: [C-") ~ Zc

106
RCC STUDY NUMBER 843788 Report Page 8

TKA 40273 (CGX PR 774)

1.8 CERTIFICATION OF GLP

AND VERIFICATION OF THE REPORT

The statement of Compliance with Good Laboratory Practice found in this report, and signed

by the Study Director is truthful and accurate, and this report as provided by the test facility

is complete and unaltered.

Signature of the Sponsor: .......................

Date:

107
ROC STUDY NUMBER 843788 Report Page 9

TKA 40273 (CGX PR 774)

1.9 ACCREDITATION

The test facility "RCC Ltd, Toxicology Division" is accredited according to EN 45001 under

accreditation number STS 085 by the Swiss Accreditation Service.

1.10 TEST GUIDELINES

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

following guidelines:

Directive 92/69 EEC, 13.5. "Acute Toxicity - Eye Irritation", July 31, 1992.

OECD Guidelines for Testing of Chemicals, Section 4, number 405 "Acute Eye Irritation/

Corrosion", adopted February 24, 1987.

1.11 ANIMAL WELFARE

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

Animal Protection Law under license no. 54.

1.12 SUMMARY OF STUDY PLAN AMENDMENT

Study Plan Amendment No. 1:

Reference to the analytical certificate was added.

108
RCC STUDY NUMBER 843788 Report Page 10

TKA 40273 (CGX PR 774)1

2 SUMMARY

The primary eye irritation potential of TKA 40273 (CGX PR 774) was investigated by

instillation of 0.1 g into one eye of each of three young adult New Zealand White rabbits.

Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test

item application. The scores of each animal at the following reading times (24, 48 and

72 hours) were used in calculating the respective mean values for each type of lesion.

The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal

opacity, grade 0.00 iris lesions, grade 0.67 redness of the conjunctivae and grade 0.00

chemosis of the conjunctivae.

The instillation of TKA 40273 (CGX PR 774) into the eye resulted in mild, early-onset and

transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and

chemosis. These effects were reversible and were no longer evident 72 hours after

treatment, the end of the observation period for all animals. No abnormal findings were

observed in the cornea or iris of any animal at any reading. No corrosion was observed at

any of the measuring intervals. No staining of the treated eyes by the test item was

observed.

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

TKA 40273 (CGX PR 774) is considered to be "not irritating" to the rabbit eye.

109
RCC STUDY NUMBER 843788 Report Page 11

TKA 40273 (CGX PR 774)

3 PURPOSE

The purpose of this primary eye irritation study was to assess the possible irritation potential

when a single dose of TKA 40273 (CGX PR 774) was placed in the conjunctival sac of rabbit

eyes.

This study should provide a rational basis for risk assessment in man as ocular contact is

one of the probable routes of human exposure.

The test item was applied at 0.1 g/animal, the dose specified in the test guidelines for a solid

test item.

4 MATERIALS AND METHODS

4.1 TEST SYSTEM

Test system New Zealand White Rabbit, SPF

Rationale Recognized by the international guidelines as the

recommended test system

Source Elevage Scientifique des Dombes

F-01400 Chatillon sur Chalaronne / France

Number of animals per test 3 (Animals of both sexes were used)

Age at treatment 17 weeks (male)

13 weeks (females)

Identification By unique cage number and corresponding ear number.

Acclimatization Under laboratory conditions after health examination. Only

animals without any visual signs of illness were used for the

study.

Allocation Male No. 25

Female Nos. 26 and 27

110
TKA ROC 40273 STUDY (CGX NUMBER PR 774) 843788 Report Page 12 1

4.2 HUSBANDRY

Room no. 106 / ROC Ltd, Fullinsdorf

Conditions Standard Laboratory Conditions

Air-conditioned with target ranges for room temperature

17-23 00, relative humidity 30-70 % and approximately

10-15 air changes per hour. Room temperature and humidity

were monitored continuously and values outside of these

ranges may have occasionally occurred, usually following

room cleaning. These transient variations are considered not

to have any influence on the study and, therefore, these data

are not reported but are retained at ROC. The animals were

provided with an automatically controlled light cycle of

12 hours light and 12 hours dark. Music was played during

the daytime light period.

Accommodation Individually in stainless steel cages equipped with feed

hoppers and drinking water bowls. Wood blocks (RCC Ltd,

Fullinsdorf) and haysticks (batch no. 0203, Provimi Klba AG)

were provided for gnawing.

Diet Pelleted standard Provimi Kliba 3418 rabbit maintenance

diet ad libitum (batch no. 25/02) provided by Provimi Kliba

AG, CH-4303 Kaiseraugst. Results of analysis are archived

at RCC Ltd, Itingen.

Water Community tap water from Fujllinsdorf, ad libiturn. Results of

bacteriological, chemical and contaminant analyses are

archived at ROC Ltd, Itingen.

4.3 TEST ITEM

The following information was provided by the sponsor:

Identification TKA 40273

Product Name CGX PR 774

Description White powder

Batch number 836-187

Purity See Analytical Certificate Study No. 14022921, Ciba

Specialty Chemicals Inc.

Stability of test item Stable under storage conditions

Expiry date 30-APR-2004

Storage conditions In the original container, at room temperature

(range of 17-23 00), away from direct sunlight.
ROC STUDY NUMBER 843788 Report Page 13

TKA 40273 (CGX PR 774)

4.4 TEST ITEM PREPARATION

0.1 g (per animal) of TKA 40273 (CGX PR 774) was weighed and applied undiluted as it was

delivered by the sponsor.

According to Directive 92/69 EEC, B.5. and OECD Guidelines 405, a test item needs not to

be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable

corrosive properties.

The pH of the test item was measured for ROC study No. 843787 and was found to be

5 to 6.

4.5 TREATMENT

The eyes of the animals were examined one day prior to test item administration.

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

interpretation of the results were not used in the test.

On the day of treatment, TKA 40273 (CGX PR 774) was placed in the conjunctival sac of the

left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were

then gently held together for about one second to prevent loss of test item. The right eye

remained untreated and served as the reference control. The treated eyes were not rinsed

after instillation.

4.6 OBSERVATIONS

Viability/Mortality Daily from acclimatization of the animals to the termination

of test.

Clinical signs Daily from acclimatization of the animals to the termination

of test.

Body weights At start of acclimatization, on the day of application and at

termination of observation.

4.6.1 IRRITATION SCORES

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

EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and

72 hours after application.

When present, corrosion and/or staining of sclera and cornea by the test item were recorded

and reported. Eye examinations were made with a Varta Cliptrix diagnostic-lamp

(A. Riegger, Basel / Switzerland).

112
RCC STUDY NUMBER 843-788 Report Page 14

TKA 40273 (CGX PR 774)

5 PATHOLOGY

5.1 NECROPSY

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

All rabbits were sacrificed by an intravenous injection of VETANARCOL into the ear vein at a

dose of at least 1 mI/kg body weight (equivalent to 162 mg sodium pentobarbitone/kg body

weight) and discarded.

6 TABULATION AND EVALUATION OF RESULTS

Data are summarized in tabular form, showing the irritation scores of each following

parameters: corneal opacity (including the area affected, where applicable), iridic effects,

conjunctival and scleral reddening and/or chemosis for each individual animal at all

observation intervals. In addition, any lesions including the degree and nature of irritation,

corrosion or reversibility, and any other toxic effects were observed.

To evaluate the irritation potential of the test item the mean values were calculated for each

individual, using the scores between 24 and 72 hours.

The Cumulative Scores for the Eye Irritation Scores represent the sum of all available

numerical scores, except area of corneal opacity and scleral reddening, for each animal at

each time point. The resulting Mean Cumulative Eye Irritation Score was calculated for all

animals at each time point.

See TABLE 1, page 18.

Mean Values represent the mean of the available numerical scores recorded for the same

parameter in each animal at 24, 48 and 72 hours, insofar as numerical scores were

assessable. The resulting Mean Score is the mean of all available numerical scores

recorded for the same parameter in all animals at 24, 48 and 72 hours.

See TABLE 2, page 18.

The primary eye irritation score was calculated by totaling the mean cumulative scores at 24,

48 and 72 hours and then divided by the number of data points.

7 DATA COMPILATION AND STATISTICAL ANALYSIS

Viability/mortality, clinical signs and ocular findings were recorded on data sheets and

transcribed for compilation and analysis.

Body weights were recorded on-line and/or on data-sheets.

No statistical analysis was performed.

113
RCC STUDY NUMBER 843788 Report Page 15

TKA 40273 (CGX PR 774)

8 RESULTS

8.1 VIABILITY/MORTALITY AND CLINICAL SIGNS

No clinical signs of systemic toxicity were observed in the animals during the study and no

mortality occurred.

8.2 IRRITATION

Application of the test item to healthy rabbit conjunctivae resulted in a primary eye irritation

score of 0.67.

The eye reactions (mean values from 24 to 72 hours) consisted of grade 0.00 corneal

opacity, grade 0.00 iris lesions, grade 0.67 redness of the conjunctivae and grade 0.00

chemosis of the conjunctivae (Table 2).

No abnormal findings were observed in the cornea or iris of any animal at any of the

measuring intervals (Table 1).

Slight to moderate reddening of the conjunctivae was apparent in all animals from 1 to 24

hours after treatment. Slight reddening continued to be observed in two animals up to the

48-hour examination (Table 1).

Slight swelling or swelling with partial eversion of lids was noted in all animals 1 hour after

treatment (Table 1).

Moderate reddening of the sclerae was observed in all animals at the 1-hour examination

and slight reddening was noted again in one animal up to the 48-hour examination (Table 1).

increased ocular discharge was present in all animals 1 hour after treatment.

No abnormal findings were observed in the treated eye of any animal 72 hours after

treatment, the end of the observation period for all animals.

8.3 COLORATION

No staining of the treated eyes by the test item was observed.

White remnants of the test item were observed in the eye or conjunctival sac of all animals

1 hour after treatment.

8.4 CORROSION

No corrosion of the cornea was observed at any of the reading times.

114
RCC STUDY NUMBER 843788 Report Page 16

TKA 40273 (CGX PR 774)

8.5 BODY WEIGHTS

The body weights of all rabbits were considered to be within the normal range of variability.

Body weight in grams______________

Animal Sex First Day of Day of Last Day of

No. _____Acclimatization Treatment Observation

25 male 2455 2621 2659

26 female 2556 2696 2746

27 female 2394 2561 2636

8.6 CONCLUSION

TKA 40273 (CGX PR 774) is considered to be "not irritating" to the rabbit eye (Table 3).

115
RCC STUDY NUMBER 843788 Report Page 17

TKA 40273 (CGX PR 774)

9 APPENDICES

9.1 EYE IRRITATION SCORES

Key to Symbols:

M Male

F Female

116
RCC STUDY NUMBER 843788 Report Page 18

TKA 40273 (CGX PR 774)

TABLE 1: EYE IRRITATION SCORES - INDIVIDUAL VALUES

Animal I Evaluation Corneal Area of COW untivae Cumulative

Number Sx Interval* Opacity Corneal Opacity Iris JRedness Chemosis IScore Mean Sclera

25 M 0 0 0 2 1 3.00 2

26 F I hour 0 0 0 2 1 3.00 3.33 2

27 F 0 0 0 2 2 4.00 2

25 M 0 0 0 1 0 1.00 0

26 F 24 hours 0 0 0 1 0 1.00 1.33 0

27 F ______ 0- 0 0 2 0 2.00 ________

25 M T0 0 0 1 0 1.000

26 F 48 hours 0 0 0 0 0 0.00 0.67 0

27 F j _____ 0 0 0 1 0 1.00- -___ 1

25 M ou$ 0 0 0 0 0 0.00 0

26 F f72bohr 0 0 0 0 0 0.00 0.00 0

27 Fl __ _ 0 0 0 0 0 0.0 0

*Examninations were performned at the specified timfes after instillation of the test item.

TABLE 2: EYE IRRITATION SCORES - MEAN VALUES AFTER 24,

___ 48 AND 72 HOURS

Animal lCorneal Conjunctivae Primary Eye

Number Sex j~aiy N Iris N Redness N Chemosis N Irritation Score

25 M 0.00 3 0.00 3 0.67 3 0.00 3

26 F 0.00 3 0.00 3 0.33 3 0.00 3 0.67

27 F 0.00 3 0.00 3 1.00 3 0.00 3 _______

Mean score 10.00 0.00 0.67 0.00

N = number of available data points

TABLE 3: EYE IRRITATION SCORES - ASSESSMENT ACCORDING

TO EEC GUIDELINES

Evaluated Corneal Conjunctivae

intervals Oacit 0 Iris Redness Cheniosis

24 hours

48 hours Not Irritating Not Irritating Not Irritating Not Irritating

72 hours

The grading scale is presented on page 23.

117
ROC STUDY NUMBER 843788

TKA 40273 (CGX PR 774) Page 19

9.2 INDIVIDUAL FINDINGS

ANIMAL NO. 25, MALE

After 1 hour: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: moderately reddened; slight swelling Discharge: increased ocular discharge

Sciera: moderately reddened

Test item: white remnants in eye or conjunctival sac

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: slightly reddened

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: slightly reddened

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

After 72 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

Observations included: cornea, conjunctivae (including nictitating membrane), sclera and iris. The

presence (or absence, as appropriate) of opacity, vascularization, reddening, oedema, discharge, staining and test item remnants were assessed.

118
ROC STUDY NUMBER 843788 Report Page 20

TKA 40273 (CGX PR 774)

INDIVIDUAL FINDINGS

ANIMAL NO. 26, FEMALE

After 1 hour: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: moderately reddened; slight swelling

Discharge: increased ocular discharge

Sclera: moderately reddened

Test item: white remnants in eye or conjunctival sac

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: slightly reddened

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

After 72 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

Observations included: cornea, conjunctivae (including nictitating membrane), sciera and iris. The

presence (or absence, as appropriate) of opacity, vascularization, reddening, oedema, discharge, staining and test item remnants were assessed.

119
RCC STUDY NUMBER 843788 Report Page 21

TKA 40273 (CGX PR 774)

INDIVIDUAL FINDINGS

ANIMAL NO. 27, FEMALE

After 1 hour: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: moderately reddened; obvious swelling with

partial eversion of lids

Discharge: increased ocular discharge

Sciera: moderately reddened

Test item: white remnants in eye or conjunctival sac

After 24 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: moderately reddened

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: slightly reddened

Test item: NO REMNANTS EVIDENT

After 48 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: slightly reddened

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: slightly reddened

Test item: NO REMNANTS EVIDENT

After 72 hours: Cornea: NO ABNORMAL FINDINGS NOTED

Iris: NO ABNORMAL FINDINGS NOTED

Conjunctivae: NO ABNORMAL FINDINGS NOTED

Discharge: NO ABNORMAL FINDINGS NOTED

Sclera: NO ABNORMAL FINDINGS NOTED

Test item: NO REMNANTS EVIDENT

Observations included: cornea, conjunctivae (including nictitating membrane), sclera and iris. The

presence (or absence, as appropriate) of opacity, vascularization, reddening, oedema, discharge,

staining and test item remnants were assessed.

120
RCC STUDY NUMBER 843788 Report Page 22

TKA 40273 (CGX PR 774)

9.3 SUMMARY OF EVALUATION CRITERIA

*EEC Commission Directive 92/69/EEC, July 31, 1992

121
ROC STUDY NUMBER 843788 Report Page 23

TKA 40273 (CGX PR 774)

EEC COMMISSION DIRECTIVE 92169/EEC, JULY 31,1992

Grading of Ocular Lesions

CORNEA

Opacity: degree of density (area most dense taken for reading)

No ulceration or opacity........................................................................ Scattered or diff use areas of opacity (other than slight dulling of normal luster),

details of iris clearly visible....................................................................1

Easily discernible translucent area, details of iris slightly obscured......................... 2

Nacreous area, no details of iris visible, size of pupil barely discernible.................... 3

opaque cornea, iris not discernible through the opacity...................................... 4

Area of cornea involved

Zero ........................................................................................... 0

One quarter (or less) but not zero.............................................................. 1

Greater than one quarter, but less than half ................................................... 2

Greater than half, but less than three quarters................................................ 3

Greater than three quarters, up to whole area ................................................ 4

IRIS

Normal............................................................................................ 0

Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia,

or injection, any of these or combination of any thereof, iris still reacting to light

(sluggish reaction is positive)................................................................... 1

No reaction to light, hemorrhage, gross destruction (any or all of these) ................... 2

CONJUNCTIVAE

Redness (refers to most severe reading of palpebral and bulbar conjunctivae

when compared with control eye)

Blood vessels normal..........................................................................

Some blood vessels definitely hyperemic (injected)........................................... 1

Diffuse, crimson color, individual vessels not easily discernible ............................. 2

Diffuse beefy red ................................................................................ 3

Chemosis: lids and/or nictitating membranes

No swelling..................................................................................... 0

Any swelling above normal (including nictitating membranes)................................ 1

Obvious swelling with partial eversion of lids.................................................. 2

Swelling with lids about half-closed............................................................. 3

Swelling with lids more than half-closed ....................................................... 4

Note: Reddening of the sclerae was assessed using the same scoring grades as conjunctivae.

122
RCC STUDY NUMBER 843788 Report Page 24

TKA 40273 (CGX PR 774)

9.4 CERTIFICATION

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

9.4.2 GLP - CERTIFICATION

123
ROC STUDY NUMBER 843788 Report Page 25

TKA 40273 (CGX PR 774)

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

SEidgenifsaachea Amrt fur Messwesen S Schweizenische Akkreditierungsatelle

Office f~ddral do m6trologie Service d'accrdditation suisse

Ufficia federale di metrologia A Servizio d'accreditamento avizzero

4 p Swiss Federal Office of Metrology 4frS Swiss Acreditation Service

ACCREDITATION

Based on the Accreditation and Designation Ordinance

dated 1r~ June 1996

the Swiss Federal Office of Metrology grants the

RCC Ltd

Toxicology Division

Zelgliweg 1

4452 Itingen

accreditation as a

Testing Laboratory for toxicological investigation of

pharmaceuticals and medical devices, agrochemnicals, industrial

chemicals, food- and feed-additives

in accordance with SN EN 45001.

The accredited scope of testing is defined in the Official

Directory of the Accredited Testing Laboratories in Switzerland.

The relevant requirements of the ISO 9002 standard

are also covered by this accreditation.

Accreditation number: STS 085

Date of the accreditation: le~ September 1994

Date of the last renewal of the accreditation: 20ei September 1999

The accreditation is valid until: 19V September 2004

Wabern, 20"' September 1999 Swiss F deral Office of Metrology

The Di ctor The hejuofSAS

Dr Wolfgang Schwitz Hanspeter lschi

M Is a aignsloy of fti rmikaal arsanis oft Euiropesan co-opasifr wAodaiiol (EA) for 0511u(5doni, 158mg and csrlIkadhal of prodWM, personnel, quality &nd erwironmensal imigemsnt systm .

'124
ROC STUDY NUMBER 843788 Report Page 26

TKA 40273 (CGX PR 774)

9.4.2 GLP - CERTIFICATION

The Swiss GLP Monitoring Authorities

Swiss Federal A Swiss Agency for the Intercantonal Offie

office of Environment. Forests /IA for the Control of

SPublic Health and Landscape Medicines

Statement of GLP Compliance

It is hereby confirmed that

during the period of August 15 - 17. 2000

the following Test Facilities of RCC Ltd

4452 Itingen

Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for the

Environment, Forests and Landscape and the Intercantonal Office for the Control of

Medicines with respect to the compliance with the Swiss legislation on Good Laboratory

Practice.

Test Facilities areas of expertise*

- Toxicology Division TOX, ACC

- Environmental Chemistry and ACC, ECT, ENIF, PCT,RES,

Pharmanalytics Division 0TH (Animal metabolism)

- Microbiological Diagnostics by 0TH (Microbiology)

Biotechnology & Animal Breeding Division

The inspection was performed in agreement with the OECD Guidelines for National GLP

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

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

the time they were inspected.

Federal Office of Public Health

The Director

Bern, November 2000 Prof. Th. Zeltner

' TOX = Toxicology; ACC - Analytical and Clinical Chemistry; ECT = Environmental toxicity on aquatic and terrestrial

organisms; ENF - Behaviour in water, soil and air. Bloacrumulation; PCT - Physical-chemical testing; RES - Residue

studies; 0TH - Other, to be specified

125
RCC Study Number 843787

TKA 40273 (CGX PR 774):

Primary Skin Irritation Study in Rabbits

(4-Hour Semi-Occlusive Application)

Report

Author: G. Arcelin

Sponsor: Ciba Specialty Chemicals Inc.

CH-4002 Basel /Switzerland

Page 1 of 25

126
RCC STUDY NUMBER 843787 Report

TKA 40273 (CGX PR 774)

Table of contents

1 . PREFACE ............................................................................ ........ 1

1.1 GENERAL .............................................................................. -4

1.2 RESPONSIBILITIES...................................................................... 4

1.3 SCHEDULE .............................................................................. 4

1.4 ARCHIVING............................................................................... 4

1.5 SIGNATURES............................................................................. 5

1.6 QUALITY ASSURANCE UNIT........................................................... 6

1.7 STATEMENT OF COMPLIANCE....................................................... 7

1.8 CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT ............... 8

1.9 ACCREDITATION ........................................................................ 9

1.10 TEST GUIDELINES ...................................................................... 9

1.11 SUMMARY OF STUDY PLAN AMENDMENT ......................................... 9

2. SUMMARY ................................................................................... 10

3. PURPOSE ...................................................................................... 11

4. MATERIALS AND METHODS ................................................................ 11

4.1 TEST SYSTEM.......................................................................... 11

4.2 HUSBANDRY............................................................................ 12

4.3 TESTITEM ............................................................................. 12

4.4 TEST ITEM PREPARATION ........................................................... 13

4.5 TREATMENT ............................................................................ 13

4.6 OBSERVATIONS........................................................................ 13

4.6.1 IRRITATION SCORES ....................................................... 13

5. PATHOLOGY................................................................................. 14

5.1 NECROPSY............................................................................. 14

6. TABULATION AND EVALUATION OF RESULTS .......................................... 14

7. DATA COMPILATION AND STATISTICAL ANALYSIS .................................... 14

8. RESULTS ....................................................................................... 15

8.1 VIABILITY/MORTALITY/CLINICAL SIGNS ........................................... 15

8.2 IRRITATION............................................................................. 15

8.3 COLORATION ........................................................................... 15

8.4 CORROSION .......................................................................... 15 i

8.5 BODY WEIGHTS ..................................................................... 15..i

8.6 CONCLUSION........................................................................... 15F

9. APPENDICES ................................................................................ 16G

9.1 SKIN IRRITATION SCORES ..........................................................

127
RCC STUDY NUMBER 843787 Report Page 3

TKA 40273 (CGX PR 774)

Table of contents (cont' d)

9.2 INDIVIDUAL FINDINGS................................................................. 18

9.3 SUMMARY OF EVALUATION CRITERIA ............................................ 21

9.4 CERTIFICATION ........................................................................ 23

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001 ............... 24

9.4.2 GLP - CERTIFICATION...................................................... 25

128
RCC STUDY NUMBER 843787 Report Page 4

TKA 40273 (CGX PR 774)

1. PREFACE

1.1 GENERAL

Title TKA 40273 (CGX PR 774):

Primary Skin Irritation Study in Rabbits

(4-Hour Semi-Occlusive Application)

Sponsor Ciba Specialty Chemicals Inc.

CH-4002 Basel / Switzerland

Study Monitor Mrs. Isabelle Frei

Test Facility RCC Ltd

Toxicology Division

W61ferstrasse 4

CH-441 4 F~llinsdorf /Switzerland

1.2 RESPONSIBILITIES

Study Director G. Arcelin

Deputy Study Director M. Ott

Technical Coordinator P. Reissbrodt

Head of RCC Quality 1. W0thrich

Assurance

1.3 SCHEDULE

Experimental Starting Date 07-MAY-2002

Experimental Completion Date 1 6-MAY-2002

Delivery of the Animals 07-MAY-2002

Acclimatization 07-MAY-2002 to 1 2-MAY-2002

Treatment 1 3-MAY-2002

Observation Throughout 72 hours after treatment.

Termination 1 6-MAY-2002

Study Completion Date 03-JUL-2002

1.4 ARCHIVING

RCC Ltd (CH-4452 Itingen /Switzerland) will retain the study plan, amendments, raw data, a

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

will be discarded without the Sponsor's consent.

129
RCC STUDY NUMBER 843787 Report Page 5

TKA 40273 (CGX PR 774)

1.5 SIGNATURES

Study Director: G. Arc in

date: )-

Management: S. Corney

d17 

4

130
RCC STUDY NUMBER 843787 Report Page 6

TKA 40273 (CGX PR 774)

1.6 QUALITY ASSURANCE UNIT

RCC Ltd, Toxicology Division, CH-4452 Itingen ISwitzerland

STATEM ENT

ROC STUDY NUMBER : 843787

TEST ITEM TKA 40273 (CGX PR 774)

STUDY DIRECTOR G. Arcelin

TITLE TKA 40273 (CGX PR 774):

Primary Skin Irritation Study in Rabbits

(4-Hour Semi-Occlusive Application)

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

the responsible person and the management.

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

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

Unit. The dates are given below.

Dates and Types of Dates of Reports to the Study Director

QAU Inspections and to Management

06-MAY-2002 Study Plan 06-MAY-2002

07-MAY-2002 Process Based 07-MAY-2002

1 2-JUN-2002 Report 1 2-JUN-2002

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

Quality Assurance: G ~~

date: o I-ZIUL -26r,2

131
RCC STUDY NUMBER 843787 Report Page 7

TKA 40273 (CGX PR 774)

GOOD LABORATORY PRACTICE

1.7 STATEMENT OF COMPLIANCE

ROC STUDY NUMBER: 843787

TEST ITEM : TKA 40273 (CGX PR 774)

STUDY DIRECTOR : G. Arcelin

TITLE : TKA 40273 (CGX PR 774):

Primary Skin Irritation Study in Rabbits

(4-Hour Semi-Occlusive Application)

The pH measurement of the test item was performed before the study initiation date and is,

therefore, also excluded from this statement.

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

Laboratory Practice, adopted February 2nd, 2000 [RS 813.01 6.5]. This Ordinance is based

on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted

November 26th, 1997 by decision of the OECD Council [C(97)186/Final].

These procedures are consistent with Good Laboratory Practice regulations specified by

regulatory authorities throughout the European Community, the United States (EPA and

FDA), and Japan (MHLW, MAFF and METI).

Study Director: G. Ar elin

132
ROC STUDY NUMBER 843787 Report Page 8

TKA 40273 (CGX PR 774)

1.8 CERTIFICATION OF GLP

AND VERIFICATION OF THE REPORT

The statemnent of Compliance with Good Laboratory Practice found in this report, and signed

by the Study Director is truthful and accurate, and this report as provided by the test facility

is complete and unaltered.

Signature of the Sponsor: .......................

Date:

133
ROC STUDY NUMBER 843787 Report Page 9

TKA 40273 (CGX PR 774)

1.9 ACCREDITATION

The test facility "ROC Ltd, Toxicology Division" is accredited according to EN 45001 under

accreditation number STS 085 by the Swiss Accreditation Service.

1.10 TEST GUIDELINES

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

following guidelines:

Directive 92/69 EEC, B3.4. "Acute Toxicity - Skin Irritation", July 31, 1992.

OECD Guidelines for Testing of Chemicals, Section 4, number 404 "lAcute Dermal IrritationI

Corrosion", adopted July 17, 1992.

1.11 SUMMARY OF STUDY PLAN AMENDMENT

Study Plan Amendment No. 1:

No signs of irritation were observed on the treated skin following the application of the test

item. For ethical reasons and in the interest of animal welfare, the animals were therefore reused for the eye irritation study (ROC Study number 843788) using the same test item.

Section 4.1 was amended accordingly.

Study Plan Amendment No. 2:

Reference to the analytical certificate was added.

134
RCC STUDY NUMBER 843787 Report Page 10

TKA 40273 (CGX PR 774)

2. SUMMARY

The primary skin irritation potential of TKA 40273 (CGX PR 774) was investigated by topical

semi-occlusive application of 0.5 g to the intact left flank of each of three young adult New

Zealand White rabbits. The duration of treatment was four hours. The scoring of skin

reactions was performed 1, 24, 48 and 72 hours after removal of the dressing.

The test item did not elicit any skin reactions at the application site of any animal at any of

the observation times (all scores 0). The individual mean score for erythemaleschar and

oedema for each of the three animals was therefore 0. The test item caused no staining of

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

the measuring intervals.

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

TKA 40273 (CGX PR 774) is considered to be "not irritating" to rabbit skin.

135
ROC STUDY NUMBER 843787 Report Page 11

TKA 40273 (CGX PR 774)

3. PURPOSE

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

when a single dose of TKA 40273 (CGX PR 774) was placed on the skin of rabbits for

approximately four hours.

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

of the probable routes of human exposure.

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

solid test item.

4. MATERIALS AND METHODS

4.1 TEST SYSTEM

Test system New Zealand White Rabbit, SPF

Rationale Recognized by the international guidelines as the

recommended test system

Source Elevage Scientifique des Dombes

F-01 400 Chatillon sur Chalaronne / France

Number of animals per test 3 (Animals of both sexes were used)

Age at treatment 15 weeks (male)

10-11 weeks (females)

Identification By unique cage number and corresponding ear number.

Acclimatization Under laboratory conditions after health examination. Only

animals without any visual signs of illness were used for the

study.

Allocation Male No. 25

Female Nos. 26 and 27

136
7 7ROC STUDY NUMBER 843787 Report Page 12 IKA 40273 (CGX PR 774)

4.2 HUSBANDRY

Room no. 106 / RCC Ltd, F~illinsdorf

Conditions Standard Laboratory Conditions

Air-conditioned with target ranges for room temperature

17-23 00, relative humidity 30-70 % and approximately

10-15 air changes per hour. Room temperature and humidity

were monitored continuously and values outside of these

ranges may have occasionally occurred, usually following

room cleaning. These transient variations are considered not

to have any influence on the study and, therefore, these data

are not reported but are retained at ROC. The animals were

provided with an automatically controlled light cycle of

12 hours light and 12 hours dark. Music was played during

the daytime light period.

Accommodation Individually in stainless steel cages equipped with feed

hoppers and drinking water bowls. Wood blocks (RCC Ltd,

Fi~llinsdorf) and haysticks (batch no. 0203, Provimi Kliba AG)

were provided for gnawing.

Diet Pelleted standard Provimi Klba 3418 rabbit maintenance

diet ad libitum (batch no. 25/02) provided by Provimi Kliba.

AG, CH-4303 Kaiseraugst. Results of analysis are archived

at RCC Ltd, Itingen.

Water Community tap water from F~llinsdorf, ad libitum. Results of

bacteriological, chemical and contaminant analyses are

archived at RCC Ltd, Itingen.

4.3 TEST ITEM

The following information was provided by the sponsor:

Identification TKA 40273

Product Name CGX PR 774

Description White powder

Batch number 836-187

Purity See Analytical Certificate Study No. 14022921, Ciba

Specialty Chemicals Inc.

Stability of test item Stable under storage conditions

Expiry date 30-APR-2004

Storage conditions In the original container, at room temperature

(range of 17-23 'C), away from direct sunlight.

137
RCC STUDY NUMBER 843787 Report Page 13

TKA 40273 (CGX PR 774)

4.4 TEST ITEM PREPARATION

0.5 g (per animal) of TKA 40273 (CGX PR 774) was weighed as delivered by the sponsor

and then moistened with approximately 0.1 ml of purified water before application.

According to Directive 92/69 EEC, B.4. and OECD Guidelines 404, a test item needs not to

be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable

corrosive properties.

The pH of the test item was measured before the study initiation date. A formulation of 1 %

in water was prepared. The pH was found to be 5 to 6.

4.5 TREATMENT

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

of approximately 100 cm2 (10 cm x 10 cm). The skin of the animals was examined one day

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

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

interpretation of the results were not used in the test.

On the day of treatment, 0.5 g of TKA 40273 (CGX PR 774) was placed on a surgical gauze

patch (ca. 4 cm x 4 cm). This gauze patch was applied to the intact skin of the clipped area.

The patch was covered with a semi-occlusive dressing. The dressing was wrapped around

the abdomen and anchored with tape.

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

flushed with lukewarm tap water to clean the application site so that any reactions

(erythema) were clearly visible at that time.

4.6 OBSERVATIONS

Viability/Mortality Daily from delivery of the animals to the termination of test.

Clinical signs Daily from delivery of the animals to the termination of test.

Body weights At start of acclimatization, on the day of application and at

termination of observation.

4.6.1 IRRITATION SCORES

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

Commission Directive 92/69/EEC, July 31, 1992 approximately 1, 24, 48 and 72 hours after

the removal of the dressing, gauze patch and test item.

138
RCC STUDY NUMBER 843787 Report Page 14

TKA 40273 (CGX PR 774)

5. PATHOLOGY

5.1 NECROPSY

The animals were not sacrificed. On completion of the observation period all animals were

re-allocated to the eye irritation study (RCC study number 843788).

6. TABULATION AND EVALUATION OF RESULTS

Data are summarized in tabular form, showing the irritation scores for erythema and oedema

for each individual animal at all observation intervals after patch removal, any lesions, a

description of the degree and nature of irritation, corrosion or reversibility, and any other

toxic effects observed.

To evaluate the irritation of the test item the mean score was calculated across 3 scoring

times (24, 48 and 72 hours after patch removal) for each animal for erythemaleschar grades

and for oedema grades, separately. An animal is positive when the mean score is 2 or

greater. The test is positive for irritation when at least 2 animals are positive for the same

endpoint (erythema/eschar or oedema).

7. DATA COMPILATION AND STATISTICAL ANALYSIS

Viability/mortality, clinical signs and dermal findings were recorded on data sheets and

transcribed for compilation and analysis.

Body weights were recorded on-line.

No statistical analysis was performed.

139
RCC STUDY NUMBER 843787 Report Page 15

TKA 40273 (CGX PR 774)

8. RESULTS

8.1 VIABILITY/MORTALITY/CLINICAL SIGNS

No clinical signs of systemic toxicity were observed in the animals during the study and no

mortality occurred.

8.2 IRRITATION

The test item did not elicit any skin reactions at the application site of any animal at any of

the observation times (all scores 0). The individual mean score for erythemaleschar and

oedema for each of the three animals was therefore 0.

8.3 COLORATION

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

8.4 CORROSION

No irreversible alterations of the treated skin were observed nor were corrosive effects

evident on the skin.

8.5 BODY WEIGHTS

Animal number 25 showed a slight body weight loss (3.6%) during the acclimatization period.

The reason for this weight loss could not be established and it is considered to be incidental.

The body weights of all other rabbits were considered to be within the normal range of

variability.

Body weight in grams

Animal Sex First Day of Day of Last Day of

No. mal Acclimatization Treatment Observation

25 male__ 2386 2301 2479

26 female 2067 2299 2399

27 female 1966 2088 2l11

8.6 CONCLUSION

TKA 40273 (CGX PR 774) is considered to be "not irritating" to rabbit skin.

140
RCC STUDY NUMBER 843787 Report Page 16

TKA 40273 (CGX PR 774)

9. APPENDICES

9.1 SKIN IRRITATION SCORES

Key to Symbols:

M Male

F Female

141
ROC STUDY NUMBER 843787 Report Page 17

TKA 40273 (CGX PR 774)

TABLE 1: SKIN IRRITATION SCORES - INDIVIDUAL VALUES

Animal Evaluation

Number Sex Interval * Erythemna Oedema

25 M 0 0

26 F 1ihour 0 0

27 F 0 0

25 M 0 0

26 F 24 hours 0 0

27 F 0 0

25 M 0 0

26 F 48 hours 0 0

27 F 0 0

25 M 0 0

26 F 72 hours 0 0

27 F ______ 0 0

*Examinations were performed at the specified intervals after removal of the dressing.

TABLE 2: SKIN IRRITATION SCORES - INDIVIDUAL MEAN VALUES

AFTER 24,48 AND 72 HOURS

Animal

Number Sex Erythema N Oedema N

25 M 0.00 3 0.00 3

26 F 0.00 3 0.00 3

27 F 0.00 3 ,0.00 3

N=number of available data points

TABLE 3: SKIN IRRITATION SCORES - ASSESSMENT ACCORDING

TO EEC GUIDELINES

Evaluated

intervals Erythema Oedema

24 hours

48 hours Not Irritating Not Irritating

72 hours

The grading scale is presented on page 22.

142
RCC STUDY NUMBER 843787 Report Page 18

TKA 40273 (CGX PR 774)

9.2 INDIVIDUAL FINDINGS

ANIMAL NO. 25, MALE

After 1 hour: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 24 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 48 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 72 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

143
ROC STUDY NUMBER 843787 Report Page 19

TKA 40273 (CGX PR 774)

INDIVIDUAL FINDINGS

ANIMAL NO. 26, FEMALE

After 1 hour: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedemna: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 24 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedemna: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 48 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 72 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedemna: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

144
ROC STUDY NUMBER 843787 Report Page 20

TKA 40273 (CGX PR 774)

INDIVIDUAL FINDINGS

ANIMAL NO. 27, FEMALE

After 1 hour: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 24 hours: Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 48 hours: Erythema: NO ABNORMAL FINDINGS NOTED

Oedema: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

After 72 hours: Erythemna: NO ABNORMAL FINDINGS NOTED

Oedemna: NO ABNORMAL FINDINGS NOTED

Flaking: NO ABNORMAL FINDINGS NOTED

Staining: NO ABNORMAL FINDINGS NOTED

145
RCC STUDY NUMBER 843787 Report Page 21

TKA 40273 (CGX PR 774)

9.3 SUMMARY OF EVALUATION CRITERIA

*EEC Commission Directive 92169/EEC, July 31, 1992

146
ROC STUDY NUMBER 843787 Report Page 22

TKA 40273 (CGX PR 774)

EEC COMMISSION DIRECTIVE 92/69/EEC, JULY 31, 1992

Grading of Skin Reactions

ERYTHEMA AND ESCHAR FORMATION

No erythema .................................................................................... 0

Very slight erythema............................................................................. 1

Well-defined erythema.......................................................................... 2

Moderate to severe erythema .................................................................. 3

Severe erythema (beet redness) or eschar formation (injuries in depth preventing erythema)

reading ........................................................................................... 4

OEDEMA FORMATION

No oedema..................................................................................... 0

Very slight oedema (barely perceptible)........................................................ 1

Slight oedema (edges of area well-defined by definite raising)............................... 2

Moderate oedema (edges raised approximately 1 mm)....................................... 3

Severe oedema (raised more than 1 mm and extending beyond the area of exposure) .... 4

147
ROC STUDY NUMBER 843787 Report Page 23

TKA 40273 (CGX PR 774)

9.4 CERTIFICATION

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

9.4.2 GLP - CERTIFICATION

148
RCC STUDY NUMBER 843787 Report Page 24

TKA 40273 (CGX PR 774)

9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

S Eidgen~tsischas Amt frjr Messweaen S Schweizerische Akktreditisrungsalle

Office fediral do metrologie Service d'accrdditation suisse

Ufficio federalse di metrologia ~ Q J A Servizia d'accreditamento sviuzero

Swiss Federal office of metrology N2Zr S Swisa Accreditation Service

ACCREDITATION

Based on the Accreditation and Designation Ordinance

dated 17" June 1996

the Swiss Federal Office of Metrology grants the

RCC Ltd

Toxicology Division

Zeigliweg 1

4452 Itingen

accreditation as a

Testing Laboratory for toxicological investigation of

pharmaceuticals and medical devices, agrochemicals, industrial

chemicals, food- and feed-additives

in accordance with SN EN 45001.

The accredited scope of testing is defined in the Official

Directory of the Accredited Testing Laboratories in Switzerland.

The relevant requirements of the ISO 9002 standard

are also covered by this accreditation.

Accreditation number: STS 085

Date of the accreditation: lew September 1994

Date of the last renewal of the accreditation: 20" September 1999

The accreditation is valid until: 19'b September 2004

Wabern, 2et September 1999 Swiss F derI Office of Metrology

The Dico The heIof SAS

Dr Wolfgang Schwitz Hanspeter lschi

SAS is a s~natory oftw i. mtala agrememnts oft ieEiopean cocopeauon for A~oduon (EA)

lor cwaian, Isetfi mi coi(cation of peftos. permo".l quaky wkd envinmontal mnaamnt systs.

149
RCC STUDY NUMBER 843787 Report Page 25

IKA 40273 (CGX PR 774)

9.4.2 GLP - CERTIFICATION

The Swiss GLP Monitoring Authorities

SisFederal Swias Agency for the Intercantonal Office

Ofic4o Environment. Forests for the Control of

PbiHelhand Landscape Medicines

Statement of GLP Compliance

It is hereby confirmed that

during the period of August 15 - 17, 2000

the following Test Facilities of RCC Ltd

4452 Itingen

Switzerland

were inspected by the Federal Office of Public Health, the Swiss Agency for the

Environment, Forests and Landscape and the Intercantonal Office for the Control of

Medicines with respect to the compliance with the Swiss legislation on Good Laboratory

Practice.

Test Facilities areas of expertise*

- Toxicology Division TOX, ACC

- Environmental Chemistry and ACC, ECT, ENF, PCT,RES,

Pharmanalytics Division 0TH (Animal metabolism)

- Microbiological Diagnostics by 0TH (Microbiology)

Biotechnology & Animal Breeding Division

The inspection was performed in agreement with the OECD Guidelines for National GLP

inspections and Audits. It was found that the aforementioned test facilities were operating in

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

the time they were inspected.

Federal Office of Public Health

The Director

Bern, November 2000 Prof. Th. Zeitner

* TOX - Toxicology; ACC -Analytical and Clinical Chemistry, ECT =Envirornental toxicity on aquatic and terrestnial

organisms :ENF - Behaviour in water, sol and air. Bioaccrsnslation PCT - Physical-chemical testing , RES - Residue

studies ;0TH - Other, to be specified

150