# TSCA NON-CONFID)ENTIAL BUSINESS INFORMATION DOCUMENT DESCRIPTION DOCUMENT CONTROL NUMBER DATE RECEIVED 

### p- 

# 17-- 3 151D200060 / , 62 

### COMMENTS: 

# DEC LASS

DOUS NOT CONTAIN CBIUNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 20460
OFFICE OF &F( CHEMICAL SAFETY AND POLLUTION PREVENTION 

## Notice ofChange inCBI 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 forthe above referenced substance was dropped bythe information submitter inthe NOC. The identified non-confidential chemical name forthis substance as provided in the NOC is: I,5-DIOXA-9-AZASPIRO[5.5]UNDECANE, 3,3,8,8, 10,10-HEXAMETHYL-9-[1

## -14- (OXIRANYLMETHOXY)PHENYL]ETHOXY]- The current TSCA Inventory name is 1,

5-Dioxa-9-azaspiro[5.5]undecane, 3,3,8,8,10,10-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 ofstaffer: 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 ofreceipt 

### PREMANUFACTURE ~f- 

### K 

# L NOTICE 

### 2 FORUNW CHEMICL SUBSTANCE 

### CompanySanitized

OCUNEN CONTROL OFFICERCE OFFICE OFPOLLUTION PREVENTION
U.S. E.P.A. 401 M STREET, SW WASHINGTON, D.C. 20460 nte. etoanubropa s Document control number EP asenumber, in the Premanufacture Notice 150 j 1X A)f?32 GENERAL INSTRUCTIONS T * Youmust provide allinformation requested in this form to the extent that it is known to orreasonably ascertainable byyou. Make reasonable estimates ifyou do nothave actual

data. * Before you complete this form you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manualis available fromthe Toxic Substances

Control Act (TSCA) Information Service bycalling 202-554-1404, or faxing 202-554-5603). * Ifa user fee has been remitted for this notice (40 CFR700.45), indicate in the boxes above the TS-user fee identification number you have generated. Remember, your user fee

ID numbermust also appear on your corresponding fee remittance, which is sent to EPA, HQAccounting Operations Branch (PM-226), P.O. 360399M, Pittsburgh, PA 15251- 6399, Attn. TSCA User fee. 

## SANITIZED
PaitI- GENERAL INFORMATION ^

## OPy TESTDATA AND OTHER DATA You must provide thecurrently correct Chemical Abstracts (CA) Name of the new chemical You arerequired tosubmit all testdata inyourposseassion or control and toprovide a substance, even ifyouclaim the identity as confidential. Youmay authorize another person to description ofall other dataknown to or reasonably ascertainable byyou, ifthese dataare submit chemicalidentity information foryou, but yosr submission will notbe complete and the related to the health and envirounmenta effects on the manufacture, processing, distribution in review willnotbegin until EPA receives this information. Aletter in support ofyour submiassion commerce, use, or disposal ofthe new chemical substance. Standard literature citations may be should reference your TS user feeidentification number. Youmust submit anoriginal and two submitted for data in theopen scientific literature. Complete teatdata (written inEnglish).not copies of this notice including all testdata. If you claimed any information as confidential, a sunmmaries ofdata. mustbesubmittediftheydo notapnarin theolen liteatr. You should single saniticed copymust also be submitted, clearly identify whether testdata ison the substance oron an analog. Also, thechemical composition ofthetested masterial should becharacterized. Following areexamples ofteat data and other data. Data shouldbe submitted according to therequirements of§720.50 ofthe Part II - HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Premanufacmure Notification Rule(40 CFR Part 720). Ifthere are several manufacture, processing, uruse operations to bedescribed is Part It,sections A and Bof this notice, reproduce the sections as needed.
Part Ill- LIST OF ATTACHMENTS Test Data (Check Below any included in this notice) Attach additional sheets ifthere isnot enough space to answersa question fully. Label each * Environmental fate data 0 Yes *Other data 0 Yes continuation sheet with the corresponding section heading. InPart Ill, listthese attachments, any test dataor otherdata and any optional information included in thenotice. * 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 thepossession or control of the submitter You may include any information thatyouwant EPA toconsider in evaluating the new substance. Onpage I1I of this form, space hasbeen provided for you todescribed pollution *A physical and chemical properties worksheet islocated on thelast page ofthis form. prevention andrecycling information youmay haveregarding thenew substance. TYPE OF NOTICE (Check OnlyOne) So-called 'binding" boxes are included throughout thisform foryou to indicate your willingness to be bound to certain statements you make inthis section. such as use, production volume,PN(reaucteNoi) protective equipment... This option isintended toreduce delays thatrutinely accompany the M (PeauctrNoi) development ofconsent orders or Significant New Use Rules. Except inthe case ofexemption applications (such as TMEA, LVE, LOREX) wherecertain information provided insuch UINTERMEDIATE PMN (submitted in sequence with final product PMN) notification is binding on thesubmitter when the Agency approves theexemption applicstion, checking abinding boxin this notice doesnot by itself prohibit the submitter from later deviating from the information (except chemical identity) reported in the form.U SNUN (Significant NewUse Notice)
CONFIDENTIALITY CLAIMS 

## ED TMEA (Test Marketing Exemption Application) C= You may claim any information inthis notice as confidential. To assert aclas J LVE (Low Volume Exemption) @40 CFR723.50(c)(1) C,. mark (X) the confidential box next to theinfurmation thatyou clam 1 0 IIA Ed claim in an attachment, circle orbracket the information you claim a~brfifilniayt yIfyoi~s~n ~ , information inthe noticesas confidential,von must also providea sanitized1 sE'P y LOREX (Low Release/Low Exposure Exemption) (40.C 723 cg notice. (includingattachments). For additional instructions on claiming sntormuf Was 

# 49 confidential, read theInstructions Manual. 

## D V oiiainLRXMfcto~ i

tLf\i Mark(x) ifany information inthis notice isclaimed 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 ofEPA Form 7710-25.Public reporting burden for this collection ofinformation is estimated to average 110 hours perresponse, including time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing thecollection of information. Send comments regarding the burden estimate or any other aspect ofthis collection ofinformation, 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 tothe Office of Management and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. CERTIFICATION I certify that to the best ofmyknowledge and belief: I. The company named in Part 1,section A, subsection I a ofthis notice form intends to manufacture or import for acommercial 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 ofthe date ofsubmission. 3. 1am submitting with this notice all test data inmypossession or control and adescription ofall other data known to or reasonably ascertainable by me asrequired by §720.50 ofthe Premanufacture Notification Rule. Additional Certification Statements: Ifyou are submitting a PMN, Intermediate PMN, Consolidated PMN, or SNUN, check the following userfee certification statement that applies:



## ZThe Company named inPart 1,Section A has remitted thefee of$2500 specified in40 CFR 700.45(b), or 

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

## LIThe Company named inPart I Section A is a small business concern under 40CFR 700.43 and has remitted a fee of$100 in accordance

with 40CFR 700.45(b), Ifyou are submitting a low volume exemption (LYE) application in accordance with 40 CFR 723.50(c)(1) ora Low release and low exposure exemption (LoRex) application in accordance with 40 CFR 723.50(c)(2), check thefollowing certification statements:



## LIThe manufacturer submidtting this notice intends to manufacture or import the new chemical substance forcommercial purposes, other

than in small quantities solely for research and development, under the terms of40 CFR 723.50. 

## l The manufacturer is familiar with the terms ofthis 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), themanufacturer intends to commence manufacture ofthe

exempted substance for commercial purposes within 1year ofthe date oftheexpiration ofthe 30 dayreview period. The accuracy ofthe statements you make inthis notice should reflect your best prediction of theanticipated facts regardingthe chemical subatance described herein. Any knowing andwillful misinterpretationIssubject to criminal penalty pursuantto 18 Usc 1001. Confidential Signature and titleiofAuthorized Offic(or ISigns Requird) /Date

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

____________ ______ Signature ofagent - (ifapplicable) Date

EPA FORM 7710-25 (Rev. 5-95) Page 2PartI -- GENERAL INFORMATION Section A - SUBMITTER IDENTIFICATION ConfiMark 0)the "Confidential" box next to any subsection you claim as confidential dential Ia. Person Name ofauthorized official Position Submitting Notice (inU.S.) Naeemn H. Madv Head, Regulatory Affairs Company

b.Agent (if aeo authoized officia l Positionio applicable)(umeran sret

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

b.ointmt (if Name of authorized official Position applicable)

ContactCinmU.S.

3. If ou hae bad a ipre ng commnictio(PC)becocnn thisnoicean EAasgeaPCimrto Ste nIotieenteron thea

number.be

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

the chemical. EPA.t Ifyoupevioul sumte aPMNzd fritissanc enteriheiPM ~~~~Malnadrs(number assine byEAsietwtdawrrinop et) **ifn e 5. f ouhae ub itted a te,f P B os deintenton toe

manfacur oruimpor fThech eical susace covee byuthois otficel enertesotctnmbrMoknX

6.hoa Typee ofacer

Noticet

- Mrk(nMnuacura2Imor 

## Elb Bindlt Ce ing COp~tion 

## l BnigOto Markin (ddes Markbe ansret
EPAytwn FOR 779-90

10-2 785. 5954Pae1PartI -- GENERAL INFORMATION -- Continued Section B -- CHEMICALIDENTITY INFORMATION: You must provide acurrently correct Chemical Abstracts (CA) name ofthe substancebased on Itheninth Collective Index (9C1) ofCA nomrenclature rules and conventions. Mark (X) the "Confidential" box next to any item you claim as confidential Complete either item 1(Class 1or 2 substances) or 2 (Polymers) as appropriate. Complete all other items. Ifanother person will submit chemiical identity information foryou (foreither Item Ior 2), mark (X) thebox at theright. ConfiIdentify the name, company, andaddress ofthatperson in a continuation sheet. C dential I.Class Ior 2 chemical substances (for definitions ofclass 1and 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 Isubstances a CA Index Name must beprovided. For Class 2 substances either a CA Index Name or CA Preferred Name must x be provided, whichever is appropriate based on CA 9CInomenclature rules and conventions).

C. Please identify which method you used to develop orobtaln thespecified chemical identity information reported in this notice: (check one). Method I(CAS Inventory Expert Service
- a copy ofthe Identification 

## LI Method 2 (Other Source) report obtained from the CAS Inventory Expert Services must be submitted as anattachment tothis notice)

e. For a class I substance, provide a complete and correct chemical structure diagram. For aclass 2 substance
- (1) Listthe immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the nature ofthe reaction orprocess. (3) Indicate the X range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative orpartial chemical structure diagram, as complete as can beknown, ifone can be reasonably ascertained.

ID Mark (X) this box ifyou attach acontinuation sheet EPA FORM 7710-25 (Rev. 5-95) Page 4PartI -- GENERAL INFORMATION -- Continued Section B -- CHEMICAL IDENTITY INFORMATION --Continued 2. Polymers (For adefinition ofpolymer, see the Instructions Manual.) Confidential a. Indicate the number-average weight ofthe lowest molecular weight composition of the polymer you intend to manufacture. Indicate maximum weight percent oflow molecular weight species (not including residual monomers, reactants, or solvents) below 500 and below 1,000 absolute molecular weight ofthat composition. Describe the methods ofmeasurement orthe basis foryour estimates: GPC

## I Other 

## II]: (Specify) i) lowestnumber average molecular weight: ii) maximum weight % below 500 molecular weight: N/A iii) maximum weight % below 1000molecular weight: 

## El Mark (X)this box ifyou attach acontinuation sheet. b You must make separate confidentiality claims for monomer or other reactant identity, composition information, and residual information. Mark (X) the

"Confidential" boxnext to any itemyou claim as confidential (1)

- Providethe specific chemical name and CAS Registiy Number (ifanumber exists) ofeach monomer or other reactant used in the manufacture ofthe polymer. (2)

- Mark (X) this column ifentry in column (1)is confidential. (3)

- Indicate the typical weight percent of each monomer or other reactant inthe polymer. (4)

- Mark (X the identity column ifyou wanta monomer or other reactant used attwo weight percent orless tobe listed as part ofthepolymer description on the TSCA Chemical Substance Inventory. (5)

- Mark (X) this columnifentries in columns (3)and (4)are confidential. (6)

- Indicate the maximum weight percent ofeach monomer or otherreactant that may be present as aresidual in thepolymer as manufactured for commercial purposes. (7)

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

______ Monomer or otherreactant 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 ifyou attach a continuation sheet.

C. Please identify which method you used to develop orobtain the specified chemical identity information reported inthis notice (check one). 

## LIMethod 1 (CAS Inventory Expert Service - a copy ofthe 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 forthe 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 beknown, ifone can be reasonably ascertaijled.

Not Applicable



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

-- GENERAL INFORMATION -- Continued Section B -- CHEMICAL IDENTITY INFORMATION -- Continued 3. Impurities (a)

- Identify each impurity thatmay bereasonably anticipated tobepresent in the chemical substance as manufactured for commercial purpose. Provide the CAS Registry Number ifavailable. Ifthere are unidentified impurities, enter "unidentified." (b)

- Estimate the maximum weight %ofeach impurity. Ifthere areunidentified impurities, estimate their total weight %.

______ ____ Impurity and CAS Registry Number Maximum Confipercent dential (a) (b)

_____

## x x x



## D Mark (X) this box ifyou attach acontinuation sheet. 4. Synonyms -Enter any chemical synonyms for the new chemical identified in subsection 1or 2. Confidential



## x 

## LI Mark (X) this box ifyou attach acontinuation sheet. 5. Trade identification -List trade names forthe new chemical substance identified in subsection Ior 2. CGX PR 774 

### F-1 Mark (X) this box ifyou attach acontinuation sheet. 6. Generic chemical name - Ifyou claim chemical identify as confidential, you must provide a generic name foryour substance that reveals the specific chemical identity ofthe new chemical substance to the maximum extent possible. Referto the TSCA Chemical Substance Inventory, 1985 Edition, Appendix B for guidance on developing generic names._____ Aromatic ether derivative 

## D Mark (X) this box ifyou attach acontinuation sheet. 7. Byproducts - Describe anybyproducts resulting from the manufacture, processing, use, or disposal ofthe new chemical substance. Provide the CAS Registry Numberif available._____ Byproduct CAS Registry Number Confi- (1) (2) dential None expected



## EI Mark (X) this box ifyou attach acontinuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 6PartI -- GENERAL INFORMATION -- Continued Section C -- PRODUCTION, IMPORT,AND USE INFORMATION: Mark (X) the "Confidential" box next to any item you claim as confidential. 1. Production volume -- Estimate the maximum production volume during the first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during thefirst three years ofproduction. Estimates should be on 100%new chemical substance basis. For aLow Volume Exemption application, ifyou choose to have your notice reviewed at a lower production volume than 10,000 kg/yr. specify the volume and mark (x) inthe binding box. Ifgranted,_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

__________________________________________ ____________________________________ Mark ar((x

2. Use Informnation -- You must make separate confidentiality claims for the description ofthe category ofuse, the percent ofproduction volume devoted to each category, the formulation of the new substance, and other useinformation. Mark (X)the "Confidential" Box next to any item youclaim asconfidential. a. (1) -- Describe each intended category ofuseofthe new chemical substance by function and application.. (2)-- Mark (X) this column ifentry column (1)is confidential business information (CBI). (3) -- Indicate your willingness to have the information provided in column (1)binding. (4) -- Estimate thepercent oftotal production for the first three years devoted to each category ofuse. (5)-- Mark (X) this column ifentry in column (4) is confidential business information (CB1). (6) -- Estimate thepercent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, or gels as manufactured for commercial purposes at sites under your control associated with each category ofuse. (7) -- Mark (X) this column ifentry in column (6) is confidential business infonmation (CBI). (8) -- Indicate %ofproduct volume expected for the listed "use" sectors. Mark more than one box ifappropriate. Mark (X) to indicate your willingness to have the use type providedin (8) binding. (9)-- Mark (X) this column ifentry(ies) in column (8) is (are) confidential business information (CR1). Category ofuse (1) CR1 Binding Produc- CRI %in CBI %of substance expected peruse 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% forthermoplastics and elastomers

___ ______

*If you have identified a "consumner" use, please provide on acontinuation sheet a detailed description ofthe use(s) ofthis chemical substance in consumerproducts. In addition include estimates oftheconcentration ofthe new chemical substance asexpected inconsumer products and describe the chemical reactions by which this substance loses its identityin the consumer product. 

# EMark(X) thisboxif you attach a continuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2a as confidential, enter a generic description ofthat category. Read the use Instructions Manual for examples ofgeneric usedescriptions. description


# LIMark (X) this box ifyou attach a continuation sheet. 3. Hazard Information -- Include inthe notice acopy ofreasonable facsimile ofany hazard warning statement, label, material safety data sheet, orother Binding Option information which will be provided to any person who is reasonably likely tobeexposed to this substance regarding protective equipment or practices Mark (x) for the safe handing,transport, use, or disposal ofthe new substance. List inpart Ill hazard information you include. 

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

EPA FORM 7710-25 (Rev. 5-95) Page 7Part11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Section A -- INDUSTRIAL SITES CONTROLLED BY THESUBMITTER Mark (X)the "Confidential" box next to anyitem you Iclaim as confidential Complete section A for each type ofmanufacture, processing, oruse operation involving the new chemical substance at industrial sites you control. Importers do nothave to complete this section for operations outside the U.S.; however, you may still have reporting requirements ifthere are furtherindustrial processing or useoperations after import. You must describe these operations. See instructions manual 1. Operation description Confi- a. Identity -- Enter the identity ofthe site atwhich 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, ZIPcode
If the same operation willoccur at more than one site, enter thenumber of sites. Identify the additional sites on a continuation sheet, and ifany ofthe sites have significantly different production rates or operations, include all the information requested in this section for those sites as attachments.



# D: Mark (X) thisbox ifyou attach acontinuation sheet. b. Type-- Mark (X) 

# [D Manufacturing Processing Use C. Amount and Duration

-- Complete 1 or 2as 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 yourprocess description binding. (1) Diagram the majorunit operation steps andchemical conversions. Include interim storage and transport containers (specify- e.g. 5gallon pails, 55 gallon drum, rail car, tank truck, etc.). (2) Provide the identity, the approximate weight (by kg/day orkg/batch on a 100%new chemical substance basis), and entry pointof all starting materials and feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, andwastes. Include cleaning chemicals (note frequency ifnot used daily orper batch.). (3) Identifyby number the points ofrelease, including small or intermittent releases, to the environment of thenew chemical substance.

Not applicable



# DMark (X)this box ifyou attach acontinuation sheet.

EPA FORM 7710-25 (Rev. 5-95) Page 8PartH-- 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 ofworker activity, physical formof the newchemical substance, number of works exposed, and duration ofactivity. Mark (X) the "Confidential" box next to anyitem 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 ifentryin column (1)is confidential business information (CBI). (3)

-- Describe anyprotective equipment and engineering controls used to protect workers. (4) and (6) --Indicate yourwillingness to have the information provided in column (3)or (5)binding. (5)

-- Indicate the physical form(s)ofthe newchemical substance (e.g., solid: crystal, granule, powder, or dust) and %new chemical substance (ifpart ofa mixture) atthe time ofexposure. (7)

-- Mark (X)this column ifentry in column (5) isconfidential business information (CBI). (8)

-- Estimate themaximum number ofworkers involved in each activity forall sites combined. (9)

-- Mark (X) this column ifentry in column (8) isconfidential businessinformation (CBI). (10) and (11) -- Estimate the maximum duration ofthe activity forany worker in hoursper day and daysper year. (12)

--Mark (X) this column ifentries 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 ifyou attach acontinuation sheet. 3. Environmental Release and Disposal -- You must make separate confidentiality claims forthe release number and the amountof the newchemical substance released and otherrelease and disposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. (1)

-- Enter the number ofeach release point identified in theprocess description, part 11, section A,subsection ld(3). (2)

-- Estimate the amount ofthe new substance released (a) directly to the environment or (b)into control technology (in kg/day orkg/batch). (3)

-- Mark (X) this column ifentries in columns (1)and (2) are confidential business information (CBI). (4)

-- Identify the media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site or off-site land or incineration, POTW, orother (specify)) towhich thenew substance willbereleased fromthatrelease point. (5)

-- a. Describe control technology, ifany, andcontrol efficiencythat willbe used to limit the release ofthe new substance to the environment. Forreleases disposed of onland, characterize the disposal method and state whether itis approved for disposal ofRCRA hazardous waste. On acontinuation sheet, for each sitedescribe any additional disposal methodsthat 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 columnifentries incolumns (4) and (5) are confidential business information (CBI). (7)

-- Identify the destination(s) ofreleases to water. Please supply NPDES (National Pollutant Discharge Elimination System) numbers fordirect discharges orNPDES numbers ofthe POTW (Publicly OwniedTretatment Works). Mark (X) ifthe POTW name orNPDES #is confidential business information (CRI). Release Amount ofnew 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 providename(s) below: CBI 

# l Navigable Other - Specify provide NPDES # CBI destination(s) of waterway releases to water.



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

EPA FORM 77 10-25 (Rev. 5-95) Page 9Part11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE -- Continued Section B-- INDUSTRIAL SITES CONTROLLED BY OTHERS Complete section Bfor typicalprocessing or use operations involving the new chemical substance at sitesyou donotcontrol. Importers do nothave to complete this section for operations outside theU.S.; however, you must report any processing oruse activities afterimport. Seethe Instructions Manual. CompleteaseparatesectionBforeachtype of processi.ng,oruseoperationinvolvingthenew chemicalsubstance. Ifthe same operation isperformed atmore than one site describe the typical operation common to these sites. Identif additional sites ona continuation sheet. 1. Operation Description --Toclalm information in this section as confidential, circle orbracket 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. 5gallon pails, 55 gallondrums, rail cars, tank trucks, etc). On the diagram, identify byletter and brieflydescribe each worker activity. (2) --Provide the identity, the approximate weight (bykg/day or kg/batch, on an 100%new chemical substance basis), and entrypoint ofall feedstocks (including reactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaning chemicals (notefrequency ifnot useddaily orperbatch). (3) --Identify bynumber the points ofrelease,including small orintermtittent releases, to the environment ofthe newchemical substance. (4) Please enter the #ofsites (remember to identify the locations ofthese siteson acontinuation sheet):
#ofsites

Please see attached continuation sheet



# ZMark (X) this box ifyou attach a continuation sheet. 2. Worker Exposure/Environmental Release (1)

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

-- Estimate the number ofworkers exposed forall sites combined. (4)

-- Estimate the typical duration ofexposure per worker in (a) hours per day and(b)days per year. (6)

-- Describe physical form of exposure and %new chemical substance (ifin mixture), and anyprotective equipment and engineering controls, ifany,used to protect workers. (7)

-- Estimate the percent ofthe newsubstance as formulated when packaged orused as a finalproduct. (9)- From the process diagram above, enter the number ofeachrelease point. Complete 9-13 for eachrelease point identified. (10) -- Estimate the amount ofthe new substancereleased (a) directiy to the environment or (b)into control technology to theenvironment (inkg/day orkg/batch). (12)

--Describe media ofrelease i.e. stack air, fugitive alr (optional-see Instructions Manual), surface water, on-site oroff-site land or incineration, POTW, or other(specify) andcontrol technology, ifany, thatwill be used to limit the release ofthe new substance to the environment. (14) --Identify byproducts which may result from the operation. (3), (5), (8), (11), (13) and (15)

--Mark (X)this columnifany oftheproceeding entries are confidential business information (CBI).-- Letter #of CR1 Duration CB1 Protective Equip. / % in CBI Release Amount of CBI Media ofRelease 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 tooffA 1 2 10.25 12 respirators 

### x 1 X Isite incinerator

(14) -- Byproducts: (15)______ None expected 

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

EPA FORM 7710-25 (Rev. 5-95) Page 10OPTIONAL POLLUTION PREVENTION INFORMATION To claim information in this section as confidential circle or bracketthe specific information thatyou claimas confidential. In this section you may provide information not reported elsewhere in this form regarding yourefforts toreduce or minimize potential risks associated with activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new information pertinent to pollution prevention, including source reduction, recycling activities and safer processes or products available due to the new chemical substance. Source reduction includes thereduction in the amount ortoxicity ofchemical wastes by technological modification, process and procedure modification, product reformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwise be treated or released as airemissions orwater discharges, or land disposal. Descriptions ofpollution prevention, source reduction and recycling should emphasize potential risk reduction subsequent to compliance with existing regulatory requirements and can be either quantitative or qualitative. The EPA is interested in the information to assess overall net reductions in toxicity or environmental releases and exposures, not the shifting ofrisks to other environmental media or non-environmental areas (e.g., occupational or consumer exposure). In addition, information on therelative cost or performance characteristics ofthe PMN substance topotential alternatives maybeprovided. All information provided inthis section will be takeninto consideration duringthe review ofthis substance. See InstructionsManual andPollution Prevention Guidance manual for guidance and examples. Describe the expected netbenefits, such as (1)an overallreduction in riskto human health or theenvironment; (2) a reduction in the volume manufactured; (3) a reduction in the generation ofwaste materials through recycling, source reduction or other means; (4) a reduction inpotential toxicity orhuman exposure and/orenvironmental release; (5)an increase inproduct performance, a decrease in the costofproduction and/or improved operation efficiency ofthenew chemical substance in comparison to existing chemical substances used in similar application; or (6) theextent to which the newchemical substance may be a substitute for an existing substance that poses a greater overall riskto human health or the environment.



### No information available



# LI Mark (X) this boxifyou attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page II

### PartIII -- 

### LIST OFATTACHMENTS Attach continuation sheets for sections ofthe form and test data and other data (including physical/chemical properties and structure/activity information), and optional information after thispage. Clearly identify the attachment and the section oftheform to which it relates, ifappropriate. Number consecutively the pages ofthe attachments. In the column below, enter the inclusive page numbers ofeach 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 inan attachment as confidential. You must include with the sanitized copy ofthe notice form asanitized version ofany attachment inwhich youclaim information as confidential. Attachment name Attachment Confipage number(s) dential MaterialSafety 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 ina48-Hour Screeninci Test 74 -80

____ TKA 40273 (CGX PR 774) Acute Oral Toxicity Study inRats 81

- 99

____ TKA 40273 (CGX PR 774) Acute Eve Irritation Study inRabbits 100 -125 TKA 40273 (CGX PR 774) Primary Skin Irritation Study inRabbits (4-Hour Semi-Occlusive Apolication) 126 -150

[D Mark (X)this box ifyou attach a continuation sheet. Enter theattachment name and number.

EPA FORM 7710-25 (Rev. 5-95) Page 12PHYSICAL AND CHEMICAL PROPERTIES WORKSHEET To assist EPA's review ofphysical andchemical properties data, please complete the following worksheet for data you provide and includeit in the notice. Identify the property measured, the page ofthenotice on whichthe property appears, the value ofthe property, the units in which the property is measured (as necessary), andwhether or notthe property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties should be forthe neat (100%pure) chemical substance. Properties that are measured for mixtures orformulations should be sonoted (%PMN substance in__). You are not required to submit this worksheet; however, EPA strongly recommends that you do so, as it will simplifyreview and ensure that confidential information isproperly protected. You should submit this worksheet as a supplement to your submission oftestdata. This worksheet is not a substitute for submission oftest data.________________ Property Mark Page Value Measured oz Col- (X if number Estimiate dentmi (a) provided (b)(c () Physical state ofneat 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 sizedistribution Octanol / waterpartition 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 13Ciba SpecialtyChemicals Corporation USA
540 White Plains Road..::* P.O. Box 2005 Tarrytown, NY 10591-9005 

## C

# iba 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 notintended for usein products for whichprolonged contact with mucous membranes or abraded skin, body fluids, or implantation within thehuman body, is specifically intended, unless the finished product has been tested in accordance with national and international applicable safetyregulations. Because ofthe wide range ofsuch potential uses, Ciba Specialty Chemicals Corporation is not able to recommend this material as safe and effective for such uses and assumes noliability 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 1of 7 

### 14CGXPR774 ***DRAFT***

Use proper grounding techniques when emptying this product from containers weighing more than 1pound. A build-up of hazardous electrostatic charges could cause a flash fire orexplosion when contents are emptied intoa flammable atmosphere. See Section 7. Environmental:

The aquatic toxicity ofthis product has not been fully assessed. Avoid spillage or leakage to a body ofwater. Health:

Limited testing information is available for this product, however, thisproduct is expected topresent little or no immediate hazard to people ifspilled orreleased. Disposal:

Sweep or shovel spilled material andplace into a sealable container. Dispose in accordance with local, state and federal regulations. This product is not a hazardous wasteunder RCRA.
PrimaryRoute ofEntry:

Dermal, inhalation, ingestion Eye:

This product is not expected to cause eye irritation. Skin:

This product is notexpected to cause skin irritation. Its ability to cause allergic skin reactions is unknown asno test data exist. Swallowing:

Small amounts, ifswallowed, are notexpected 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. FIRSTAID MEASURES First Aid for Swallowing:

Ifswallowed, give atleast 3-4 glasses ofwater, but DONOT induce vomiting. Do notgive anything by mouth to anunconscious or convulsingperson. First Aid for Skin:

Following skin contact, wipe away excess material with a drytowel. Then wash affected areas with plenty ofwater and soap, if available, for several minutes. Get medical attention ifirritation occurs. First Aid forInhalation:

Ifinhaled, remove from area to fresh air. Get medical attention ifrespiratory irritation develops, or ifbreathing becomes difficult. FirstAid forEye:

Following eye contact, flush eyes with plenty ofwater for at least 15 minutes. Get medical attention ifirritation occurs. Noteto Physician:

Specific identity ofthis product or component(s) ofthisproduct is withheld as a trade secret. Amore complete description will be provided to a doctor or nurse in the event ofa medical emergency. Aggravated Medical Conditions: None known. Printed: 07/09/2002 Page 2 of 7 15CGX PR774 ***DRAFT***

5. FIREFIGHTING 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 ofexplosion ifan air-dust mixture forms

- Avoid creating dusty conditions

- Empty only into grounded containers

- Ifcontainer is larger than 550US gallons (2m3)in volume, orwhen flammable solvents are present, the container must be inerted or the system otherwise designed to prevent or contain an explosion
- seek expert advise. * Inaddition, forproducts packagedin fused-lined(coated) fiberdrums,fiberdrumswith conductive liners,steeldrums, steelpails,andCibasuppliedbulkbags,the following instructionsalsoapply:

- Always ground this package before emptying * For productswhich haveexternal protectivepackaging,discharge productonly from theprimaryproduct packa ging,not from the externalcontaineroritsliner. Theuser is responsible for designing a system thatsafely handles solid additives andto ensure propertrainingof employees in the system's use.

Printed: 07/09/2002 Page 3of 7 

### 16CGX PR 774 ***DRAFT*** 8. EXPOSURE CONTROLS /PERSONAL PROTECTION Engineering Controls:

Work in well-ventilated areas. Do notbreathe dust. General Protection: Wear coveralls. Eye/Face Protection:

In cases where there is likelihood ofeye 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 otherMaterials:

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 

### V7CGX PR774 ***DRAFT **

SkinIrritation: Confidential Aromatic ether derivative (Rabbit) Not an irritant Eye Irritation: Confidential Aromatic etherderivative (Rabbit) Not an irritant Sensitization: Notknown Mutagenicity: Confidential Aromatic etherderivative 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 notregulated by any means oftransport.
15. REGULATORY INFORMATION US FederalRegulations: Clean AirAct -Hazardous Air Pollutants (HAP):

This product contains no hazardous air pollutants (HAP), as defined by theU.S. Clean Air Act. Clean AirAct
- Ozone Depleting Substances (ODS):

This product neither contains, nor was manufactured with, a ClassI orClass II ozone depleting substance (ODS). We have relied on our suppliers labeling their products. None have done so. CleanWaterAct
- PriorityPollutants (PP): This product contains no chemicals listed under the U.S. Clean Water Act Priority Pollutant List. Occupational Safety andHealth Act (OSHA):

This product isconsidered 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 5of 7 

### 18CGX PR 774 ***DRAFT*** This product is not considered to bea hazardous waste under RCRA (40CFR 261). SARA Title III: Section 302
- Extremely Hazardous Substances (EHS):

This product contains no chemicals regulated underSection 302as extremely hazardous substances. SARA Title 

### M: Section 304
- CERCLA:

This productcontains no chemicals regulated under Section 304 as extremely hazardous chemicals for emergency release notification ("CERCLA" List). SARA TitleIII: 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) ConsentOrder 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 notbe used, processed or distributed forcommercial purposes. TSCA Section 12(b)
- ExportNotification:

This product does notcontain any chemicals subject to Section 12(b) export notification. Additional Federal Information:

Thisproduct is not subject toa Section 5(f0/6(a) rule. International Remulations: CanadianInventory Status:

Not all components listed on the Domestic Substance List (DSL) EuropeanInventory Status (ElNECS):

This product contains one or more chemicals currently not on theEuropean Inventory ofExisting Commercial Chemical Substances (ELNECS) orPre-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 anychemicals currently on the California list ofKnown Carcinogens and Reproductive Toxins. Massachusetts Right-to-Know:

This product does notcontain 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 19CGX 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 etherderivative CASRN: Confidential Comment: Noton Pennsylvania Hazardous Substance List
16. OTHER INFORMATION
MSDS No: 7340
Disclaimer:

The information and recommendations contained herein are based upon data believed tobe correct. However, no guarantee or warranty ofanykind expressed or implied is made with respectto 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 Page7 of 7 

### 20

# crsInvyentory ExpertService

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 ischecked, 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 StudyTitle: 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 ofCiba Specialty Chemicals Inc 1402292WA 

### 23_______p 2 of 12- Study Number: 14022921 1



### Tableof 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 ofthe 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 Kby 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 bytitration 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 2pages Appendix 4: IRSpectrum 1page

1402292 lAZProperty ofCiba speciary Chemnicals Inc. 14022921AZ 

### 24Study Number: 14022921 

## 3. Abstract

1402292 I AZProperty of Ciba Specialty Chemicals In.10221A 25Study 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-1059.4.15 

### 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 inthe 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 OfCiba Specialty Chemicals Inc 14022921AZ 

### 26Study Number: 14022921 'i 

### 5. Materials and Methods 5.1. IdentificationofTest 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 ofcomponents, by-products and the IR spectrum (see appendix 4). The main methods are:

- Reversed phase liquid chromatographic determination ofthe 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 inthe proton spectrum, by El-S and ESI-MS, by IRand by elemental composition.

1402292IAZproperIWof Ciba Specialty Chemicals Inc. 14022927AZ 

### 27Study Number: 14022921 NMR: The NMR spectra were acquired on an UNITYINOVA 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 ODC1 3 (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 inthe positive ionization mode on a Time-of-Flight mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA). Sample was introduced as a solution inMethanol /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 Awell-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 forthe determination of 

### C, Hand N. Operating parameters for Leco CHNS-932 Furnace Oxidation Reduction Temperature [00] 1000 650 Sample preparation About 1to 3mg of the test sample is wrapped inacapsule consisting ofAg and directly combusted. Calibration The calibration is done using pure organic substances containing C, Hand Nwith known elementary composition: e.g. EDTA (Ethylenediaminetetraacetic acid; C40.96 %, H5.50 % and N9.56 %).

1402292 1 AZProperty of Ciba Specialty Chemicals Inc. 14022921 AZ 

### 28Study Number: 14022921


### Oxygen: Conversion /Cracking to

### CO. Principle Awell defined amount ofthetest sample iscombusted at about 1

3000

### Cunder an atmosphere containing nitrogen/hydrogen. The formed combustion gas (CO) isthen quantitatively detected by infrared absorption. Equipment Leco RO-478 or equivalent Sample About 1to3mg ofthe testsample are wrappedinacapsule Preparation consistingofSn anddirectlycombusted. 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) afew milligram ofthe test sample tobe analysed are completely burned inan Erlenmeyer flaskfilledwith oxygen. An absorbent solution provides the absorption of the reaction gases. Afterwards, this solution is being analysed by argentometric titration todetermine the contentof halogens inthe test sample.
Potassium: ICP-OES at 766.491 nm Copper: ICP-OES at 327.395 nm

14022921lAZPropefly of Ciba Specialty Chemicals Inc 14022921AZ 29Study Number: 14022921 

## 6. Results 6.1 Idniyb NRadM

30* Study Number: 14022921c'

1402292 IAZPropefly ol CobaSpecialty Chemicals Inc ?022A 31Study Number: 14022921
6.2 Identity by JR The identity isconfirmed by JR spectrum. See appendix 4 for spectrum.

14022921lAZProperty of Ciba Specialty Chemicals Inc. 14022921AZ 32Study Number: 14022921
6.4 Content of Main Component and BV Products by HPLC

33Study Number: 14022921



### 7. Appendices Appendix 1 Copyofthe analytical method KBB-493/1 12 pages Appendix 2 'H-NMR and 13

### C-NMR Spectra 4pages Appendix 3 El and ESl-MS Spectra 2pages Appendix 4 IRSpectrum 1page

1402292 lAZProperty ot Ciba Specialty Chemicals Inc 1402221AZ 

### 34

# Analytical Method 

# Ciba
Revision: 00 

### CGX PR774 KBB-493/1 Date: -- Initial: Page 1 of 12 Distributed by Analytics Process Development Schweizerhalle Issue: 01.MAY 2002
Thisdocuimentis generatedfrom an electronic file whichis controlledin compliance Print 12.06.2002 withthe ISO 9001 Qiialit standard, date: Authors:1 0JonManacrement oftest 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 ofCl, Cu and Kby elemental analysis. 4. Content ofmain 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 ofepoxide by titration

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

### 35

## Analytical Method& 

# Ciba.. Revision: 00 

## CGX PR774 KBB-493/1 Date:

--- ________ initial: Page 2 of 12 

### Structures ofstarting materials

PropneiaryiftrmiUilof or Ciba Specialty Chernicals Inc. Not tobe disclosed Value beyond chemistry to third panilc-' without previous consent ofCiba Specialty Chemnicals Inc. 

### 36

## Analytical Method 

# Ciba. . Revision: 00 

## CGX PR774 KBB-493/1 Initial:Page 3 of 12

Structures ofby-products

Propriearzy informfationl of Ciba Spe±cialty Chemicals Inc. Not to be disclosed Value beyond chemistry tothird 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, CH4002 Basle.
2. Identity 

### byIR-Spectrumn Check IR spectrum of the sample for qualitative congruence with the reference spectrum below. FT-IR-Spectrometer: Nicolet FT-1R 510 Range ofspectrum: 4000
- 550 cm-' Transmission: 0 -100 % Resolution: 4 cm'1 Reference: air Recording technique: ATR No. ofscans: 64

Waveriumbers (cm-i)

Proprietary information of Ciba Specialty Chemicals Inc. Not tobe disclosed value beyond chemistry to third panics without previous consent of Ciba Specialty Chemicals Inc 

### 38*Ciba SpecialtyChemicals Inc. 

## Analytical Method

____ ____ ____ ___ ____ ___ 

### Ciba Revision: 00 

## CGX PR774 KBB-493/1 Date: -- Initial: Page 

### 5 of 12 3. Elemental analysis Determination ofthe 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 ofmain component and by products by HPLC Principle ofseparation: liquid chromatography Calculation :external standard method for Ethyiphenylacetate, CA 32-0099, CG41-1212, 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, C18, 5pm 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: 250C Injection volume: 5pl Detector: UV- detector at 230 nm

Proprietary information ofCiba Specilty Chemicals Inc. Not to be disclosed Value beyond chemistry to third panics without previousconsent 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 of0.5
- 1.5 mgI25ml of 4-Ethylphenylacetate, CA 32-0099, CG 41-12 12, CG41-0350, CG41-1742 and 20.0- 25.0mg ofCGX PR 774 (batch FF226/9 F1 purifi6). Table ofdata

Typical chromatogramns: see on page 7

Propriary information ofCiba Specialty Chemicals Inc. Not to be disclosed Valuc beyond chemistry tothird panics without previous consent ofCiba Specially Chemicals Inc. 

### 40

# Analytical Method 64 

# Ciba :? 

### Revision: 

### 00 

### CGX PR774 KBB-493/1 Date: -- Initial: Page 7 of 12

Proprieary information ofCiba Spcialty Checalsi Inc. Not to be disclosed Value beyond chemistry to third panics without previous consent ofCiba Specialty Chec.als Inc. 

### 41Ciba Specialty Chemicals Inc. 

## Analytical Method

___ ___ ___ ___ ___ 

# C ba Revision: 00 

## CGX PR774 KBB-493/1 Date: -- Initial: Page 8 of 12 

### 5. Content ofresidual 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: Ijil Carrier gas: He, 2mI/mmn Temperatures: Injector: 0.5 min at 320*C Oven: 2min at40'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 of0.2
- 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 ofdata____________ component retention time
Residual methanol 1.9___________ Ethyl acetate5.

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

### 42*CibaSpecialtyChemicals Inc. 

# A ayia AnaytialMethod 

# Ciba Revision: 00 

### CGX PR774 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: IA.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 of0.05
- 0.2 mg/mI each. Test solution Prepare asolution in methanol with a final sample concentration of80.00
- 120.00 mg/mI.

Table ofdata

__________ 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 ofCiba Specialty Chemicals Inc. 

### 43'CibaSpecialty Chemicals Inc. 

# Analytical Method 

# Ciba Revision: 00 

### CGX PR774 KBB-493/1 Date: -- Initial: Page 10 of 12
7. Content of epoxide by titration 7.1 Content ofamine 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 of20 ml ofglacial acetic acid and 20 ml ofacetone 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 ofAmine in the sample is calculated as: a-*0.1 - f W

where A = content of amine in Eq/Kg a = consumption ofperchloric acid-solution 

### I ml] for the sample after temperature correction f =factor ofthe perchloric acid solution* W =sample weight [a]

Proprietry informiationofciba Specialiy Chemicals Inc Not to be disclosed Value beyond chemistry it)third pantics without previousconsent ofciba Specialty Chemicals Inc.'Ciba SpecialtyChemicals Inc. 

# A ayia AnaytialMethod z

____ ____ _ __ ___ ____ ____ ___Ciba. 

# . Revision: 00 

### CGX PR774 KBB-493/1 Date: -- Initial: Page 11 of 12 7.2 Content of the sum epoxide +amine: Principle ofdetermination: potentiometric titration of the epoxide functional group with perchioric acid after reaction with TAAB-reagent*. Preparation oftheTAAB-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. Ifit 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 GPTitrino, 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 tomethod '5DET U, Epoxid" Calculation The sum ofthe content ofEpoxide and Amine in the sample is calculated as: a-0.1 *f S - 

## w

where S = sum ofepoxide and amine in Eq/Kg a = consumption ofperchloric acid-solution [ml] for the sample after temperature correction f = factor ofthe 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 ofciba Specialty Chemicals Inc 45Ciba Specialty Chemicals Inc. 

# M to Analytical Mto 

# Ciba Revision: 00 

## CGX PR774 KBB-493/1 Date: -- Initial: 

### Page 12 of 12

7.3 Content 

### ofepoxide in the sample The content of epoxide (Eq/Kg) in the sample is calculated as the difference ofthe two titrations carried out according to pos. 7.2 and 7.1.

Propritry information ofCiba Specialty Chemicals Inc Not to be disclosed Value beyond chemistry to third panics without previous consent of Ciba Specialty Chemicals Inc. 46

### Appendix2 Study Number: 14022921 

## 'H-NMR and 13

## C-NMR spectrum

Product Designation: TK A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187



### 47

### Appendix 2 Study Number: 14022921

Property of'Ciba Specialty Chemicals Inc. 

### 48Appendix_2 Page 3ofW-- 

### 4---- Study Number: 14022921 1

### H-NMR spectrum (in CDCI3) expanded

Product Designation: TK A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187



# e4

Property ofCiba Specialty Chemicals Inc. 

### 49

### Appendix 2-ag P 

### f 4~ Study Number: 14022921 13

### C-NMR 

## in CDC13

### :

## overview

Product Designation: TK 

### A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187

PropertyofCiba SpecialtyChemnicals Inc. 

### 50

### Appendix 3 ~L I~ 

### Study Number: 14022921 

### MS spectra

Product Designation: TK A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187 Chemical Structure Icomposition:

Propertyof Ciba Specialy Chovicals Inc.


### 51

## Appendix3 PStudy Number: 14022921



### Product Designation: TK A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187

Property ofCiba Specialty Chemnicals Inc. 

### 52Study Number: 14022921 

### IR spectrum Product Designation: TK A40273 Product Name: CGX PR 774 Batch Number: Vs. 836-187 Chemical Structure Icomposition:

Property ofCiba SpecialtyChemicals Inc.


### 53'~ Li~ f~ 

### / ~(J *7% S./



## RCC-CCR PROJECT 692300
PROJECT IDOF THE CONTRACTING INSTITUTE: RCC PROJECT 803542 SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY SCREENING VERSION
WITH
TKA 40231 REPORT
STUDY COMPLETION DATE: March 09, 2001

54Test Report RCC-CCR Project 692300


## CONTENTS
PREFACE 3

General 3 Project Staff 3 Schedule 3 Project StaffSignatures 4 Guidelines 4 Archiving 4 Distribution of the Report 4 OBJECTIVE 5

Aims ofthe 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 ofthe 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

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55Test 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 Inden 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: TKA40231 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

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56Test 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 studyfollowed 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, L1362000, Annexe V,B13/14 dated May 19, 2000 

### Archiving RCC Cytotest Cell Research GmbH, D-64380 Roldorfwill archive the following data for 2years: Raw data, protocol and copy ofreport, and sample ofthe test item. No raw data or material relating to the study will be discarded without the sponsor's prior consent. 

### Distributionofthe Report Contracting Institute 2x (original, copy) Study Director lx (copy)

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-


### 57Test Report RCC-CCR Project 692300 

### OBJECTIVE Aims ofthe 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 aplate incorporation assay. 

### Reasons forthe 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 inthe absence ofthat amino acid (reversion from auxotrophy to prototrophy) isthe 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 concentrationswere tested. The maximum dose level was 5000 pg/plate. To validate the test, reference mutagens are tested inparallel tothe test item.

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58Test 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 inSolvent: not indicated bythesponsor Storage: room temperature Expiration Date: February 01, 2003 On the day ofthe experiment, the test item TKA 40231 was dissolved inacetone (purity> 99 

### %,MERCK, 0-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative non-toxicity tothe bacteria. No precipitation ofthe test item occurred up tothe highest investigated dose.

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### 59Test Report RCC-CCR Project 692300 

### Controls Negative Controls Concurrent untreated and solvent controls were performed. PositiveControl Substances Without metabolic activation Strains: TA 1535,TA 100 Name: sodium azide, NaN3 Supplier: SERVA, D-69042 Heidelberg Catalogue No.: 30175 Purity: at least 99 % Dissolved in: waterdeionised Concentration: 10 pg/plate Strains: TA 1537, TA 98 Name: 4-nitro-o-phenylene-diamine, 4-NOPD Supplier: SIGMA, D-82041 Deisenhofen Catalogue No.: N9504 Purity: > 99.9 % Dissolved in: DMVSO (purity >99 %,MERCK, D-64293 Darmstadt) Concentration: 50 pg/plate inTA 1537, 10 pg/plate inTA98 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.: A1381 Purity: 97.5 % Dissolved in: DMSO (purity >99 %,MERCK, 0-64293 Darmstadt) Concentration: 2.5 pg/plate (10.0 pg/plate inTA 102) The stability of the positive control substances insolution was unknown but amutagenic response inthe expected range issufficient evidence of biological stability.

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60Test Report RCC-CCR Project692300 

### Test System Characterisation ofthe Salmonella typhimurium Strains The histidine dependent strains are derived from S.typhimuriumn strain LT2 through a mutation inthe histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess afaulty lipopolysaccharide envelope which enables substances to penetrate the cellwall more easily. Afurther mutation causes an inactivation of the excision repair system. The latter alteration includes mutational processes inthe nitrate reductase and biotin genes produced ina UV-sensitive area of the gene named "uvrB-minus'. Inthe strainsTA 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 isexcision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQI carrying the hisG428 mutation (ochre mutation inthe hisG gene)and atetracycline resistance gene. Insummary, the mutations of the TA strains used inthis study can be described as follows: Salmonella typhimurium TA 1537: his C3076; rfa*; uvrB': frame shift mutations TA 98: his D3052; rfa-; uvrB';R-factor: frame shift mutations TA 1535: his G46; rfa-; uvrB-: base-pair substitutions TA 100: his G46; rfa-; uvrB-;R-factor: base-pair substitutions TA 102: his G428; 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 inRCCOCR according to Ames et al. (1). Inthis way itwas ensured that the experimental conditions setdown byAmes were fulfilled. The bacterial strains TA 1535, TA 1537, TA 1 Ooand TA 102 were obtained from Ames (University ofCalifornia, 94720 Berkeley, U.S.A.). The bacterial strainTA 98was obtained from von E.Merck (D-64293 Darmstadt). 

### Storage The strain cultures were stored as stock cultures inampoules with nutrient broth + 5 % DMVSO (MERCK, D-64293 Darmstadt) inliquid nitrogen.

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### 61Test Report RCC-CCR Project 692300
Precultures From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flaskscontaining 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: 8gMerck Nutrient Broth (MERCK, D-64293 Darmstadt) 5gNaCI (MERCK, D-64293 Darmstadt) The bacterial culture was incubated inashakingwater bath for 4hours at 370 C. Selective Agar The plates with the minimal agarwere obtainedfrom E.Merck, D-64293 Darmstadt. Overlay Agar The overlay agar contains per litre:

6.0 gMERCK Agar Agar* 6.0 gNaCI* 10.5mg L-HistidinexHClxH2 O* 12.2mg Biotin* * (MERCK, D-64293 Darmstadt) Sterilisations were performed at 1210 Cinan autoclave. 

### Mammalian Microsomal Fraction S9 Mix The bacteria used inthese assays do not possess the enzyme systems which, inmammals, are known to convert promutagens into active DNA damaging metabolites. Inorder 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 incorn oil (Aldrich, 0-89555 Steinheim). The livers were prepared 24 hours afterthe lasttreatment.

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### 62Test 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 inKCI and centrifuged at 9,000 gfor 25 minutes at 40 C.Astock of the supernatant containing the microsomes was frozen inampoules of 2,3or 5ml and stored at -800 C.Small numbers ofthe ampoules are kept at -20* Cfor 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 inthe 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 ofthe cofactor solution was chosen to yieldthe following concentrations inthe S9 mix: 8MM MgCI2 33 mM KCI

5mM Glucose-6-phosphate 5mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment theS9 mix was stored inan ice bath. The S9 mix preparation was performed according toAmes et al.(2). Dose Selection The maximum concentration was 5000 pg/plate. The concentration range covered two logarithmic decades. Inthis study eight adequately spaced concentrations were tested. Thetest item was tested atthe following concentrations: 3; 10; 33; 100; 333; 1000;2500; and 5000 pg/plate

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### 63Test Report RCC-CCR Project 692300 

### Experimental Performance For each strain and dose level, including the controls three plates were used as aminimum. The following materials were mixed inatest 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 (fortestwith metabolic activation) or S9 mix substitution buffer (fortest without metabolic activation), 100 pl Bacteria suspension (cf.test system, pre-culture ofthe strains), 20 00pl Overlay agar After solidification the plates were incubated upside down for at least 48 hours at 370 Cin the dark. 

### Data Recording The colonies were counted using theAUTOCOUNT (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 concentrationtogether with standard deviations andenhancement factors as compared to the spontaneous reversion rates (see tables of results). 

### Acceptability ofthe Assay The Salmonella typhimurium reverse mutation assay isconsidered acceptable if itmeets thefollowing criteria:

- regular background growth inthe negative and solvent control

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

our historical data

- the positive control substances should produce asignificant increase inmutant colony

frequencies

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### 64Test Report RCC-CCR Project 692300 

## EVALUATION OF RESULTS Atest item isconsidered positive ifadose related increase inthe number of revertants or abiologically relevant increase for at least onetest concentration isinduced. Atest item producing neither no adose related increase inthe number of revertants nor a biologically relevant positive response at any one of the test points isconsidered nonmutagenic inthis system. Abiologically relevant response isdescribed asfollows: Atest item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate instrains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537 (3,4). Also, adose-dependent increase inthe number ofrevertants isregarded 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.

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65Test 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 intriplicate. 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 inthe number of revertants, were observed inall strains with andwithout metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without S9 mix inall strains used. No relevant substantial increase inrevertant colony numbers of thefive tester strains was observed following treatment with TKA 40231 at any concentration level, neither inthe presence nor absence ofmetabolic activation (S9 mix). Instrain TA 1535 aweak increase ofthe revertant colony numbers was observed inthe highest concentration with metabolic activation. Since the threshold of threewas not reached the test item isconsidered to benon-mutagenic inthis assay. Appropriate reference mutagens were used as positive controls. They showed a distinct increase ininduced revertant colonies. 

## CONCLUSION Inconclusion, itcan 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 inthe genome ofthe strains used. Therefore, TKA 40231 isconsidered to be non-mutagenic inthis Salmonella typhimurium reverse mutation assay.

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### 66Test Report RCC-CCR Project 692300 

## REFERENCES 1

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

Carcinogens are mutagens: asimple test system combining liver homogenatesfor activation and bacteriafor 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 MutagenicityTest Procedures" Elsevier, Amsterdam, 1-17 3.Claxton, L.D., Allen, J.,Auletta, A., Mortelmans, K., Nestmann, E.and Zeiger, E. (1987) Guideforthe Salmonella typhimurium/mammalian microsome tests or bacterial mutagenicity Mutation Res. 189, 83-91 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 ofthe Ames assay: ).Results and general interpretation Mutation Res. 85, 391-410

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67Test 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 SolventControl 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 SolventControl 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. testitem I revertants/solvent control

'Sodium azide 10.0 pg/plate S2-aminoanthracene 2.5 pg/plate

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### 68Test Report RCC-CCR Project692300 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. testitem fa Irrevertants/solvent control 4nitro-o-phenylene diamine 50.0 pg/plate S2-aminoanthracene 2.5 pg/plate

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69Test Report RCC-CCR Project 692300 Main-Experiment: Plate Incorporation Test Test item: TKA40231 S9 mixfrom: Rat liver (Batch 190101) Teststrain: TA98 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

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70Test Report RCC-CCR Project 692300 Main-Experiment: Plate Incorporation Test Test item: TKA 40231 S9 mixfrom : Rat liver (Batch 190101) Test strain: TA 100 withoutS9 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* NegativeControl 143 139 155 146 8.3 Solvent Control 132 171 144 149 20.0 1.0 

# IPositiveControl" 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 of20-



### 71Test Report RCC-CCR Project 692300 Main-Experiment: Plate Incorporation Test Test item: TKA 40231 S9 mix from: Ratliver (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 SolventControl 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 /solventcontrol

'Methyl methane sulfonate 5.0 p1/plate S2-aminoanthracene 10.0 pg/plate

st4r -Page 19 of20-

72Test 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 20of20-
73

## REPORT
StudyTitle: 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 SpecialtyChemicals Inc. Additives Division P.O. Box CH-4002 Basel/Switzerland RCC Study Number: 803621 Page 1of 7



### 74ROC Study Number 803621 REPORT Page 2 TKA 40231 

## SIGNATURES

Study Director: Dr. Birgit Seyfried

.........

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

Management: Mr. Markus Arenz

-

Dat:



### 75RCC StudyNumber 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 inthe test media atthe start and end of the test............................... 7 Table 3: Appearance ofthetest media during the test period ......................... 7

76RCC Study Number 803621 REPORT Page 4 TKA 40231 

## PREFACE
GENERAL StudyTitle: Acute toxicityofTKA 40231 to Daphnia magna ina 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



### 77ROC Study Number 803621 REPORT Page 5 TKA 40231 

## 1 MATERIALS AND METHODS
Test Item: TKA 40231 (CGX PR774) Batch No.: 783-068 Purity: >95% Expiry Date: February 02, 2003 Storage conditions: Closed container, room temperature, inthe dark


### 1.1 EXPERIMENTAL CONDITIONS The acute toxicity of the test item TKA 40231 to Daphnia magna was determined ina48- hour static test based on the Commission Directive 92/69/EEC, C.2 (1992) and the OECD Guideline for Testing ofChemicals, No. 202, Part 1, (1984). Ineach treatment and the control, 20 daphnids were tested, divided into two groups of ten animals, each group in50 ml test medium ina 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 thetest period Light conditions: The test was performed as far as possible inthe dark to avoid photolytic degradation ofthe test item Test duration: 48 hours Test water: Reconstituted water according tothe test guidelines



### 78RCC StudyNumber 803521 REPORT Page6 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 in3000 mI test water

- 

### 10.0 mg suspended in 1000 ml test water

- 31.0 mg suspended in 300 ml testwater The test item was dispersed inthe test media by ultrasonic treatment for 15 minutes. Then the mixtures were stirred byamagnetic stirrer at room temperature inthe dark over 96 hours to dissolve respectively disperse amaximum concentration of the test item inthetest media. No auxiliary solventor 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 acontrol (test water without test item) were tested inparallel.



### 79RCC Study Number 803621 REPORT Page 7 TKA 40231 

## 2 RESULTS
Table 1: Influence of TKA 40231 onthe mobility of Daphnia magna Loading rate/ No. of No. of %of immobilized

Treatment daphnids immobilized daphnids after tested daphnids after 24h I48h 24h 48h Control 20 0 0 0 0 1.0 mg/I filtered 20 0 0 0 0 10mg/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 inthe test media at the start and end ofthe test Loading rate/ Start (0hours) 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 thetest period Loading rate /Appearance ofthe test item

Treatment inthe 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

TKA40273 (CGX PR 774): Acute Oral Toxicity Study inRats Report 

## Author: G.Arcelin Sponsor: Ciba Specialty Chemicals Inc. CH-4002 Basel/Switzerland

Page 1of 19



### 81RCC STUDY NUMBER 843786 Report Page 2 TKA 40273 (CGX PR 774) 

## Table ofcontents
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 TESTSYSTEM.......................................................................... 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 BODYWEIGHTS........................................................................ 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



### 82RCC 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



### 83RCC STUDY NUMBER 843786 Report Page4 TKA 40273 (CGX PR 774) 

### 1 

## PREFACE 1.1 GENERAL Title TKA 40273 (CGX PR 774): Acute Oral Toxicity Study inRats Sponsor Ciba SpecialtyChemicals 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 ofAnimals 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 (firstdose) 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 to07-JUN-2002 (males, 2000 mg/kg) Study Completion Date 04-JUL-2002



### 84RCC STUDY NUMBER 843786 Report Page5 TKA 40273 (CGX PR 774) 

### 1.4 ARCHIVING ROC Ltd (CH-4452 Itingen / Switzerland) will retain the study plan, rawdata, amendment, a sample of test item(s) and the final report of the present studyfor at least ten years. No data will be discardedwithout the Sponsor's consent.



### 85RCC STUDY NUMBER 843786 Report Page 6 TKA 40273 (CGX PR774) 

### 1.5 SIGNATURE PAGE Study Director: G.Arce*

date
Management: S.Corney

dat



### 86RCC 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 ToxicityStudy inRats The general facilities and activities are inspected periodically and the results are reported to the responsible person andthe management. Study procedures, with the exception ofthe trialformulation, 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 tothe 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 alsoconfirms that this final report reflectsthe raw data. 

### QualityAssurance: 

# (b) S aqren

.............. date: 04LIU

### L-2co 

## 2

87RCC 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 inRats The stability of the test item inthe vehicle isunknown and is,therefore, excluded from this statement. The trial formulation was performed before the study initiation date and isalso excluded from this statement. This study has been performed incompliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd,2000 [RS 813.016.5].This Ordinance isbasedon the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted th November 26

, 1997 bydecision of the OECD Council [C(97)186/Final]. These procedures were consistent with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), andJapan (MHLW, MAFF and METI). Study Director: G.ArceJin-)

. . ... e. . ... date: rU- z~



### 88RCC STUDY NUMBER 843786 Report Page 9 TKA 40273 (CGX PR 774) 

### 1.8 ACCREDITATION The test facility ORCC Ltd, Toxicology Division' isaccredited according to EN 45001 under accreditation number STS 085 bythe 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 OralToxicity-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.



### 89RCC 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 invehicle (PEG 300) at a concentration of 0.2 g/ml and administered at a volume dosageof 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 1,2,3and 5hours after treatment on day 1and 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 thestudy period. Slightly ruffled fur and hunched posture was noted in one female animal and all male animals treated at 2000 mg/kg from 1to 5hours afterthetest 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 aperiod of 14 days is: LD 50 (RAT): GREATER THAN 2000 mg/kg BODY WEIGHT



### 90ROC 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 3males or3females Total number ofanimals 3males 3females Age when treated Males: 8weeks 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 anyvisiblesigns of illness were usedfor 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.



### 91RCC STUDY NUMBER 843786 Report Page 12 TKA 40273 (CGX PR 774) Accommodation Ingroups of three per sex in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (tignocel' SchillAG, 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 analysesfor 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 836-187 Purity SeeAnalytical Certificate No. 14022921, Ciba Specialty Chemicals Inc. Expiry date 30-APR-2004 Stability oftest item dilution Unknown inPEG 300; isexcludedfrom the statement of compliance. Storage conditions Inthe original container, at room temperature (range of20 ± 3'C), away from direct sunlight. Safety precautions Routine hygienic procedures were used to ensure the health and safetyofthe 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 Stabilityof vehicle Stable under storage conditions; expiration date: 28-FEB-2003



### 92RCC STUDY NUMBER 843786 Report Page 13 TKA 40273 (CGX PR 774) Storage conditions Inthe original container, at room temperature (range of20 ±30 C), away from direct sunlight. Safety precautions Routine hygienic procedures were used to ensure the health and safetyof the personnel. PEG 300 was found to be asuitable vehicle. The vehicle was chosen after a non-GLP solubility trial which was performed before the study initiation date. Thistrial formulation isexcluded from the GLP statement of compliance. 

### 5.5 DOSE FORMULATION Dose levels are in terms of the test item as supplied. The doseformulations were made shortlybefore each dosing occasion. The test item was weighed into atared glass beaker on asuitable precision balance and the vehicleadded (weight:volume). Theformulations were prepared using amagnetic stirrer. Homogeneity of the test item inthe 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). Foodwas provided again 3hours afterdosing. The application volume was 10 mI/kg bodyweight. 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 andtwice daily during days 1 -15. Body weights On testdays 1(prior toadministration), 8and 15. Clinical signs Daily during acclimatization and at approximately 1,2,3and 5 hours after administration on test day 1.Once dailyduring days 2-15. All abnormalities were recorded.



### 93ROC STUDY NUMBER 843786 Report Page 14 IKA 40273 (CGX PR 774) 

## 6 PATHOLOGY 6.1 NECROPSY Necropsy was performed inone male animal found dead on testday 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 examinationswere performed. No organs or tissues were retained. 

## 7 STATISTICAL ANALYSIS No statistical analysis was used. 

## 8 DATA COMPILATION Bodyweights were recorded on-line. Clinical signswere 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.



### 94RCC STUDY NUMBER 843786 Report Page 15 TKA40273 (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 at2000 mg/kg from 1to5hours afterthe test item administration. 

### 9.3 BODY WEIGHTS The body weight of the animals was within the range commonly recorded forthis strain and age. 

### 9.4 MACROSCOPIC FINDINGS No macroscopic findingswere 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 aperiod of 14days is: LD50 (RAT): GREATER THAN 2000 mg/kg BODY WEIGHT



### 95ROC 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 Noclinical 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 Noclinical 4 4 4 4 4 4 4 signs Ruffledfur 1 1 1 1 Hunched 4444 posture 2000 4 M Noclinical 4 4 4 4 4 4 4 signs Ruffledfur 1 1 1 1 Hunched 4444 posture 5 M No clinical 4 + signs Ruffled fur 1 1 1 1 Hunched 4444 posture 6 M Noclinical 4 4 4 4 4 4 4 signs Ruffled fur 1 1 1 1 Hunched 4444 posture I I I II I I Key:1slight, founddead,4noted * Examination performed approximately 1,2,3and5 hours after treatment. No clinical signs were evident in any animal during the acclimatization period.



### 96ROC STUDY NUMBER 843786 Report Page 17 TKA 40273 (CGX PR 774) 

### 10.2 BODY WEIGHTS

Dose An. Sex Dayl1 Day8 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 ingrams 

### 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, Dfound dead



### 97RCC STUDY NUMBER 843786 Report Page 18 TKA 40273 (GGX PR 774) 

### 11 

## CERTIFICATIONS 11.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

fh Eidgen6ssischesAmrt for Mesawesen S Schweizenische Akkreditierungssalle

Ih Office f~dhral de mizroiogie Service d'accriditationsuisse Ufficia federale di metrologia A~cServizio d'accreditamento avizzero Swiss Federal Office ofMetrology S Swiss Accreditation Service 

# ACCREDITATION

Based onthe 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 ofthe Accredited Testing Laboratories in Switzerland. The relevant requirements ofthe ISO 9002 standard are also covered by this accreditation. Accreditation number: STS 085 Date ofthe accreditation: 16"e September 1994 Date ofthe last renewal ofthe accreditation: 20"' September 1999 The accreditation isvalid 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 asign" of ato mulfiaeal agivrenit olto Europea co-operadmorcr edlall (EA) for

calibralion. wsWQn and

cerdlication ol pro*"t. personnel. quality anid awnvlrientl mianagernent sylim.



### 98RCC 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 Publichealth an Enciont FMet 

# )frecntro o 

# Statement of GLP Compliance

Itishereby confirmed that during theperiod of August 15- 17, 2000 the following Test Facilitiesof 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 inagreement with the OECD Guidelines for National GLP inspections and Audits. Itwas 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 theywere inspected. Federal Office ofPublic Health The Director
Bemn, November 2000 Prof. Th. Zeltner

*TOX - Toxicology
. ACC -Analytical and Clinical Chemistry , ECT *Environmental toxicity on aquaticandterrestrial organisms
. ENF - Behaviour inwater, soil and air.Bicaccumulation PCTa Physical-chemical tasting ;RES aResidue studies 0TH - Other, tobespectfied



### 99

## RCC Study Number 843788

TKA 40273 (CGX PR 774): Primary Eye Irritation Study inRabbits Report
Author: 

## G.Arcelin Sponsor: Ciba Specialty Chemicals Inc. CH-4002 Basel /Switzerland

Page 1of 26



### 100ROC STUDY NUMBER 843788 Report Page2 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 BODYWEIGHTS........................................................................ 16 8.6 CONCLUSION........................................................................... 16 9 APPENDICES ................................................................................ 17



### 101RCC 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



### 102RCC 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 inRabbits Sponsor Ciba SpecialtyChemicals Inc. CH-4002 Basel/ Switzerland Study Monitor Mrs. Isabelle Frei Test Facility RCC Ltd Toxicology Division Wdlferstrasse 4 CH-4414 F0llinsdorf ISwitzerland 

### 1.2 RESPONSIBILITIES Study Director G.Arcelin Deputyfor 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 usedfor RCC Study 843787) Acclimatization 23-MAY-2002 to 27-MAY-2002 Treatment 28-MAY-2002 Observation Throughout 72 hours aftertreatment. 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 oftest item(s) and the final report of the present studyfor at least ten years. No data will be discardedwithout the Sponsor's consent.



### 103ROC 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



### 104RCC 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 inRabbits 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 bythe QualityAssurance Unit. The datesare given below. Dates and Types of Dates of Reports to the Study Director

QAU Inspections and to Management 1

6-MAY-2002 Study Plan 16-MAY-2002 22-MAY-2002 Process Based 22-MAY-2002 1

9-JUN-2002 Report 1 9-JUN-2002
This statement also confirms that thisfinal report reflectsthe raw data. Quality Assurance:G.Ht
date: 03.

## j~UL-2eD62

105ROC 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 PR774): Primary Eye Irritation Study inRabbits This study has been performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2"', 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted th November26 , 1997 bydecision ofthe 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

106RCC STUDY NUMBER 843788 Report Page 8 TKA40273 (CGX PR 774) 

### 1.8 CERTIFICATION OF GLP AND VERIFICATION OF THE REPORT

The statement of Compliance with Good Laboratory Practice found inthis report, and signed by the Study Director istruthful and accurate, and this report as provided by the test facility iscomplete and unaltered.

Signature ofthe Sponsor: .......................
Date:



### 107ROC STUDY NUMBER 843788 Report Page 9 TKA40273 (CGX PR 774) 

### 1.9 ACCREDITATION The test facility "RCC Ltd, Toxicology Division" isaccredited according to EN 45001 under accreditation number STS 085 bythe 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 inan AAALAC-approved laboratory inaccordance with the Swiss Animal Protection Law under license no.54. 

### 1.12 SUMMARY OF STUDY PLAN AMENDMENT Study PlanAmendment No. 1: Reference to the analytical certificate was added.



### 108RCC STUDY NUMBER 843788 Report Page 10 

### TKA40273 (CGX PR774)1 

## 2 SUMMARY The primary eye irritation potential of TKA 40273 (CGX PR 774) was investigated by instillation of 0.1 ginto 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 incalculating 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 grade0.00 chemosis of the conjunctivae. The instillation of TKA 40273 (CGX PR 774) into the eye resulted inmild, early-onset and transient ocular changes, such as reddening ofthe 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, thetest item did not induce significant or irreversible damage to the rabbit eye. TKA 40273 (CGX PR 774) isconsidered to be "not irritating" tothe rabbit eye.



### 109RCC 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 asingle dose ofTKA 40273 (CGX PR 774) was placed intheconjunctival sac of rabbit eyes. This study should provide a rational basis for risk assessment inman as ocular contact is one ofthe probable routes of human exposure. The test item was applied at 0.1 g/animal, the dose specified inthe test guidelinesfor asolid test item. 

### 4 MATERIALS AND METHODS 4.1 TEST SYSTEM Testsystem New Zealand White Rabbit, SPF Rationale Recognized by the international guidelines as the recommended test system Source Elevage Scientifique des Dombes F-01400 Chatillon surChalaronne/ France Number of animals per test 3(Animals of both sexeswere 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 illnesswere used for the study. Allocation Male No. 25 Female Nos. 26 and 27



### 110

### ROC STUDY NUMBER 843788 Report Page 12 

# 1

TKA 40273 (CGX PR 774) 

### 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 lightperiod. 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, adlibiturn. 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 Inthe 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) ofTKA 40273 (CGX PR 774) was weighed and applied undiluted as itwas delivered by the sponsor. According to Directive 92/69 EEC, B.5. and OECD Guidelines 405, atest 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 5to6. 

### 4.5 TREATMENT The eyes of the animalswere examined one dayprior totest item administration. Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation ofthe results were not used inthetest. On the dayoftreatment, TKA 40273 (CGX PR 774) was placed inthe conjunctival sac of the left eye of each animal after gently pullingthe lower lid awayfrom the eyeball. The lidswere 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 ofthe animals tothe termination of test. Clinical signs Dailyfrom acclimatization of the animals to the termination of test. Bodyweights At start of acclimatization, onthe day of applicationand at termination of observation. 4.6.1 IRRITATION 

### SCORES The ocular reaction was assessed according to the numerical scoring system listed inthe EEC Commission Directive 92/69/EEC, July31, 1992 at approximately 1, 24, 48 and 72 hours after application. When present, corrosion and/or staining of sclera and cornea bythe test item were recorded and reported. Eye examinations were made with a Varta Cliptrix diagnostic-lamp (A.Riegger, Basel/ Switzerland).



### 112RCC STUDY NUMBER 843-788 Report Page 14 TKA 40273(CGX PR 774) 

## 5 PATHOLOGY 5.1 NECROPSY No necropsywas performed onthe animals sacrificed attermination ofobservation. All rabbits were sacrificed byan intravenous injection of VETANARCOL into the earvein at a dose of at least 1mI/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. Inaddition, any lesions including the degree and nature of irritation, corrosion or reversibility, and any othertoxic effects were observed. To evaluate the irritation potential of the test item the mean valueswere calculated for each individual, using the scores between 24 and72 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 forthe same parameter inall animals at24, 48 and 72 hours. See TABLE 2,page 18. The primary eye irritation score was calculated bytotaling 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. Bodyweights were recorded on-line and/or on data-sheets. No statistical analysiswas performed.



### 113RCC 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 inthe animals during the study and no mortality occurred. 

### 8.2 IRRITATION Application of the test item to healthy rabbit conjunctivae resulted inaprimary 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 1to24 hours after treatment. Slight reddening continued to be observed intwo animals up to the 48-hour examination (Table 1). Slight swelling or swelling with partial eversion of lids was noted inall animals 1hour after treatment (Table 1). Moderate reddening of the sclerae was observed inall animals at the 1-hour examination and slight reddening was noted again inone animal uptothe 48-hour examination (Table 1). increased ocular discharge was present inall animals 1hour 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 Nostaining of the treated eyes by the testitem was observed. White remnants of the test item were observed inthe eye or conjunctival sacof all animals 1hourafter treatment. 

### 8.4 CORROSION No corrosion of the cornea was observed at anyof the reading times.



### 114RCC STUDY NUMBER 843788 Report Page 16 TKA 40273 (CGX PR 774) 

### 8.5 BODY WEIGHTS The bodyweights of all rabbits were considered to be within the normal range ofvariability.

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) isconsidered to be "not irritating" tothe rabbit eye (Table3).

115RCC STUDY NUMBER 843788 Report Page 17 TKA 40273 (CGX PR 774) 

### 9 APPENDICES 9.1 EYE IRRITATION SCORES Keyto Symbols: M Male F Female



### 116RCC STUDY NUMBER 843788 Report Page 18 TKA 40273 (CGX PR 774) 

### TABLE 1: EYE IRRITATION SCORES

- 

### INDIVIDUAL VALUES

Animal I Evaluation Corneal Area of COWuntivae 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 Ihour 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 24hours 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 48hours 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 afterinstillation ofthe testitem.


### 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 ofavailable data points


### TABLE 3: EYE IRRITATION SCORES

- 

### ASSESSMENT ACCORDING TO EEC GUIDELINES Evaluated Corneal Conjunctivae

intervals 0 Oacit Iris Redness Cheniosis 24 hours 48 hours NotIrritating NotIrritating NotIrritating Not Irritating 72 hours The grading scale is presented on page 23.



### 117ROC STUDY NUMBER 843788 TKA 40273 (CGX PR774) Page 19 

### 9.2 INDIVIDUAL FINDINGS ANIMAL NO. 25, MALE After 1hour: Cornea: NO ABNORMAL FINDINGS NOTED Iris: NOABNORMAL FINDINGS NOTED Conjunctivae: moderately reddened; slightswelling Discharge: increased ocular discharge Sciera: moderately reddened Test item: white remnants ineye 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, asappropriate) of opacity,vascularization, reddening, oedema, discharge, staining andtest item remnants were assessed.



### 118ROC STUDY NUMBER 843788 Report Page20 TKA 40273 (CGX PR 774) 

### INDIVIDUAL FINDINGS ANIMAL NO. 26, FEMALE After 1hour: 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 ineyeor 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, asappropriate) of opacity, vascularization, reddening, oedema, discharge, staining andtest item remnants were assessed.



### 119RCC STUDY NUMBER 843788 Report Page 21 TKA 40273 (CGX PR 774) 

### INDIVIDUAL FINDINGS ANIMAL NO. 27, FEMALE After 1hour: Cornea: NO ABNORMAL FINDINGS NOTED Iris: NOABNORMAL FINDINGS NOTED Conjunctivae: moderately reddened; obvious swelling with partial eversion of lids Discharge: increased ocular discharge Sciera: moderately reddened Test item: white remnants ineyeor 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: NOABNORMAL 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: NOABNORMAL FINDINGS NOTED Sclera: NO ABNORMAL FINDINGS NOTED Test item: NO REMNANTS EVIDENT

Observations included:cornea, conjunctivae (including nictitatingmembrane), sclera and iris. The presence (or absence, asappropriate) ofopacity, vascularization, reddening, oedema, discharge, staining andtest item remnants were assessed.

120RCC STUDY NUMBER 843788 Report Page 22 TKA 40273 (CGX PR774) 

### 9.3 SUMMARY OF EVALUATION CRITERIA

*EEC Commission Directive 92/69/EEC, July 31, 1992



### 121ROC STUDY NUMBER 843788 Report Page23 TKA40273 (CGX PR 774) EEC COMMISSION DIRECTIVE 92169/EEC, JULY31,1992 Grading of Ocular Lesions CORNEA Opacity: degree of density (area most dense taken for reading) No ulceration or opacity........................................................................ Scattered or diffuse areas of opacity (otherthan slight dulling of normal luster), details of iris clearlyvisible....................................................................1 Easily discernible translucent area, details of iris slightlyobscured......................... 2 Nacreous area, no details of iris visible,size of pupil barely discernible.................... 3 opaque cornea, iris not discernible through theopacity...................................... 4 Area of cornea involved Zero........................................................................................... 0 One quarter (or less) but notzero.............................................................. 1 Greaterthan one quarter, but less than half................................................... 2 Greaterthan half, but lessthan three quarters................................................ 3 Greater than three quarters, upto whole area................................................ 4 IRIS Normal............................................................................................ 0 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection, any ofthese or combination of anythereof, iris still reacting to light (sluggish reaction ispositive)................................................................... 1 No reaction to light, hemorrhage, gross destruction (any or all ofthese)................... 2 CONJUNCTIVAE Redness (refers to most severe reading of palpebral and bulbar conjunctivae when compared with control eye) Bloodvessels normal.......................................................................... Some blood vessels definitelyhyperemic (injected)........................................... 1 Diffuse, crimson color, individualvessels not easily discernible............................. 2 Diffuse beefy red................................................................................ 3 Chemosis: lids and/or nictitating membranes No swelling..................................................................................... 0 Anyswelling above normal (including nictitating membranes)................................ 1 Obviousswelling with partialeversion of lids.................................................. 2 Swellingwith lids about half-closed............................................................. 3 Swellingwith lids more than half-closed....................................................... 4 Note: Reddening ofthe sclerae was assessed usingthe same scoring grades asconjunctivae.

122RCC 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



### 123ROC STUDY NUMBER 843788 Report Page25 TKA 40273 (CGX PR 774) 9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001
SEidgenifsaachea Amrt fur Messwesen S Schweizenische Akkreditierungsatelle Office f~ddraldo m6trologie Serviced'accrdditation suisse Ufficia federale di metrologia A Servizio d'accreditamento avizzero 4p Swiss Federal Office of Metrology 4frS SwissAcreditation Service



# ACCREDITATION

Based on the Accreditation and Designation Ordinance dated 1r~June 1996 the Swiss Federal 

### Office of Metrology grantsthe 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 inaccordance with SN EN 45001. The accredited scope oftesting isdefined in the Official Directory ofthe Accredited Testing Laboratories in Switzerland. The relevant requirements ofthe ISO 9002 standard are also covered bythis accreditation. Accreditation number: STS 085 Date ofthe accreditation: le~September 1994 Date ofthe last renewal ofthe accreditation: 20ei September 1999 The accreditation isvalid until: 19V September 2004 Wabern, 20"' September 1999 Swiss F deral Office of Metrology The Di ctor The hejuofSAS
DrWolfgang Schwitz Hanspeter lschi
M Isaaignsloyofftirmikaal arsanis oft Euiropesan co-opasifr wAodaiiol(EA) for

0511u(5doni, 158mg and csrlIkadhal of prodWM, personnel, quality &nderwironmensal imigemsnt systm .

'124ROC STUDY NUMBER 843788 Report Page 26 TKA 40273 (CGX PR 774) 9.4.2 GLP

- CERTIFICATION

The Swiss GLP Monitoring Authorities
Swiss Federal A SwissAgency forthe Intercantonal Offie officeof Environment. Forests /IA for the Control of SPublic Health and Landscape Medicines 

# Statement of GLP Compliance

Itishereby confirmed that duringthe 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 ofexpertise*

-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 inagreement with the OECD Guidelines for National GLP Inspections and Audits. Itwas found that the aforementioned test facilitieswere operating in compliance withthe Swiss Ordinance relating to Good Laboratory Practice [RS 813.016.5] at the timethey were inspected. Federal OfficeofPublic Health The Director
Bern, November 2000 Prof. Th. Zeltner 

### 'TOX =Toxicology; ACC - Analytical and Clinical Chemistry; ECT =Environmental toxicityon aquatic andterrestrial organisms; ENF - Behaviour inwater, soiland air. Bloacrumulation; PCT - Physical-chemicaltesting; RES - Residue studies; 0TH - Other, tobe 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 SpecialtyChemicals Inc. CH-4002 Basel/Switzerland

Page 1of25



### 126RCC 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 ..........................................................



### 127RCC 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



### 128RCC STUDY NUMBER 843787 Report Page 4 TKA 40273 (CGX PR774) 

### 1. PREFACE 1.1 GENERAL Title TKA 40273 (CGX PR 774): Primary Skin Irritation Study inRabbits (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-4414F~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 theAnimals 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 tenyears. No data will be discarded withoutthe Sponsor's consent.



### 129RCC 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



### 130RCC STUDY NUMBER 843787 Report Page 6 TKA 40273 (CGX PR774) 

### 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 inRabbits (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 QualityAssurance Unit. The dates are given below. Dates andTypes of Dates of Reports tothe Study Director

QAU Inspections andto 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. QualityAssurance: G~~
date: oI-ZIUL 

### -26r,2

131RCC STUDY NUMBER 843787 Report Page7 TKA 40273 (CGX PR 774) GOOD LABORATORY PRACTICE 

### 1.7 STATEMENT OF COMPLIANCE ROC STUDY NUMBER: 843787 TEST ITEM : TKA 40273 (CGX PR774) STUDY DIRECTOR : G.Arcelin TITLE : TKA 40273 (CGX PR 774): Primary Skin Irritation Study inRabbits (4-HourSemi-Occlusive Application) The pH measurement of the test item was performed before the study initiation date and is, therefore, also excludedfrom this statement. This study has been performed incompliance with the Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 bydecision ofthe 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



### 132ROC 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 inthis report, and signed by the Study Director istruthful and accurate, and this report as provided by the test facility iscomplete and unaltered.

Signature ofthe Sponsor: .......................
Date:



### 133ROC STUDY NUMBER 843787 Report Page 9 TKA 40273 (CGX PR 774) 

### 1.9 ACCREDITATION The test facility "ROC Ltd, Toxicology Division" isaccredited according to EN 45001 under accreditation number STS 085 by the SwissAccreditation 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 forTesting of Chemicals, Section4,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 inthe interest of animalwelfare, 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 PlanAmendment No. 2: Reference to the analytical certificate was added.

134RCC 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 gto 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 ofthe 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 anyof the measuring intervals. Thus, the test item did not induce significantor irreversible damage tothe skin. TKA 40273 (CGX PR774) isconsideredto be "not irritating" to rabbit skin.



### 135ROC STUDY NUMBER 843787 Report Page 11 TKA 40273 (CGX PR 774) 

### 3. PURPOSE The purpose ofthis 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 arational basis for risk assessment inman as skin contact isone of the probable routes of human exposure. The test item was administered at 0.5 g/animal, the dosespecified inthe test guidelinesfor a solid test item. 

### 4. MATERIALS AND METHODS 4.1 TEST SYSTEM Test system New Zealand White Rabbit, SPF Rationale Recognized bythe 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 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 anyvisual signs of illness were used forthe study. Allocation Male No. 25 Female Nos. 26 and 27



### 136

# 77ROC 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 haveany influence on the studyand, 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, adlibitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, Itingen. 

### 4.3 TEST ITEM The following information was provided bythe 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 oftest item Stable understorage conditions Expiry date 30-APR-2004 Storage conditions Inthe original container, at room temperature (range of 17-23 'C), awayfrom direct sunlight.



### 137RCC 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, atest 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. Aformulation of 1% inwater was prepared. The pH was found to be 5to 6. 

### 4.5 TREATMENT Four days before treatment, one flank was clipped with an electric clipper, exposing an area of approximately 100 cm 2 (10 cm x10 cm). The skin ofthe animals was examined one day before treatment, and regrown fur of all animals was again clipped. Animals with overtsigns of skin injuryor marked irritation which may have interfered with the interpretation ofthe results were not used inthe test. On the day of treatment, 0.5 gof TKA 40273 (CGX PR 774) was placed on asurgical gauze patch (ca. 4 cm x4cm). 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 theabdomen 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 clearlyvisible atthat time. 

### 4.6 OBSERVATIONS Viability/Mortality Dailyfrom delivery ofthe animals tothe termination oftest. Clinical signs Dailyfrom delivery ofthe animals to the termination of test. Body weights At start of acclimatization, on the day ofapplication and at termination of observation. 

### 4.6.1 IRRITATION SCORES The skin reaction was assessed according to the numerical scoring system listed inthe 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.



### 138RCC STUDY NUMBER 843787 Report Page 14 TKA40273 (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 intabularform, showing the irritation scoresfor 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 ispositive 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. Bodyweights were recorded on-line. No statistical analysiswas performed.



### 139RCC 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 inthe 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 wastherefore 0. 

### 8.3 COLORATION No staining bythe test item ofthe treated skin was observed. 

### 8.4 CORROSION No irreversible alterations of the treated skin were observed nor were corrosive effects evident onthe skin. 

### 8.5 BODY WEIGHTS Animal number 25 showed aslight bodyweight loss (3.6%) during the acclimatization period. The reason for this weight losscould not be established and it isconsidered to be incidental. The body weights of all other rabbits were considered to be within the normal range of variability. Body weight ingrams

Animal Sex First Day of Dayof Last Dayof

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) isconsidered to be "not irritating" to rabbit skin.



### 140RCC STUDY NUMBER 843787 Report Page 16 TKA 40273 (CGX PR 774) 

### 9. APPENDICES 9.1 SKIN IRRITATION SCORES Key toSymbols: M Male F Female



### 141ROC 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 24hours 0 0 27 F 0 0 25 M 0 0 26 F 48hours 0 0 27 F 0 0 25 M 0 0 26 F 72hours 0 0 27 F

______ 0 0 *Examinations were performed atthe specified intervals after removal ofthe dressing.


### TABLE 2: SKIN IRRITATION SCORES

- 

### INDIVIDUAL MEAN VALUES AFTER 24,48AND 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 ofavailable datapoints


### TABLE 3: SKIN IRRITATION SCORES

- 

### ASSESSMENT ACCORDING TO EEC GUIDELINES Evaluated

intervals Erythema Oedema 24 hours 48 hours Not Irritating Not Irritating 72hours The grading scale is presented on page 22.



### 142RCC STUDY NUMBER 843787 Report Page 18 TKA 40273 (CGX PR 774) 

### 9.2 INDIVIDUAL FINDINGS
ANIMAL NO. 25, MALE After 1hour: 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



### 143ROC 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



### 144ROC 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

145RCC STUDY NUMBER 843787 Report Page 21 TKA 40273 (CGX PR 774) 

### 9.3 SUMMARY OF EVALUATION CRITERIA

*EEC Commission Directive 92169/EEC, July31, 1992



### 146ROC STUDY NUMBER 843787 Report Page 22 TKA 40273 (CGX PR 774) EEC COMMISSION DIRECTIVE 92/69/EEC, JULY31, 1992 Grading of Skin Reactions ERYTHEMA AND ESCHAR FORMATION No erythema.................................................................................... 0 Veryslight erythema............................................................................. 1 Well-defined erythema.......................................................................... 2 Moderate to severe erythema .................................................................. 3 Severe erythema (beet redness) or escharformation (injuries indepth 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 1mm)....................................... 3 Severe oedema (raised more than 1mm and extending beyond the area ofexposure)....4



### 147ROC 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



### 148RCC STUDY NUMBER 843787 Report Page24 TKA40273 (CGX PR 774) 9.4.1 ACCREDITATION / EUROPEAN STANDARD EN 45001

S Eidgen~tsischas Amt frjrMessweaen S Schweizerische Akktreditisrungsalle Officefediral dometrologie Service d'accrdditation suisse Ufficio federalse dimetrologia ~ 

### Q J A Servizia d'accreditamento sviuzero SwissFederal office of metrology N2Zr S SwisaAccreditation 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 oftesting isdefined inthe Official Directory ofthe Accredited Testing Laboratories inSwitzerland. The relevant requirements ofthe ISO 9002 standard are also covered bythis accreditation. Accreditation number: STS 085 Date of the accreditation: lew September 1994 Date ofthe last renewal ofthe accreditation: 20" September 1999 The accreditation isvalid until: 19'b September 2004 Wabern, 2et September 1999 Swiss F derI Office of Metrology The Dico The heIofSAS
DrWolfgang Schwitz Hanspeter lschi SAS isas~natory oftw i. mtala agrememnts oftieEiopean cocopeauon forA~oduon(EA) lorcwaian, Isetfi mi coi(cationofpeftos.permo".l quaky wkd envinmontal mnaamnt systs.

149RCC 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

Itisherebyconfirmed that duringthe periodof 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 ofexpertise*

- 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 inagreement with the OECD Guidelines for National GLP inspections andAudits. Itwasfound that the aforementioned test facilitieswere operating in compliancewith the Swiss Ordinance relating toGood Laboratory Practice [RS 813.016.5] at thetimethey were inspected. Federal Office of Public Health The Director
Bern, November 2000 Prof. Th. Zeitner

*TOX - Toxicology; ACC -Analytical andClinicalChemistry, ECT=Envirornental toxicity on aquatic andterrestnial organisms :ENF - Behaviour inwater, sol and air.Bioaccrsnslation PCT - Physical-chemical testing ,RES - Residue studies ;0TH - Other, to be specified



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