### SANITIZEDVERSION FormA 

### U.S. ENVIRONMENTALPROTECTION AGENCY 

### AGENCY USE ONLY 

## PREMANUFACTURE NOTICE Date ofreceipt



### CES 

# 03 OCT

# 27 Aii 6: 11 Company Sanitized

: 

## , Ih ,I, ,'. 

# li 

# iIII 

# n It 

# II II Ii ;l

elll!;;r til!;; lUlal lIumoer 01 pages in the PremanufactureNotice 

### INS1'RUCTIONS You mustprovide all information requested in this form to theextent that it is known to orreasonably ascertainable by you. Make reasonable estimates ifyou do not have actual data. Beforeyou complete this form, you should read the "Instructions Manual for Premanufacture Notification" (the Instructions Manual is available from theToxic Substances Control Act (TSCA) Information Service by calling 202-554-1404, orfaxing 202-554-5603). • Ifa user fee has been remitted for this notice (40CFR 700.45), indicate in the boxes above theTS-userfee identification numberyou have generated. Remember, your user fee 10numbermust also appear on your correspondingfee remittance, which is sent to EPA, HQ Accounting Operations Branch (PM·226), P.O. 360399M, Pittsburgh, PA 15251-6399, Attn. TSCA User fee.
Part I-GENERALINFORMATION You must provide the currently correctCbemical Abstracts (CA) Name ofthe new chemical substance, even ifyouclaim tbe identity as confidential. You may authorize another person to submit chemical identity information for you, but your submission will not becomplete andthe review will not begin until EPA receives this information. A letterin support ofyour submission should reference yourTS userfee identification number. You must submit an original and two copies ofthis notice including all test data. Ifyou claimed any information as confidential, a single sanitized copy must also be submitted.
Part II

- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE Ifthere are several manufacture, processing, oruse operations to be describedin Part II, sections AandBofthis notice, reproduce the sections as needed.
Part Ill-LIST OF ATTACHMENTS Attach additional sheets ifthere is not enough space to answer aquestion fully. Label each continuation sheet with the corresponding section heading. In Part III, listthese attachments, any testdata orotherdata and any optional information included in the notice.

OPTIONAL INFORMATION You may include any information that you want EPAto considerinevaluating the new substance. On page II ofthis form, space has been providedforyou to described pollution prevention and recycling information you may have regarding the new substance. So-called "binding"boxes are included throughoutthis form foryou to indicate your willingness to be bound to certain statementsyou make in this section, such asuse, production volume, protective equipment
.. This option is intendedto reduce delays that routinely accompany the development ofconsent orders orSignificantNew Use Rules. Except in the case ofexemptionapplications (such as TMEA, LVE, LOREX) where certain information provided in such notification is binding onthe submitter when the Agency approves the exemption application, checking abinding box in this notice by itselfprohibit the submitterfrom laterdeviating from the infonnation (except chemical identity) reported in the form. CONFIDENTIALITY CLAIMS You may claim any information in this notice as confidential. To assert a claimon the form, mark (X) the confidential box next to the infonnation that you claimas confidential. To assert a claim in an attachment, circle orbracket the information you claim as confidential. Ifvou claim information in the notices asconfidential you must also provide a sanitized version ofthe notice (including attachments), For additional instructions onclaiming information asconfidential, read the Instructions Manual. Mark (x) ifany infonnation in this notice is claimed as confidential. TEST DATA AND OTHER DATA You arerequired to submitall test data in your possession or control andto provide a description ofall otherdata known to or reasonably ascertainable by you, ifthese data are related to thehealth and environmental effects on the manufacture, processing, distribution in commerce, use, ordisposal ofthe newchemical substance. Standard literature citations maybe submitted for data in the open scientific literature. Complete testdata(written in English). not summaries ofdata, must be submitted [ftheydo not appear in the open literature. You should clearly identifY whether test data is on the substance or on an analog. Also, the chemical composition ofthe tested material should be characterized. Following are examples oftest data and otherdata. Data should be submitted accordingto the requirements of§720.50 ofthe Premanufacture Notification Rule (40 CFR Part 720).

Test Data (Check Below any included in this notice) Environmental fate data 0 Yes Otherdata 181 Yes Health effects data 0 Yes Risk assessments Environmental effectsdata 0 Yes Structure/activity relationships PhysicaVChemical Properties· 181 Yes Test data not in the possession orcontrol ofthe submitter Aphysical and chemical properties worksheet is locatedon the last page ofthis form. TYPE OF NOTICE (Check Only One)

### ~ PMN (Premanufacture Notice) 

## :;g <::":) 

# o INTERMEDIATE PMN (submitted in sequence with final product**P~** C') --I 

# o SNUN (SignificantNew Use Notice) t., 

# - o TMEA (Test Marketing Exemption Application) 

# o LVE (Low Volume Exemption) @40CFR723.50(c)(l) 

# o LOREX (Low ReleaselLow Exposure Exemption)@40CFR 723]!!t)(2) 

# o LVE Modification WREXModification IS THIS ACONSOLIDATED PMN? Yes # ofchemicals .,.--::-

__ 2-::--::-_--,::­ (Prenotice Communication #required, enter# on page 3) EPAFORM 7710·25 (Rev. 5·95) Replaces previous editionsofEPA Form 7710-25. 

## Cl70!50bPublic reporting burden for this collection ofinformation is estimated to average 110 hours per response, including time for reviewing instructions, searching existing datasources, gathering and maintainingthe data needed, and completing and reviewing the collection of information. Send comments regarding the burden estimate or any other aspect ofthis collection ofinformation, including suggestions for reducing thisburden, to Chief, Information Policy Branch, PM-223, U.S. Environmental Protection Agency, 401 M. St., S.W., Washington, D.C. 20460; andto the Office ofManagement and Budget, Paperwork Reduction Act (2070-0012), Washington, D.C. 20503. 

### CERTIFICATION I certify that to the best ofmy knowledge andbelief: I. The company named in Part I, section A, subsection 1 a ofthis notice fonn intends tomanufacture or import for a commercial purpose, otherthan in small quantities solely for research and development, the substance identified in Part I, Section 

### B. 2. All infonnation provided in this notice is complete and truthful as ofthe date ofsubmission. 3. I am submitting with this notice all test data in my possession or control and a description ofall other data known to or reasonably ascertainable by me as required by §720.50 ofthe PremanufactureNotification Rule.
Additional Certification Statements: Ifyou are submitting a PMN, Intennediate PMN, Consolidated PMN, or SNUN, check the following user fee certification statementthat applies: 

### I2$'J TheCompanynamed in Part I, SectionA has remitted the fee of$2500 specified in 40 CFR 700.45(b), or 

# o The Company named in PartI, Section A hasremitted the fee of$1000for an lntennediate PMN (defined @40 CFR 700.43) in accordance with 40 CFR 700.45(b), or 

# o The Company named in Part I Section A is a small business concern under40 CFR700.43 andhas remitted a fee of$100 in accordance with40 CFR 700.45(b). Ifyou are submitting a low volume exemption (LVE) application in accordance with 40 CFR 723.50(c)(l) ora Low release and low exposure exemption (LoRex) application in accordance with 40 CFR723.50(c)(2), checkthe following certification statements: 

# o The manufacturer submittingthis notice intends to manufacture or import the new chemical substance for commercial purposes, otherthan in small quantities solelyfor research anddevelopment, underthe tenns of40 CFR 723.50. 

# oThe manufacturer is familiar with the tennsofthis section and will complywith those tenns; and 

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

# o Ifthis application is for an LVE in accordance with 40 CFR 723.50(c)(I), the manufacturer intends to commence manufacture ofthe exempted substance for commercial purposes within 1yearofthe date ofthe expiration ofthe 30 day review period. The accuracy ofthe statementsyou makeinthis notice should renectyourbest prediction ofthe anticipated facts regardingthe chemical substance described herein. Any knowinl! andwillful misinteruretationis subjectto criminal penally pursuantto 18 USC 1001. Signature andtitle ofAuthorized Official (Original Signature Required) Dale Confidential 

### x Signature ofagenl

- (ifapplicable) Dale

EPAFORM 7710·25 (Rev. 5-95) Page 2 

### xSubmitting Notice (in U.S.)

b. Agent (if applicable) Company
address (number street) State, ZIP Code
Name ofauthorized official

Mailing address (numberand street) City, State, ZIP Code Telephone

c.

## IfyouareSul)miitt~~~~~ea~W1OfaJ<~~hm~km~rnrl~0t~~~::::::::::::::::::::::::::::~::::::::::::~lC:r Joint Submitter (if applicable) Name of

3. Contact (in U.S.) Mailing address (number and street)

Company (number and street) City, State, Code
notice andEPA assigned aPC Numberto the notice. enter the number.
the chemical substance covered by this notice, enter the exemption number assigned by EPA. Ifyou previously submitteda PMN for this substanceenterthe PMNnumber assigned by EPA (i.e. withdrawn orincomplete). .. 5. have submitted anotice Bona

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

Type ofNotice

- Mark (X) I. Manufacture Only 

# D Binding Option Mark EPA FORM 7710-25 (Rev. 5-95) Position Confidential 

### x

Mark (X) 

# • D X ifnone Mark (X) X 

# • D ifnone Mark (X) X 

# • D ifnone 2. Import Only 3. Both 

# D Binding Option Mark Page 3 

# DChemical Abstracts (CA) name ofthe substance based on nomenclature rules and conventions.

a. b.

c. name (Currently correct Chemical Abstracts (CA) Name that is consistent with TSCA Inventory listings for similar substances. ForClass I substances a CAIndexName must be provided. ForClass 2 substances either a CA Index Name or CA Preferred Name must be provided, which ever is appropriate based on CA 9CI nomenclature rules and conventions).

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

# D Method I (CAS Inventory Expert Service - a copy ofthe Identification 

# D Method 2 (Other Source) report obtainedfrom theCAS Inventory Expert Services must be submitted as an attachment to this 

### x 

## x

CAS# 

### x

x

e. For a class correct structure
- (I) precursor substances with theirrespective CAS Registry Numbers. (2) Describe the nature ofthe reaction orprocess. (3) Indicate the range ofcomposition and the typical composition (where appropriate). (4) Provide a correct representative orpartial chemical structure 

### x diallram.**a~** comnlete**a~** can he known. ifone can he**rea~nahlv a~certained.** 

this box ifyou attach a continuation sheet.

EPA FORM 7710-25 (Rev. 5-95) Page 4- (on

I. a. Class ofsubstance

- Mark (X) name correct

c. you

as an d.

e.

Mark (X) this box if Ifanother person will submit chemical identity information for you (foreither Item I or 2), mark (X) the box atthe right. that in a sheet. Class I or2 chemical substances (for definitionsofclass 1 and class 2 substances, see the Instructions Manual) 

# D D I Class I or 2 Class 2 Inventory ForClass I substances aCA Index Name must be provided. ForClass 2 substances either a CA Index Name or CAPreferred Name must be provided, which ever is appropriate basedon CA 9CI nomenclature rules and conventions). Confidential



### x x

Method 1(CAS Inventory Expert Service - a copyofthe Identification report obtained from the CAS Inventory Expert Services mustbe Molecular formula andCAS RegistryNumber(ifa number alreadyexists for the substance) 

## x

x and correct structure diagram. Fora class 2 substance - (I) List the immediate precursor substances with their respective CAS Registry Numbers. (2) Describe the natureofthe reaction orprocess. (3) Indicate the 

### x range ofcomposition and thetypical composition (where appropriate). (4) Provide a correct representative orpartial chemical structure di3.llfam. as comnlete**a~** can he known. ifone can he**rea~onahlv a~certained,** 

attach a continuation sheet.

EPA FORM 7710-25 (Rev. 5-95) Page4-2a. Indicate the number-average weight ofthe lowest molecularweight composition ofthe polymer you intend to manufacture. Indicate maximum weight percent oflowmolecularweight species (not includingresidual monomers, reactants, orsolvents) below 500 and below 1,000 absolute molecular weightofthat composition. Describe the methods ofmeasurement or the basis for your estimates: OPC

# 0 Other 

# 0:(Specify) i) lowest number average molecular weight: ii) maximum weight % below 500 molecular weight: iii) maximum weight % below 1000 molecular weight: attach a continuation sheet. You must make separate ity claims for monomeror and residual information. "Confidential" box next to any item you claim as confidential Confidential

(1)

- Provide the specificchemical name and CAS Registry Number (ifa number exists) ofeach monomerorotherreactant used in the manufacture of the polymer. Mark (X) this column ifentry in column (1) is confidential. Indicate the typical weight percent ofeach monomer orother reactant in the polymer. (2)

- (3)

- (4)

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

- (6)

- Indicate the maximum weight percentofeach monomerorotherreactant that may bepresent as a residual in the polymer as manufactured for commercial purposes. umn if in column is confidential. Monomer orotherreactant and CAS RegistryNumber
% % % % % % %

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

# o Method 1 (CAS Inventory Expert Service - a copyofthe identification report 

# 0 Method 2 (other source) obtainedfrom CAS Inventory Expert Service must be submitted as d. Thecurrently correct Chemical Abstracts (CA) name for the polymer that is consistent with TSCAInventory listings for similar polymers.

e. a correctrepresentative orpartial chemical structure diagram, as complete as can be known, ifone can be reasonably ascertained.

attach a continutation sheet. % % %

% % %

EPA FORM 7710-25 (Rev. 5-95) Page 5substance. Provide the CAS Number 

### x (a) Identify each impurity that may be reasonably anticipated to bepresent in the chemical substance as manufactured for commercial purpose. Providethe CAS RegistryNumber ifavailable. Ifthere are unidentified impurities, enter ·'unidentified." Estimatethe maximum % ofeach
.. %. Maximum percent dential % X
% X
% X
% X
%
%
%



# D Mark (X) this box ifyou attach a continuation sheet 5. 1m 

# D Mark (X) this box ifyou attach a continuation sheet. you must provide a generic name your substancethat reveals the specific chemical identity ofthe newchemical substanceto the maximum extent possible. Refer to the TSCAChemical Substance Inventory, 1985 Edition, Appendix B for guidanceon developing generic names. Halogen-substituted oxetane

EPA FORM 7710-25 (Rev. 5-95) Page 6 X

## PartI

-- GENERAL INFORMATION

-- Continued Section C

-- PRODUCTION, IMPORT,AND USE INFORMATION: Mark (X)the "Confidential"box nextto any itemyou claim as confidential. 1. Production volume

-- Estimate the maximum production volume duringthe first 12 months ofproduction. Also estimate the maximum production volume for any consecutive 12-month period during the first three years ofproduction. Estimates should be on 100% new chemical substance basis. For a Low 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) in the bindingbox. Ifgranted, you are bound to this volume Maximum first 12-month production (kglyr) Maximum 12-monthproduction (kglyr) Confi- Binding (100% new chemical substance basis) (100% new chemical substance basis) dential Option Mark (x) X 2. Use Information

-- You must make separate confidentiality claims for the description ofthe category ofuse, the percent ofproduction volume devoted to each category, the formulation of the new substance, and other use information. Mark (X) the "Confidential" Box next to any item you claim as confidential. a. (I)

-­ Describe each intended category ofuse ofthenew chemical substance by function and application
.. (2)

-­ Mark (X) this column ifentry column (1) is confidential business information (CBI). (3)

-­ Indicate your willingness to have the information provided in column (1) binding. (4)

-­ Estimate the percent oftotalproduction for the first three years devoted to each category ofuse. (5)

-­ Mark (X) this column ifentry in column (4) is confidential business information (CBI). (6)

-­ Estimate the percent ofthe new substance as formulated in mixtures, suspensions, emulsions, solutions, orgels as manufactured for commercial purposes at sites under yourcontrol associated with each category ofuse. (7)

-­ Mark (X) this column ifentry in column (6) is confidential business information (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 provided in (8) binding. (9) -­ Mark (X) this column ifentr (ies) in column (8) is (are) confidential business information (CBI). Category ofuse (1) CBI Binding Produc- CB] %in CBI %ofsubstance expected peruse CBI Option tion% Form­ (8) (by function and application i.e. adispersive dye Mark (x) ulation Site- Con-'" Indus- Com- Binding (3) forfinishing polyesterfibers) (2) (4) (5) (6) (7) limited sumer trial mercial Option (9) Monomer used in the production of 

### x x x reactive oolvmers for surface % % coating materials and other 

## - - -% 

## - -% 

## - - 

# - - - - 

## - oolvmer intermediates [open 

### I non-<lispersive use] % % % % % % % % % % '" Ifyou have identified a "consumer" use, please provide on acontinuation sheet adetailed description ofthe use(s) ofthis chemical substance in consumerproducts.

In addition include estimates ofthe concentrationofthe new chemical substance as expected in consumer products and describe the chemicalreactions bywhich this substance loses its identity in the consumer product. DMark(X) this box ifyou attach acontinuation sheet. b. Generic Ifyou claim any category ofuse description in subsection 2aas confidential, enterageneric description ofthatcategory. Read the use Instructions Manual forexamples ofgeneric use descriptions. description N/A 

### I

I

. D Mark (X) thIS box Ifyou attach acontmuatlOn sheet. Binding Option 3. Hazard Information

- Include in the notice acopy ofreasonable facsimile ofany hazard warning statement, label, material safety data sheet, orother

information which will be provided to any person who is reasonably likely to be exposed to this substanceregarding protective equipmentor practices Mark (x) forthe safehanding, transport, use, ordisposal ofthe new substance. List in part III hazard informationyou include.

Mark (X) this box ifyou attach hazard information.

EPAFORM 77\0-25 (Rev. 5-95) Page 7box next to any you Complete section A for eachtype of1II(I",U"".,u.",**pro(:es~,In:g,** operation Involvingthe new chemical substance at industrial sites you control. Importers do not have to completethis**"n,~r,,·tl"""** outsidethe U.S.; however, you may still have reporting requirements if there are further industrial ou must these See in"·trll,,ti£\nC Confiofthe site at which the on,,,·,:,t,on will occur. 1. dential 

# x Site address (numberand street) City, County, State,ZIP code
Ifthe same operation will occur at morethan one site, enterthe numberofsites. IdentifY the additional sites on a continuation sheet, and ifany ofthe sites have significantly different production rates oroperations, include all the information requested in this section for those 

# .. sites as attachments. 

## x Batches/year 

## x 

# x
d. Process description Mark (X) to indicate yourwillingness to have your process description binding. 

## x (I) Diagram the major unit operation steps and chemical conversions. Include interim storage and transport containers (specify- e.g. 5 gallon pails, 55 gallon drum, rail car, tanktruck, etc.). (2) Provide the identity, the approximate weight (by kg/day orkg/batch on a 100"!. new chemical substance basis), and entry point ofall starting materials and feedstocks (including reactants, solvents, catalysts, etc.), and ofall products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily or perbatch.). (3) Identify by numberthe points ofrelease, including small or intermittentreleases, to the environment ofthenew chemical substance. 2. Continuous

this box ifyou attach acontinuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 8

## Part 11-- HUMAN EXPOSURE AND ENVIRONMENTAL RELEASE

-- Continued Section A

-- INDUSTRIALSITESCONTROLLED BY mESUBMITTER

-- Continued 2. Occupational Exposure

-- You mustmake separate confidentiality claims for the description ofworker activity, physical form ofthe new chemical substance, number ofworks exposed, and duration ofactivity. Mark (X) the "Confidential" box next to any item you claim as confidential. (l)

-- Describe the activities (i.e. bag dumping, tote filling, unloadingdrums, sampling, cleaning, etc.) in which workers may be exposed tothe substance. (2)

-- Mark (X) this column ifentry in column (I) is confidential business information (CBl). (3)

--Describe any protective equipment and engineering controls used to protect workers. (4) and (6) --Indicate your willingness tohave the information provided in column (3) or (5) binding. (5) --Indicate the physical form(s) ofthe new chemical substance (e.g., solid: crystal, granule, powder, ordust) and % new chemical substance (ifpartofamixture) at the time ofexposure. (7)

--Mark (X) this column ifentry in column (5) is confidential business information (CSI). (8)

-- Estimate the maximum numberofworkers involved in each activity for all sites combined. (9)

-- Mark (X) this column ifentry in column (8) is confidential business information (CSl). (10) and (II) ­ Estimate the maximum duration ofthe activity for any worker in hours per day and days peryear. (\2)

-- Mark (X) this column ifentries in columns (10) and (II) are confidential business information (CSI). Worker activity CBl Protective Equipment! Binding Physical fonns(s) Binding CBI #of CBl Maxirnu duration CBl rn (i.e., bagdumping, filling drums) Engineering Controls Option and %new Option Worker Hrslday Dayslyr Mark (x) substance Mark (x) s (I) (2) (3) (4) (5) (6) (7) Expose (9) (\0) (\I) (12) d (8) i X X

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

3. Environmental Release and Disposal

-- You must make separate confidentiality claimsfor the release numberand the amount ofthe new chemical substance released and otherrelease and disposal information. Mark (X) the "Confidential" box next to each item you claim as confidential. (I)

-- Enterthe numberofeach release point identified in the process description, part II, section A, subsection Id(3). (2)

--Estimate the amountofthe new substancereleased (a) directly to the environment or (b) into control technology (in kg/day or kg/batch). (3)

--Mark (X) this column ifentries in columns (1) and (2) are confidential business information (CSI). (4) --Identifythe media (stack air, fugitive air (optional-see Instruction Manual), surface water, on-site oroff-site land or incineration, POTW, orother(specify» to which the new substance will be released from that release point. (5)

-- a. Describe control technology, ifany, and control efficiencythat will be used to limit the release ofthe new substance to the environment. For releases disposed ofon land, characterizethe disposal method and statewhether it is approved for disposal ofRCRA hazardous waste. On a continuation sheet, for each sitedescribe any additional disposal methods that will be used and whether the waste is subject to secondaryortertiaryon-site treatment. b. Estimate the amountreleased to theenvironment after control technology (in kg/day). (6)

-- Mark (X) this column ifentries in columns (4) and (5) are confidential business information (CSI). (7) --Identifythedestination(s) ofreleases to water. Please supplyNPDES (National Pollutant Discharge Elimination System) numbers for direct discharges or NPDES numbers ofthe POTW (Publici Owned Treatment Works). Mark (X) ifthe POTW nameor NPDES # is confidential business information (CBI). Release Amount ofnew substance CSI Media of Control technology and efficiency (you may wish to optionally attach efficiency data) CBI Number released release

(I) (2a) (2b) e.g, stacksir Binding (3) (4) (Sa) Mark (Xl (5b) (6) X
X

(7) Mark (X) the 

# D POTW provide name(s) below: CBI 

# D Navigable 

# D Other

- Specify provide NPDES# CSI destination(s) of ,--­ waterway releases towater.
Mark (X) this box ifyou attach a continuation sheet. EPA FORM 7710-25 (Rev. 5-95) Page 9

## Part 11-- HUMAN EXPOSURE AND ENVIRONMENTALRELEASE

-- Continued INDUSTRIAL SITES CONTROLLEDBY OTHERS Complete section Bfor typical processing oruse operations involvingthe new chemical substance at sites you do not control. Importers do not have to complete this section for operations outside the U.S.; however, you must report any processing oruse activities afterimport. See the Instructions Manual. Complete aseparate section B for each type of processing, or use operation involVing the new chemicalsubstance. Ifthe same operation is performed at more than one site describe the typical operation common to these sites. Identify additional sites on acontinuation sheet. 1. Operation Description

-- To claim information in this section as confidential,circle or bracketthe specific information that you claim as confidential. (I)

-- Diagram the major unit operation steps and chemical conversions, including interim storage and transportcontainers (specify

-e.g. 5gallon pails, 55 gallon drums, rail cars, tank trucks, etc). On the diagram, identify by letterand briefly describeeach worker activity. (2)

-- Provide the identity, the approximate weight (by kg/day orkglbatch, on an 100% new chemical substance basis), and entry pointofall feedstocks (including reactants, solvents and catalysts, etc) and all products, recycle streams, and wastes. Include cleaning chemicals (note frequency ifnot used daily orperbatch). (3)

- Identify by number the points ofrelease, including small or intermittentreleases, to the environmentofthe new chemical substance. (4) Please enter the # ofsites (rememberto identify the locations of these sites on acontinuation sheet): [ 

### ] # ofsites 

### CONFIDENTIAL BUSINESS INFORMATION: 

# [ ]

I

# D Mark (X) this box ifyou attach acontinuation sheet.

2. WorkerExposurelEnvironmental Release (I) -­From the diagram above, provide the letterforeachworker activity. Complete 2-8 for each worker activity described. (2) --Estimate the numberofworkers exposed for all sites combined. (4) --Estimatethe typical duration ofexposure per worker in (a) hours perday and (b) days per year. (6) -- Describe physical form ofexposure and %new chemical substance (ifin mixture), and any protective equipment and engineering controls, ifany, used to protect workers. (7)

-- Estimate the percentofthe new substance as formulated when packaged orused as afinal product. (9)

--From the process diagram above, enterthe number ofeach release point. Complete 9-13 for each release point identified. (10)

-- Estimate the amount ofthenew substance released (a) directly to the environment or(b) into control technology to the environment (in kg/day or kglbatch). (12)

-­ Describe media ofrelease i.e. stack air, fugitive air(optional-see Instructions Manual), surfacewater, on-siteoroff-site land or incineration, POTW, orother (specify) and control technology, ifany, that will be used to limit the release ofthe new substance to the environment. (14)

-- Identify byproducts which may result from the operation.

(3), (5), (8), II), (13) and (IS) ­ Mark (X) this column ifany ofthe nroceedin!!. entries are confidential business information (CBI). Letter # of CBI Duration CBI Protective Equip. I "loin CBI Release Amount of CBI Media ofRelease CBI

of Workers of Engineering Controls! Fonn· Number New & Control Act· Exposed Exposure Physical Form and % ulation Substance Technology ivity new substance Released (I) (2) (3) (4a) (4b) (5) (6) (7) (8) (9) (lOa) (lOb) (Ill (12) (13) 

# I

oducts: (15) (X)this box ifyou attach acontinuation sheet, EPA FORM 7710-25 (Rev. 5-95) Page 10

## OPTIONALPOLLUTIONPREVENTIONINFORMATION To claim infonnation in this section as confidential circle orbracket the specific infonnationthat you claim as confidential. 

### Inthis sectionyoumayprovide informationnotreported elsewhere inthis form regardingyour efforts to reduce orminimizepotential risks associatedwith activities surrounding manufacturing, processing, use and disposal ofthe PMN substance. Please include new informationpertinent to pollutionprevention, including source reduction, recycling activities and saferprocesses orproducts available due to thenewchemical substance. Source reductionincludes the reduction inthe amount ortoxicity ofchemical wastes bytechnological modification,process and procedure modification, productreformulation, raw materials substitution, and/or inventory control. Recycling refers to the reclamation ofuseful chemical components from wastes that would otherwisebe treated orreleased as air emissions orwater discharges, orland disposal. Descriptions ofpollution prevention, sourcereduction and recycling should emphasizepotential riskreduction subsequentto compliance with existingregulatory requirements and can be either quantitative orqualitative. The EPAis interested inthe informationto assess overall netreductions in toxicity orenvironmental releases and exposures, notthe shifting ofrisks to otherenvironmental mediaornon-environmental areas (e.g., occupational orconsumer exposure). In addition, informationontherelative cost orperformance characteristics ofthePMN substance to potential alternatives maybe provided. 

## All information providedin this section will be taken into consideration duringthe review ofthis substance. SeeInstructions Manualand Pollution Prevention Guidancemanual for guidanceand examples. Describethe expected net benefits, such as (1) an overall reduction in riskto human health orthe environment; (2) a reduction in the volume manufactured; (3) a reduction inthe generation ofwaste materials through recycling, source reduction orothermeans; (4) a reduction in potentialtoxicity orhuman exposure and/orenvironmental release; (5) an increase in product perfonnance, a decrease in the cost ofproduction and/or improved operation efficiency ofthe new chemical substance in comparison to existing chemical substances used in similar application; or(6) the extent to whichthe new chemical substance may be a substitute for an existing substancethat poses a greater overall riskto human health orthe environment. 

### See Attachment #13 - Pollution Prevention Information.

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

EPA FORM 7710-25 (Rev. 5-95) Page 11

## Part III -- LIST OFATTACHMENTS Attach continuation sheets for sections ofthefonn and test data and other data (includingphysical/chemical properties and structure/activity infonnation), and optional infonnation afterthis page. Clearly identifY the attachment and the section ofthefonn to which it relates, ifappropriate. Numberconsecutively thepages 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 howto claimany infonnation inan attachment as confidential. You must include with the sanitized copy ofthe notice fonn a sanitized version ofany attachment in which you claim infonnation as confidential. Attachment name 

### Material SafetvData Sheet (MSDS) Physical and Chemical Properties Worksheets Attachment #1 - CAS Inventory Expert Service Reports Attachment#2 Attachment#3 Attachment

### #4 Attachment#5 Attachment

### #6 Attachment #7 Attachment #8 Attachment

### #9 Attachment #10 Attachment#11 Attachment#12 Attachment #13 - Pollution Prevention Information Attachment #14 - Toxicological Data on Two Structurally Similar Compounds (Ames Tests) Attachment Confi· page number(s) dentiaJ 13 - 24 25 -26 27 - 29 30 - 33 x 34 x 35 x 36 x 37 - 38 x 39 -41 x 42 -43 x 44 -46 x 47 x 48 -49

• x 50 - 52 x 53 - 54 55 - 101



# l 

# J Mark (X) this box ifyou attach acontinuation sheet. Enterthe attachment name and number.
EPA FORM 7710·25 (Rev. 5·95) Page 12

### ISSUED: MARCH 2000 PAGE 1OF6 

# [ ] MATERIAL SAFETY DATA SHEET Section 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION 

## MANUFACTURER: [ ] [ ] [ 

## ] [ ] 

## EMERGENCY TELEPHONE: CHEMTREC

### ® (800) 424-9300** 24 hour info line ** CHEMTREC 

## ® numberto be used only in the eventofa chemical emergency 

## ISSUE DATE: March 7,2001 

## REVISED: INFORMATION TELEPHONE NUMBER: 

# [ ) Monday-Friday, 8:00 a.m. - 5:00 p.m. 

## PRODUCT NAME: [ 

## ] CHEMICAL FAMILY: Halogen substituted oxetane CHEMICAL FORMULA: Proprietary DOT PROPER SHIPPING NAME: 

## N/A 10 NUMBER: 

## N/A DOT HAZARD CLASSIFICATION: 

## N/A



# 030013

### ISSUED: MARCH 2000 PAGE20F6
SECTION 2: COMPOSITIONIINFORMATION ON INGREDIENTS 

## COMPONENT PERCENT WEIGHT EXPOSURE LIMIT UNITS AGENCY TYPE Halogen substituted oxetane 

### CAS# N/A 

### 100% None Established *Indicates toxic chemicals subject to the reporting requirements ofsection 313 oftitle III and of40 CFR 372. 

## HAZARD EMERGENCY OVERVIEW May bea mildskin, eye and respiratory irritant.
SECTION 3: HAZARDS IDENTIFICATION HEALTH HAZARDS/ROUTES OF ENTRY EYE CONTACT: YES, MAY BE MILDLY IRRITATING TO EYES. SKIN CONTACT: YES, MAY BE MILDLY IRRITATING TO SKIN. INHALATION (BREATHING): YES, MAY BE MILDLY IRRITATING TO RESPIRATORY TRACT. INGESTION: POSSIBLE CARCINOGENICITY STATUS: NOT LISTED BY NTP, IARC OR OSHA 

# I SECTION 4: FIRSTAID MEASURES EYE CONTACT: FLUSH WITH WATER FOR 15 MINUTES. GET MEDICAL ATTENTION. SKIN CONTACT: FLUSH WITH WATER FOR 15 MINUTES. REMOVE CONTAMINATED CLOTHING. GET MEDICAL ATTENTION. INHALATION: REMOVE TO FRESH AIR. GET MEDICAL ATTENTION. INGESTION: INDUCE VICTIM TO VOMIT. GET MEDICAL ATTENTION. SECTION 5: FIRE FIGHTING MEASURES

### ISSUED: MARCH 2000 PAGE30F6 EXTINGUISHING MEDIA: WATER, CARBON DIOXIDE, DRY CHEMICAL OR FOAM. UNUSUAL FIRE AND EXPLOSION HAZARD: Mayform [ ] when decomposing in heat orfire. Material is reactive with Lewis Acids/Strong Acids. SPECIAL FIRE FIGHTING PROCEDURES: WEAR POSITIVE PRESSURE SELFCONTAINED BREATHING APPARATUS (SCBA). AVOID COMBUSTION PRODUCTS. CONTROL RUN-OFF.
SECTION 6: ACCIDENTAL RELEASE MEASURES SPILL AND LEAK PROCEDURES WEAR PERSONAL PROTECTIVE EQUIPMENTTOAVOID EXPOSURE. CONTAIN SPILLS; KEEP MATERIAL FROM BEING DISCHARGED TO LAND, WATERWAYS ORAIR. PICK UP WITH INERTABSORVENT; PLACE INTO WASTE CONTAINERS. 

# I SECTION 7: HANDLING AND STORAGE CONTAINS LIQUIDS. USE GLOVES (NEOPRENE OR NITRILE). STORE IN A COOL AND DRY LOCATION. 

# I SECTION 8: EXPOSURE CONTROUPERSONAL PROTEC"nON VENTILATION: GENERAL ROOM VENTILATION IS ADEQUATE. RESPIRATORY PROTECTION: NONE REQUIRED PROTECTIVE GLOVES: NEOPRENE OR NITRILE EYE PROTECTION: SAFETY GLASSES OTHER PROTECTION: N/A



# 000015

### ] ISSUED: MARCH 2000 

### ] PAGE40F6
SECTION 9: PHYSICALAND CHEMICAL PROPERTIES BOILING POINT RANGE: 300 

### C @ 0.2 mm 

# R!:!: NEUTRAL SOLUBILITY IN WATER: < 

### 1

## % PERCENTVOLATILE: < 

### 1

## % APPEARANCE: Colorless viscous oil ODOR: Sweet odor. VAPOR DENSITY: 

## N/A VAPOR PRESSURE: 

## N/A EVAPORATION RATE (BUOAC=100): 

## N/A FLASH POINT: Not Established LEL: 

## N/A UEL: 

## N/A 

# I SECTION 10: STABILITYAND REACTIVITY STABILITY: STABLE INCOMPATIBILITY (MATERIALS TO AVOID): WILL REACT WITH LEWIS ACIDS AND STRONG ACIDS. CONDITIONS TO AVOID: TEMPERATURE ABOVE 1500 

### C HAZARDOUS DECOMPOSITION PRODUCTS: [ HAZARDOUS POLYMERIZATION: MAY OCCUR 

# I SECTION 11: TOXICOLOGICAL INFORMATION NEGATIVE RESULTS FOR MUTAGENICITY ON AMES TEST FOR SIMILAR SUBSTANCES. 

# I SECTION 12: ECOLOGICAL INFORMATION NOTAVAILABLE AT THIS TIME. 

# I SECTION 13: DISPOSAL INFORMATION DISPOSE IN ACCORDANCE WITH FEDERAL, STATE AND LOCAL REGULATIONS. 

# GG0016

### ] ISSUED: MARCH 2000 PAGE50F6 SECTION 14: TRANSPORT INFORMATION 

### NOTA HAZARDOUS MATERIALAS REGULATED BY DOT. 

# I SECTION 15: REGULATORY INFORMATION OSHA HAZARD COMMUNICATION RULE 29 CFR 1910.1200: 

### Compounds listed are defined as hazardous ingredients according to OSHA (Occupational Safety and Health Administration) Regulations. Routine handling ofthis material is not expected to generate airborne concentrations in excess ofOSHA Permissible Exposure Limits orACGIH (American Conference of Government Industrial Hygienists) TLV (Threshold Limit Value). However, each use ofthis product should be evaluated to determine ifexposure hazard exists. CERCLAISUPERFUND, 40 CFR 117 PART 302: 

### N/A TOXIC SUBSTANCES CONTROL ACT (TSCA): 

### This is a proprietary product of [ ] and is currently being managed under a Low Volume Exemption to TSCA, (40 CFR 723.50(c)(1». 40 CFR 723 limits the use ofthis material tothe subsequent production of reactive polymers for coating material and other polymer intermediates. EUROPEAN INVENTORY OF EXISTING COMMERCIAL CHEMICAL SUBSTANCES (EINECS) AND EUROPEAN LIST OF NOTIFIED CHEMICAL SUBSTANCES (ELlNCS): 

### This is a proprietary product of [ ] and is currently being managed in the European Community underArticle 13 paragraph 2. of Substances Directive 

### (67/548/EEC) as a substance intended solely for purposes of scientific research and development carried out under controlled conditions. This product is to be used within the European Community only for research and development as permitted by EC and other appropriatejurisdictional regulations. CANADIAN ENVIRONMENTAL PROTECTION ACT(CEPA): 

### This is a proprietary product of [ ] and is currently being managed in Canada under the provisions ofthe Research and Development Special Use Category as a substance intended solely for purposes ofscientific research and development carried out under controlled conditions. It is not on the DSL or NDSL. This product is to be used only for research and development as permitted by the CEPA and other appropriate jurisdictional regulations.


# G0001?

### ISSUED: MARCH

## 2000 PAGE60F6
SEC1"ION 16: OTHER INFORMATION HAZARD RATINGS: NFPA HAZARD CLASS 2 

### HMIS HAZARD CLASS 3 

### HAZARD RANKING Health Hazard: 2 Health Hazard: 1 

### 0- Least Flammability: 

## 0 Flammability: 0 1 - Slight Reactivity: 2 Reactivity: 2 2 - Moderate Other: - PPE: 3 - High 4 - Extreme 2 _ 

### National Fire Protection Association (NFPA) Rating identifies the severity of hazards of material during a fire (i.e. "on fire"). 3_ 

### Hazardous Materials Identification System (HMIS) National Paint and Coatings Association ratings apply to product "as Packaged". Information on this form is furnished solely for the purpose ofcompliance with the Occupational Safety and Health Act of 1970, and applicable regulations, and should not 

### be used for any other purpose. No warranty express or implied, of merchantability, fitness for purpose, or ofany other nature regarding this data orthe product is made herein. 

### Issue date: March 7, 

## 2001 Revision date: April 4, 

## 2001 Written by: [ Reviewed by: Awaiting independenttoxicological review. Approved by: [ OUTSIDE THE UNITED STATES, THIS MATERIAL IS PROVIDED FOR RESEARCH AND DEVELOPMENT USE ONLY BY THE ORIGINAL RECIPIENT. THIS MATERIAL MAY NOT BE TRANSFERRED TO ANY OTHER INDIVIDUAL, CONCERN OR ORGANIZATION WITHOUT THE EXPRESSWRITTEN APPROVAL OF [ ]. THE U.S. TOXIC SUBSTANCE CONTROLACT, 40 CFR 723, THE EUROPEAN COMMUNITY SUBSTANCES DIRECTIVE 

### (675/548/EEC) AND CANADA'S CEPA REGULATIONS LIMITS THE USE OF THIS MATERIAL.



# G00013

### Current Issue: December 18, 2002 PAGE 1OF6 

# [ ] MATERIAL SAFETY DATA SHEET Section 1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATION 

## MANUFACTURER: [ ] 

## [ ] [ ] 

### [ ] 

## EMERGENCY TELEPHONE: CHEMTREC 

## ® (800) 424-9300** 24 hour info line ** CHEMTREC ® numberto be used only in the eventofa chemical emergency 

## ISSUE DATE: December 18,2002 

## REVISED: Original INFORMATION TELEPHONE NUMBER: [ ] Monday-Friday, 8:00 a.m. - 5:00 p.m. 

## PRODUCT NAME: [ ] CHEMICAL FAMILY: Halogen substituted oxetane CH EMICAL FORMULA: Proprietary DOT PROPER SHIPPING NAME: 

## N/A 10 NUMBER: 

## N/A DOT HAZARD CLASSIFICATION: 

## N/A



# G00019

### Current Issue: December 18, 2002 

# 1 ] PAGE20F6 SECTION 2: COMPOSrnON/INFORMATION ON INGREDIENTS COMPONENT PERCENT

### EXPOSl~RE 

# I UNITS AGENCY TYPE WEIGHT LIMIT Halogen substituted 

### 100 

### % None oxetane 

### Established CAS# N/A 

### *Indicates toxic chemicals subjectto the reporting requirements ofsection 313 oftitle III and of40 CFR 372. 

## HAZARD EMERGENCY OVERVIEW May be a mild skin, eye and respiratory irritant.
SECTION 3: HAZARDS IDENTIFICATION HEALTH HAZARDS/ROUTES OF ENTRY EYE CONTACT: YES, MAY BE MILDLY IRRITATING TO EYES. SKIN CONTACT: YES, MAY BE MILDLY IRRITATING TO SKIN. INHALATION (BREATHING): YES, MAY BE MILDLY IRRITATING TO RESPIRATORY TRACT. INGESTION: POSSIBLE CARCINOGENICITY STATUS: NOT LISTED BY NTP, IARC OR OSHA 

# I SECTION 4: FIRST AID MEASURES EYE CONTACT: FLUSH WITH WATER FOR 15 MINUTES. GET MEDICAL ATTENTION. SKIN CONTACT: FLUSH WITH WATER FOR 15 MINUTES. REMOVE CONTAMINATED CLOTHING. GET MEDICAL ATTENTION. INHALATION: REMOVE TO FRESH AIR. GET MEDICALATTENTION. INGESTION: INDUCE VICTIM TO VOMIT. GET MEDICAL ATTENTION. SECTION 5: FIRE FIGHTING MEASURES**Gaoo~~o**

### Current Issue: December 18, 

## 2002 PAGE30F6 EXTINGUISHING MEDIA: WATER, CARBON DIOXIDE, DRY CHEMICAL OR FOAM. UNUSUAL FIRE AND EXPLOSION HAZARD: Mayform [ ] when decomposing in heat orfire. Material is reactive with Lewis Acids/Strong Acids. SPECIAL FIRE FIGHTING PROCEDURES: WEAR POSITIVE PRESSURE SELFCONTAINED BREATHING APPARATUS (SCBA). AVOID COMBUSTION PRODUCTS. CONTROL RUN-OFF. SECTION 6: ACCIDENTAL RELEASE MEASURES SPILLAND LEAK PROCEDURES WEAR PERSONAL PROTECTIVE EQUIPMENT TO AVOID EXPOSURE. CONTAIN SPILLS; KEEP MATERIAL FROM BEING DISCHARGED TO LAND, WATERWAYS OR AIR. PICK UP WITH INERT ABSORVENT; PLACE INTO WASTE CONTAINERS. 

# I SECTION 7: HANDLING AND STORAGE CONTAINS LIQUIDS. USE GLOVES (NEOPRENE OR NITRILE). STORE IN A COOL AND DRY LOCATION. 

# I SECTION 8: EXPOSURE CONTROUPERSONAL PROTECTION VENTILATION: GENERAL ROOM VENTILATION IS ADEQUATE. RESPIRATORY PROTECTION: NONE REQUIRED PROTECTIVE GLOVES: NEOPRENE OR NITRILE EYE PROTECTION: SAFETY GLASSES OTHER PROTECTION: N/A**G~0021**

### [ Current Issue: December 18, 2002 

### [ PAGE40F6 SECTION 9: PHYSICAL AND CHEMICAL PROPERTIES BOILING POINT RANGE: 300 

### C @ 0.2 mm 

# .Il!:!: NEUTRAL SOLUBILITY IN WATER: < 

### 1% PERCENTVOLATILE: < 

### 1% APPEARANCE: Colorless viscous oil ODOR: Sweet odor. VAPOR DENSITY: N/A VAPOR PRESSURE: N/A EVAPORATION RATE (BUOAC=100): N/A FLASH POINT: Not Established LEL: N/A UEL: N/A SECTION 10: STABILITYAND REACTIVITY STABILITY: STABLE INCOMPATIBILITY (MATERIALS TOAVOID): WILL REACT WITH LEWIS ACIDS AND STRONG ACIDS. CONDITIONS TO AVOID: TEMPERATURE ABOVE 1500 

### C HAZARDOUS DECOMPOSITION PRODUCTS: [ HAZARDOUS POLYMERIZATION: MAY OCCUR 

# I SECTION 11: TOXICOLOGICAL INFORMATION NEGATIVE RESULTS FOR MUTAGENICITY ON AMES TEST FOR SIMILAR SUBSTANCES. SECTION 12: ECOLOGICAL INFORMA"rlON NOTAVAILABLE AT THIS TIME. SECTION 13: DISPOSAL INFORMATION DISPOSE IN ACCORDANCE WITH FEDERAL, STATE AND LOCAL REGULATIONS. 

## I

I

## ] Current Issue: December 18, 2002 ] PAGE50F6 SECTION 14: TRANSPORT INFORMATION NOTA HAZARDOUS MATERIALAS REGULATED BY DOT. TION 15: REGULATORY INFORMATION OSHA HAZARD COMMUNICATION RULE 29 CFR 1910.1200: Compounds listed are defined as hazardous ingredients according to OSHA (Occupational Safety and Health Administration) Regulations. Routine handling ofthis material is not expected to generate airborne concentrations in excess of OSHA Permissible Exposure Limits orACGIH (American Conference of Government Industrial Hygienists) TLV (Threshold Limit Value). However, each use ofthis product should be evaluated to determine ifexposure hazard exists. CERCLAISUPERFUND, 40 CFR 117 PART 302: N/A TOXIC SUBSTANCES CONTROL ACT (TSCA): This product is currently being managed underthe R&D Exemption to TSCA, (40 CFR 720.36). It is not on the TSCA list orthe Confidential TSCA list. This product is provided in small quantities onlyfor Research and Development and must be used underthe direct supervision ofa Technically Qualified Individual as defined by 40 CFR 720.3 (ee). The recipient ofthis material is expected to conduct its research and development activities in accordance with Prudent Laboratory Practices and to inform employees who may be exposed to this material ofthe hazards associated therewith. For use by original recipient only. EUROPEAN INVENTORY OF EXISTING COMMERCIAL CHEMICAL SUBSTANCES (EINECS) AND EUROPEAN LIST OF NOTIFIED CHEMICAL SUBSTANCES (ELlNCS): This product is currently being managed in the European Community under Article 13 paragraph 2. ofSubstances Directive 

## (67/548/EEC) as a substance intended solely for purposes ofscientific research and development carried out under controlled conditions. It is not on the EINECS or ELINCS lists. This product is to be used within the European Community only for research and development as permitted by EC and other appropriatejurisdictional regulations. CANADIAN ENVIRONMENTAL PROTECTION ACT(CEPA): This product is currently being managed in Canada underthe provisions ofthe Research and Development Special Use Category as a substance intended solely for purposes ofscientific research and development carried out under controlled conditions. It is not on the DSL or NDSL. This product is to be used only for research and development as permitted by the CEPA and other appropriatejurisdictional regulations.


# Ga0023

### Current Issue: December 18, 

## 2002 ] PAGE60F6 

### OtherJurisdictions: This product has not been notified. registered or listed on any new chemical substance list orsimilar document outside the United States. Use ofthis product in countries otherthan those listed above shall be in accordance with the appropriatejurisdictional regulations concerning new chemical substances. SECTION 16: OTHER INFORMATION HAZARD RATINGS: NFPA HAZARD CLASS 2 

### HMIS HAZARD CLASS 3 

### HAZARD RANKING Health Hazard: 2 Health Hazard: 1 

### 0- Least Flammability: 1 Flammability: 1 1 - Slight Reactivity: 2 Reactivity: 2 2 - Moderate Other: - PPE: 3 - High 4 - Extreme 2 • 

### National Fire Protection Association (NFPA) Rating identifies the severity ofhazards of material during a fire (i.e. "on fire").
3. 

### Hazardous Materials Identification System (HMIS) National Paint and Coatings Association ratings apply to product "as Packaged". Information on this form is furnished solely for the purpose ofcompliance with the Occupational Safety and Health Act of 1970, and applicable regulations, and should not be used for any other purpose. No warranty express or implied, ofmerchantability, fitness for purpose, orofany other nature regarding this data orthe product is made herein. 

### Issue date: October 18. 

## 2002 Revision date: December 18, 

## 2002 Written by: [ 

### ] Reviewed by: Awaiting independenttoxicological review. Approved by: [ 

### ]



# G00024PHYSICAL AND CHEMICALPROPERTIESWORKSHEET Toassist EPA'sreview ofphysical and chemical properties data, please completethe following worksheetfor datayou provide and include it in thenotice. Identitythe property measured, the page ofthe notice on which the property appears, the value ofthe property, the units in whichtheproperty is measured (as necessary), and whether or not the property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties should be for the neat ( I00%pure) chemical substance. Properties that are measured for mixtures or formulations should be sonoted (% PMN substance in

# ->. You are not required to submitthis worksheet; however, EPA strongly recommends that you do so, as it will simplity review and ensure that confidential information is properly protected. You should submit this worksheet as a supplement to your submission oftest data. This worksheet is not a substitute for submission oftest data. Property Mark Page Value 

### [ 

## 1 (Xl if number provided (b) (c) (a) Physical state ofneat substance X 

### 25 (s) x (I) (g) Vaporpressure @Temperature 

### °C Torr Density/relativedensity X 

### 25 1.2 g/cm3 Solubility @ Temperature 

### °C

Solvent gil Solubility in water @ Temperature 

### 25 

### °C X 

### 25 <1% gil:, Meltingtemperature 

### °C Boiling/ sublimationtemperature(a) 

### 0.2 torrpressure X 

### 25 30 

### °C Spectra Dissociationconstant Particle size distribution Octanol / water partition coefficient Henry's Law constant Volitalizationfrom water Volitalization from soil pH@ concentration 100% X 

### 25 

### -7 Flammability Explodability Adsorption / coefficient Other

- SpecifY Estimate (MorE) N/A

M

M

M

I !

M I 

# I I



# &00025 ATTACHMENT 

### PAC!I! 13PHYSICAL AND CHEMICALPROPERTIES WORKSHEET To assist EPA's review ofphysical and chemical properties data, please complete the following worksheet for datayou provide and include it in the notice. Identify the property measured, the page ofthe notice on which the property appears, the value ofthe property, the units in which the property is measured (as necessary), and whether or not the property is claimed as confidential. The physical state ofthe neat substance should be provided. These measured properties should be for the neat (100% pure) chemical substance. Properties that are measured for mixtures orformulations shoUld be so noted (%PMN substance**in~.** You are not required to submitthis worksheet; however, EPA strongly recommends that you do so, as it will simplify review and ensure that confidential information is properly protected. You should submitthis worksheet as a supplementto your submission oftest data. This worksheet is not a substitute for submission oftest data. Property Mark Page Value lMeasured 0 Confidential 

### [ J (X) if provided (a) Physical state ofneat substance X Vaporpressure 

### @ Temperature 

### °C Density/relative density X Solubility 

### @ Temperature 

### °C

Solvent Solubility in water (QJ Temperature 

### 25 

### °C X Meltingtemperature Boiling 

### I sublimationtemperature(a) 0.2 torrpressure X Spectra Dissociationconstant Particle size distribution Octanol 

### Iwaterpartition coefficient Henry's Law constant Volitalization from water 

### I Volitalization from soil pH(QJ concentration ]00% X Flammability Explodability Adsorption 

### I coefficient Other

- Specify number (b) (c) 

### 26 (s) 

### x (I)



### 26 1.4



### 26 <1%
I 

### 26 30



### 26**~7** (g)
Torr g/cm3

gIL**~** 

### °C °C Estimate Mark (X) (Mor E) (d) 

### N/A

M

M

M

M



## G00026 ATTACHMENT P'I)"(;I& 

### 13

### Consolidated PMN TS-IHlSlM 

### Attachment#1 PartI,SectionB.l 

### PMNp.4 

# asInventoryExpertServiceReports



# G00027

# ern InventoryExpertService

A division of theAmerican Chemical Society Phone: 800-631-1884. 614-447·3600 (ext. 2308) 

## Fax: 614-447·3747 E-mail: answers@cas.org



### INVENTORY EXPERT SERVICE REPORT

IES-ORDER NUMBER: [ ] REGISTRY NUMBER:[ 

# l

CA INDEX NAME 

# [ ]

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

# o If this box is checked, CAS has made correction(s) marked in red to your IES order. Please make the same correction(s) to your PMN before submitting it to the EPA.



# G00028 2540 Olentangy River Road P.O. Box 3343 Columbus. Ohio 43210..()334

# ern InventoryExpertService

A division oftheAmerican Chemical Society Phone: 800-631-1884, 614-447-3600 (ext. 2308) Fax:614w447-3747 E-mail: answers@cas.org



### INVENTORY EXPERT SERVICE REPORT

IES-ORDER NUMBER: 

# ]
REGISTRY

# NUMBER:~ 

# ]

CA INDEX NAME 

# [ ]

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

# o If this box is checked, CAS has made correction(s) marked in red to your lES order. Please make the same correction(s) to your PNN before submitting it to the EPA.



## <;00029

2540 Olencangy River Road P.O. Sox 3343 Columbus. Ohio 43210-0334

### Consolidated PMN TS-IHlSIM 

### Attachment#2 PartI, SectionsA andB 

### PMNp.4



# caOO2:0

# G00031

# G00032

### Consolidated PMN TS-IHISIM 

### Attachment#3 PartI, SectionC.l 

### PMNp.7

### ConsolidatedPMN TS·IHlS1M 

### Attachment#4
PartII, SectionA.t.c 

### PMNp.8

I I

I

I

,

I



# CGG035

### Consolidated PMN TS-IHISIM 

### Attachment#5
PartII, SectionA.1 

### PMNp.8



# G00036

### Consolidated PMN TS"lHlSlM 

### Attachment#6
PartII, SectionA.l.d PMNp.8**GOOO~'7**TS-1H1S1M



# G00038

### Consolidated PMN TS-IHISIM 

### Attachment#7
PartII, SectionsA.2 andA.3 

### PMNp.9



# G00039

### TS-1H1S1M 

## Attachment#7

Worker Activity

(I) CBI

(2) Protective Equipment! Engineering

Controls
(3) Binding Option Mark

(4) Physical form(s) and

% new chemical substance (5) Binding Option Mark

(6) CBI



## m Number of Workers Exposed
(8) CBI

(9) Maximum duration CBI

(12) Hrs/day

(10) Days/year (11) X X X X

X X X X

X X X X

I X X X X

X X X X

X X X X

### TS-IHISIM



### Attachment

## #7

Release Number (1) AmountofNew Substance Released CUI 

### (3) Media ofRelease 

### (4) Control Technology and Efficiency CDI 

### (6) (2a) (2b) (Sa) Bindi ng Mark (5b) X X

X X

X X

X X

X X

X X

X X

X X
(7) Mark (X)the 

# o POTW provide name(s) below: destination(s) of releases to water. CBI 

### r--­ 

# o Navigable waterway 

# o Other Provide NPDES # CBI



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

### Consolidated PMN 

### TS-IHISIM 

### Attachment#8
PartII, SectionA.l.d 

### PMNp.8



## GGOO<12TS-IHlSlM



# L C00043

### Consolidated PMN TS-IHISIM 

### Attachment#9
PartII,Sections A.2 andA.3 

### PMNp.9



## COOO,14

### TS-IHISIM 

### Attachment#9

. 

### [ 

### ] Worker Activity

(1) CBI



### (2) Protective Equipmentl Engineering

Controls 

### (3) Binding Option Mark



### (4) Physical formes) and

0/

.. new chemical substance 

### (5) Binding Option Mark



### (6) CBI



### (7) Number of Workers Exposed 

### (8) CBI

(9) Maximum duration CBI

(12) Hrs/day

(10) Days/year (11) X X X X

X X X X

X X X X

X X X X

X X X X

X X X X



# G()0045 I

### TS-IHlSlM


## Attachment#9

[Release

Number (J) AmountofNew Substance Released CDI (3) Media ofRelease 

### (4) Control Technology and Efficiency CHI (6) (2a) (2b) (58) Bind;

### ~a~ (5b) X X

X X



## I X X

X X

X X

X X

X X

X X
(7) Mark (X) the 

# o POTWprovide narne(s) below: destination(s) of releases to water. CBI 

### r--­ 

# o Navigable waterway 

# o Other Provide NPDES # CBl

• D Mark (X)this box jfyou attach a continuation sheet.

### Consolidated PMN TS-IHISIM 

### Attachment#10
PartII, SectionA.1 PMNp.8



# 000047

### Consolidated PMN 

### TS-IHISIM 

### Attachment#11


### PartII SectionA.1.d 

### PMNp.8



# G00048TS-IHISIM2 

### Consolidated PMN TS-IHISIM 

### Attachment#12
PartII, SectionsA.2 andA.3 

### PMNp.9



# GOC050

### TS-IHISIM 

## Attachment#12

[ 

## 1 Worker Activity

(1) CBI

(2) Protective Equipment! Engineering

Controls


### (3) Binding Option Mark



### (4) Physical form(s) and

0/

.. new chemical substance 

### (5) Binding Option Mark



### (6) CBI



### (7) Number of Workers Exposed


### (8) CBI



### (9) Maximum duration CBI

(12) HrsJday

(10) Days/year (11) X X X X

X X X X

X X X X

X X X X

X X X X

X X X X

I



# 000051

### TS-IHISIM



### Attachment

### #12

[ ]

Release Number (1) AmountofNew Substance Released CDI (3) Media ofRelease (4) Control Technology and Efficiency eBI (6) (2a) (2b) (Sa) Bindi 

# n, Mark (5b) X X

X X

X X

X X

X X

(7) Mark: (X) the 

# o POTW provide name(s) below: destination(s) of releases to water. CBl

- 

# o Navigable waterway 

# o Other ProvideNPDES # eB!

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



# 000052

### Consolidated PMN 

### TS-IHISIM 

### Attachment#13 PMNp.ll 

### Pollution PreventionInfonnation



# G000534 

### Consolidated PMN 

### TS-IHISIM



# 000054

### Consolidated PMN TS-IHlSlM 

### Attachment#14
OtherInformation 

### ToxicologicalData on Two StructurallySimilar Compounds (Ames Tests)**G~0055**

### MLH 0011984844 

### CONFIDENTIAL



### SUBJECT CHEMICAL#1 BACTERIAL MUTATION ASSAY

Sponsor 

### Research Laboratory HuntingdonLife Sciences Ltd., P.O. Box 2, Huntingdon, Cambridgeshire, 

# I l

PE186ES, 

# L ENGLAl'rD. Report issued 9 December 1998 

### Page 1of23 

## G00056

### I'v1LH 0011984844 CONTENTS Page COrvrPLlANCEWITH GOOD LABORATORYPRACTICE STANUARDS
............... . 

### 3 QUALnY ASSURANCE STATEMENT
.............. 4 

### SUMMARy
..................................................... 5
INTRODUCTION
...................................................................................................................... 6
TESTSUBSTANCE
................................................................................................................... 8
EXPERllvIENTAL PROCEDURE
............................................................................................. 9
ASSESSMENT OF RESULTS
.................................................................................................. 13
MAlNTINANCE OFRECORDS
.............................................................................................. 14
RESULTS
.................................................................................................................................... 15
CONCLUSION.
.......................................................................................................................... 15
REFERENCES
............................................................................................................................ 16
TABLES 1. SubjectChemica] 

### #1 Mutation test 1
........................................................................ 

### 17 

### 2. Positive control compOlmds

- Mutationtest 1
................ ......... 

### 19 3. Subject Chemica] #1

- Mutationtest 2
......................... 

### 20 4. Positive control compounds

- Mutationtest 2
..... 

### 22
APPENDIX Historical control data
. 

### 23

2 

## 000057

### MLH 001/984844 COMPLIANCEWITH GOOD LABORATORY PRACTICE STANDARDS

The study described 

### in this report was conducted 

### in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid. The UK Good Laboratory Practice Regulations 1997 (Statutory Instrument No 654). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MCICHEM(98)17. EC Council Directive 

### 871l8IEEC of l8 December 1986 (Official JournalNo L 

### 15129)



# .1
..**f).g,~** .

# .t.j.1.8
.. Date DepartmentofGenetic Toxicology, Huntingdon Life Sciences Ltd
..



## G00058

### MLH 0011984844 QUALITY ASSUR;\NCE STATEMENT
The foIloVving have been inspected orauclited in relation to this study Study Phases Inspected Date ofInspection Date ofReporting Protocol review 3November 1998 26 November 1998 Process Based Inspections Preparation & 

### labelling } 5June 1998 } 5 June 1998 Agar preparation Test material preparation } 30 June 1998 } 10 Ju1y 1998 Dosing Plate counting 3 July 1998 10 July 1998 Record keeping 9july 1998 10 July 1998 S9 prepamtion 4

- 10 November 1998 4

- 10 November 1998 Report 23 November 1998 26 November 1998
Protocol: An 

### audit ofthe protocol for this study was conducted and reported to the Study Directorand Company Management as inclicated above, Process based inspections: At or about the time this study 

### was in progress inspections and audits ofroutine and repetitive procedures employed on this type of study were carried out. Thesewere conducted and reported to appropriate Company Management as indicated above, Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the rawdata,

"(**~".-"',** .1

__ .::>. IG,qb



### .........**~~~** ........ " .. 

### Vanessa E. Grant, F.LA.T., Dip. R.Q.A., Date Quality Assurance Unit Head, Department of Quality Assurance, Huntingdon Life Sciences Ltd.

4 

## cn0059

### MLH 0011984844 

### SUMMARY

In this in vitro 

### assessment of the mutagenic potential of Subject Chemical #I, histidine dependent auxotrophic mutants ofSalmonella t}phimurium (strains TA1535, TAl TA98 and TAJOO) and a tryptophan dependent mutant of Escherichia coli (strain CM89l) were exposed to the test substance diluted in dimethyl sulphoxide, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence ofliver preparations from Aroclor 1254-induced rats (S9 mix), The first was a standard plate incorporation assay, the second involved a pre-incubation stage, Dose levels ofup to 5000 /-Lg/plate were tested in 

### the mutation tests, This is the standard limit dose reconunended in the regulatory guidelines this assay follows, Other dose levels used were a series ofca half-loglO dilutions ofthe highest concentration, No signs oftoxicity were observed towards the tester strains ill 

### eithermutation test, No evidence ofmutagenic activity was seen at any dose level ofSubject Chemical #

### I in either mutation test, The concurrent positive controls demonstrated the sensitivity ofthe assay and the metabolising activity ofthe liverpreparations, 

### It is concluded 

### that, when tested in dimethyl sulphoxide, Subject Chemical #1 shows no evidence of mutagenic activity in this test system,



### 5 

# C00060

### MLH 0011984844 INTRODUCTION

TIlls report describes a study designed to assess the mutagenic potential of Subject Chemical #

### I ill 

### a bacterial system The study was conducted in compliance with the following guidelines OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guidelines 471. EEC Annex to Directive 92/69fEEC. 

### (1992) Part B Methods for Determination of Toxicity,B.13. Other effects Mutagenicity 

### Escherichia coli

- Reverse Mutation Assay. OJ No. L 

### 383 A, 157. EEC Annex to Directive 92/69fEEC. (1992) 

### Part B . Methods for Determination of Toxicity, B.14. Other effects

- Mutagenicity: 

### Salmonella typhimurium

- Reverse Mutation Assay. OJ No. L 383 A, 

### 160. US EPA 40 CFR Part 799 (1997) Toxic Substances Control Act Test Guidelines

­ Sub-section 799.9510, TSCA bacterial reverse mutation test. Federal Register, Vol. 62, No. 158. The method described was also designed to comply with ICH (1996 & 1997), and followed the recommendations ofthe United Kingdom Environmental Mutagen Society (Gatehouse et al 1990). 

### The 

### in vitro technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, Maron and Ames 1983) enables the mutagenic effect ofa test substance to be determined by exposing specially selected strains of

### Salmonella typhimurium to the test substance. Normally 

### S. typhimurium is capable of synthesising the essential amino acid, histidine, but the mutant strains used in this test are incapable of this function. When these strains are exposed to a mutagen, reverse mutation to the original histidine independent form takes place in a proportion ofthe population. These are referred to as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient medium. A techniquebased on similarprinciples has alsobeen described by Green (1984). This system employs mutant strains of 

### Escherichia coli which are incapable of synthesising the amino acid tryptophan requiredfor growth. The strains used carry additional mutations which render them more sensitive to mutagens. The S 

### typhimurium strains have a defective cell coat which allows greater permeability oftest substances into the celL All the strains are deficient in normal DNA repairprocesses. 

### In addition three ofthem possess a plasmid (pKt\l[1 01) which introduces an error-prone repair process, resulting in increased sensitivity to somemutagens. Many substances do not exert a mutagenic effect until the}' have been metabolised by enzyme systems not available in the bacterial cell. Therefore the bacteria and test substance are incubated in 

### both the absence and presence ofa supplemented liver fraction (S9 mix) prepared from rats previously treated with a substance (Aroelor 1254) known to induce a high level ofenzymic activity.



### 6 

## COOOS!

### IVILH 001/984844 The protocol was approved by Huntingdon Life Sciences Management on 14 September 1998, the Sponsor on 5 October 1998 and 

### by the Study Director on 27 October 1998. The experimental phase ofthe study was conducted between 28 October and 9 November 1998

7 

## CC3062

### Identity
Alternative name:
Chemical name:

CAS number: Appearance: Storage conditions:
Batchnumber: Expiry date:
Purity:
Date received: 

### MLH 001/984844 TEST SUBSTANCE
Subject Chemical #

### 1 

# [ 

# J ]

Clear colourless 

### liquid
Room temperature 

### in the dark, desiccated
CP077-62B
Stated to be stable for 

### 3 months on 

### the 5 October 1998
>99.5%
22 October 1998



### : 8 : 

## C00063MLH 0011984844 EXPERIMENTAL PROCEDURE

BACTERIAL STRAINS The following strains were used:­ 

### S typhimunum TAI535 

### hisG46 rfa uvrB 

# s. typhlmurium 

### TAI537 

### hisC3076 rfa uvrB S. 

### typhlmurium TA98 

### hlsD3052 'fa uvrB pKMIOI 

### S. typhimurium TAIOO 

### hisG46 

## ria uvrB pKMIOI 

### E. coli CM891 WP2 

### trp uvrApKM101 The strains of S. 

### typhimurium were obtained from Professor 

### B.X Ames, University of California, Berkeley, California, USA. The strain of

### E. coli was obtained from the National Collections of Industrial and Marine Bacteria. Aberdeen, Scotland. Batches ofthe strains were obtained from master stocks held in liquid nitrogen. The test batches were aliquots ofnutrient broth cultures and were stored at 

### -80°C. Dimethyl sulphoxide (DMSO) was added to the cultures at 

### 8% vivas a cryopreservative. Each batch of frozen strain was tested, where applicable, for cell membrane penneability 

### (rfa mutation), sensitivity to 

### UV light and the pKM101 plasmid which confers resistance to ampicillin. The responses ofthe strains to a series ofdiagnostic mutagens were also assessed. For use in tests an aliquot of frozen culture was added to 25 

### ml ofnutrient broth (Merck No.2) and incubated, with shaking, at 37°C for 10 hours. These cultures provided at least 2 

### x 109 cells per ml which were measured photometrically.

POSITIv""E CONTROLS 

### In the absence ofS9 mix Identity: N-Ethyl-N'-nitro-N-nitrosoguanidine Supplier: Sigma Chemical Batch number: 67F-3700 Appearance: Pale yellow crystalline powder Solvent: DMSO Concentration: 5 !J.g/plate for strain T A

### IS35 3 flg/plate for strain TAl00 2 llg/plate for strain CM891

9 

## G30064MLH 001/984844 Identity Supplier: Batchnumber Appearance Solvent: Concentration: Identity: Supplier: Batch number: Appearance: Solvent: Concentration: In the presence of

### S9 mix Identity: Supplier Batch number: Appearance: Solvent: Concentration: Identity: Supplier: Batch nmnber: Appearance: Solvent: Concentration: 9-Arrunoacridine Sigma Chenucal 35H1408 Yellow powder DMSO 30 tJ.g/plate for strain TAl537 2-Nitrofluorene Aldrich Cherllical Company 75108 Beige powder DMSO 1 t-tg/plate for strain TA98

2-Arrunoanthracene Aldrich Chemical Company 0013406 Green powder DMSO 2 tJ.g/plate for strain TAIS35 10 fJ.g/plate for strain CM891 Benzo[a]pyrene Aldrich Chemical Company 05915DY Yellow powder DMSO 5 tJ.gJplate for strains TA1537, TA98 andTAIOO
PREPAR>\TION OFS9 FRACTION Species: Sex: Strain: Source: Age: Weight: Diet: 

### Rat Male Sprague-Dawley derived Harlan Olac 

### Ltd 7-8 weeks <300 

### g SDS R+M no.I (modified) nuts S9 fraction was prepared from a group of

### co 20 animals. Mixed function oxidase systems in the rat livers were stimulated by Arodor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mglkg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 0.15 M KCI (3 ml KCl : 1 g liver) before being transferred to an ultra-Turrax homogeniser. Following preparation, the homogenate was centrifuged at 9000 

### g for 10 minutes. The supernatant fraction
10 

# GOOOS5

### MLH 001/984844 (59 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy ofeach batch of S9 fraction was tested in a bacterial mutation assay with the mutagenic precursors 7,12­ dimethylbenzanthracene and 2-aminoanthracene before use. The sterility was also checked.



### PREPARATION OF S9 MIX S9 mix contained: 59 fraction (10% v/v), MgCI" (8 mM), KCI (33 mM), sodium orthophosphate buffer pH 7.4 (l00 mM), glucose-6-phosphate (5 mM). NADP (4 mM). All the cofactors were filtersterilised before use.



### SELECT!ON OF SOLVENT Priorto commencing testing, the solubility ofthe test substance was assessed at 50 mglml in dimethyl sulphoxide, in which 

### it dissolved. Water was not assessed as information provided by the Sponsor indicated that the test substance was insoluble in this solvent. Therefore dimethyl sulphoxide was used as the solvent for this study.



### MUTATION TEST PROCEDURE Firsttest Thetest substance was added to cultures ofthe five tester strains at seven concentrations separated by ca half-loglo intervals. The highest concentration of Subject Chemical #1 tested was 50 mglml in the chosen solvent, which provided a final concentration of5000 /lg/plate. This is the standard limit dose recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, dimethyl sulphoxide. The appropriate positive controls were also included. 

### An aliquot of0.1 ml ofa 10 hour bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed 

### in glass bottles. 

### An aliquot of 100 III ofthe test solution was added, followed immediately by 2 

### m1 ofhistidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Each petri dish was individually labelled with a unique code corresponding to a sheet, identifying the dish's contents. Three petri dishes wereused for each dose level. Plates were also prepared withoutthe addition ofbacteria in order to assess the sterility of the test substance, S9 mix and phosphate buffer. All plates were incubated at 37°C for ca 72 hours. After this period the appearance ofthe background bacterial lawn was examined and revertant colonies counted using a Seescan automated colony counter. 

### Any toxic effects ofthe test substance would be detected by a substantial reduction in revertant colony counts or by the absence ofa complete background bacterial lawn. 

### In the absence ofany toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed a lower concentration may be chosen. It should be ensured that if a lower concentration was chosen, signs of bactenal inhibition are present at the top concentration. Ideally a minimum ofthree non-toxic concentrations should be obtained.
II 

# GOOOSG

### MLH0011984844 Second test As a clear negative response was obtained in the first test. a variation to the test procedure was used for the second. The variation used was the pre-incubation assay in which the bottles were incubated at 37°C for 30 minutes with shaking before the addition ofthe agar overlay 5000**~g/plate** 

### was agam chosen as the top concentration but onl} five dose levels were used as it was known from the first test that the test substance was non-toxic

STABILITY AND FORMULATION ANALYSIS The stability 

### ofthetest substance and ofthe test substance in 

### the solvent were not determined as part of this study
..Analysis ofachieved concentration was not perfonned as part ofthis study.

12 

## oa0067

### :MLH 001/984844 

### ASSESSMENT OF RESLTLTS

For a test to be considered valid the mean ofthe solvent control revertant colony numbers for each strain should be in the range stated in the appropriate Standard Operating Procedure. These ranges are based on the laboratory's historical control values
..Also, the positive control compounds must cause at least a doubling ofmean revertant colony numbers overthe solvent controL The mean number ofrevertant colonies for all treatment groups is compared with those obtained for the solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria: (a) Iftreatment with a test substance produces an increase in revertant colony nwnbers ofat least twice the concurrent solvent controls, vvith some e-v'idence ofa positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence ofS9 mix, it is considered to show evidence ofmutagenic activity in this test system. (b) Iftreatment with a test substance does not produce reproducible increases ofat least 1.5 times the concurrent solvent controls, in either mutation test, it is considered to show no evidence of mutagenic activity in this test system. 

### In addition the test data were subjected to analysis to determinethe statistical significance ofany observed increases in revertant colony numbers. The statistical procedures used were analysis of variance followed by Durmett's test (testing at p:s; 0.01 level), as described by Mahon et al 

### (1989). The biological significance ofany observed statistically significant increases were also considered.

13 

# &00068

### :tvlLH 001/984844 MAINTENANCE OF RECORDS

All experimental data arising from the study (including documentary raw data, records and other materials: collectively defined as the "materials") \'Vill remain the property ofthe Sponsor Huntingdon Life Sciences shan retain the materials in its archive for a period offive years from the date ofissue ofthe final 

### report. After such time, the Sponsor will be contacted and their adVIce sought on the return, disposal or further retention ofthe materials. If requested, Huntingdon Life Sciences will continue to retain the materials, subjectto a reasonable fee being agreed with the Sponsor.

14 

## G00069

### MLH 001/984844 

### RESULTS

The mean number of revertant colorues, together with the individual plate counts for Subject Chemical # I obtained in the first mutation test with the tester strains are shovvn in Table 1. Positive control mutability checks are shown in Table 2. No toxicity was observed towards thetester strains. The mean number ofrevertant colonies. together with the individual plate counts for Subject Chemical #I obtained in the second mutation test with the tester strains are shown in Table 3. Positive control mutabilIty checks are shown in Table 4 No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Subject Chemical #1 at any dose leveL in the presence or absence of S9 mix, in either mutation test. VVhen the test data was statistically analysed using Dunnett's test a few single significant values were shown (marked in results tables) However, none of these values were greater than 1.5 times the concurrent negative control and all fell well 

### within the laboratories historical negative control range. As there was also no evidence ofany dose relationship in the observed significant increases they were not seen as having anybiological significance. The mean revertant colony counts for the solvent controls were within the historical range. The concurrent positive controls demonstrated the sensitivity ofthe assay and the metabolising activity of the liver preparations by causing increases overdouble the concurrent solvent controL



### CONCLUSION

It is concluded that, when tested in dimethyl sulphoxide. Subject Chemical #1 shows no evidence of mutagenic activity in this test system.

15 

## G00070

### lv1LH 001/984844 REFERENCES
AMES, B.N., McCANN, J., and YAMASAKI. E. (1975) Methods for detecting carcinogens and mutagens WIth the 

### Salmonella/mammalian mIcrosome mutagenicity test. 

### Mutation Research, 31,347. GATEHOUSE, D.G., ROWLAND, I.R., WILCOX, P., CALLANDER, R.D. and FORSTER, R. (1990) Bacterial mutation assays in: KIRKLAND, DJ. (Ed.). 

### UKEMS Sub-committee on GuidelinesjorMutagenicity Testing. Report. Part I revised. Basic Mutagenicity Tests: UKEMS Recommended Procedures, p.13. Cambridge University Press, Cambridge. GREEN, M.H.L. (1984) Mutagen testing using lrp+ reversion in 

### Eschen'chia coli in KILBEY, B.L, LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). 

### Handbook ojMutagenicity Test Procedures. Second edition, p.161. Elsevier Science Publishers BV, Amsterdam. ICH (1996) Genotoxicity: Guidance on Specific Aspects ofRegulatory Genotoxicity Tests. ICH (1997) Genotoxicity: A Standard Battery of Genotoxicity Testing ofPharmaceuticals. MAHON, G.AT., GREEN, M.H.L., MIDDLETON, B., MITCHELL, I.de G., ROBINSON, W.D. and TWEATS, OJ. (1989) Analysis ofdata from microbial colony assay in: 

### KIRKLAND, D.J. (Ed.) 

### UKEMS Subcommittee 

### on Guidelines for Mutagenicity Testing Report, Part Ill. Statistical Evaluation of MutageniCity Test Data, p.26. Cambridge University Press, Cambridge. MARON, D.M. and AMES, B.N. (1983) Revised methods for the 

### Salmonella mutagenicity test. 

### Mutation Research, 113, 173.

16 

## G00071

### JVlLH 0011984844 TABLE 1 Mutation Test 1 Subject Chemical #1 - revertant colony counts obtained per plate using bacterial strains TA1535, TA1537 and TA98
Strain Dose level Liver Mean SD Individual revertant (j.lg/plate) S-9 revertant colony counts colony counts TA 1535 5000
- 15 1 .7 17, 14, 14 1500

- 14 4 .6 11, 11, 19

500

- 17 1.5 16, 17, 19 150
- 14 2.1 12, 13, 16

50

- 15 0.6 14, 15, 15 15

- 14 2.5 14, 12, 17

5
- 14 2.3 17, 13, 13 Solvent
- 13 1.5 15, 12, 13 5000 + 14 1.2 13, 15, 13 1500 + 13 3.1 16, 12, 10

500 + 18 2.9 21, 16, 16 150 + 15 0.6 15, 15, 14

50 + 14 0.0 14, 14, 14 15 + 18 1.0 17, 18, 19

5 + 18 2.1 16, 17, 20 Solvent + 14 2.1 16, 13, 12 TA 1537 5000
- 8 0.6 8, 9, 8 1500
- 10 2.0 10, 12, 8

500
- 8 1.5 6, 9, 8 150
- 9 3.5 7, 7, 13

50 

### - 13 1.2 14, 12, 12 15
- 6 2.0 4, 6, 8

5
- 9 1.5 7, 10, 9 Solvent 

## - 11 1.0 10, 12, 11 5000 + 8 1.0 7, 8, 9 1500 + 9 2.5 7, 9, 12

500 + 11 1.0 11, 10, 12 150 + 12 2.5 12, 10, 15

50 + 11 1.5 9, 11, 12 15 + 9 3.2 8, 7, 13

5 + 11 1.2 10, 10, 12 Solvent + 14 3.6 17, 15, 10 TA 98 5000
- 23 3.8 19, 25, 26 1500
- 22 1.5 22, 21, 24

500
- 26 3.1 23, 25, 29 150
- 23 2.1 25, 21, 22

50 - 20 1.5 19, 22, 20 15
- 22 2.0 22, 24, 20

5
- 27 3.5 31, 25, 25 Solvent

- 25 5.8 22, 22, 32 5000 + 23 2.3 22, 26, 22 1500 + 27 4 .2 32, 26, 24

500 + 27 2.5 27, 30, 25 150 + 22 1.0 22, 23, 21

50 + 31 0.6 31, 31, 30 15 + 27 4.5 22, 27, 31

5 + 24 1. "7 25, 25, 22 Solvent + 28 4 .5 33, 28, 24 Absence + Presence SD Standard deviation

17 

## 000072

### lv1LH 001/984844 TABLE 1 (continued) Mutation Test 1 Subject Chemical #1 revertant colony counts obtained

perplate using bacterial strains TA100 and CM891
Dose level Liver Mean SD Individual revertant Strain (llg/plate) 5-9 revertant colony counts s 5000**13~1** 145, 130, 119 TA 100 

## - ** 131 1500 147 14.5 132, 147, 161 ** 500 107 3.5 109, 103, 109 

## - 150 107 95, 117, 109 11 1 50 ll2 114 111, 110 2 1 15 4.2 , 137, 129 ** 134

- 5 ll7 9.1 107, 121, 124

- Solvent 7.5 92, 107, 101 100

CM 891 5000 1500

500 150

50 15

5 Solvent 5000 1500

500 150

50 15

5 Solvent 5000 1500

500 150

50 15

5 Solvent + + + + + + + +

-



## -

-



## - + + + + + + + + 

### i 114 100 III 107 113 106 115 120 234 333 322 327 330 294 316 290 261 362 361 357 348 371 365 324 9.7 103, 116, 122 6.1 93, 105, 101 2.1 109, 112, 113 5.1 113, 103, 106 6.1 120, 109, 110 5.0 105, ll1, 101 2.3 116, 116, 112 9.0 121, 129, 111 41.5 276, 193, 234 27.5 341, 302, 355 21. 9 338, 331, 297 21. 4 318, 351, 311 15.3 318, 324, 347 50.3 281, 252, 350 25.5 344, 310, 294 13.6 285, 279, 305 48.1 316, 237, 229 25.7 369, 334, 384

7.0 354, 360, 368 16.3 344, 375, 351 15.0 363, 348, 333 43.6 413, 326, 374 19.5 351, 356, 387 19.9 310, 316, 347 Absence 

### + Presence SD Standard deviation Significant at p:::;O.O 1using Dunnett's test.

18 

## G00073

### MLH 0011984844 TABLE 2 

### Mutation Test 1 Revertant colony counts obtained per plate with positive controls
Strain Compound Dose level ().lg/plate) Liver 5-9 Mean revertant:

colony 5D Individual revertalCt colony counts counts TA TA TA TA CM 1535 1537 98 100 891 ENNG 9 AC NF ENNG ENNG :; 30

1 3 2 -

-

- 141 92 1 433 1877 6 

# ° 6. 20.7 25.2 20.0 135, 141, 147 87£ 100, 90 143 184 1 159 45 437 406 1 , 6, 1898 TA TA TA TA CM 1535 1537 98 100 891 AA BlalP B[a]P B[al£' Af'.. 2 5 5 5 10 .,. .,. + + 86 39 139 601 1527 14.8 8.0 34.7 32.1 21. 9 70, 99, 90 31, 39, 47 99, 157, 161 584, 581, 638 1511 , 1552, 1518 Absence + Presence SD Standard deviation ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-Aminoacridine NF 2-Nitrofluorene AA 2-Aminoanthracene B[aJP Benzo[a

### Ipyrene

19

### :tvILH 0011984844 

### TABLE 3 Mutation Test 2 Subject Chemical #1

- revertant colony counts obtained per plate using bacterial strains TA1535, TA1537 and TA98
Str-ain Dose level Liver Mean SD Individua-1 revertant (]lg/plate) S-9 revertant colony counts colony counts TA 1535 5000 15 4.2 12, 20, 14 1500
- 13 1.5 12, 15, 13

500
- 18 3**~** 1 21, 15, 19 150
- 14 2. 12, 12, 17

50
- 15 2.3 16, 16, 12 Solvent

- 18**1~2** 19, 19, 17 5000 + 15 1.2 14, 16, 14 1500 + 16 2.0 16, 14, 18 500 + 18 5.6 24, 17, 13 150 + 15 0.0 15, 15, 15

50 + 16 0.6 16, 17, 16 Solvent + 20 2.3 19, 19, 23 TA 1537 5000
- 8 2.9 10, 10, 5 1500 

### - 9 3.8 13, 7, 6

500 7 0.6 7, 7, 6 150 8 7, 6, 12

50 3.2 8 3.5 10, 4, 10 Solvent 10 4.7 6, 8, 15 

## - 5000 + 10 .1 8, 11, 12 1500 + 9 2 5 11, 6, 9

500 + 10 3.6 11, 6, 13 150 + 13 2.1 11, 15, 14

50 + 9 2.1 8 7, 11 Solvent + 12 5.5 , 9, 8 TA 98 5000 23 2.1 22, 25, 21 1500 

## - 24 5
.. 6 19, 30, 23

500
- 24 2.6 27, 23, 22 150 - 24 6. 19, 22, 31

50 

## - 23 5.1 17, 27, 24 Solver:t

- 23 3.1 26, 20, 22


### ., 5000 + 25 _.L-**~** 23, 24, 29 1500 + 26 3.2 25, 24, 30

500 + 24 2.6 23, 22, 27 150 + 28 3.2 30, 29, 24

50 + 26 4.7 24, 31 Solvent + 28 

### i 4.4 26, , 33 i Absence 

### + Presence SD Standard deviation

20 

## G00075

### l\1LH 001/984844 TABLE 3 

### (continued) Mutation Test 2 

### Subject Chemical #1

- revertant colony counts obtained

per plate using bacterial strains TAl00 and 

### CM891
Strain Dose level (Ilg/plate) 

## I Liver S-9 Mean revertant

colony counts SD Individual revertant colony counts
TA 100 5000 1500

500 150

50 Solvent 

### -

-



## -

- ** 123 105 109 108 106

98 9.5 7.1 :J.6 2.1 4.0 7.2 117, :34, 18 106, 97, III 109, 109, 108 106, 109, 110 105, 110, 102 90, 102, 103 5000 1500

500 150

50 Solvent + +**~** 

# .. 

# .. + 118 ::'16 125 120 138 108 14.8 2.0 4.6 9.7 12.7 16.6 102, 114, 128, 131, 124, 106, 122, 131 116, 118 120, 128 112, 118 148, 143 93, 126 CM 891 5000 1500

500 150

50 Solvent 

## -

-



# -

-



# -

- 159 186 207 189 169 184 5.0 16.1

6.0 14.5 23.7

5.8 160, 168, 207, 204, 154, 177, 164, 191, 201, 175, 196, 187, 154 199 213 188 156 187 5000 1500

500 150

50 Solvent + +
+ + + + 150 198 209 174 166 172 14.0 7.6 3.6 12.1 17 .0 11.0 134, 195, 212, 17:J, 157, 168, 156, 207, 205, 165, 186, 184, 160 193 210 188 156 163 Absence + Presence SD Standard deviation 

### Significant at

### psO.OI using Dunnett's test.



# 000076 21

### Iv1LH 001/984844 TABLE 4 Mutation Test 2 Revertant colony counts obtained per plate with positive controls
Strai:1 Compou:1d Dose level (]Jg/plate) Liver S-9 Mean revertant

colony counts SD Individua.i revertant colony counts
TA 1535 ENNG 5 230 9.2 238, 232, 220 TA 1537 9 AC 30**~** 442 9.1 438, 4Cl5, 452 TA 98 NF 1 - 89 6.1 84, 96, 88 TA 100 ENNG 3 437 18.7 4 431, 422 CM 891 ENNG 2 185
. 92.6 18 , 1910, :744 TA 1535 AA 2 + 447 61.1 404, 517, 420 TA 1537 B[a]P 5 + i 59 17 .7 50, 47, 79 TA 98 B[a]P 5 + 287 28. 303, 254, 305 T}l. 100 B[a]P 5 + 454 30.4 429, 446, 488 CM 891 AA 

### Ie 

## - I 2094 I 1.2 2095, 2093, 2093 

### Absence + Presence SD Standarddeviation 

### ENNG N-EthyI-N'-nitro-N-nitrosoguanidine 9AC 9-Aminoacridine NF 2-Nitrofluorene AA 2-Aminoanthracene B[aJP Benzo[a]pyrene



### 22 

## GG0077

### rvrLH 001/984844 APPENDIX Historical control data

Presented below are the historical control data from thetwo years priorto commencing this study 

### Dimethyl sulphoxide solvent controls Strain TA1535 TA1537 TA98 TAIGO CM891 S9 mix + + + + + Minimum 9 10 5 6 17 20 75 76 134 101 Maximum 21 22 14 16 33 34 158 154 376 373 Mean 14.8 16.2 9.9 11.2 24.5 26.5 116.1 122.5 225.8 208.0 Values 154 162 151 162 152 163 157 167 78 77


### Positive controls Strain TAl535 TAl537 TA98 TAlOO CM891 S9 mix + + + + + Minimum 80 31 75 28 68 71 178 159 436 351 Maximum 2489 2171 2094 326 681 747 2564 2137 2221 2192 Mean 470.2 156.5 497.l 74.4 304.1 192.3 496.0 452.7 1466.4 1256.8 Values 224 235 39 233 221 239 228 246 III 114

23 

## 000078

### CONFIDENTIAL MLH 002/984845



### SGBJECT CHEMICAL#2 BACTERIALMUTATION ASSAY

Sponsor Research Laboratory 

### Huntingdon Life SciencesLtd., P.O. Box 2, Huntingdon, Cambridgeshire, PE186ES, ENGLAt'ID. 

### Report issued 9December 1998 Page 1of23 

## G00079

### MLH 002/984845 

### CONTENTS Page 

### COMPLIANCEWITH GOOD LABORATORY PRACTICE STANDARDS
......... ,....... . 3



### TABLES 

### QUALm'ASSURANCE STATEMEl\TT
..."
.. 4 

### SlJMl\1ARY ,.......,...',
..,...............,..,.........,....,..... "
......,',,,
..,....,,.,'.,.,"'
.......,.............."
...,.."
..,..... 5


### INlRODUCTION
...,.......,........,.....,....................",.,
.....,.."
..... , 6


### TEST SUBSTANCE
...... "
....'.,
....,..,.......,.,''
........,.......,.....,..............,..........'
..........",.,
..,.....,...... 8


### EXPERIMENTAL PROCEDURE
............,............,'
....,....,....,...........,....................................... 9


### ASSESSMENT OFRESULTS
..............,..................,...............,..............,.......,.......................... 13


### MAINTENANCE OF RECORDS ,.,...........,..........,......,.........,................,.."
...........................,.. 14


### RESULTS
.........,.....,""',
....,....,.......,.."
..,..."
......"
...........,..'
...........,........."
......,...,...,..,.....,........., 15


### CONCLUSION
.,..............,...'
......'
..,.,',
....,.....,..'
...,..,...................,...........,..,.....,............,.....,....., 15
REFERENCES
................"
......................". ,,'
......................"
...,...."
.......,..,.........',
.....,',.,....,...... 16

1. Subject Chemical#2

- Mutation test L.,
.. 

### 17 2. Positive control compolU1ds

- Mutationtest 1
........ 19 3, Subject Chemical #2

- Mutationtest2
..,.. "" 

### 20 4, Positive control compounds

- Mutation test2 22 

### APPENDIX Historical control data
...,...........,.............

: 2 

## G00080

### 1vILH 002/984845 

### COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

The study described 

### in this report was conducted 

### in compliance with the following Good Laboratory Practice standards and I 

### consider the data generated to be valid. The UK Good LaboratoryPractice Regulations 1997 (Statutory Instrument No 654). OEeD Principles of Good Laboratory Practice (as 

### revised in 

### 1997), 

### ENVIMCICHEM(98)17. EC Council Directive 

### 87/181EEC of 18 December 1986 (Official Journal No L 

### 15/29)



# ..**~.F-"?~:dpL/.!'J.~** J Kite Date Study Director, Department ofGenetic Toxicology, Huntingdon Life Sciences Ltd
..

3 : 

## G00081

### :MLH 002i984845 

### QUALITY ASSURANCE STATEMENT
The follo\',ing have been inspected or audited in relatIOn to this study Study Phases Inspected Date ofInspection Date ofReporting Protocol review 3 :'\Iovember 1998 26 November 1998 Process Based Inspections Preparation & 

### labelling } 5 June 1998 } 5 June 1998 Agar preparation Test material preparation } 30 June 1998 } 10 July 1998 Dosing Plate counting 3 July 1998 10 July 1998 Record keeping 9July 1998 10 July 1998 S9 preparation 4 - 10 November 1998 4 - 10 November 1998 Report 25 November 1998 26 November 1998
Protocol: 

### An audit ofthe protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study 

### was in progress inspections and audits ofroutine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above. Report Audit: This report has 

### been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.
The methods, procedures and observations were found to be accurately described and the reported results to reflect the raw data
.

--,**~~** \9'="t**~** 

### ......

# ~.~ ...........

...........**~** ••••**~** ....... w •••••••••••••• 

### Vanessa E. Grant, F.LA.T., Dip. R.Q.A., Date Quality Assurance Unit Head, Department ofQuality Assurance, Huntingdon Life Sciences Ltd.



### : 4 

## C00082

### :MLH 002/984845 SUMMARY



### In this 

### in vitro assessment of the mutagenic potential of Subject Chemical #2, histidine dependent auxotrophic mutants of

### Salmonella typhimurium (strams TA1535. TA1537, TA98 and TAlOO) and a tryptophan dependent mutant of

### Escherichia cob (stram CM891) were exposed to the test substance diluted in dimethyl sulphoxide, which was also used as a negative control Two independent mutation tests were performed in the presence and absence ofliver preparations from ArocIor 1254-induced rats (S9 

### mix). The first was a standard plate incorporation assay, the second involved a pre-incubation stage. Dose levels ofup to 5000 IJ-g/plate were tested in the mutation tests. This is the standard limit dose recommended in the regulatory guidelines this assay follows Other dose levels used were a series of

### co half-loglO 

### dilutions ofthe highest concentration. No signs oftoxicity were observed towards the tester strains in eithermutation test. No evidence ofmutagenic activity was seen at any dose level ofSubject Chemical #2 in either mutation test. The concurrent positive controls demonstrated the sensitivity ofthe assay and the metabolising activity ofthe liverpreparations. It is concluded 

### that., when tested in dimethyl sulphoxide, Subject Chemical #2 shows no evidence of mutagenic activity in this test system.



### :5: 

## G00083

### MLH 002/984845 

### INTRODUCTION

This report describes a study designed to assess the mutagenic potential of Subject Chemical #2 in a bacterial system. The study was conducted in compliance \A.lth the following guidelines: OECD Guideline for the Testing 

### of Chemicals. (1997) Genetic Toxicology Bacterial Reverse MutationTest, Guidelines 471. EEC Annex to Directive 92/691EEC. 

### (1992) Part B : Methods for Determination of Toxicity, R13. Other effects

- Mutagenicity: Escherichia coli

- 

### Reverse Mutation Assay O.J 

### No. L 

### 383 A, 

### 157. EEC Annex to Directive 92/69IEEC. 

### (1992) Part B : Methods for Determination of Toxicity, B.14. Other effects

- Mutagenicity: Salmonella typhimurium

- 

### Reverse Mutation Assay. OJ 

### No. L 

### 383 A, 160. US EPA 40 CFR Part 799 (1997) Toxic Substances Control Act Test Guidelines

­ Sub-section 799.9510, TSCA bacterial reverse mutation test. Federal Register, Vol. 

### 62, No. 158. The method described was also designed to comply with lCH (1996 & 

### 1997), and followed the recommendations ofthe United Kingdom Environmental Mutagen Society (Gatehouse et al 1990). 

### The in vitro 

### technique described by Ames and his co-workers, (Ames, McCann and Yamasaki 1975, Maron and Ames 1983) enables the mutagenic effect ofa test substance to be determined by exposing specially selected strains ofSalmonella typhimurium 

### tothe test substance. Normally S typhimurium 

### is capable ofsynthesising the essential amino acid, histidine, but the mutant strains used in this test are incapable of this function. 'When these strains are exposed to a mutagen, reverse mutation to 

### the original histidine independent form takes place in a proportion ofthe population. These are referred to as revertants, and are readily detected by their ability to grow and form colonies on a histidine deficient 

### medium. A technique based on similar principles has also been described by Green (1984). This system employs mutant strains of Escherichia coli 

### which are incapable of synthesising the amino acid tryptophan required for groVvth. The strains used carry additional mutations which render them more sensitive to mutagens. The S lyphimurium 

### strainshave a defective cell coat which allows greater permeability oftest substances into the celL All the strains are deficient in normal DNA repair processes. In 

### addition three ofthem possess a plasmid 

### (pKMIOI) which introduces an error-prone repair process, resulting in increased sensitivity to some mutagens. Many substances do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial celL Therefore the bacteria and test substance are incubated in both the absence and presence ofa supplemented liver fraction (S9 mix) prepared from rats previously treated with a substance (Aroelor 1254) known to induce a high level ofenzymic activity.



### 6 :

### MLH 002/984845 The protocol was approved by Huntingdon Life Sciences Management on 14 September 1998, the Sponsor on 5 October 1998 and by 

### the Study Duector on 27 October 1998. The experimental phase ofthe study was conducted between 28 October and 9November 1998.



### 7 

## G00085

### Identity
Alternative name:
Chemical name:

CAS number: Appearance: Storageconditions:
Batchnumber: Expiry date: Purity:
Datereceived: 

### I'v1LH 002/984845 TEST SUBSTANCE
Subject Chemical #2

## [~ 

# J [ ]

Clearcolourless liquid
Room temperature 

### in the dark, desiccated
CP077-62A
Statedtobe stable for 

### 3 months on 

### 5 October 1998
>99.5%
22 October 1998



# G00086 8

### 1v1LH 002/984845 

### EXPERIMENTAL PROCEDURE

BACTERIAL STRAINS The following strains were used

### S. typhimurium TAl535 

### hlSG46 ria uvrB 

### S. typhimurium TAl537 

### hisC3076 ria uvrB 

### S. typhimurium TA98 

### hisD3052 ria uvrB pKMlOI 

### S. typhimurium TAlOO 

### hisG46 ria uvrB pKMIOl 

### E coli CM891 WP2 

### trp uvrApKMlOI The strains of 

### S. typhimurium were obtained from Professor RN Ames, University of California, Berkeley, California, USA. The strain of

### E coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland. Batches ofthe strains were obtained from master stocks held in liquid nitrogen. The testbatches were aliquots ofnutrientbroth cultures and were stored at -80°C. Dimethyl sulphoxide (DMSO) was added to the cultures at 

### 8% vivas a cryopreservative. Each batch of frozen strain was tested, where applicable, for cell membrane permeability 

### (ria mutation), sensitivity to 

### UV light and the pKMIOl plasmid which confers resistance to ampicillin. The responses ofthe strains to a series ofdiagnostic mutagens were also assessed. For use in tests an 

### aliquot offrozen culture was added to 25 

### ml of nutrient broth (Merck No.2) and incubated, with shaking, at 37°C for 10 hours. These cultures provided at least 2 

### x 109 

### cells per 

### ml which were measured photometrically.



### POSITIVE COl\'TROLS In the absence ofS9 mix Identity: N-Ethyl-N'-nitro-N-nitrosoguanidine Supplier Sigma Chemical Batch number: 67F-3700 Appearance: Pale yellow crystalline powder Solvent: DMSO Concentration: 

### S flg/plate for strain TAIS35 3 flg/plate for strain TAIOO 2 flg/plate for strain CM891



### : 9 

# G00087

### 1vfLH 002/984845 Identity Supplier Batch number: Appearance Solvent: Concentration: Identity: Supplier: Batchnumber: Appearance: Solvent: Concentration: In the presenceofS9 mix Identity: Supplier: Batch number: Appearance: Solvent: Concentration: Identity: Supplier: Batchnumber: Appearance: Solvent: Concentration: 9-Amlnoacridine Sigma Chemical 35H1408 Yellow powder DMSO 30 f.Lg/plate for stram T A153 7 2-Nitrofluorene Aldrich Chemical Company 75108 Beige powder DMSO I 

### f.Lg/plate for strain TA98

2-Aminoanthracene Aldrich Chemical Company 0013406 Green powder DMSO 2 lJ.g/plate for strain TA1535 10 f.Lg/plate for strain CM89 I 

### Benzo[a ]pyrene Aldrich Chemical Company 05915DY Yellowpowder DMSO 5 IJ.g/plate for strains TA1537, TA98 and TAl00

PREPARATlON OF S9 FRACTION Species: Sex: Strain Source: Age: Weight: Diet: Rat Male Sprague-Dawley derived Harlan Olac Ltd 7-8 weeks <300 

### g SDS R-+-M no. 

### 1(modified) nuts S9 fraction was prepared from a group of ca 

### 20 animals. Mixed function oxidase systems 

### in the rat livers were stimulated

### by Aroclor 1254, administered as 

### a single intra-peritoneal injection in Arachis oil at a dosage of 500 rnglkg bodyweight. 

### On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The follo'W1.ng steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 

### 0.15 M KCl (3 ml 

### KCl : I 

### g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction
10 

## {}00088

### MLH 002/984845 (59 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy ofeach batch of 59 fraction was tested in a bacterial mutation assay \-vith the mutagenic precursors 7.12­ dimethylbenzanthracene and 2-aminoanthracene before use. The sterility was also checked



### PREPARATION OF S9 MIX 59 mix contained: S9 fraction (10% v/v), MgCI: (8 mM), 

### KCl (33 mM). 

### sodium orthophosphate buffer pH 7.4 (100 roM), 

### glucose-6-phosphate (5 mM), 

### NADP (4 mM). 

### .tVI the cofactors were filtersterilised before use.



### SELECTION OFSOLVENT Prior to commencing testing, the solubility ofthe test substance was assessed at 50 mglml in dimethyl sulphoxide, in which it dissolved. Water was not assessed as information provided by the Sponsor indicated that the test substance was insoluble in this solvent. Therefore dimethyl sulphoxide was used as the solvent for this study.



### MUTATION TEST PROCEDURE Firsttest The test substance was added to cultures ofthe five tester strains at seven concentrations separated by ca 

### half-Ioglo intervals. The highest concentration ofSubject Chemical #2 tested was 50 mglml in the chosen solvent, which provided a final concentration of5000 IJ.glplate. This is the standard limit dose recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, dimethyl sulphoxide. The appropriate positive controls were also included. An aliquot of0.1 ml ofa 10 hour bacterial culture and 0.5 ml S 9 mix or 0.5 ml 

### 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 100 IJ.I ofthe test solution was added, followed immediately by 2 ml ofhistidineltryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml 

### minimal agar. Each petri dish was individually labelled with a umque code corresponding to a sheet. identifying the dish's contents. Three petri dishes were used for each dose level. Plates were also prepared without the addition ofbacteria in order to assess the sterility of the test substance, S9 mix and phosphate buffer. luI 

### plates were incubated at 37°C for ca 

### 72 hours. After this period the appearance ofthe background bacterial lavm was examined and revertant colonies counted using a Seescan automated colony counter. Any toxic effects ofthetest substance would be detected by a substantial reduction in revertant colony counts or by the absence ofa complete background bacterial lawn. In 

### the absence ofany toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed a lower concentration may be chosen. It 

### should be ensured that if a lower concentration was chosen, signs ofbacterial inhibition are present at the top concentration. Ideally a minimum ofthree non-toxic concentrations should be obtained.

11: 

## 000089

### l'v1LH 002/984845 

### Secondtest 

### As a clear negative response was obtained in the first test. a variation to the test procedure was used for the second. The variation used was the pre-incubation assay in which the bottles were incubated at 37°C for 30 minutes WIth shaking before the addition ofthe agar overlay. 5000**~g/plate** 

### was chosen as the top concentration but only five dose levels were used as It was known from the first test thatthe test substance was non-to;-;:lc.



### STABILITY AND FORMULATION ANALYSIS The stability ofthe test substance and ofthe test substance in the solvent were not determined as part of this study. Analysis ofachieved concentration was not performed as partofthis study.



## GO{)090 : 12

### :MLH 002/984845 

### ASSESSMENT OF 

### RESULTS

For a test to be considered vahd the mean ofthe solvent control revertant colony nwnbers for each strain should be in the range stated ill

### the appropriate Standard Operating Procedure, These ranges are based on the laboratory's historical control values, 

### Also, the positive control compounds must cause at least a doubling ofmean revertant colony nwnbers over the solvent control. The mean nwnber ofrevertant colonies for all treatment groups is compared with those obtained for the solvent control groups, The mutagenic activity of a test substance is assessed by applying the following criteria: (a) Iftreatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence ofa positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence ofS9 mix, it is considered to show evidence ofmutagenic activity in 

### this test system, (b) Iftreatment with a test substance does not produce reproducible increases ofat least 1.5 times the concurrent solvent controls, in either mutation test, it is considered to show no evidence of mutagenic activity in this test system. 

### In addition the test data were subjected to analysis to determine the statistical significance ofany observed increases in revertant colony numbers. The statistical procedures used were analysis of variance followed by Durmett's test (testing at p::::: 0.01 level), as described by Mahon et al 

### (1989). The biological significance ofany observed statistically significant increases were also considered.



## 000091 : 13

### MLH 002/984845 MAINTENANCE OF RECORDS



### All experimental data arising from the study (including documentary raw data, records and other materials: collectively defined as the "matenals") will 

### remain the property ofthe Sponsor. Huntingdon Life Sciences shall retain the materials m its archive for a period offive years from the date ofissue ofthe final report. After such time, the Sponsor will be contacted and their advice sought on the return, disposal or further retention ofthe materials. If

### requested, Huntingdon Life Sciences will continue to retain the materials, subject to a reasonable fee being agreed with the Sponsor.

14 : 

## G00092

### IvlLH002/984845 RESULTS

The mean number ofrevertant colorues. together with the individual plate counts for Subject Chemical #2 obtained in the first mutation test with the tester strains are shown in Table 1. Positive control mutability checks are shown in Table 2. No toxicity was observed towards the tester strains. The mean number ofrevertant colonies, together with the individual plate counts for Subject Chemical #2 obtained in the second mutation test with the tester strains are shown in Table 3. Positive control mutability checks are shown in Table4. No substantial increases in revertant colony numbers of any of the tester strains were observed following treannent with Subject Chemical #2 at any dose level, in the presence or absence ofS9 mix, in either mutation test. When the test data was statistically analysed using Dunnett's test a few single significant values were shown (marked in results tables). However, none of these values were greater than 1.5 times the concurrent negative control and all fell well 

### v,lthin the laboratories historical negative control range. As there was also no evidence ofany dose relationship in the observed significant increases they were not seen as having any biological significance. The mean revertant colony counts for the solvent controls were within the historical range. The concurrent positive controls demonstrated the sensitivity ofthe assay and the metabolising activity of the liverpreparations by causing increases over double theconcurrent solventcontrol.



### CONCLUSION

It is concluded that, when tested in dimethyl sulphoxide, Subject Chemical #2 shows no evidence of mutagenic activity in this test system.



# C00093 IS

### MLH 002/984845 

### REFERENCES
AMES, B.N
.. McCANN, 

### L and YAMA.SAKL E. 

### (1975) Methods for detecting carcinogens and mutagens with the 

### Saimonellalmammalian rrucrosome mutagenicity test, 

### Mutation Research, 31, 347, 

### GATEHOUSE, D.G., ROWLAND, l.R., WILCOX, P., CALLANDER, R.D. and FORSTER, R. 

### (1990) Bacterial mutation assays in: KIRKLAND, OJ. (Ed.). 

### UKEMS Sub-committee on Guidelinesfor Mutagenicity Testing. Repon. Pan1revised. Basic Mutagenicity Tests: UKEMS Recommended Procedures, p.13. Cambridge University Press, Cambridge. GREEN, M.H.L. (1984) 

### Mutagen testing using 

### trp+ reversion in 

### Escherichia coli in KILBEY, BJ., LEGATOR, M., NICHOLS, W. and RAMEL, C. (Eds.). 

### Handbook of Mutagenicity Test Procedures. Second edition, p.161. Elsevier Science Publishers BV, Amsterdam. ICH (1996) Genotoxicity: Guidance on Specific Aspects ofRegulatory Genotoxicity Tests. ICH (1997) Genotoxicity: A Standard Battery ofGenotoxicity Testing of Pharmaceuticals. MAHON, GAT., GREEN, M,H.L, MIDDLETON, B" MITCHELL, I.de G., ROBINSON, W.O, and TWEATS, OJ, (1989) Analysis ofdata from microbial colony assay 

### in: KIRKLAND, OJ, (Ed,) 

### UKEMS Subcommittee on Guidelines for Mutagenicity Testing. Report. Part 

# m, Statistical Evaluation of Mutagenicity TestData, p.26. CambridgeUniversity Press, Cambridge. MARON, O.M. and AMES, B.N. (1983) Revised methods for the 

### Saimonella mutagenicity test. 

### Mutation Research, 113, 

### 173.



## C00094 16

### MLH 002/984845 

### TABLE 1 Mutation Test 1 Subject Chemical #2 - revertant colony counts obtained per plate using bacterial strains TA1535, TA1537 and TA98
Dose level Liver Mear! SD 

## I Strain Individual revertant (jlgiplate) S-9 revertant colony counts
TA 1535 5000 13 4.4 18, 11, 10 

## - 1500 15 15, 14, 17 1.5 500 

## - 14 4.4 19, 12, 11 150 12 1.2 13, 11, 11 

## - 50 

## - 17 0.0 17, 17, 17 15
- 10 1.2 11, 11, 9

5 15 1.5 15, 17, 14 

## - Solvent
- 13 1.5 15, 12, 13 5000 16 + 2.0 14, 18, 16 1500 + 16 3.6 20, 15, 13

500 + 15 14, 16, 14 1.2 150 + 12 0.6 12, 12, 13

50 + 16 0.6 16, 17, 16 15 + 12 3.6 11, 9, 16

5 17 + 1.0 17, 16, 18 Solvent + 14 2.1 16, 13, 12 TA 1537 5000 7 6, 8, S 1.2 1500 7 0.0 7, 7 7

500 13 0.6 12, , 13 150 

## - 11 0.6 11, 11 12

50 

## - 10 3.1 7, 9, 15
- 12 2.3 13, 13, 9

5
- 10 2.9 7, 12, 12 Solvent
- 11 1.0 10, 12, 11 5000 + 10 2.0 8, 12, 10 1500 + 10 4.2 13, 5, 11

500 + 9 2.1 7, 10, 11 150 + 11 1.2 10, 10, 12

50 + 14 1.0 15, 13, 14 15 + 9 2.1 7, 11, 10

5 + 10 0 0 10, 10, 10 Solvent + 14 3.6 17, 15, 10 TJl. 98 5000
- 23 1.5 23, 25 1500 25 0.6 2 , 25, 24

500 24 5.5 18, 28, 27 150 27 6.4 23, 23, 34

50
- 23 4.9 29, 20, 21 15 

## - 20 2 0 18, 22, 20

5
- 20 2.5 17 22, 20 Solvent
- 25 5.8 22, 32 5000 + 21 3.6 24, 17 1500 + 27 2.1 2 26, 25

500 + 25 3.8 29, 23, 22 150 + 26 6.0 32, 20, 25

50 + 22 .5 22, 24, 21 15 + 24 0.6 24, 24, 23

5 + 25 :2.5 25 27, 22 Solvent + 28 4 .5 28, 24 Absence + Presence SD Standard deviation

17 

# G00095 

# i

### tvlLH 002/984845 

### TABLE 1 (continued) Mutation Test I Subject Chemical #2

- revertant colony counts obtained

per plate usingbacterial strains TAl00 and CM891
Strain Dose level Liver Mean SD Individual revertant ().1g/plate) $-9 revertant colony coun1:s colony counts

...... 'fA 100 5000 134 6.7 131, 142, 130 1500 

### - 114 2.3 111, 115, 115


# .. 500 138 13 .3 129, 131, 153 150 115 14.8 99, 128 119

50 :25 11.3 131, 1 112 15 

# - :00 10.8 97, 91

5 ** 145 15.€ 161, , 143 Solvent 100 7.5 92, 107, 101 

# - 5000 + 109 7.8 104, 105, 118 1500 + 127 26.8 120, 105, 157

500 + 119 35.0 79, 141 150 + :11 6.1 114, 1 , 104

50 + 132 22.9 12O, 158, 117 15 + 104 13 .1 114, 108

5 + 119 42.6 83, 166 Solvent + 120 9.0 121, 111 CM 891 5000 300 52.4 360, 262, 279 1500 293 9.8 299, 299, 282

500 358 14.7 371, 361, 342 150 287 22.7 306, 262, 294

50 272 35.8 252, 313, 250 15 

### - 241 13.6 233, 257, 234

5 264 lC2 267, 249, 277 Solvent 290 13.6 285, 279, 305 5000 + 323 38.4 316, 364 1500 + 330 33.5 317, 3 305

500 + 393 12.5 399, 402 150 + 338 2.6 337, 341

50 + 301 22.1 299, 280, 324 15 + 290 27.2 265, 319, 286

5 + 329 30.6 300, 361 Solvent + 324 19.9 310, 347 Absence + Presence SD Standard deviation Significant at p;:;O.Ol using Dunnett's test.

18 

## CG0096

### MLH 002/984845 

### TABLE 2 Mutation Test 1 Revertant colony counts obtained per plate with positive controls
Strain Compound Dose level (pg/plate) Liver S-9 Mean revertanT:

colony SD Individual revertant colony C:OU:1,"'.::s i
counts 1'A TA 1'A TA CM 1535 1537 98 100 891 ENNG 9 AC NF ENNG ENNG 5 30

1 3 2 -

-

- 141 92 162 433 1877 6.0 6.8 20.7 25.2 20.0 135, 141, 147 87, 100, 90 143, 184, 159 456, 437, 406 1858, 1876, 1898 TA l'JI. l'JI. TA CM 1535 1537 98 100 891 All. B[a]P B[ajP B[ajP AA 2 5 5 5 10 + + + + + 86 39 139 601 1527 14.8 8.0 34.7 32.1 21. 9 70, 99, 90 31, 39, 47 99, 157, 161 584, 581, 638 1511 , 1552, 1518 Absence 

### + Presence SD Standard deviation ENNG N-Ethyl-N'-nitro-N-nitrosoguanidine 9 AC 9-Aminoacridine NF 2-Nitrofluorene AA 2-Aminoanthracene BlalP Benzola]pyrene

19 

## G00097

### MLH 002/984845 TABLE 3 Mutation Test 2 Subject Chemical #2 - revertant colony counts obtained per plate using bacterial strains TA1535, TAl537 and TA98
Strain Dose level (Ilgi plate) Liver S-9 Mean revertant SD Individual revertant colony counts
TA 1535

TA 1537

TA 98 5000 1500

500 150

50 Solvent 5000 1500

500 150

50 Solvent 5000 1500

500 150

50 Solvent 5000 1500

500 150

50 Solvent 5000 1500

500 150

50 Solvent 

### sOCIa

. 500

500 150

50 Solvent 

## -

-

-

-



# - + + + + + +



### -



# -

-

-

- + + + + + +



# -

-

-

- + + + + + + 14 12 16 14 17 18 18 17 14 18 14 20

7 8 9 9 7 10 10 10 11 11 13 12 19 28 27 26 25 23 30 25 23 27 26 28 2.9 1.7 1.7 6.4 2.6 1.2 1.2 0.6 4.6 2.1 2.6 2.3 2.5 1.5 3.2 1.5 2.1 4.7 3.5 3.5 2.3 3.0 1.0 5.5 4.7 6.7 4.7 3.2 1.0 3.1 2.5 5.1 1.5 1.5 3.6 4.4 16, 11, 16 10, 13, 13 15, 15, 18 21, 10, 10 14, 19, 18 19, 19, 17 19, 17, 19 16, 17, 17 13, 10, 19 17, 20, 16 17, 13, 12 19, 19, 23 7, 5, 10 6, 8, 9 10, 5, 11 9, 11, B 5, 9, 6 6, 8, 15 lC1, 14, 7 14, 8, S 12, 12, S 11, 8, :4 13, 12, 14 :8, 9, 8 23, 14, 21 32, 31, 20 22, 31. 29 28, 27, 22 26, 25, 24 26, 20, 22 30, 27, 32 31, 21, 24 24, 21, 23 27, 29, 26 22, 29, 27 26, 25, 33 Absence + Presence SD Standard deviation



## G00098 20

### 1v1LH 002/984845 

### TABLE 3 (continued) Mutation Test2 Subject Chemical #2 revertant colony counts obtained

per plate using bacterial strains TAtoO and CM891
Strain Dose level ().lg/plate) Liver $-9 Mean revertant SD Individual revertant colony counts
Tl', :00 5000 1500

500 150 50 Solvent 5000 1500

500 150

50 Solvent -

-

-



# -

-
+ + + + + + ** 120 128 112 108 111

98 110 146 121 128 116 108 12.5 12.3

8.4 7.6 7.8 7.2 7.2 9.5 8.1 8.4 17.7 16 .6 106, 124, 130 119, 142, 123 :07, 108, 122 99, 113, 111 106, 107, 120 90, 102, 103 102, 114, 115 141, 157, 140 130, 116, 116 124, 123, 138 128, 125, 96 106, 93, 126 CM 891 5000 1500

500 150

50 Solvent 5000 1500

500 150

50 Solvent 

### -

-

- + + + + + + 175 203 195 209 202 184 189 194 201 189 185 172 13.1 30.5

8.5 15.3 16.4

5.8 2.6 44.1 29.7 15.7

9.5 lLO 165, 173, 186, 222, 198, 177, 186, 151, 176, 201, 175, 168, 190, 201, 196, 2:2, 188, 187, 19:, 191, 234, 171, 194, 184, 171 234 203 192 220 187 190 239 194 194 185 163 Absence Presence SD Standard deviation Significant atpsO.Ol using Dunnett's test.



## ca0099 2l

### MLH 

### 002/984845 TABLE 4 Mutation Test 2 Revertant colony counts obtained per platewith positive controls
Strain Compound Dose level (1l9 Iplate) Liver: $-9 Mean reverta:;t

cOlony SD Individual ::-evertant colony counts I counts 'fA 'fA ":A ":A eM 1535 1537 98 100 891 ENNG 9 AC NF ENNG ENNG 5 30

1 3 2 

## -



## -

- 230 442

89 437 1851 9.2 9.1 6.1 18.7 92.6 238, 232, 220 438, 435, 452 84 96, 88 4 , 431 1 422 1898, 1910, 1744 TA TA 'fA 'fA eM 1535 1537 98 100 891 AA B[a]P B[a]P B[a]!' AA 2 5 5 5 10 + + + + + 447 59 287 454 2094 61.1 17.7 28.9 30.4

1.2 404, 517, 420 50, Q7, 79 303, 254, 305 429, 446, 488 2Q 95, 2093, 2093 

### Absence + Presence SD Standarddeviation 

### ENNG N-Ethyl-N'-nitro-N-nitrosoguanicline 9AC 

### 9-Aminoacridine NF 

### 2-Nitrofluorene AA 

### 2-Aminoanthracene B[a]P Benzo[a]pyrene



### : 22 

## (J00100

### MLH 002/984845 APPENDIX Historical control data

Presented below are the historical control data from the two years prior to commencing this study. Dimethyl sulphoxide solvent controls Strain 

### TAl535 TAIS37 TA9S TAIOO CM891 89 mix + + + + + Minimum 

### 9 10 5 6 17 20 75 76 134 101 Maximum 

### 21 22 14 16 33 34 158 154 376 373 Mean 14.8 16.2 9.9 11.2 24.5 26.5 116.1 122.5 225.8 208,0 Values 

### 154 162 151 162 152 163 157 167 78 77


### Positive controls Strain TA1535 TA1537 TA98 TA100 CM891 S9 mix + + + + + Minimum 

### 80 31 75 28 68 71 178 159 436 351 Maximum 

### 2489 2171 2094 326 681 747 2564 2137 2221 2192 Mean 470.2 156.5 497.1 74.4 304.0 192.3 496.0 452.7 1466.4 1256.8 Values 

### 224 235 39 233 221 239 228 246 111 

### 114

23 

## 000101